Note: Descriptions are shown in the official language in which they were submitted.
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1
LAUNDRY DETERGENT COMPOSITIONS COMPRISING A SACCHARIDE
GUM DEGRADING ENZYME
10
Field of the Invention
The present invention relates to laundry detergent compositions
2o comprising a saccharide gum degrading enzyme.
Background of the invention
Detergent compositions include nowadays a complex combination of
active ingredients which fulfill certain specific needs. In particular,
current
detergent formulations generally include detergent enzymes providing cleaning
and fabric care benefits and more specificaNy cellulase and amylase enzymes.
3o The efficiency of cellulytic enzymes, i.e. cellulases, in terms of textile
cleaning and harshness-reducing agent for fabrics has been recognized for some
time. The activity of cellulase is one in which cellulosic fibres or
substrates are
hydrolysed by the cellulase and depending on the particular function of the
cellulase, which can be endo- or exo- cellulase and the respective
hemicellulases. The cellulose structures are depolymerized or cleaved into
smaller and thereby more soluble or dispersible fractions. This activity in
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particular on fabrics provides a cleaning, rejuvenation, softening and
generally
improved handfeel characteristics to the fabric structure.
Amylases are known in the art to provide stain removal performance
benefits on naturally present or added starch containing food stainslsoils or
added as a finishing agent.
Food stains/soils represent the majority of consumer relevant stains/soils
and often comprise food additives. Neutraceuticals, acidulants, antioxidants,
~o preservatives, sweeteners, enzymes, thickener/stabiliser agents such as
hydrocolloids and emulsifiers are commonly used food additives. In particular,
the consumer demand for reductions in fat and calories is driving growth in
texturing agents as fat replacers. The market for hydrocolloid texturing /
stabiliser agents also called food gums, is expected to grow about 4% a year,
~5 xanthan gum growth should register gains of 6% to 8%/year and carrageenan
about 3%/year (Chemical week, June 19 (1996) pp32-34).
The term "gum" denotes a group of industrially useful polysaccharides
(long chain polymer) or their derivatives that hydrate in hot or cold water to
from
2o viscous solutions, dispersions or gels. Gums are classified as natural and
modified. Natural gums include seaweed extracts, plant extrudates, gums from
seed or root, and gums obtained by microbial fermentation. Modified
{semisynthetic) gums include cellulose and starch derivatives and certain
synthetic gums such as low methoxyl pectin, propylene glycol alginate, and
25 carboxymethyl and hydropropyl guar gum (Gums in Encyclopedia Chemical
Technology 4th Ed. Vol. 12, pp842-862, J. Baird, Kelco division of Merck). See
also Carbohydrate Chemistry for Food Scientists (Eagan Press - 1997) by R. L.
Whistler and J.N. BeMiller, Chap 4, pp63-89 and Direct Food Additives in Fruit
Processing by P. Laslo, Bioprinciples and Applications, Vol1, Chapter II,
pp313
30 325 (1996) Technomie publishing.
Some of these gums, such as xanthan gum (E 415, CEE number), gelian
gum {E416), guar gum (E412), locust bean (E410) and tragacanth (E413) are
widely used alone or in combinations in many food applications (Gums in ECT
35 4th Ed., Vol. 12 pp842-862, J. Baird, Kelco division of Merck). In
particular, guar
gum is often used in food as a thickener and a binder of free water in sauces
and
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salad dressings. Guar gum is also used as a binder of free water and
stabiliser in
ice cream and frozen desserts. Free water in ice cream mix causes a grainy
texture, ice crystals, poor meltdown properties and poor heat-shock resistance
in
the finished ice cream. The incorporation of a stabiliser containing guar gum
in
s quantities up to about 0.3% of the ice cream mix yields a smooth-textured,
chewy
product with slow-melting down properties and good heat shock resistance. It
is
also particularly suitable for flash pasteurisation because of its rapid
hydration
properties. Other food items that can be stabilised with guar gum because of
its
ability to bind water, are frozen foods, cheeses, pie fillings, icings and pet
foods.
o Other examples include : algin gums are known to be used in sherbet, canned
and fabricated food, tragacanth gums used in salad dressings, xanthan for
dairy
products and beverages. Gellans are found in icing, frosting and dairy
products
and locust bean and agar in ice creams.
~5 The specificity of these food gums is that they give a high to very high
viscosity solution when hydrated in water. Some of these gums such as guar,
algin, arabic, karaya, methyl cellulose locus bean gums are also used in the
paper industry and chosen for their high affinity for cellulosic fibres
(Industrial
gums by R. L. Whistler and J.N. BeMiller (Academic Press - 1973). Their
2o potential to flocculate clays and other inorganic materials such as calcium
salts,
are used in other applications such as water treatment. The high viscosity of
these food gums is desirable for all the above mentioned food and other
applications.
25 However, it has been surprisingly found that these food gums adsorb
strongly onto the cotton fibers of the fabric, thereby gluing the stains/soils
on the
fabric. This even when the gum is present at a very low level in the food
compositions, such as 0.01 % to 5%, more usually between 0.01 % to 0.8%.
3o It has been also surprisingly found that the capability of these food gums
to flocculate clays results in the dinginess and yellowing of the fabric. This
is
particularly important since, the overall performance of a detergent is judged
by
not only its ability to remove soils and stains but also its ability to
prevent
redeposition of the soils, or the breakdown products of the soils or of any
35 insoluble salt, on the article washed. Redeposition effects results in the
articles
being coated in an unseemly film, appearing streaked or being covered in
visible
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spots which remain intact at the end of the wash process. These residues build
up on the fabric leading to dinginess and yellowing.
As can be seen from the above, there is a continuous need to formulate
laundry detergent compositions which provide excellent overall cleaning
performance. Accordingly it is an object of the present invention to provide a
laundry detergent composition which delivers superior cleaning and whiteness
performance benefits, especially excellent food stain/soil removal, dingy
cleaning and whiteness maintenance.
0
The above objective has been met by formulating laundry detergent
compositions comprising a saccharide gum degrading enzyme.
It has been surprisingly found that the laundry detergent composition of
the present invention comprising a saccharide gum degrading enzyme, provides
excellent food stain/soil removal, dingy cleaning and whiteness maintenance
resulting from the hydrolysis of the food saccharide gums binding food or
clays
stains/soils to cotton fabrics. It has been further found that the performance
of
the laundry detergent compositions of the present invention is enhanced by the
2o addition of selected surfactants, another enzyme, a builder and/or a bleach
system.
GB2-169-393 describes a method for removing cellulose contaminant and
other vegetable contaminants from fabrics, using the conventional machinery
and
equipment of dye-house and finishing mills by treatment with an enzymatic
preparation containing celluloiytic and pectinolytic enzymes that allow for a
reduction of H2S04 concentration below 2% during fabric carbonisation.
W096/06532 relates to a composition capable of killing or inhibiting
3o growing microbial cells by means of a basic protein or peptide of
biological origin,
e.g. protamine or protamine sulphate. For certain bacteria or fungi, these
composition furhter comprise an oxidoreductase or cell-wall degrading enzyme
such as an endoglycosidase type li, a lysozyme and/or a chitinase.
W095/35362 describes to cleaning compositions, including laundry,
dishwashing and especially household cleaning compositions, comprising cell
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wall degrading enzymes having pectinases and/or hemicellulases and optionally
cellulases. These compositions are particularly suitable for removing stains
of
vegetable origin and soil and dirt having a similar structure. These plant
cell wall
degrading enzymes degrade the structural components of the plant cell walls
5 such as the structured polysaccharides (cellulose, hemicellulose, pectins)
and
encompass cellulolytic, pectin degrading and hemicellulose degrading enzymes.
A large number of plant cell wall degrading enzyme exist. Cellulolytic enzymes
have divided into three classes : endoglucanases, exoglucanases or
cellobiohydrolases and (i-glucosidases. A large number of enzymes are known to
o degrade pectins; examples are pectin esterase, pectin lyase, pectate lyase,
and
endo- or exo-polygalacturonase. In addition to these enzymes degrading the
smooth regions, enzymes degrading hairy regions such as rhamnogalacturonase
and accessory enzymes have also been found. A multitude of enzymes is
available to degrade the hemicellulose structures such as xylanase,
galactanase,
~ 5 arabinase, lichenase and mannanase.
However, the use of saccharide gum degrading enzymes for excellent
cleaning performance on cotton fabrics in laundry detergent compositions, has
never been previously recognised.
Summar~r of the invention
The present invention relates to laundry detergent compositions
comprising a saccharide gum degrading enzyme, providing excellent cleaning
performance on cotton fibers, especially food stain/soil removal, dingy
cleaning
and whiteness maintenance benefits.
3o Detailed description of the invention
An essential component of the laundry detergent compositions of the
present invention is a saccharide gum degrading enzyme. These enzymes are
able to hydrolyse non-starch non-cellulose food polysaccharides having a
viscosity higher than 800 cps at 1 % solution (Measured in water at
25°C,
Brookfield Synchro-Lectic viscosimeter at 20 rpm).
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It has been surprisingly found that the laundry detergent compositions of
the present invention provide excellent cleaning and whiteness performance and
especially significant food stain/soil removal benefits and dingy stain/soil
cleaning.
Without wishing to be bound by theory, it is believed that saccharide gum
degrading enzymes hydrolyse the food gums additives present in food
stains/soils, that glue the stains/soils to the cotton fibres. Indeed, it has
been
o found that these non-starch non-cellulose food polysaccharides have a high
affinity for the cotton fibres thereby bind the stains/soils to the fabric.
The
4
hydrolysis of these non-starch non-cellulose food polysaccharides release
therefore the stains/soils from the cotton textile.
Moreover, it has been surprisingly found that the laundry detergent
composition of the present invention provide significant dingy cleaning and
whiteness maintenance. Without wishing to be bound by theory, it is believed
that
the saccharides, being breakdown products of the partial cleaning of these
food
stains/soils by current detergent formulations, redeposit on the fabric and
react
2o with particulate soils such as clay compounds, leading to the dinginess of
the
cotton fabric. The saccharide gum degrading enzymes of the present invention,
hydrolyse the film of saccharides deposited onto the fabrics and thereby
prevent
the flocculation of these compounds with particulate soils.
Without wishing to be bound by theory, it is also believed that the
enzymatic action of the saccharide gum degrading enzymes of the present
invention renders the food and dingy stains/soils more accessible to the other
detergent components of the laundry detergent composition. Especially, it has
been found that the performance of the laundry detergent composition of the
so present invention is enhanced by the combination with a selected
surfactant,
another enzyme, a builder and/or a bleach system.
The enzymes of the present invention have a main or side activity on the
non-starch non-cellulose food polysaccharides having a viscosity higher than
800cps at 1 % solution, such as agar, algin, karawa, tragacanth, guar gum,
locus
beam, xathan and/or mixtures thereof.
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Examples of the industrial gums used separately of in combination as food
additives are
- Seed Gums such as Guar Gum, Locust Bean Quince seed Psylium, Flax seed
and Okra Gums, Tamarin, Larch Arabinogalactan;
- Plant exudates such as Arabic, Ghatti, Karaya, Tragacanth;
- Seaweed extracts such as Algin, Agar, Carrageenan, Fucoidan, Furcellaran,
and - Biosynthetic gums such as Xanthan.
Suitable enzymes for the purpose of the present invention have the following
main or side enzymatic activity
- Arabinases : Endo Arabanase (E.C. 3.2.1.99), such as endo a-1,5-
arabinosidase, exo Arabanase (E.C. 3.2.1.55), exo A (a-1,2; a-1,3)
arabinofuranosidase, exo B (a-1,3; a-1,5) arabinofuranosidase;
- (a-1,2; a-1,3) fucosidase, a-1,6-fucosidase (E.C. 3.2.1.127);
~5 - ~i-1,2-Galactanase, (3-1,3-Galactanase {E.C. 3.2.1.90), ~-1,4-
Galactanase, ~i-
1,6-Galactanase, Galactanase are a also called Arabino galactan galactosidase
(E.C. 3.2.1.89), a and ~i galactosidase (E.C. 3.2.1.22 & 23), (E.C. 3.2.1.102)
(E.C. 3.2.1.103)
- ~i-Mannosidase (3.2.1.25), a-Mannosidase (3.2.1.24), (i-1,2-Mannosidase, a
20 1,2-Mannosidase (E.C. 3.2.1.113) (E.C. 3.2.1.130), a-1,2-1.6 -Mannosidase
(3.2.1.137), ~i-1,3-Mannosidase (E.C. 3.2.1.77), (i-1,4-Mannosidase (E.C.
3.2.1.78), ~3-1,6-Mannosidase (E.C. 3.2.1.101), a-1,3-1,6-Mannosidase (E.C.
3.2.1.114), ~i-1,4-Mannobiosidase (E.C. 3.2.1.100),
- Glucuronosidase (E.C. 3.2.1.131 ), glucuronidase (E.C. 3.2.1.31 ). exo 1,2
or 1,4
25 glucuronidase,
- Agarase (E.C. 3.2.1.81), Carrageenase (E.C. 3.2.1.83), a-1,2-, Xanthan
lyase;
Poly(a-L guluronate) lyase , also called Alginase II (E.C. 4.2.2.11)
Preferred saccharide gums degrading enzymes are
- Mannosidase : (i-mannosidase, endo 1,4-~i-D mannosidase, endo 1,2-~i-D
mannosidase, and exo 1,3-~i-D mannosidase;
- Galactosidase : exo 1,6-~i-D-galactosidase and 1,3-~3-D-galactosidase;
- Glucuronidase, glucuronosidase and exo 1,2 or 1,4 glucuronidase;
- Arabinase : endo a-1,5-arabinosidase, exo Arabanase, exo A (a-1,2; a-1,3)
35 arabinofuranosidase, exo B {a-1,3; a-1,5) arabinofuranosidase;
- Xanthan lyase; Poly(a-L guluronate) lyase; Agarase, and Carrageenase.
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In particular, the following enzymes are preferred saccharide gum
degrading enzymes for specific non-starch non-cellulose food polysaccharide
having a viscosity higher than 800 cps at 1 % solution : Enzymes hydrolysing
guar gum (Guar flour , Jaguar Gum), locust bean gums and carob bean gum
(known as food additive E 410, E 412 and 21 CFR 184.1339 and 13423) are
mannosidase, galactomannosidase, preferentially endo mannosidase and
galactomannosidase enzyme such as Gamanase~ being a galactomannanase
from Aspergillus niger. Preferred enzymes for degrading xanthan gums are
o mannosidase, glucuronosidase and glucosidase. Preferred enzymes are
galactosidase, rhamnogalacturonase to degrade Karaya gum. Preferred
enzymes are galacturonase, galactosidase, fucosidase, arabanase to degrade
Tragacanth gums. Preferred enzymes for degrading gellan, agar and carageenan
gums are respectively, glucosidase, rhamnosidase and glucuronidase; agarase
~5 and carrageenase. Preferred enzymes are mannuronase and guluronase that
degrade the mannopyranosyluronic and gulopyranosyluronic moiety contained in
alginate.
Encompassed in the present invention are the following three mannans-
2o degrading enzymes : EC 3.2.1.25 : ~-mannosidase, EC 3.2.1.78 : Endo-1,4-~3-
mannosidase, referred therein after as "mannanase" and EC 3.2.1.100 : 1,4-(i-
mannobiosidase (IUPAC Classification- Enzyme nomenclature, 1992 ISBN 0-12-
227165-3 Academic Press).
25 More preferably, the laundry detergent compositions of the present
invention
comprise a ~i-1,4-Mannosidase (E.C. 3.2.1.78) referred to as Mannanase. The
term "mannanase" or "galactomannanase" denotes a mannanase enzyme
defined according to the art as officially being named mannan endo-1,4-beta-
mannosidase and having the alternative names beta-mannanase and endo-1,4-
3o mannanase and catalysing the reaction: random hydrolysis of 1,4-beta-D-
mannosidic linkages in mannans, galactomannans, glucomannans, and
galactoglucomannans.
In particular, Mannanases (EC 3.2.1.78) constitute a group of polysaccharases
35 which degrade mannans and denote enzymes which are capable of cleaving
polyose chains contaning mannose units, i.e: are capable of cleaving
glycosidic
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bonds in mannans, glucomannans, galactomannans and galactogluco-mannans.
Mannans are polysaccharides having a backbone composed of ~3-1,4- linked
mannose; glucomannans are polysaccharides having a backbone or more or less
regularly alternating (3-1,4 linked mannose and glucose; galactomannans and
galactoglucomannans are mannans and glucomannans with a-1,6 linked
galactose sidebranches. These compounds may be acetylated.
The degradation of galactomannans and galactoglucomannans is
facilitated by full or partial removal of the galactose sidebranches. Further
the
degradation of the acetylated mannans, glucomannans, galactomannans and
galactoglucomannans is facilitated by full or partial deacetylation. Acetyl
groups
can be removed by alkali or by mannan acetylesterases. The oligomers which
are released from the mannanases or by a combination of mannanases and a-
galactosidase and/or mannan acetyl esterases can be further degraded to
~5 release free maltose by ~i-mannosidase and/or ~3-glucosidase.
Mannanases have been identified in several Bacillus organisms. For example,
Talbot et al., Appl. Environ. Microbiol., Vo1.56, No. 11, pp. 3505-3510 (1990)
describes a beta-mannanase derived from Bacillus stearothermophilus in dimer
2o form having molecular weight of 162 kDa and an optimum pH of 5.5-7.5.
Mendoza et al., World J. Microbiol. Biotech., Vol. 10, No. 5, pp. 551-555
(1994)
describes a beta-mannanase derived from Bacillus subtilis having a molecular
weight of 38 kDa, an optimum activity at pH 5.0 and 55C and a pl of 4.8. JP-
0304706 discloses a beta-mannanase derived from Bacillus sp., having a
25 molecular weight of 373 kDa measured by gel filtration, an optimum pH of 8-
10
and a pl of 5.3-5.4. JP-63056289 describes the production of an alkaline,
thermostable beta-mannanase which hydrolyses beta-1,4-D-mannopyranoside
bonds of e.g. mannans and produces manno-oligosaccharides. JP-63036774
relates to the Bacillus microorganism FERM P-8856 which produces beta-
3o mannanse and beta-mannosidase at an alkaline pH. JP-08051975 discloses
alkaline beta-mannanases from alkalophilic Bacillus sp. AM-001. A purified
mannanase from Bacillus amyloliquefaciens useful in the bleaching of pulp and
paper and a method of preparation thereof is disclosed in WO 97/11164. WO
91/18974 describes a hemicellulase such as a glucanase, xylanase or
35 mannanase active at an extreme pH and temperature. WO 94/25576 discloses
an enzyme from Aspergillus aculeatus, CBS 101.43, exhibiting mannanase
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activity which may be useful for degradation or modification of plant or algae
cell
wall material. WO 93124622 discloses a mannanase isolated from Trichoderma
reseei useful for bleaching lignocellulosic pulps. An hemicellulase capable of
degrading mannan-containing hemicellulose is described in W091/18974 and a
5 purified mannanase from Bacillus amyloliquefaciens is described in
W097111164.
In particular, this mannanase enzyme will be an alkaline mannanase as defined
below, most preferably, a mannanase originating from a bacterial source.
o Especially, the laundry detergent composition of the present invention will
comprise an alkaline mannanase selected from the mannanase from the strain
Bacillus agaradherens and/or Bacillus subtilisis strain 168, gene yght
The terms "alkaline mannanase enzyme" is meant to encompass an enzyme
having an enzymatic activity of at least 10%, preferably at least 25%, more
preferably at least 40% of its maximum activity at a given pH ranging from 7
to
12, preferably 7.5 to 10.5.
Most preferably, the laundry detergent composition of the present invention
will
2o comprise the alkaline mannanase from Bacillus agaradherens. Said mannanase
is
i) a polypeptide produced by Bacillus agaradherens, NCIMB 40482, or
ii) a polypeptide comprising an amino acid sequence as shown in positions 32-
343 of SEQ ID N0:2 or
iii) an analogue of the polypeptide defined in i) or ii) which is at least 70%
homologous with said polypeptide, or is derived from said polypeptide by
substitution, deletion or addition of one or several amino acids, or is
immunologically reactive with a polyclonal antibody raised against said
poiypeptide in purified form.
The present invention also encompasses an isolated polypeptide having
mannanase activity selected from the group consisting of
(a) polynucleotide molecules encoding a polypeptide having mannanase
activity and comprising a sequence of nucleotides as shown in SEQ ID NO:
1 from nucleotide 97 to nucleotide 1029;
(b) species homologs of (a);
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(c) polynucleotide molecules that encode a polypeptide having mannanase
activity that is at least 70% identical to the amino acid sequence of SEQ ID
NO: 2 from amino acid residue 32 to amino acid residue 343;
(d) molecules complementary to (a), {b) or (c); and
(e) degenerate nucleotide sequences of (a), (b), (c) or (d).
The plasmid pSJ1678 comprising the polynucleotide molecule (the DNA
sequence) encoding a mannanase of the present invention has been
transformed into a strain of the Escherichia coli which was deposited by the
1o inventors according to the Budapest Treaty on the International Recognition
of
the Deposit of Microorganisms for the Purposes of Patent Procedure at the
Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH,
Mascheroder Weg 1 b, D-38124 Braunschweig, Federal Republic of Germany, on
18 May 1998 under the deposition number DSM 12180.
A second most preferred enzyme is the mannanase from the Bacillus subtilisis
strain 168, which mannanase:
i) is encoded by the coding part of the DNA sequence shown in SED ID No. 5
or an analogue of said sequence and/or
2o ii) a polypeptide comprising an amino acid sequence as shown SEQ ID N0:6
or
iii) an analogue of the polypeptide defined in ii) which is at least 70%
homologous with said polypeptide, or is derived from said poiypeptide by
substitution, deletion or addition of one or several amino acids, or is
immunologically reactive with a polyclonal antibody raised against said
polypeptide in purified form.
The present invention also encompasses an isolated polypeptide having
mannanase activity selected from the group consisting of
(a) polynucleotide molecules encoding a polypeptide having mannanase
activity and comprising a sequence of nucleotides as shown in SEQ ID
N0:5
(b) species homologs of (a);
(c) polynucleotide molecules that encode a polypeptide having mannanase
activity that is at least 70% identical to the amino acid sequence of SEQ ID
NO: 6;
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(d) molecules complementary to (a), (b) or (c); and
(e) degenerate nucleotide sequences of (a), (b), (c) or (d).
DEFINITIONS
Prior to discussing this invention in further detail, the following terms will
first be
defined
The term "ortholog" (or "species homolog") denotes a polypeptide or protein
obtained from one species that has homology to an analogous polypeptide or
o protein from a different species.
The term "paralog" denotes a polypeptide or protein obtained from a given
species that has homology to a distinct polypeptide or protein from that same
species.
The term "expression vector" denotes a DNA molecule, linear or circular, that
~5 comprises a segment encoding a polypeptide of interest operably linked to
additional segments that provide for its transcription. Such additional
segments
may include promoter and terminator sequences, and may optionally include one
or more origins of replication, one or more selectable markers, an enhancer, a
polyadenylation signal, and the like. Expression vectors are generally derived
2o from plasmid or viral DNA, or may contain elements of both. The expression
vector of the invention may be any expression vector that is conveniently
subjected to recombinant DNA procedures, and the choice of vector will often
depend on the host cell into which the vector it is to be introduced. Thus,
the
vector may be an autonomously replicating vector, i.e. a vector which exists
as
25 an extra chromosomal entity, the replication of which is independent of
chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one
which, when introduced into a host cell, is integrated into the host cell
genome
and replicated together with the chromosomes) into which it has been
integrated.
3o The term "recombinant expressed" or "recombinantly expressed" used herein
in connection with expression of a polypeptide or protein is defined according
to
the standard definition in the art. Recombinantly expression of a protein is
generally performed by using an expression vector as described immediately
above.
35 The term "isolated", when applied to a polynucleotide molecule, denotes
that
the polynucleotide has been removed from its natural genetic milieu and is
thus
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13
free of other extraneous or unwanted coding sequences, and is in a form
suitable
for use within genetically engineered protein production systems. Such
isolated
molecules are those that are separated from their natural environment and
include cDNA and genomic clones. Isolated DNA molecules of the present
invention are free of other genes with which they are ordinarily associated,
but
may include naturally occurring 5' and 3' untranslated regions such as
promoters
and terminators. The identification of associated regions will be evident to
one of
ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-
78,
1985).
o The term "an isolated polynucleotide" may alternatively be termed "a cloned
polynucleotide". When applied to a protein/polypeptide, the term "isolated"
indicates that the protein is found in a condition other than its native
environment.
In a preferred form, the isolated protein is substantially free of other
proteins,
particularly other homologous proteins (i.e. "homologous impurities" (see
below)).
~5 It is preferred to provide the protein in a greater than 40% pure form,
more
preferably greater than 60% pure form. Even more preferably it is preferred to
provide the protein in a highly purified form, i.e., greater than 80% pure,
more
preferably greater than 95% pure, and even more preferably greater than 99%
pure, as determined by SDS-PAGE.
2o The term "isolated protein/polypeptide may alternatively be termed
"purified
protein/polypeptide".
The term "homologous impurities" means any impurity (e.g. another
polypeptide than the polypeptide of the invention) which originate from the
homologous cell where the polypeptide of the invention is originally obtained
25 from.
The term "obtained from" as used herein in connection with a specific
microbial
source, means that the polynucleotide and/or poiypeptide produced by the
specific source, or by a cell in which a gene from the source have been
inserted.
The term "operably linked", when referring to DNA segments, denotes that the
3o segments are arranged so that they function in concert for their intended
purposes, e.g. transcription initiates in the promoter and proceeds through
the
coding segment to the terminator.
The term "polynucleotide" denotes a single- or double- stranded polymer of
deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
35 Polynucleotides include RNA and DNA, and may be isolated from natural
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14
sources, synthesized in vitro, or prepared from a combination of natural and
synthetic molecules.
The term "complements of polynucleotide molecules" denotes polynucleotide
molecules having a complementary base sequence and reverse orientation as
s compared to a reference sequence. For example, the sequence 5'
ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
The term "degenerate nucleotide sequence" denotes a sequence of
nucleotides that includes one or more degenerate codons (as compared to a
reference polynucleotide molecule that encodes a polypeptide). Degenerate
codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and GAC
triplets
each encode Asp).
The term "promoter" denotes a portion of a gene containing DNA sequences
that provide for the binding of RNA pofymerase and initiation of
transcription.
~ 5 Promoter sequences are commonly, but not always, found in the 5' non-
coding
regions of genes.
The term "secretory signal sequence" denotes a DNA sequence that encodes a
polypeptide (a "secretory peptide") that, as a component of a larger
polypeptide,
directs the larger polypeptide through a secretory pathway of a cell in which
it is
2o synthesized. The larger peptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
HOW TO USE A SEQUENCE OF THE INVENTION TO GET OTHER RELATED
SEQUENCES:
25 The disclosed sequence information herein relating to a polynucleotide
sequence
encoding a mannanase of the invention can be used as a tool to identify other
homologous mannanases. For instance, polymerase chain reaction (PCR) can
be used to amplify sequences encoding other homologous mannanases from a
variety of microbial sources, in particular of different Bacillus species.
ASSAY FOR ACTIVITY TEST
A polypeptide of the invention having mannanase activity may be tested for
mannanase activity according to standard test procedures known in the art,
such
as by applying a solution to be tested to 4 mm diameter holes punched out in
agar plates containing 0.2% AZCL galactomannan (carob), i.e. substrate for the
assay of endo-1,4-beta-D-mannanase available as CatNo.l- AZGMA from the
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company Megazyme for US$110.00 per 3 grams (Megazyme's Internet address:
http://www.megazyme.com/Purchase/index.html).
5
POLYNUCLEOTIDES:
An isolated polynucleotide of the invention will hybridize to similar sized
regions of SEQ ID No. 1, or a sequence complementary thereto, under at least
o medium stringency conditions.
In particular polynucleotides of the invention will hybridize to a denatured
double-stranded DNA probe comprising either the full sequence shown in
positions 97-1029 of SEQ ID N0:1 or any probe comprising a subsequence of
SEQ ID N0:1 having a length of at least about 100 base pairs under at least
~5 medium stringency conditions, but preferably at high stringency conditions
as
described in detail below. Suitable experimental conditions for determining
hybridization at medium, or high stringency between a nucleotide probe and a
homologous DNA or RNA sequence involves presoaking of the filter containing
the DNA fragments or RNA to hybridize in 5 x SSC (Sodium chloride/Sodium
2o citrate, Sambrook et al. 1989) for 10 min, and prehybridization of the
filter in a
solution of 5 x SSC, 5 x Denhardt's solution (Sambrook et al. 1989), 0.5 % SDS
and 100 Ng/ml of denatured sonicated salmon sperm DNA (Sambrook et al.
1989), followed by hybridization in the same solution containing a
concentration
of 10ng/ml of a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal.
Biochem. 132:6-13), 32P-dCTP-labeled (specific activity higher than 1 x 109
cpm/Ng ) probe for 12 hours at ca. 45°C. The filter is then washed
twice for 30
minutes in 2 x SSC, 0.5 % SDS at least 60°C (medium stringency), still
more
preferably at least 65°C (medium/high stringency), even more preferably
at least
70°C (high stringency), and even more preferably at least 75°C
(very high
3o stringency).
Molecules to which the oligonucleotide probe hybridizes under these
conditions are detected using a x-ray film.
As previously noted, the isolated pofynucleotides of the present invention
include DNA and RNA. Methods for isolating DNA and RNA are well known in
the art. DNA and RNA encoding genes of interest can be cloned in Gene Banks
or DNA libraries by means of methods known in the art.
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Polynucleotides encoding polypeptides having mannanase activity of the
invention are then identified and isolated by, for example, hybridization or
PCR.
The present invention futher provides counterpart polypeptides and
polynucleotides from different bacterial strains (orthologs or paralogs). Of
particular interest are mannanase polypeptides from gram-positive alkalophilic
strains, including species of Bacillus.
Species homologues of a polypeptide with mannanase activity of the
invention can be cloned using information and compositions provided by the
present invention in combination with conventional cloning techniques. For
example, a DNA sequence of the present invention can be cloned using
chromosomal DNA obtained from a cell type that expresses the protein. Suitable
sources of DNA can be identified by probing Northern blots with probes
designed
from the sequences disclosed herein. A library is then prepared from
chromosomal DNA of a positive cell line. A DNA sequence of the invention
~5 encoding an polypeptide having mannanase activity can then be isolated by a
variety of methods, such as by probing with probes designed from the sequences
disclosed in the present specification and claims or with one or more sets of
degenerate probes based on the disclosed sequences. A DNA sequence of the
invention can also be cloned using the polymerase chain reaction, or PCR
20 (Mullis, U.S. Patent 4,683,202), using primers designed from the sequences
disclosed herein. Within an additional method, the DNA library can be used to
transform or transfect host cells, and expression of the DNA of interest can
be
detected with an antibody (mono-clonal or polyclonal) raised against the
mannanase cloned from B.agaradherens, NCIMB 40482, expressed and purified
25 as described in Materials and Methods and Example 1, or by an activity test
relating to a polypeptide having mannanase activity.
The mannanase encoding part of the DNA sequence cloned into plasmid
pSJ1678 present in Escherichia coli DSM 12180 and/or an analogue DNA
sequence of the invention may be cloned from a strain of the bacterial species
3o Bacillus agaradherens, preferably the strain NCIMB 40482, producing the
enzyme with mannan degrading activity, or another or related organism as
described herein.
Alternatively, the analogous sequence may be constructed on the basis of
the DNA sequence obtainable from the plasmid present in Escherichia coli DSM
35 12180 (which is believed to be identical to the attached SEQ ID N0:1), e.g
be a
sub-sequence thereof, and/or by introduction of nucleotide substitutions which
do
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17
not give rise to another amino acid sequence of the mannanase encoded by the
DNA sequence, but which corresponds to the codon usage of the host organism
intended for production of the enzyme, or by introduction of nucleotide
substitutions which may give rise to a different amino acid sequence (i.e, a
variant of the mannan degrading enzyme of the invention).
POLYPEPTIDES:
The sequence of amino acids nos. 32-343 of SEQ ID NO: 2 is a mature
mannanase sequence.
o The present invention also provides mannanase polypeptides that are
substantially homologous to the polypeptide of SEQ ID N0:2 and species
homologs (paralogs or orthologs) thereof. The term "substantially homologous"
is
used herein to denote polypeptides having 70%, preferably at least 80%, more
preferably at least 85%, and even more preferably at least 90%, sequence
~5 identity to the sequence shown in amino acids nos. 32-343 of SEQ ID N0:2 or
their orthologs or paralogs. Such polypeptides will more preferably be at
least
95% identical, and most preferably 98% or more identical to the sequence shown
in amino acids nos. 32-343 of SEQ ID N0:2 or its orthologs or paralogs.
Percent
sequence identity is determined by conventional methods, by means of computer
2o programs known in the art such as GAP provided in the GCG program package
(Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics
Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 ) as
disclosed in Needleman, S.B. and Wunsch, C.D., (1970), Journal of Molecular
Biology, 48, 443-453, which is hereby incorporated by reference in its
entirety.
25 GAP is used with the following settings for polypeptide sequence
comparison:
GAP creation penalty of 3.0 and GAP extension penalty of 0.1.
Sequence identity of polynucleotide molecules is determined by similar
methods using GAP with the following settings for DNA sequence comparison:
GAP creation penalty of 5.0 and GAP extension penalty of 0.3.
The enzyme preparation of the invention is preferably derived from a
microorganism, preferably from a bacterium, an archea or a fungus, especially
from a bacterium such as a bacterium belonging to Bacillus, preferably to an
alkalophilic Bacillus strain which may be selected from the group consisting
of
the species Bacillus agaradherens and highly related Bacillus species in which
all species preferably are at least 95%, even more preferably at least 98%,
homologous to Bacillus agaradherens based on aligned 16S rDNA sequences.
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Substantially homologous proteins and polypeptides are characterized as
having one or more amino acid substitutions, deletions or additions. These
changes are preferably of a minor nature, that is conservative amino acid
substitutions (see Table 2) and other substitutions that do not significantly
affect
the folding or activity of the protein or polypeptide; small deletions,
typically of
one to about 30 amino acids; and small amino- or carboxyl-terminal extensions,
such as an amino-terminal methionine residue, a small linker peptide of up to
about 20-25 residues, or a small extension that facilitates purification (an
affinity
tag), such as a poly-histidine tract, protein A (Nilsson et al., EMBO J.
4:1075,
0 1985; Nilsson et al., Methods Enzymol. 198:3, 1991. See, in general Ford et
al.,
Protein Expression and Purification 2: 95-107, 1991, which is incorporated
herein
by reference. DNAs encoding affinity tags are available from commercial
suppliers (e.g., Pharmacia Biotech, Piscataway, NJ; New England Biolabs,
Beverly, MA).
~5 However, even though the changes described above preferably are of a
minor nature, such changes may also be of a larger nature such as fusion of
larger polypeptides of up to 300 amino acids or more both as amino- or
carboxyl-
terminal extensions to a Mannanase polypeptide of the invention.
2o Table 1
Conservative amino acid substitutions
Basic : arginine, lysine, histidine
Acidic : glutamic acid, aspartic acid
Polar : glutamine, asparagine
Hydrophobic : leucine, isoleucine, valine
Aromatic : phenylalanine, tryptophan, tyrosine
Small : glycine, alanine, serine, threonine,
methionine
In addition to the 20 standard amino acids, non-standard amino acids (such
2s as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline
and a-
methyl serine) may be substituted for amino acid residues of a polypeptide
according to the invention. A limited number of non-conservative amino acids,
amino acids that are not encoded by the genetic code, and unnatural amino
acids may be substituted for amino acid residues. "Unnatural amino acids" have
3o been modified after protein synthesis, and/or have a chemical structure in
their
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19
side chains) different from that of the standard amino acids. Unnatural amino
acids can be chemically synthesized, or preferably, are commercially
available,
and include pipecolic acid, thiazolidine carboxylic acid, dehydroproiine, 3-
and 4-
methylproline, and 3,3-dimethylproline.
Essential amino acids in the mannanase polypeptides of the present
invention can be identified according to procedures known in the art, such as
site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and
Wells, Science 244: 1081-1085, 1989). In the latter technique, single alanine
mutations are introduced at every residue in the molecule, and the resultant
mutant molecules are tested for biological activity (i.e mannanase activity)
to
identify amino acid residues that are critical to the activity of the
molecule. See
also, Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. The active site of
the
enzyme or other biological interaction can also be determined by physical
analysis of structure, as determined by such techniques as nuclear magnetic
15 resonance, crystallography, electron diffraction or photoaffinity labeling,
in
conjunction with mutation of putative contact site amino acids. See, for
example,
de Vos et al., Science 255:306-312, 1992; Smith et al., J. Mol. Biol. 224:899-
904,
1992; Wlodaver et al., FEES Lett. 309:59-64, 1992. The identities of essential
amino acids can also be inferred from analysis of homologies with polypeptides
2o which are related to a polypeptide according to the invention.
Multiple amino acid substitutions can be made and tested using known
methods of mutagenesis, recombination and/or shuffling followed by a relevant
screening procedure, such as those disclosed by Reidhaar-Olson and Sauer
(Science 241:53-57, 1988), Bowie and Sauer (Proc. Natl. Acad. Sci. USA
25 86:2152-2156, 1989), W095/17413, or WO 95/22625. Briefly, these authors
disclose methods for simultaneously randomizing two or more positions in a
polypeptide, or recombination/shuffling of different mutations (W095/17413,
W095/22625), followed by selecting fur functional a polypeptide, and then
sequencing the mutagenized polypeptides to determine the spectrum of
3o allowable substitutions at each position. Other methods that can be used
include
phage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991; Ladner et
al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and
region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al.,
DNA 7:127, 1988).
35 Mutagenesis/shuffling methods as disclosed above can be combined with
high-throughput, automated screening methods to detect activity of cloned,
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mutagenized polypeptides in host cells. Mutagenized DNA molecules that
encode active polypeptides can be recovered from the host cells and rapidly
sequenced using modern equipment. These methods allow the rapid
determination of the importance of individual amino acid residues in a
5 polypeptide of interest, and can be applied to polypeptides of unknown
structure.
Using the methods discussed above, one of ordinary skill in the art can
identify and/or prepare a variety of polypeptides that are substantially
homologous to residues 32 to 343 of SEQ ID NO: 2 and retain the mannanase
activity of the wild-type protein.
PROTEIN PRODUCTION:
The proteins and polypeptides of the present invention, including full-length
proteins, fragments thereof and fusion proteins, can be produced in
genetically
engineered host cells according to conventional techniques. Suitable host
cells
are those cell types that can be transformed or transfected with exogenous DNA
and grown in culture, and include bacteria, fungal cells, and cultured higher
eukaryotic cells. Bacterial cells, particularly cultured cells of gram-
positive
organisms, are preferred. Gram-positive cells from the genus of Bacillus are
especially preferred, such as from the group consisting of Bacillus subtilis,
2o Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus
alkalophilus,
Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus
lautus,
Bacillus thuringiensis, Bacillus licheniformis, and Bacillus agaradherens, in
particular Bacillus agaradherens.
Techniques for manipulating cloned DNA molecules and introducing
exogenous DNA into a variety of host cells are disclosed by Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY, 1989; Ausubel et al. (eds.), Current Protocols
in
Molecular Bioloav, John Wiley and Sons, Inc., NY, 1987; and "Bacillus subtilis
and Other Gram-Positive Bacteria", Sonensheim et al., 1993, American Society
3o for Microbiology, Washington D.C., which are incorporated herein by
reference.
In general, a DNA sequence encoding a mannanase of the present
invention is operably linked to other genetic elements required for its
expression,
generally including a transcription promoter and terminator within an
expression
vector. The vector will also commonly contain one or more selectable markers
and one or more origins of replication, although those skilled in the art will
recognize that within certain systems selectable markers may be provided on
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21
separate vectors, and replication of the exogenous DNA may be provided by
integration into the host cell genome. Selection of promoters, terminators,
selectable markers, vectors and other elements is a matter of routine design
within the level of ordinary skill in the art. Many such elements are
described in
the literature and are available through commercial suppliers.
To direct a polypeptide into the secretory pathway of a host cell, a secretory
signal sequence (also known as a leader sequence, prepro sequence or pre
sequence) is provided in the expression vector. The secretory signal sequence
may be that of the polypeptide, or may be derived from another secreted
protein
0 or synthesized de novo. Numerous suitable secretory signal sequences are
known in the art and reference is made to "Bacillus subtilis and Other Gram-
Positive Bacteria", Sonensheim et al., 1993, American Society for
Microbiology,
Washington D.C.; and Cutting, S. M.(eds.) "Molecular Biological Methods for
Bacillus", John Wiley and Sons, 1990, for further description of suitable
secretory
~5 signal sequences especially for secretion in a Bacillus host cell. The
secretory
signal sequence is joined to the DNA sequence in the correct reading frame.
Secretory signal sequences are commonly positioned 5' to the DNA sequence
encoding the polypeptide of interest, although certain signal sequences may be
positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al.,
2o U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).
Transformed or transfected host cells are cultured according to
conventional procedures in a culture medium containing nutrients and other
components required for the growth of the chosen host cells. A variety of
suitable
media, including defined media and complex media, are known in the art and
25 generally include a carbon source, a nitrogen source, essential amino
acids,
vitamins and minerals. Media may also contain such components as growth
factors or serum, as required. The growth medium will generally select for
cells
containing the exogenously added DNA by, for example, drug selection or
deficiency in an essential nutrient which is complemented by the selectable
3o marker carried on the expression vector or co-transfected into the host
cell.
PROTEIN ISOLATION:
When the expressed recombinant polypeptide is secreted the polypeptide
may be purified from the growth media. Preferably the expression host cells
are
3s removed from the media before purification of the polypeptide (e.g. by
centrifugation).
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When the expressed recombinant polypeptide is not secreted from the host
cell, the host cell are preferably disrupted and the polypeptide released into
an
aqueous "extract" which is the first stage of such purification techniques.
Preferably the expression host cells are collected from the media before the
cell
disruption (e.g. by centrifugation).
The cell disruption may be performed by conventional techniques such as
by fysozyme digestion or by forcing the cells through high pressure. See
(Robert
K. Scobes, Protein Purification, Second edition, Springer-Verlag) for further
description of such cell disruption techniques.
o Whether or not the expressed recombinant polypeptides (or chimeric
polypeptides) is secreted or not it can be purified using fractionation and/or
conventional purification methods and media.
Ammonium sulfate precipitation and acid or chaotrope extraction may be
used for fractionation of samples. Exemplary purification steps may include
~5 hydroxyapatite, size exclusion, FPLC and reverse-phase high performance
liquid
chromatography. Suitable anion exchange media include derivatized dextrans,
agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI,
DEAE,
QAE and Q derivatives are preferred, with DEAE Fast-Flow Sepharose
(Pharmacia, Piscataway, NJ) being particularly preferred. Exemplary
2o chromatographic media include those media derivatized with phenyl, butyl,
or
octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650
(Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or
polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
Suitable solid supports include glass beads, silica-based resins, cellulosic
resins,
25 agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked
polyacrylamide resins and the like that are insoluble under the conditions in
which they are to be used. These supports may be modified with reactive groups
that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl
groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling
3o chemistries include cyanogen bromide activation, N-hydroxysuccinimide
activation, epoxide activation, sulfhydryl activation, hydrazide activation,
and
carboxyl and amino derivatives for carbodiimide coupling chemistries. These
and
other solid media are well-known and widely used in the art, and are available
from commercial suppliers.
35 Selection of a particular method is a matter of routine design and is
determined in part by the properties of the chosen support. See, for example,
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23
Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology,
Uppsala, Sweden, 1988.
Polypeptides of the invention or fragments thereof may also be prepared
through chemical synthesis. Polypeptides of the invention may be monomers or
multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and
may or may not include an initial methionine amino acid residue.
Based on the sequence information disclosed herein a full length DNA
sequence encoding a mannanase of the invention and comprising the DNA
o sequence shown in SEQ ID No 1, at least the DNA sequence from position 97
to position 1029, may be cloned.
Cloning is performed by standard procedures known in the art such as
by,
~ preparing a genomic library from a Bacillus strain, especially the strain 8.
~5 agaradherens, NCIMB 40482;
~ plating such a library on suitable substrate plates;
~ identifying a clone comprising a polynucfeotide sequence of the invention by
standard hybridization techniques using a probe based on SEQ ID No 1; or
by
20 ~ identifying a clone from said Bacillus agaradherens NCIMB 40482 genomic
library by an Inverse PCR strategy using primers based on sequence
information from SEQ ID No 1. Reference is made to M.J. MCPherson et al.
("PCR A practical approach" Information Press Ltd, Oxford England) for
further details relating to Inverse PCR.
25 Based on the sequence information disclosed herein (SEQ ID No 1,
SEQ ID No 2) is it routine work for a person skilled in the art to isolate
homologous polynucleotide sequences encoding homologous mannanase of
the invention by a similar strategy using genomic libraries from related
microbial
organisms, in particular from genomic libraries from other strains of the
genus
3o Bacillus such as alkalophilic species of Bacillus.
Alternatively, the DNA encoding the mannan or galactomannan-
degrading enzyme of the invention may, in accordance with well-known
procedures, conveniently be cloned from a suitable source, such as any of the
above mentioned organisms, by use of synthetic oligonucleotide probes
35 prepared on the basis of the DNA sequence obtainable from the plasmid
present in Escherichia coli DSM 12180.
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24
Accordingly, the polynucleotide molecule of the invention may be
isolated from Escherichia coli, DSM 12180, in which the plasmid obtained by
cloning such as described above is deposited. Also, the present invention
relates to an isolated substantially pure biological culture of the strain
Escherichia coli, DSM 12180.
In the present context, the term "enzyme preparation" is intended to mean
either a conventional enzymatic fermentation product, possibly isolated and
purified, from a single species of a microorganism, such preparation usually
comprising a number of different enzymatic activities; or a mixture of
monocomponent enzymes, preferably enzymes derived from bacterial or fungal
species by using conventional recombinant techniques, which enzymes have
been fermented and possibly isolated and purified separately and which may
originate from different species, preferably fungal or bacterial species; or
the
fermentation product of a microorganism which acts as a host cell for
expression
~o of a recombinant mannanase, but which microorganism simultaneously produces
other enzymes, e.g. pectin degrading enzymes, proteases, or cellulases, being
naturally occurring fermentation products of the microorganism, i.e. the
enzyme
complex conventionally produced by the corresponding naturally occurring
microorganism.
A method of producing the enzyme preparation of the invention, the method
comprising culturing a microorganism, eg a wild-type strain, capable of
producing
the mannanase under conditions permitting the production of the enzyme, and
recovering the enzyme from the culture. Culturing may be carried out using
2o conventional fermentation techniques, e.g. culturing in shake flasks or
fermentors
with agitation to ensure sufficient aeration on a growth medium inducing
production of the mannanase enzyme. The growth medium may contain a
conventional N-source such as peptone, yeast extract or casamino acids, a
reduced amount of a conventional C-source such as dextrose or sucrose, and an
inducer such as guar gum or locust bean gum. The recovery may be carried out
using conventional techniques, e.g. separation of bio-mass and supernatant by
centrifugation or filtration, recovery of the supernatant or disruption of
cells if the
enzyme of interest is intracellular, perhaps followed by further purification
as
described in EP 0 406 314 or by crystallization as described in WO 97/15660.
IMMUNOLOGICAL CROSS-REACTIVITY:
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Polyclonal antibodies to be used in determining immunological cross-
reactivity may be prepared by use of a purified mannanase enzyme. More
specifically, antiserum against the mannanase of the invention may be raised
by
immunizing rabbits (or other rodents) according to the procedure described by
N.
5 Axelsen et al. in: A Manual of Quantitative Immunoelectrophoresis, Blackwell
Scientific Publications, 1973, Chapter 23, or A. Johnstone and R. Thorpe,
Immunochemistry in Practice, Blackwell Scientific Publications, 1982 (more
specifically p. 27-31 ). Purified immunoglobulins may be obtained from the
antisera, for example by salt precipitation ((NH4)2 S04), followed by dialysis
and
o ion exchange chromatography, e.g, on DEAE-Sephadex. Immunochemical
characterization of proteins may be done either by Outcherlony double-
diffusion
analysis (O. Ouchterlony in: Handbook of Experimental Immunology (D.M. Weir,
Ed.), Blackwell Scientific Publications, 1967, pp. 655-706), by crossed
immunoelectrophoresis (N. Axelsen et al., s_u~ra, Chapters 3 and 4), or by
rocket
~5 immunoelectrophoresis (N. Axelsen et al., Chapter 2).
Examples of useful bacteria producing the enzyme or the enzyme
preparation of the invention are Gram positive bacteria, preferably from the
BacilluslLactobacillus subdivision, preferably a strain from the genus
Bacillus,
2o more preferably a strain of Bacillus agaradherens, especially the strain
Bacillus
agaradherens, NCIMB 40482.
The present invention includes an isolated mannanase having the properties
described above and which is free from homologous impurities, and is produced
using conventional recombinant techniques.
DETERMINATION OF CATALYTIC ACTIVITY (ManU) OF MANNANASE
Colorimetric Assay:Substrate:0.2% AZCL-Galactomannan (Megazyme, Australia)
from carob in 0.1 M Glycin buffer, pH 10Ø The assay is carried out in an
Eppendorf Micro tube 1.5 ml on a thermomixer with stirring and temperature
3o control of 40°C. Incubation of 0.750 ml substrate with 0.05 ml
enzyme for 20 min,
stop by centrifugation for 4 minutes at 15000 rpm. The color of the
supernatant is
measured at 600 nm in a 1 cm cuvette. One ManU (Mannanase units) gives 0.24
abs in 1 cm.
OBTENTION OF THE BACILLUS AGARADHERENS MANNANASE NCIMB
40482
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WO 99!09127 PCTNS98/11995
26
Strains
Bacillus agaradherens NCIMB 40482 comprises the mannanase enzyme
encoding DNA sequence.
E. coli strain: Cells of E. coli SJ2 {Diderichsen, B., Wedsted, U.,
Hedegaard, L., Jensen, B. R., Sjmholm, C. (1990) Cloning of aldB, which
encodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis.
J. Bacteriol., 172, 4315-4321), were prepared for and transformed by
electroporation using a Gene PuIserTM electroporator from BIO-RAD as
described by the supplier.
B.subtilis PL2306. This strain is the B.subtilis DN1885 with disrupted apr
and npr genes (Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen, B. R.,
Sjwholm, C. (1990) Cloning of aldB, which encodes alpha-acetolactate
decarboxylase, an exoenzyme from Bacillus brevis. J. Bacteriol., 172, 4315-
4321) disrupted in the transcriptional unit of the known Bacillus subtilis
cellulase
~5 gene, resulting in cellulase negative cells. The disruption was performed
essentially as described in ( Eds. A.L. Sonenshein, J.A. Hoch and Richard
Losick
(1993) Bacillus subtilis and other Gram-Positive Bacteria, American Society
for
microbiology, p.618).
Competent cells were prepared and transformed as described by Yasbin,
2o R.E., Wilson, G.A. and Young, F.E. (1975) Transformation and transfection
in
lysogenic strains of Bacillus subtilis: evidence for selective induction of
prophage in
competent cells. J. Bacteriol, 121:296-304.
Plasmids
2s pSJ1678 (as described in detail in WO 94/19454 which is hereby incorporated
by
reference in its entirety).
pMOL944: This plasmid is a pUB110 derivative essentially containing elements
making the plasmid propagatable in Bacillus subtilis, kanamycin resistance
gene
and having a strong promoter and signal peptide cloned from the amyl gene of
3o B.licheniformis ATCC14580. The signal peptide contains a Sacll site making
it
convenient to clone the DNA encoding the mature part of a protein in-fusion
with
the signal peptide. This results in the expression of a Pre-protein which is
directed towards the exterior of the cell.
The plasmid was constructed by means of conventional genetic
s5 engineering techniques which are briefly described in the following.
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27
Construction of pMOL944:
The pUB110 plasmid (McKenzie, T. et al., 1986, Plasmid 15:93-103) was
digested with the unique restriction enzyme Ncil. A PCR fragment amplified
from
the amyl promoter encoded on the plasmid pDN1981 (P.L. Jr~rgensen et
a1.,1990, Gene, 96, p37-41.) was digested with Ncil and inserted in the Ncil
digested pUB110 to give the plasmid pSJ2624.
The two PCR primers used have the following sequences:
# LWN5494 5'-
GTCGCCGGGGCGGCCGCTATCAATTGGTAACTGTATCTCAGC -3'
~o # LWN5495 5'-
GTCGCCCGGGAGCTCTGATCAGGTACCAAGCTTGTCGACCTGCAGAA
TGAGGCAGCAAGAAGAT-3'
The primer #LWN5494 inserts a Notl site in the plasmid.
~5 The plasmid pSJ2624 was then digested with Sacl and Notl and a new
PCR fragment amplified on amyl promoter encoded on the pDN1981 was
digested with Sacl and Notl and this DNA fragment was inserted in the Sacl-
Notl
digested pSJ2624 to give the plasmid pSJ2670.
This cloning replaces the first amyl promoter cloning with the same
2o promoter but in the opposite direction. The two primers used for PCR
amplification have the following sequences:
#LWN5938 5'-
GTCGGCGGCCGCTGATCACGTACCAAGCTTGTCGACCTGCAGAATG
25 AGGCAGCAAGAAGAT -3'
#LWN5939 5'-GTCGGAGCTCTATCAATTGGTAACTGTATCTCAGC -3'
The plasmid pSJ2670 was digested with the restriction enzymes Pstl and
Bcll and a PCR fragment amplified from a cloned DNA sequence encoding the
3o alkaline amylase SP722 (disclosed in the International Patent Application
published as W095/26397 which is hereby incorporated by reference in its
entirety) was digested with Pstl and Bcll and inserted to give the plasmid
pMOL944. The two primers used for PCR amplification have the following
sequence:
s5 #LWN7864 5' -AACAGCTGATCACGACTGATCTTTTAGCTTGGCAC-3'
#LWN7901 5' -AACTGCAGCCGCGGCACATCATAATGGGACAAATGGG -3'
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28
The primer #LWN7901 inserts a Sacli site in the piasmid.
Cloning of the mannanase Gene from Bacillus agaradherens
Genomic DNA preparation:
Strain Bacillus agaradherens NCIMB 40482 was propagated in liquid
medium as described in W094101532. After 16 hours incubation at 30°C
and 300
rpm, the cells were harvested, and genomic DNA isolated by the method
described by Pitcher et al. (Pitcher, D. G., Saunders, N. A., Owen, R. J.
(1989).
Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett.
Appl. Microbiol., 8, 151-156).
~o Genomic library construction:
Genomic DNA was partially digested with restriction enzyme Sau3A, and
size-fractionated by electrophoresis on a 0.7 % agarose gel. Fragments between
2 and 7 kb in size was isolated by electrophoresis onto DEAE-cellulose paper
(Dretzen, G., Bellard, M., Sassone-Corsi, P., Chambon, P. (1981) A reliable
~s method for the recovery of DNA fragments from agarose and acrylamide gels.
Anal. Biochem., 112, 295-298).
Isolated DNA fragments were ligated to BamHl digested pSJ1678 plasmid
DNA, and the ligation mixture was used to transform E. coli SJ2.
Identification of positive clones:
20 A DNA library in E. coli, constructed as described above, was screened on
LB agar plates containing 0.2% AZCL-galactomannan (Megazyme) and 9 Ng/ml
Chloramphenicol and incubated overnight at 37oC. Clones expressing
mannanase activity appeared with blue diffusion halos. Plasmid DNA from one of
these clone was isolated by Qiagen plasmid spin preps on 1 ml of overnight
25 culture broth (cells incubated at 37°C in TY with 9 Ng/ml
Chloramphenicol and
shaking at 250 rpm).
This clone {MB525) was further characterized by DNA sequencing of the
cloned Sau3A DNA fragment. DNA sequencing was carried out by
primerwalking, using the Taq deoxy-terminal cycle sequencing kit (Perkin-
Elmer,
3o USA), fluorescent labelled terminators and appropriate oligonucleotides as
primers.
Analysis of the sequence data was performed according to Devereux et al.
(1984) Nucleic Acids Res. 12, 387-395. The sequence encoding the mannanase
is shown in SEQ ID No 1. The derived protein sequence is shown in SEQ ID
35 No.2.
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29
Subcloning and expression of mannanase in B.subtilis:
The mannanase encoding DNA sequence of the invention was PCR
amplified using the PCR primer set consisting of these two oligo nucleotides:
Mannanase.upper.Sacll
5'-CAT TCT GCA GCC GCG GCA GCA AGT ACA GGC TTT TAT GTT GAT GG-
3'
Mannanase.lower.Notl
5'-GAC GAC GTA CAA GCG GCC GCG CTA TTT CCC TAA CAT GAT GAT
ATT TTC G -3'
~o Restriction sites Sacll and Notll are underlined.
Chromosomal DNA isolated from B.agaradherens NCIMB 40482 as described
above was used as template in a PCR reaction using Amplitaq DNA Polymerase
(Perkin Elmer) according to manufacturers instructions. The PCR reaction was
set up in PCR buffer (10 mM Tris-HCI, pH 8.3, 50 mM KCI, 1.5 mM MgCl2, 0.01
% (w/v) gelatin) containing 200 NM of each dNTP, 2.5 units of AmpIiTaq
polymerase (Perkin-Elmer, Cetus, USA) and 100 pmol of each primer.
The PCR reaction was performed using a DNA thermal cycler (Landgraf,
Germany). One incubation at 94°C for 1 min followed by thirty cycles
of PCR
performed using a cycle profile of denaturation at 94°C for 30 sec,
annealing at
60°C for 1 min, and extension at 72°C for 2 min. Five-pl
aliquots of the
amplification product was analysed by electrophoresis in 0.7 % agarose gels
(NuSieve, FMC). The appearance of a DNA fragment size 1.4 kb indicated
proper amplification of the gene segment.
Subcloning of PCR fragment.
Fortyfive-NI aliquots of the PCR products generated as described above
were purified using QIAquick PCR purification kit (Qiagen, USA) according to
the
manufacturer's instructions. The purified DNA was eluted in 50 NI of lOmM Tris-
HCI, pH 8.5.
5 Ng of pMOL944 and twentyfive-NI of the purified PCR fragment was digested
3o with Sacll and Notl, electrophoresed in 0.8% low gelling temperature
agarose
(SeaPlaque GTG, FMC) gels, the relevant fragments were excised from the gels,
and purified using QIAquick Gel extraction Kit (Qiagen, USA) according to the
manufacturer's instructions. The isolated PCR DNA fragment was then ligated to
the Sacll-Notl digested and purified pMOL944. The ligation was performed
3s overnight at 16°C using 0.5Ng of each DNA fragment, 1 U of T4 DNA
ligase and
T4 ligase buffer (Boehringer Mannheim, Germany).
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The ligation mixture was used to transform competent B.subtilis PL2306.
The transformed cells were plated onto LBPG-10 Ng/ml of Kanamycin plates.
After 18 hours incubation at 37°C colonies were seen on plates. Several
clones
were analysed by isolating plasmid DNA from overnight culture broth.
5 One such positive clone was restreaked several times on agar plates as
used above, this clone was called MB594. The clone MB594 was grown
overnight in TY-10 Ng/ml kanamycin at 37°C, and next day 1 ml of cells
were
used to isolate plasmid from the cells using the Qiaprep Spin Plasmid Miniprep
Kit #27106 according to the manufacturers recommendations for B.subtilis
~o plasmid preparations. This DNA was DNA sequenced and revealed the DNA
sequence corresponding to the mature part of the mannanase, i.e. positions 94-
1404 of the appended SEQ ID N0:3. The derived mature protein is shown in
SEQ ID N0:4. It will appear that the 3' end of the mannanse encoded by the
sequence of SEQ ID N0:1 was changed to the one shown in SEQ ID N0:3 due
~ to the design of the lower primer used in the PCR. The resulting amino acid
sequence is shown in SEQ ID N0:4 and it is apparent that the C terminus of the
SEQ ID N0:2 (SHHVREIGVQFSAADNSSGQTALYVDNVTLR) is changed to the
C terminus of SEQ ID N0:4 (IIMLGK).
Media:
2o TY (as described in Ausubel, F. M. et al. (eds.) "Current protocols in
Molecular
Biology". John Wiley and Sons, 1995).
LB agar (as described in Ausubel, F. M. et al. (eds.) "Current protocols in
Molecular Biology". John Wiley and Sons, 1995).
LBPG is LB agar (see above) supplemented with 0.5% Glucose and 0.05
25 M potassium phosphate, pH 7.0
BPX media is described in EP 0 506 780 (WO 91/09129).
Expression, purification and characterisation of mannanase from Bacillus
agaradherens
3o The clone MB 594 obtained as described above under Materials and
Methods was grown in 25 x 200m1 BPX media with 10 Ng/ml of Kanamycin in
500m1 two baffled shakeflasks for 5 days at 37°C at 300 rpm.
6500 mI of the shake flask culture fluid of the clone MB 594 (batch #9813)
was collected and pH adjusted to 5.5. 146 ml of cationic agent (C521 ) and 292
ml of anionic agent (A130) was added during agitation for flocculation. The
flocculated material was separated by centrifugation using a Sorval RC 3B
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31
centrifuge at 9000 rpm for 20 min at 6°C. The supernatant was clarified
using
Whatman glass filters GFID and C and finally concentrated on a filtron with a
cut
off of 10 kDa.
750 ml of this concentrate was adjusted to pH 7.5 using sodium hydroxide.
The clear solution was applied to anion-exchange chromatography using a 900
ml Q-Sepharose column equilibrated with 50 mmol Tris pH 7.5. The mannanase
activity bound was eluted using a sodium chloride gradient.
The pure enzyme gave a single band in SDS-PAGE with a molecular
weight of 38 kDa. The amino acid sequence of the mannanase enzyme, i.e. the
~o translated DNA sequence, is shown in SEQ ID No.2.
Determination of kinetic constants:
Substrate: Locust bean gum (carob) and reducing sugar analysis
(PHBAH). Locust bean gum from Sigma (G-0753).
Kinetic determination using different concentrations of locust bean gum
and incubation for 20 min at 40°C at pH 10 gave
Kcat: 467 per sec.
Km: 0.08 gram per I
MW: 38kDa
pl {isoelectric point): 4.2
2o The temperature optimum of the mannanase was found to be 60°C.
The pH activity profile showed maximum activity between pH 8 and 10.
DSC differential scanning calometry gives 77°C as melting point at
pH 7.5
in Tris buffer indicating that this enzyme is very thermostable.
Detergent compatibility using 0.2% AZCL-Galactomannan from carob as
substrate and incubation as described above at 40°C shows excellent
compatibility with conventional liquid detergents and good compatibility with
conventional powder detergents.
OBTENTION OF THE BACILLUS SUBTILISIS MANNANASE 168
3o The Bacillus subtilisis (3-mannanase was characterised and purified as
follows : The Bacillus subtilis genome was searched for homology with a known
Bacillus sp ~-Mannanase gene sequence (Mendoza et al., Biochemica et
Bioa~hysica Acta 1243:552-554, 1995). The coding region of ydhT, whose
product was unknown, showed a 58% similarity to the known Bacillus (i-
Mannanase. The following oligonucleotides were designed to amplify the
sequences coding for the mature portion of the putative ~-Mannanase: 5'-GCT
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32
CAA TTG GCG CAT ACT GTG TCG CCT GTG-3' and 5'-GAC GGA TCC CGG
ATT CAC TCA ACG ATT GGC G-3'. Total genomic DNA from Bacillus subtilis
strain 1A95 was used as a template to amplify the ydhT mature region using the
aforementioned primers. PCR is performed using the GENE-AMP PCR Kit with
AMPLITAQ DNA Polymerase (Perkin Elmer, Applied Biosystems, Foster City,
CA). An initial melting period at 95°C for 5 min was followed by 25
cycles of the
following program: melting at 95°C for 1 min, annealing at 55°C
for 2 min, and
extension at 72°C for 2 min. After the last cycle, the reaction was
held at 72°C for
min to complete extension. The PCR products were purified using QIAquick
o PCR purification kit (Qiagen, Chatsworth, CA).
The ydhT mature region amplified from Bacillus subtilis strain 1A95 was
inserted into the expression vector pPG1524 (previously described) as follows.
The amplified 1028bp fragment was digested with Mfe I and BamH I. The
expression vector pPG1527 was digested with EcoR I and BamH I. The
~5 restriction products were purified using QIAquick PCR purification kit
(Qiagen,
Chatsworth, CA). The two fragments were ligated using T4 DNA ligase {13 hr,
16°C) and used to transform competent E. coli strain DH5-a. Ampiciiin
resistant
colonies were cultured for DNA preparations. The DNA was then characterized
by restriction analysis. Plasmid pPG3200 contains the mature region of the
ydhT
2o gene. Plasmid pPG3200 was then used to transform competent Bacillus
subtilis
strain PG 632 (Saunders et al., 1992).
Seven kanamycin resistant Bacillus subtilis clones and one PG 632
control clone were picked and grown in 20m1 of 20/20/5 media ( 20g/I tryptone,
20g/l yeast extract, 5g/I NaCI ) supplemented with 1 ml 25% maltrin, 1201 1
OmM
25 MnCl2, and 201 of 50 mg/ml kanamycin. Clones were grown overnight in 250m1
baffled flasks shaking at 250 rpm at 37°C for expression of the
protein. Cells
were spun out at 14,OOOrpm for 15 minutes. One ~,I of each supernatant was
diluted in 991 of 50mM sodium acetate (pH 6.0). One ~I of this dilution was
assayed using the endo-1,4-~3-Mannanase Beta-Mannazyme Tabs (Megazyme,
3o Ireland) according to the manufacturers instructions. Absorbance was read
at
590 nm on a Beckman DU640 spectrophotometer. Clone 7 showed the highest
Absorbance of 1.67. The PG632 control showed no Absorbance at 590nm.
Supernatant was analyzed by SDS-PAGE on a 10-20% Tris-Glycine gel
(Novex, San Diego, Ca) to confirm expected protein size of 38kDa. Samples
35 were prepared as follows. A 5001 sample of ydhT clone 7 and PG 632
supernatants were precipitated with 55.5u1 100% Trichloroacetic acid (Sigma),
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33
washed with 1001 5% Trichloroacetic, resuspended in 501 of Tris-glycine SDS
sample buffer(Novex) and boiled for five minutes. One pl of each sample was
electrophoresed on the gel at 30 mA for 90minutes. A large band of protein was
observed to run at 38kDa for ydhT clone 7.
A 10 I fermentation of Bacillus subtilis ydhT clone 7 was performed in a
B.Braun Biostat C fermentator. Fermentation conditions were as follows. Cells
were grown for 18h in a rich media similar to 20/20/5 at 37°C. At the
end of the
fermentation run, the cells were removed and the supernatant concentrated to 1
liter using a tangential flow filtration system. The final yield of ~i-
Mannanase in the
concentrated supernatant was determined to be 3 gll.
The purification of the ~i-Mannanase from the fermentation supernatant
was performed as follows: 500m1 of supernatant was centrifuged at 10,000 rpm
for 10 min at 4°C. The centrifuged supernatant was then dialyzed
overnight at 4°
C in two 4 I changes of 10 mM potassium phosphate (pH 7.2) through
Spectrapor 12,000-14,000 mol.wt. cutoff membrane (Spectrum). The dialyzed
supernatant was centrifuged at 10,000 rpm for 10 min at 4°C. A 200 ml Q
Sepharose fast flow (Pharmacia) anion exchange column was equilibrated with 1
liter of 10 mM potassium phosphate (pH 7.2) at 20°C and 300m1 of
supernatant
was loaded on column. Two flow through fractions of 210 ml (sample A) and 175
2o ml (sample B) were collected. The two fractions were assayed as before,
except
that the samples were diluted with 199 ~I of 50 mM sodium acetate (pH 6.0),
and they showed Absorbance of .38 and .52 respectively. Two ~I of each
sample was added to 8pl of Tris-glycine SDS sample buffer (Novex, CA) and
boiled for 5 min. The resulting samples were electrophoresed on a 10-20% Tris-
25 Glycine gel (Novex, Ca) at 30 mA for 90minutes. A major band corresponding
to
38kDa was present in each sample and comprised greater than 95% of the total
protein. A BCA protein assay (Pierce) was performed on both samples according
to the manufacturers instructions, using bovine serum albumin as standard.
Samples A and B contained 1.3 mg/ml and 1.6 mg/ml of ~i-Mannanase
3o respectively. The identity of the protein was confirmed by ion spray mass
spectrometry and amino terminal amino acid sequence analysis.
The purified ~3-Mannanase samples were used to characterize the
enzymes activity as follows. All assays used endo-1,4-~-Mannanase Beta-
Mannazyme Tabs (Megazyme, Ireland) as described earlier. Activity at pH range
35 3.0-9.0 were performed in 50 mM citrate phosphate buffer, for activity
determination at pH 9.5, 50 mM CAPSO (Sigma), and for pH 10.0-11.0 range 50
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34
mM CAPS buffer was employed. The optimum pH for the Bacillus subtilis ~i-
Mannanase was found to be pH 6.0-6.5. Temperature activity profiles were
performed in 50mM citrate phosphate buffer (pH 6.5). The enzyme showed
optimum activity at 40-45°C. The Bacillus subtilis ~i-Mannanase
retained
significant activity at less than 15°C and greater than 80°C.
Specific activity
against ~3-1,4-Galactomannan was determined to be 160,000 ~mol/min~mg (3-
Mannanase using endo-1,4-~i-Mannanase Beta-Mannazyme Tabs (Megazyme,
Ireland) according to the manufacturers directions. The nucleotide and amino
acid sequences of the Bacillus subtilisis ~3-mannanase are shown in SEQ. ID.
No.
5and6.
The saccharide gum degrading enzyme is incorporated into the laundry
detergent compositions of the present invention generally at a level of from
0.0001 % to 2%, more preferably from 0.0001 % to 0.1 %, most preferred from
~5 0.0006% to 0.02% pure enzyme by weight of the composition.
The saccharide gum degrading enzyme of the invention can in addition to the
enzyme core comprising the catalytically domain, also comprise a cellulose
binding domain (CBD), the cellulose binding domain and enzyme core (the
2o catalytically active domain) of the enzyme being operably linked. The
cellulose
binding domain (CBD) may exist as an integral part of the encoded enzyme, or a
CBD from another origin may be introduced into the enzyme thus creating an
enzyme hybrid. In this context, the term "cellulose-binding domain" is
intended to
be understood as defined by Peter Tomme et al. "Cellulose-Binding Domains:
25 Classification and Properties" in "Enzymatic Degradation of Insoluble
Carbohydrates", John N. Saddler and Michael H. Penner (Eds.), ACS
Symposium Series, No. 618, 1996. This definition classifies more than 120
cellulose- binding domains into 10 families (I-X), and demonstrates that CBDs
are found in various enzymes such as cellulases, xylanases, mannanases,
3o arabinofuranosidases, acetyl esterases and chitinases. CBDs have also been
found in algae, e.g. the red alga Porphyra purpurea as a non-hydrolytic
polysaccharide-binding protein, see Tomme et al., op.cit. However, most of the
CBDs are from cellulases and xylanases, CBDs are found at the N and C termini
of proteins or are internal. Enzyme hybrids are known in the art, see e.g. WO
35 90/00609 and WO 95/16782, and may be prepared by transforming into a host
cell a DNA construct comprising at least a fragment of DNA encoding the
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cellulose- binding domain ligated, with or without a linker, to a DNA sequence
encoding the saccharide gum degrading enzyme and growing the host cell to
express the fused gene. Enzyme hybrids may be described by the following
formula:
5 CBD-MR-X
wherein CBD is the N-terminal or the C-terminal region of an amino acid
sequence corresponding to at least the cellulose- binding domain; MR is the
middle region (the linker), and may be a bond, or a short linking group
preferably
of from about 2 to about 100 carbon atoms, more preferably of from 2 to 40
1o carbon atoms; or is preferably from about 2 to to about 100 amino acids,
more
preferably of from 2 to 40 amino acids; and X is an N-terminal or C-terminal
region of the enzyme of the invention.
The above-mentioned enzymes may be of any suitable origin, such as
~5 vegetable, animal, bacterial, fungal and yeast origin. Origin can further
be
mesophilic or extremophilic (psychrophilic, psychrotrophic, thermophilic,
barophilic, alkalophilic, acidophilic, halophilic, etc.). Purified or non-
purified forms
of these enzymes may be used. Nowadays, it is common practice to modify wild-
type enzymes via protein / genetic engineering techniques in order to optimise
2o their performance efficiency in the cleaning compositions of the invention.
For
example, the variants may be designed such that the compatibility of the
enzyme
to commonly encountered ingredients of such compositions is increased.
Alternatively, the variant may be designed such that the optimal pH, bleach or
chelant stability, catalytic activity and the like, of the enzyme variant is
tailored to
25 suit the particular cleaning application.
In particular, attention should be focused on amino acids sensitive to
oxidation in the case of bleach stability and on surface charges for the
surfactant
compatibility. The isoelectric point of such enzymes may be modified by the
3o substitution of some charged amino acids, e.g. an increase in isoelectric
point
may help to improve compatibility with anionic surfactants. The stability of
the
enzymes may be further enhanced by the creation of e.g. additional salt
bridges
and enforcing metal binding sites to increase chelant stability.
Detergient components
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36
The laundry detergent compositions of the invention may also contain
additional detergent components. The precise nature of these additional
components, and levels of incorporation thereof will depend on the physical
form
of the composition, and the nature of the cleaning operation for which it is
to be
used.
The laundry detergent compositions of the present invention preferably
further comprise a detergent ingredient selected from a selected surfactant,
1o another enzyme, a builder and/or a bleach system.
The laundry detergent compositions according to the invention can be
liquid, paste, gels, bars, tablets, spray, foam, powder or granular forms.
Granular
compositions can also be in "compact" form, the liquid compositions can also
be
~ 5 in a "concentrated" form.
One preferred type of gel detergent is a heavy duty gel laundry detergent
composition comprising from 15% to 40% by weight of an anionic surfactant
component which comprises : (i) from 5% to 25% by weight of alkyl
2o polyethoxylate sulfates wherein the alkyl group contains from about 10 to
about
22 carbon atoms and the polyethoxylate chain contains from 0.5 to about 15,
preferably from 0.5 to about 5, more preferably from 0.5 to about 4, ethylene
oxide moieties; and (ii) from 5% to 20% by weight of fatty acids.
Gel compositions herein may further contain one or more additional
25 detersive additives selected from the group consisting of non-citrate
builders,
optical brighteners, soil release polymers, dye transfer inhibitors, polymeric
dispersing agents, enzymes, suds suppressers, dyes, perfumes, colorants,
filler
salts, hydrotropes, antiredeposition agents, antifading agent, dye fixative
agents,
prill/fuzzing reducing agents, and mixtures thereof.
3o The gel compositions herein have a viscosity at 20 s-1 shear rate of from
about 100 cp to about 4,000 cp, preferably from about 300 cp to about 3,000
cp,
more preferably from about 500 cp to about 2,000 cp and are stable upon
storage.
Without being limited by theory, it is believed that the presence of
35 electrolytes acts to control the viscosity of the gel compositions. Thus,
the gel
nature of the compositions herein are affected by the choice of surfactants
and
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WO 99/09127 PCTNS98/11995
37
by the amount of electrolytes present. In preferred embodiments herein, the
compositions will further comprise from 0% to about 10%, more preferably from
about 2% to about 6%, even more preferably from about 3% to about 5%, of a
suitable electrolyte or acid equivalent thereof. Sodium citrate is a highly
preferred electrolyte for use herein.
The compositions herein may optionally contain from about 0% to about
10%, by weight, of solvents and hydrotropes. Without being limited by theory,
it
is believed that the presence of solvents and hydrotropes can affect the
structured versus isotropic nature of the compositions; By "solvent" is meant
the
~o commonly used solvents in the detergent industry, including alkyl
monoalcohol,
di-, and tri-alcohols, ethylene glycol, propylene glycol, propanediol,
ethanediol,
glycerine, etc. By "hydrotrope" is meant the commonly used hydrotropes in the
detergent industry, including short chain surfactants that help solubilize
other
surfactants. Other examples of hydrotropes include cumene, xylene, or toluene
~5 sulfonate, urea, Cg or shorter chain alkyl carboxyiates, and Cg or shorter
chain
alkyl sulfate and ethoxylated sulfates.
Fatty acids of use herein include saturated and/or unsaturated fatty acids
obtained from natural sources or synthetically prepared. Examples of fatty
acids
include capric, lauric, myristic, palmitic, stearic, arachidic, and behenic
acid.
2o Other fatty acids include palmitoleic, oleic, linoleic, linolenic, and
ricinoleic acid.
The compositions of the invention may be formulated hand and machine
laundry detergent compositions including laundry additive compositions and
compositions suitable for use in the soaking and/or pretreatment of stained
25 fabrics, rinse added fabric softener compositions.
When formulated as compositions suitable for use in a laundry machine
washing method, the compositions of the invention preferably contain both a
surfactant and a builder compound and additionally one or more detergent
3o components preferably selected from organic polymeric compounds, bleaching
agents, additional enzymes, suds suppressors, dispersants, lime-soap
dispersants, soil suspension and anti-redeposition agents and corrosion
inhibitors. Laundry compositions can also contain softening agents, as
additional
detergent components. Such compositions containing an enzyme hydrolysing
35 saccharides gums, can provide fabric cleaning, stain removal, whiteness
maintenance, softening, color appearance and dye transfer inhibition.
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38
The compositions of the invention can also be used as detergent additive
products. Such additive products are intended to supplement or boost the
performance of conventional detergent compositions.
If needed the density of the laundry detergent compositions herein ranges
from 400 to 1200 g/litre, preferably 500 to 950 gllitre of composition
measured at
20°C.
The "compact" form of the compositions herein is best reflected by density
1o and, in terms of composition, by the amount of inorganic filler salt;
inorganic filler
salts are conventional ingredients of detergent compositions in powder form;
in
conventional detergent compositions, the filler salts are present in
substantial
amounts, typically 17-35% by weight of the total composition. In the
compact compositions, the filler salt is present in amounts not exceeding 15%
of
~5 the total composition, preferably not exceeding 10%, most preferably not
exceeding 5% by weight of the composition. The inorganic filler salts, such as
meant in the present compositions are selected from the alkali and alkaline-
earth-metal salts of sulphates and chlorides. A preferred filler salt is
sodium
sulphate.
2o Liquid detergent compositions according to the present invention can also
be in a "concentrated form", in such case, the liquid detergent compositions
according the present invention will contain a lower amount of water, compared
to conventional liquid detergents. Typically the water content of the
concentrated
liquid detergent is preferably less than 40%, more preferably less than 30%,
most
25 preferably less than 20% by weight of the detergent composition.
Surfactant syrstem
The laundry detergent compositions according to the present invention
3o generally comprise a surfactant system wherein the surfactant can be
selected
from nonionic and/or anionic and/or cationic and/or ampholytic and/or
zwitterionic
andlor semi-polar surfactants. Preferably, the laundry detergent compositions
of
the present invention will comprise a nonionic, an anionic and/or a cationic
surfactant.
35 It has been surprisingly found that the laundry detergent compositions of
the present invention further comprising a nonionic, an anionic surfactant
and/or
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39
a cationic surfactant, provide enhanced food stain/soil removal, dingy
cleaning
and whiteness maintenance.
Without wishing to be bound by theory, it is believed that the enzymatic
hydrolysis results in small particles being more easily removed by nonionic
surfactants known to focus on particulate soiling. Preferred nonionic
surfactants
are alkyl ethoxylate AE3 to AE7. It is also believed that the combination of
the
fabric substantive cationic surfactant with the enzymatic hydrolysis of the
saccharide gums degrading enzyme provides improved performances.
The surfactant is typically present at a level of from 0.1 % to 80% by
weight. More preferred levels of incorporation are 1 % to 35% by weight, most
preferably from 1 % to 30% by weight of laundry detergent compositions in
accord
with the invention.
The surfactant is preferably formulated to be compatible with enzyme
components present in the composition. In liquid or gel compositions the
surfactant is most preferably formulated such that it promotes, or at least
does
not degrade, the stability of any enzyme in these compositions.
Nonionic surfactants
Polyethylene, polypropylene, and polybutylene oxide condensates of alkyl
phenols are suitable for use as the nonionic surfactant of the surfactant
systems
of the present invention, with the polyethylene oxide condensates being
preferred. These compounds include the condensation products of alkyl phenols
having an alkyl group containing from about 6 to about 14 carbon atoms,
preferably from about 8 to about 14 carbon atoms, in either a straight-chain
or
branched-chain configuration with the alkylene oxide. In a preferred
embodiment,
the ethylene oxide is present in an amount equal to from about 2 to about 25
3o moles, more preferably from about 3 to about 15 moles, of ethylene oxide
per
mole of alkyl phenol. Commercially available nonionic surfactants of this type
include IgepaITM CO-630, marketed by the GAF Corporation; and TritonTM X
45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company. These
surfactants are commonly referred to as alkylphenol alkoxylates (e.g., alkyl
phenol ethoxylates).
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The condensation products of primary and secondary aliphatic alcohols
with from about 1 to about 25 moles of ethylene oxide are suitable for use as
the
nonionic surfactant of the nonionic surfactant systems of the present
invention.
The alkyl chain of the aliphatic alcohol can either be straight or branched,
primary
s or secondary, and generally contains from about 8 to about 22 carbon atoms.
Preferred are the condensation products of alcohols having an alkyl group
containing from about 8 to about 20 carbon atoms, more preferably from about
10 to about 18 carbon atoms, with from about 2 to about 10 moles of ethylene
oxide per mole of alcohol. About 2 to about 7 moles of ethylene oxide and most
o preferably from 2 to 5 moles of ethylene oxide per mole of alcohol are
present in
said condensation products. Examples of commercially available nonionic
surfactants of this type include TergitolTM 15-S-9 (the condensation product
of
C11-C15 linear alcohol with 9 moles ethylene oxide), TergitoITM 24-L-6 NMW
(the condensation product of C12-C14 primary alcohol with 6 moles ethylene
5 oxide with a narrow molecular weight distribution), both marketed by Union
Carbide Corporation; NeodoITM 45-9 (the condensation product of C14-C15
linear alcohol with 9 moles of ethylene oxide), NeodoITM 23-3 (the
condensation
product of C12-C13 linear alcohol with 3.0 moles of ethylene oxide), NeodoITM
45-7 (the condensation product of C14-C15 linear alcohol with 7 moles of
2o ethylene oxide), NeodoITM 45-5 (the condensation product of C14-C15 linear
alcohol with 5 moles of ethylene oxide) marketed by Shell Chemical Company,
KyroTM EOB (the condensation product of C13-C15 alcohol with 9 moles
ethylene oxide), marketed by The Procter & Gamble Company, and Genapol LA
030 or 050 (the condensation product of C12-C14 alcohol with 3 or 5 moles of
25 ethylene oxide) marketed by Hoechst. Preferred range of HLB in these
products
is from 8-11 and most preferred from 8-10.
Also useful as the nonionic surfactant of the surfactant systems of the
present invention are the alkylpolysaccharides disclosed in U.S. Patent
30 4,565,647, Llenado, issued January 21, 1986, having a hydrophobic group
containing from about 6 to about 30 carbon atoms, preferably from about 10 to
about 16 carbon atoms and a polysaccharide, e.g. a polyglycoside, hydrophilic
group containing from about 1.3 to about 10, preferably from about 1.3 to
about
3, most preferably from about 1.3 to about 2.7 saccharide units. Any reducing
35 saccharide containing 5 or 6 carbon atoms can be used, e.g., glucose,
galactose
and galactosyl moieties can be substituted for the glucosyl moieties
(optionally
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41
the hydrophobic group is attached at the 2-, 3-, 4-, etc. positions thus
giving a
glucose or galactose as opposed to a glucoside or galactoside). The
intersaccharide bonds can be, e.g., between the one position of the additional
saccharide units and the 2-, 3-, 4-, and/or 6- positions on the preceding
saccharide units.
The preferred alkylpolyglycosides have the formula:
R2~(CnH2n~)t(9lYcosyl)x
o wherein R2 is selected from the group consisting of alkyl, alkylphenyl,
hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which the alkyl
groups
contain from about 10 to about 18, preferably from about 12 to about 14,
carbon
atoms; n is 2 or 3, preferably 2; t is from 0 to about 10, preferably 0; and x
is from
about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably
from
~5 about 1.3 to about 2.7. The glycosyl is preferably derived from glucose. To
prepare these compounds, the alcohol or alkylpolyethoxy alcohol is formed
first
and then reacted with glucose, or a source of glucose, to form the glucoside
(attachment at the 1-position). The additional glycosyl units can then be
attached
between their 1-position and the preceding glycosyl units 2-, 3-, 4- and/or 6-
2o position, preferably predominately the 2-position.
The condensation products of ethylene oxide with a hydrophobic base
formed by the condensation of propylene oxide with propylene glycol are also
suitable for use as the additional nonionic surfactant systems of the present
25 invention. The hydrophobic portion of these compounds will preferably have
a
molecular weight of from about 1500 to about 1800 and will exhibit water
insolubility. The addition of polyoxyethylene moieties to this hydrophobic
portion
tends to increase the water solubility of the molecule as a whole, and the
liquid
character of the product is retained up to the point where the polyoxyethylene
3o content is about 50% of the total weight of the condensation product, which
corresponds to condensation with up to about 40 moles of ethylene oxide.
Examples of compounds of this type include certain of the commercially-
available
PlurafacTM LF404 and PluronicTM surfactants, marketed by BASF.
35 Also suitable for use as the nonionic surfactant of the nonionic surfactant
system of the present invention, are the condensation products of ethylene
oxide
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42
with the product resulting from the reaction of propylene oxide and
ethylenediamine. The hydrophobic moiety of these products consists of the
reaction product of ethylenediamine and excess propylene oxide, and generally
has a molecular weight of from about 2500 to about 3000. This hydrophobic
moiety is condensed with ethylene oxide to the extent that the condensation
product contains from about 40% to about 80% by weight of polyoxyethylene and
has a molecular weight of from about 5,000 to about 11,000. Examples of this
type of nonionic surfactant include certain of the commercially available
TetronicTM compounds, marketed by BASF.
~o
Preferred for use as the nonionic surfactant of the surfactant systems of
the present invention are polyethylene oxide condensates of alkyl phenols,
condensation products of primary and secondary aliphatic alcohols with from
about 1 to about 25 moles of ethylene oxide, alkylpolysaccharides, and
mixtures
thereof. Most preferred are Cg-C14 alkyl phenol ethoxylates having from 3 to
15
ethoxy groups and Cg-C1g alcohol ethoxylates (preferably C10 avg.) having from
2 to 10 ethoxy groups, and mixtures thereof.
Highly preferred nonionic surfactants are polyhydroxy fatty acid amide
2o surfactants of the formula:
R2-C-N-Z,
O R1
wherein R1 is H, or R1 is C1_4 hydrocarbyl, 2-hydroxy ethyl, 2-hydroxy propyl
or
a mixture thereof, R2 is C5_31 hydrocarbyl, and Z is a polyhydroxyhydrocarbyl
having a linear hydrocarbyl chain with at least 3 hydroxyls directly connected
to
the chain, or an alkoxylated derivative thereof. Preferably, R1 is methyl, R2
is a
straight C11-15 alkyl or C16-18 alkyl or alkenyl chain such as coconut alkyl
or
3o mixtures thereof, and Z is derived from a reducing sugar such as glucose,
fructose, maltose, lactose, in a reductive amination reaction.
Anionic surfactants
Preferred anionic surfactants for the purpose of the present invention are
alkyl esters sulfates and linear alkyl benzene surfactants. Suitable anionic
surfactants to be used are linear alkyl benzene sulfonate, alkyl ester
sulfonate
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43
surfactants including linear esters of Cg-C2p carboxylic acids (i.e., fatty
acids)
which are sulfonated with gaseous S03 according to "The Journal of the
American Oil Chemists Society", 52 (1975), pp. 323-329. Suitable starting
materials would include natural fatty substances as derived from tallow, palm
oil,
etc.
The preferred alkyl ester sulfonate surfactant, especially for laundry
applications,
comprise alkyl ester sulfonate surfactants of the structural formula:
O
I
1o R3 - CH - C - OR4
S03M
wherein R3 is a Cg-C20 hydrocarbyl, preferably an alkyl, or combination
thereof,
~5 R4 is a C1-Cg hydrocarbyl, preferably an alkyl, or combination thereof, and
M is
a cation which forms a water soluble salt with the alkyl ester sulfonate.
Suitable
salt-forming cations include metals such as sodium, potassium, and lithium,
and
substituted or unsubstituted ammonium cations, such as monoethanolamine,
diethanolamine, and triethanoiamine. Preferably, R3 is C10-C1g alkyl, and R4
is
2o methyl, ethyl or isopropyl. Especially preferred are the methyl ester
sulfonates
wherein R3 is C10-C1g alkyl.
Other suitable anionic surfactants include the alkyl sulfate surfactants
which are water soluble salts or acids of the formula ROS03M wherein R
25 preferably is a C10-C24 hydrocarbyl, preferably an alkyl or hydroxyalkyl
having a
C10-C20 alkyl component, more preferably a C12-C1g alkyl or hydroxyalkyl, and
M is H or a cation, e.g., an alkali metal cation (e.g. sodium, potassium,
lithium), or
ammonium or substituted ammonium (e.g. methyl-, dimethyl-, and trimethyl
ammonium cations and quaternary ammonium cations such as tetramethyl-
3o ammonium and dimethyl piperdinium cations and quaternary ammonium cations
derived from alkylamines such as ethylamine, diethylamine, triethylamine, and
mixtures thereof, and the like). Typically, alkyl chains of C12-C16 are
preferred
for lower wash temperatures (e.g. below about 50°C) and C16-18 alkyl
chains
are preferred for higher wash temperatures (e.g. above about 50°C).
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44
Other anionic surfactants useful for detersive purposes can also be
included in the laundry detergent compositions of the present invention. These
can include salts (including, for example, sodium, potassium, ammonium, and
substituted ammonium salts such as mono-, di- and triethanoiamine salts) of
s soap, Cg-C22 primary of secondary alkanesulfonates, Cg-C24 olefinsulfonates,
sulfonated polycarboxylic acids prepared by sulfonation of the pyrolyzed
product
of alkaline earth metal citrates, e.g., as described in British patent
specification
No. 1,082,179, Cg-C24 alkylpolyglycolethersulfates {containing up to 10 moles
of
ethylene oxide); alkyl glycerol sulfonates, fatty acyl glycerol sulfonates,
fatty oleyl
o glycerol sulfates, alkyl phenol ethylene oxide ether sulfates, paraffin
sulfonates,
alkyl phosphates, isethionates such as the acyl isethionates, N-acyl taurates,
alkyl succinamates and sulfosuccinates, monoesters of sulfosuccinates
(especially saturated and unsaturated C12-C1g monoesters) and diesters of
sulfosuccinates {especially saturated and unsaturated Cg-C12 diesters), acyl
~5 sarcosinates, sulfates of alkylpolysaccharides such as the sulfates of
alkylpolyglucoside (the nonionic nonsulfated compounds being described below),
branched primary alkyl sulfates, and alkyl polyethoxy carboxylates such as
those
of the formula RO(CH2CH20)k-CH2C00-M+ wherein R is a Cg-C22 alkyl, k is
an integer from 1 to 10, and M is a soluble salt-forming cation. Resin acids
and
2o hydrogenated resin acids are also suitable, such as rosin, hydrogenated
rosin,
and resin acids and hydrogenated resin acids present in or derived from tall
oil.
Further examples are described in "Surface Active Agents and
Detergents" (Vol. l and II by Schwartz, Perry and Berch). A variety of such
25 surfactants are also generally disclosed in U.S. Patent 3,929,678, issued
December 30, 1975 to Laughlin, et al. at Column 23, line 58 through Column 29,
line 23 (herein incorporated by reference).
When included therein, the laundry detergent compositions of the present
invention typically comprise from about 1 % to about 40%, preferably from
about
30 3% to about 20% by weight of such anionic surfactants.
Highly preferred anionic surfactants include alkyl alkoxylated sulfate
surfactants hereof are water soluble salts or acids of the formula RO(A)mS03M
wherein R is an unsubstituted C10-C24 alkyl or hydroxyalkyl group having a C10-
35 C24 alkyl component, preferably a C12-C20 alkyl or hydroxyalkyl, more
preferably C12-C1g alkyl or hydroxyalkyl, A is an ethoxy or propoxy unit, m is
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greater than zero, typically between about 0.5 and about 6, more preferably
between about 0.5 and about 3, and M is H or a cation which can be, for
example, a metal cation (e.g., sodium, potassium, lithium, calcium, magnesium,
etc.), ammonium or substituted-ammonium cation. Alkyl ethoxylated sulfates as
5 well as alkyl propoxylated sulfates are contemplated herein. Specific
examples
of substituted ammonium cations include methyl-, dimethyl, trimethyl-ammonium
cations and quaternary ammonium cations such as tetramethyl-ammonium and
dimethyl piperdinium cations and those derived from alkylamines such as
ethylamine, diethylamine, triethylamine, mixtures thereof, and the like.
Exemplary
1o surfactants are C12-C1g alkyl polyethoxylate (1.0) sulfate (C12-
C18E(1.0)M),
C12-C1g alkyl polyethoxylate {2.25) sulfate {C12-C18E(2.25)M), C12-C1g alkyl
polyethoxylate (3.0) sulfate (C12-C18E{3.0)M), and C12-C1g alkyl
polyethoxylate
(4.0) sulfate (C12-C18E(4.0)M), wherein M is conveniently selected from sodium
and potassium.
Cationic surfactants
Cationic detersive surfactants suitable for use in the laundry detergent
compositions of the present invention are those having one long-chain
hydrocarbyi group. Examples of such cationic surfactants include the ammonium
2o surfactants such as alkyltrimethyiammonium halogenides, and those
surfactants
having the formula
jR2(OR3)y)IR4{OR3)y)2R5N+X-
wherein R2 is an alkyl or alkyl benzyl group having from about 8 to about 18
carbon atoms in the alkyl chain, each R3 is selected from the group consisting
of
-CH2CH2-, -CH2CH(CH3)-, -CH2CH(CH20H)-, -CH2CH2CH2-, and mixtures
thereof; each R4 is selected from the group consisting of C1-C4 alkyl, C1-C4
hydroxyalkyl, benzyl ring structures formed by joining the two R4 groups, -
3o CH2CHOH-CHOHCOR6CHOHCH20H wherein R6 is any hexose or hexose
polymer having a molecular weight less than about 1000, and hydrogen when y
is not 0; R5 is the same as R4 or is an alkyl chain wherein the total number
of
carbon atoms of R2 plus R5 is not more than about 18; each y is from 0 to
about
10 and the sum of the y values is from 0 to about 15; and X is any compatible
a5 anion.
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46
Quaternary ammonium surfactant suitable for the present invention has
the formula (I):
R2 ~+ ~ 3,,,,,,Rq
R~ N'~
~O R5 X-
Formula I
whereby R1 is a short chainlength alkyl (C6-C10) or alkylamidoalkyl of the
formula (II)
Cs C.~ N
~CH~
0
Formula II
y is 2-4, preferably 3.
whereby R2 is H or a C1-C3 alkyl,
whereby x is 0-4, preferably 0-2, most preferably 0,
whereby R3, R4 and R5 are either the same or different and can be either a
short
chain alkyl (C1-C3) or alkoxylated alkyl of the formula III,
whereby X- is a counterion, preferably a halide, e.g. chloride or
methylsulfate.
zo
Rs
~H
0 z
Formula III
R6 is C1-C4 and z is 1 or 2.
Preferred quat ammonium surfactants are those as defined in formula I
whereby
R1 is Cg, C1p or mixtures thereof, x=o,
R3, R4 = CHg and R5 = CH2CH20H.
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47
Highly preferred cationic surfactants are the water-soluble quaternary
ammonium compounds useful in the present composition having the formula
R1 R2R3R4N+X- (i)
wherein R1 is Cg-C1g alkyl, each of R2, Rg and R4 is independently C1-C4
alkyl,
C1-C4 hydroxy alkyl, benzyl, and -(C2H40)xH where x has a value from 2 to 5,
and X is an anion. Not more than one of R2, Rg or R4 should be benzyl.
The preferred alkyl chain length for R1 is C12-C15 particularly where the
alkyl
o group is a mixture of chain lengths derived from coconut or palm kernel fat
or is
derived synthetically by olefin build up or OXO alcohols synthesis. Preferred
groups for R2Rg and R4 are methyl and hydroxyethyl groups and the anion X
may be selected from halide, methosulphate, acetate and phosphate ions.
Examples of suitable quaternary ammonium compounds of formulae (i) for use
~5 herein are
coconut trimethyf ammonium chloride or bromide;
coconut methyl dihydroxyethyl ammonium chloride or bromide;
decyl triethyl ammonium chloride;
decyl dimethyl hydroxyethyl ammonium chloride or bromide;
2o C12-15 dimethyl hydroxyethyl ammonium chloride or bromide;
coconut dimethyl hydroxyethyl ammonium chloride or bromide;
myristyl trimethyl ammonium methyl sulphate;
lauryl dimethyl benzyl ammonium chloride or bromide;
lauryl dimethyl (ethenoxy)4 ammonium chloride or bromide;
25 choline esters (compounds of formula (i) wherein R1 is
CH2-CH2-O-C-C12-14 alkyl and R2R3R4 are methyl).
I
O
di-alkyl imidazolines [compounds of formula (i)].
Other cationic surfactants useful herein are also described in U.S. Patent
4,228,044, Cambre, issued October 14, 1980 and in European Patent
Application EP 000,224.
Typical cationic fabric softening components include the water-insoluble
quaternary-ammonium fabric softening actives or thei corresponding amine
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48
precursor,
the most
commonly
used having
been di-long
alkyl chain
ammonium
chloride methyl sulfate.
or
Preferred
cationic
softeners
among these
include
the following:
1) ditallow dimethylammonium chloride (DTDMAC);
2) dehydrogenated tallow dimethylammonium chloride;
3) dehydrogenated tallow dimethylammonium methylsulfate;
4) distearyl dimethylammonium chloride;
5) dioleyl dimethylammonium chloride;
6) dipalmityl hydroxyethyl methylammonium chloride;
7) stearyl benzyl dimethylammonium chloride;
8) tallow trimethylammonium chloride;
9) hydrogenated tallow trimethylammonium chloride;
10) 012-14 alkyl hydroxyethyl dimethylammonium chloride;
11) 012-18 alkyl dihydroxyethyl methylammonium chloride;
~5 12) di(stearoyloxyethyl) dimethylammonium chloride (DSOEDMAC);
13) di(tallow-oxy-ethyl) dimethylammonium chloride;
14) ditallow imidazolinium methylsulfate;
15) 1-(2-tallowylamidoethyl)-2-tallowyl imidazolinium
methylsulfate.
zo Biodegradable quaternary ammonium compounds have been presented
as alternatives to the traditionally used di-long alkyl chain ammonium
chlorides
and methyl sulfates. Such quaternary ammonium compounds contain long chain
alk(en)yl groups interrupted by functional groups such as carboxy groups. Said
materials and fabric softening compositions containing them are disclosed in
25 numerous publications such as EP-A-0,040,562, and EP-A-0,239,910.
The quaternary ammonium compounds and amine precursors herein have the
formula (I) or (II), below
3 2 R3 R3
R NR(~2~ Q~ 1 X- +\i~ (CH2)n-CH -CHZ X -
R3 Q
RI Tt T2
30 or
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49
wherein Q is selected from -O-C(O)-, -C(O)-O-, -O-C(O)-O-, -NR4-C(O)-, -C(O)-
N R4-;
R1 is (CH2)n-Q-T2 or T3;
R2 is (CH2)m-Q-T4 or T5 or R3;
R3 is C1-C4 alkyl or C1-C4 hydroxyalkyl or H;
R4 is H or C1-C4 alkyl or C1-Cq, hydroxyalkyl;
T1, T2, T3, T4, T5 are independently C11-C22 alkyl or alkenyl;
n and m are integers from 1 to 4; and
X- is a softener-compatible anion. Non-limiting examples of softener-
compatible
anions include chloride or methyl sulfate.
The alkyl, or alkenyl, chain T1, T2, T3, T4, T5 must contain at least 11
carbon
atoms, preferably at least 16 carbon atoms. The chain may be straight or
~5 branched. Tallow is a convenient and inexpensive source of long chain alkyl
and
alkenyl material. The compounds wherein T1, T2, T3, T4, Tb represents the
mixture of long chain materials typical for tallow are particularly preferred.
Specific examples of quaternary ammonium compounds suitable for use in the
2o aqueous fabric softening compositions herein include
1) N,N-di(tallowyl-oxy-ethyl)-N,N-dimethyl ammonium chloride;
2) N,N-di(tallowyl-oxy-ethyl)-N-methyl, N-(2-hydroxyethyl) ammonium methyl
sulfate;
3) N,N-di(2-tallowyl-oxy-2-oxo-ethyl)-N,N-dimethyl ammonium chloride;
25 4) N,N-di(2-tallowyl-oxy-ethylcarbonyl-oxy-ethyl)-N,N-dimethyl ammonium
chloride;
5) N-(2-tallowyl-oxy-2-ethyl)-N-(2-tallowyl-oxy-2-oxo-ethyl)-N,N-dimethyl
ammonium
chloride;
3o 6) N,N,N-tri(tallowyl-oxy-ethyl)-N-methyl ammonium chloride;
7) N-(2-tallowyl-oxy-2-oxo-ethyl)-N-(tallowyl-N,N-dimethyl-ammonium chloride;
and
8) 1,2-ditallowyl-oxy-3-trimethylammoniopropane chloride;
and mixtures of any of the above materials.
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When included therein, the laundry detergent compositions of the present
invention typically comprise from 0.2% to about 25%, preferably from about 1
to about 8% by weight of such cationic surfactants.
5 Conventional detergent enzymes
The laundry detergent compositions will preferably comprise in addition to
the saccharide gum degrading enzyme one or more enzymes which provide
cleaning performance, fabric care and/or sanitisation benefits, preferably a
~o cellulase, and/or amylase.
It has been surprisingly found that the laundry detergent compositions of
the present invention further comprising another enzyme, especially a
cellulase
and/or an amylase provide enhanced food stain/soil removal, dingy cleaning and
whiteness maintenance. In particular, it has been found that cellulolytic
enzymes
~5 are particularly useful in degrading cellulose polysaccharide food
additives and
thereby useful in helping the cleaning of food stain/soil from cotton fabrics.
Without wishing to be bound by theory, it is believed that this improved
performance is resulting from the combined enzymatic hydrolysis's of the
2o cellulase enzyme on the cotton fabric support and of the saccharide gum
degrading enzyme on the polysaccharide binding the stain onto the cotton
fabric
support. Similarly, the combined action of the amylase on the starch-based
finishing agent covering the surface of the cotton fabric and of the
saccharide
gum degrading enzyme on the polysaccharide binding the stain onto the cotton
25 fabric, gives enhanced pertormance:
Said enzymes include enzymes selected from cellulases, hemicellulases,
peroxidases, proteases, gluco-amylases, amylases, xylanases, lipases,
phospholipases, esterases, cutinases, pectinases, keratanases, reductases,
30 oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases,
tannases,
pentosanases, f3-glucanases, arabinosidases, hyaluronidase, chondroitinase,
laccase or mixtures thereof.
The cellulases usable in the present invention include both bacterial or
35 fungal cellulases. Preferably, they will have a pH optimum of between 5 and
12
and a specific activity above 50 CEVU/mg (Cellulose Viscosity Unit). Suitable
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51
celluiases are disclosed in U.S. Patent 4,435,307, Barbesgoard et al,
J61078384
and W096/02653 which discloses fungal cellulase produced respectively from
Humicola insolens, Trichoderma, Thielavia and Sporotrichum. EP 739 982
describes cellulases isolated from novel Bacillus species. Suitable cellulases
are
s also disclosed in GB-A-2.075.028; GB-A-2.095.275; DE-OS-2.247.832 and
W095/26398.
Examples of such cellulases are cellulases produced by a strain of
Humicola insolens (Humicola grisea var. thermoidea), particularly the Humicola
strain DSM 1800.
o Other suitable cellulases are cellulases originated from Humicola insolens
having
a molecular weight of about 50KDa, an isoelectric point of 5.5 and containing
415
amino acids; and a -43kD endoglucanase derived from Humicola insolens, DSM
1800, exhibiting cellulase activity; a preferred endogiucanase component has
the
amino acid sequence disclosed in PCT Patent Application No. WO 91/17243.
Also suitable cellulases are the EGIII cellulases from Trichoderma
longibrachiatum described in W094/21801, Genencor, published September 29,
1994. Especially suitable cellulases are the cellulases having color care
benefits.
Examples of such cellulases are cellulases described in European patent
application No. 91202879.2, filed November 6, 1991 (Novo). Carezyme and
2o Celluzyme (Novo Nordisk A/S) are especially useful. See also W091/17244 and
W091/21801. Other suitable cellulases for fabric care and/or cleaning
properties
are described in W096/34092, W096/17994 and W095/24471.
Said cellulases are normally incorporated in the detergent composition at
levels from 0.0001 % to 2% of pure enzyme by weight of the detergent
25 composition.
Amylases (a and/or f3) can be included for removal of carbohydrate-based
stains. W094/02597, Novo Nordisk A/S published February 03, 1994, describes
cleaning compositions which incorporate mutant amylases. See also
3o W095/10603, Novo Nordisk A/S, published April 20, 1995. Other amylases
known for use in cleaning compositions include both a- and ~i-amylases. a-
Amylases are known in the art and include those disclosed in US Pat. no.
5,003,257; EP 252,666; WO/91/00353; FR 2,676,456; EP 285,123; EP 525,610;
EP 368,341; and British Patent specification no. 1,296,839 (Novo). Other
suitable
35 amylases are stability-enhanced amylases described in W094/18314, published
August 18, 1994 and W096/05295, Genencor, published February 22, 1996 and
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52
amylase variants having additional modification in the immediate parent
available
from Novo Nordisk A/S, disclosed in WO 95/10603, published April 95. Also
suitable are amylases described in EP 277 216, W095/26397 and W096/23873
(all by Novo Nordisk).
Examples of commercial a-amylases products are Purafect Ox Am~ from
Genencor and Termamyl~, Ban~ ,Fungamyl~ and Duramyl~, all available from
Novo Nordisk A/S Denmark. W095/26397 describes other suitable amylases : a-
amylases characterised by having a specific activity at least 25% higher than
the
specific activity of Termamyl~ at a temperature range of 25°C to
55°C and at a
o pH value in the range of 8 to 10, measured by the Phadebas~ a-amylase
activity
assay. Suitable are variants of the above enzymes, described in W096/23873
(Novo Nordisk). Other amylolytic enzymes with improved properties with respect
to the activity level and the combination of thermostability and a higher
activity
level are described in W095/35382.
~5 The amylolytic enzymes are incorporated in the detergent compositions of
the present invention a level of from 0.0001 % to 2%, preferably from 0.00018%
to 0.06%, more preferably from 0.00024% to 0.048% pure enzyme by weight of
the composition.
2o A preferred combination is a laundry detergent composition having
cocktail of conventional applicable enzymes like protease, amylase, lipase,
cutinase and/or cellulase in conjunction with one or more plant cell wall
degrading enzymes.
25 Peroxidase enzymes are used in combination with oxygen sources, e.g.
percarbonate, perborate, persulfate, hydrogen peroxide, etc and with a
phenolic
substrate as bleach enhancing molecule. They are used for "solution
bleaching",
i.e., to prevent transfer of dyes or pigments removed from substrates during
wash operations to other substrates in the wash solution. Peroxidase enzymes
3o are known in the art, and include, for example, horseradish peroxidase,
iigninase
and haloperoxidase such as chloro- and bromo-peroxidase. Peroxidase-
containing detergent compositions are disclosed, for example, in PCT
International Application WO 89/099813, W089/09813 and in European Patent
application EP No. 91202882.6, filed on November 6, 1991 and EP No.
35 96870013.8, filed February 20, 1996. Also suitable is the laccase enzyme.
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53
Enhancers are generally comprised at a level of from 0.1 % to 5% by
weight of totai composition. Preferred enhancers are substitued phenthiazine
and
phenoxasine 10-Phenothiazinepropionicacid (PPT), 10-ethylphenothiazine-4-
carboxylic acid (EPC), 10-phenoxazinepropionic acid (POP) and 10-
methylphenoxazine (described in WO 94/12621) and substitued syringates (C3-
C5 substitued alkyl syringates) and phenols. Sodium percarbonate or perborate
are preferred sources of hydrogen peroxide.
Said peroxidases are normally incorporated in the detergent composition
at levels from 0.0001 % to 2% of pure enzyme by weight of the detergent
o composition.
Other preferred enzymes that can be included in the detergent
compositions of the present invention include iipases. Suitable lipase enzymes
for detergent usage include those produced by microorganisms of the
~5 Pseudomonas group, such as Pseudornonas stutzeri ATCC 19.154, as disclosed
in British Patent 1,372,034. Suitable lipases include those which show a
positive
immunological cross-reaction with the antibody of the lipase, produced by the
microorganism Pseudomonas fluorescent IAM 1057. This lipase is available from
Amano Pharmaceutical Co. Ltd., Nagoya, Japan, under the trade name Lipase P
20 "Amano," hereinafter referred to as "Amano-P". Other suitable commercial
lipases include Amano-CES, lipases ex Chromobacter viscosum, e.g.
Chromobacter viscosum var. lipolyticum NRRLB 3673 from Toyo Jozo Co.,
Tagata, Japan; Chromobacter viscosum lipases from U.S. Biochemical Corp.,
U.S.A. and Disoynth Co., The Netherlands, and lipases ex Pseudomonas
25 gladioli. Especially suitable lipases are lipases such as M1 LipaseR and
LipomaxR (Gist-Brocades) and LipolaseR and Lipolase UItraR(Novo) which have
found to be very effective when used in combination with the compositions of
the
present invention. Also suitables are the lipolytic enzymes described in EP
258
068, WO 92/05249 and WO 95/22615 by Novo Nordisk and in WO 94/03578,
3o WO 95/35381 and WO 96/00292 by Unilever.
Also suitable are cutinases [EC 3.1.1.50] which can be considered as a special
kind of lipase, namely lipases which do not require interfacial activation.
Addition
of cutinases to detergent compositions have been described in e.g. WO-A
88/09367 (Genencor); WO 90/09446 (Plant Genetic System) and WO 94/14963
35 and WO 94/14964 (Unilever).
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The lipases and/or cutinases are normally incorporated in the detergent
composition at levels from 0.0001 % to 2% of pure enzyme by weight of the
detergent composition.
Suitable proteases are the subtilisins which are obtained from particular
strains of B. subtilis and 8. licheniformis (subtilisin BPN and BPN'). One
suitable
protease is obtained from a strain of Bacillus, having maximum activity
throughout the pH range of 8-12, developed and sold as ESPERASE~ by Novo
Industries A/S of Denmark, hereinafter "Novo". The preparation of this enzyme
and analogous enzymes is described in GB 1,243,784 to Novo. Other suitable
proteases include ALCALASE~, DURAZYM~ and SAVINASE~ from Novo and
MAXATASE~~ MAXACAL~, PROPERASEO and MAXAPEM~ (protein
engineered Maxacal) from Gist-Brocades. Proteolytic enzymes also encompass
modified bacterial serine proteases, such as those described in European
Patent
~5 Application Serial Number 87 303761.8, filed April 28, 1987 (particularly
pages
17, 24 and 98), and which is called herein "Protease B", and in European
Patent
Application 199,404, Venegas, published October 29, 1986, which refers to a
modified bacterial serine protealytic enzyme which is called "Protease A"
herein.
Suitable is the protease called herein "Protease C", which is a variant of an
2o alkaline serine protease from Bacillus in which lysine replaced arginine at
position 27, tyrosine replaced valine at position 104, serine replaced
asparagine
at position 123, and alanine replaced threonine at position 274. Protease C is
described in EP 90915958:4, corresponding to WO 91/06637, Published May 16,
1991. Genetically modified variants, particularly of Protease C, are also
included
25 herein.
A preferred protease referred to as "Protease D" is a carbonyl hydrolase
variant
having an amino acid sequence not found in nature, which is derived from a
precursor carbonyl hydrolase by substituting a different amino acid for a
plurality
of amino acid residues at a position in said carbonyl hydrolase equivalent to
3o position +76, preferably also in combination with one or more amino acid
residue
positions equivalent to those selected from the group consisting of +99, +101,
+103, +104, +107, +123, +27, +105, +109, +126, +128, +135, +156, +166, +195,
+197, +204, +206, +210, +216, +217, +218, +222, +260, +265, and/or +274
according to the numbering of Bacillus amyloliquefaciens subtilisin, as
described
35 in W095I10591 and in the patent application of C. Ghosh, et al, "Bleaching
Compositions Comprising Protease Enzymes" having US Serial No. 08/322,677,
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WO 99/09127 PCT/US98/11995
filed October 13, 1994. Also suitable is a carbonyl hydrolase variant of the
protease described in W095/10591, having an amino acid sequence derived by
replacement of a plurality of amino acid residues replaced in the precursor
enzyme corresponding to position +210 in combination with one or more of the
5 following residues : +33, +62, +67, +76, +100, +101, +103, +104, +107, +128,
+129, +130, +132, +135, +156, +158, +164, +166, +167, +170, +209, +215,
+217, +218, and +222, where the numbered position corresponds to naturally-
occurring subtilisin from Bacillus amyloliquefaciens or to equivalent amino
acid
residues in other carbonyl hydrolases or subtilisins, such as Bacillus lentus
subtilisin (co-pending patent application US Serial No. 60/048,550, filed June
04,
1997).
Also suitable for the present invention are proteases described in patent
applications EP 251 446 and WO 91/06637, protease BLAP~ described in
W091/02792 and their variants described in WO 95/23221.
~5 See also a high pH protease from Bacillus sp. NCIMB 40338 described in WO
93/18140 A to Novo. Enzymatic detergents comprising protease, one or more
other enzymes, and a reversible protease inhibitor are described in WO
92103529 A to Novo. When desired, a protease having decreased adsorption
and increased hydrolysis is available as described in WO 95/07791 to Procter &
2o Gamble. A recombinant trypsin-like protease for detergents suitable herein
is
described in WO 94/25583 to Novo. Other suitable proteases are described in
EP 516 200 by Unilever.
The proteolytic enzymes are incorporated in the detergent compositions of
the present invention a level of from 0.0001 % to 2%, preferably from 0.001 %
to
25 0.2%, more preferably from 0.005% to 0.1 % pure enzyme by weight of the
composition.
The above-mentioned enzymes may be of any suitable origin, such as
vegetable, animal, bacterial, fungal and yeast origin. Origin can further be
3o mesophilic or extremophilic (psychrophilic, psychrotrophic, thermophilic,
basophilic, alkalophilic, acidophilic, halophilic, etc.). Purified or non-
purified forms
of these enzymes may be used. Nowadays, it is common practice to modify wild-
type enzymes via protein / genetic engineering techniques in order to optimise
their performance efficiency in the cleaning compositions of the invention.
For
35 example, the variants may be designed such that the compatibility of the
enzyme
to commonly encountered ingredients of such compositions is increased.
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56
Alternatively, the variant may be designed such that the optimal pH, bleach or
chelant stability, catalytic activity and the like, of the enzyme variant is
tailored to
suit the particular cleaning application.
In particular, attention should be focused on amino acids sensitive to
oxidation in the case of bleach stability and on surface charges for the
surfactant
compatibility. The isoelectric point of such enzymes may be modified by the
substitution of some charged amino acids, e.g. an increase in isoelectric
point
may help to improve compatibility with anionic surfactants. The stability of
the
1o enzymes may be further enhanced by the creation of e.g. additional salt
bridges
and enforcing calcium binding sites to increase chelant stability. Special
attention
must be paid to the cellulases as most of the cellulases have separate binding
domains (CBD). Properties of such enzymes can be altered by modifications in
these domains.
Said enzymes are normally incorporated in the detergent composition at
levels from 0.0001 % to 2% of pure enzyme by weight of the detergent
composition. The enzymes can be added as separate single ingredients (grills,
granulates, stabilized liquids, etc... containing one enzyme ) or as mixtures
of two
or more enzymes ( e.g. cogranulates ).
Other suitable detergent ingredients that can be added are enzyme
oxidation scavengers which are described in Co-pending European Patent
application 92870018.6 filed on January 31, 1992. Examples of such enzyme
oxidation scavengers are ethoxylated tetraethylene polyamines.
A range of enzyme materials and means for their incorporation into
synthetic detergent compositions is also disclosed in WO 9307263 A and WO
9307260 A to Genencor International, WO 8908694 A to Novo, and U.S.
3,553,139, January 5, 1971 to McCarty et al. Enzymes are further disclosed in
U.S. 4,101,457, Place et al, July 18, 1978, and in U.S. 4,507,219, Hughes,
March 26, 1985. Enzyme materials useful for liquid detergent formulations, and
their incorporation into such formulations, are disclosed in U.S. 4,261,868,
Hora
et al, April 14, 1981. Enzymes for use in detergents can be stabilised by
various
techniques. Enzyme stabilisation techniques are disclosed and exemplified in
U.S. 3,600,319, August 17, 1971, Gedge et al, EP 199,405 and EP 200,586,
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57
October 29, 1986, Venegas. Enzyme stabilisation systems are also described,
for example, in U.S. 3,519,570. A useful Bacillus, sp. AC13 giving proteases,
xylanases and cellulases, is described in WO 9401532 A to Novo.
Bleaching agent
It has been surprisingly found that the laundry detergent compositions of
the present invention further comprising a bleaching agent, especially a
bleach
activator bleaching system, provide enhanced food stain/soil removal, dingy
cleaning and whiteness maintenance. Without wishing to be bound by theory, it
is
believed that the smaller chromophoric particles resulting from the saccharide
gums degrading enzyme hydrolysis are more easily attacked by the bleach
activated bleaching systems, especially at low temperature.
~5 Additional optional detergent ingredients that can be included in the
laundry detergent compositions of the present invention include bleaching
agents
such as hydrogen peroxide, PB1, PB4 and percarbonate with a particle size of
400-800 microns.
2o These bleaching agent components can include one or more oxygen
bleaching agents and, depending upon the bleaching agent chosen, one or more
bleach activators. When present oxygen bleaching compounds will typically be
present at levels of from about 1 % to about 25%.
25 The bleaching agent component for use herein can be any of the
bleaching agents useful for laundry detergent compositions including oxygen
bleaches as well as others known in the art. The bleaching agent suitable for
the
present invention can be an activated or non-activated bleaching agent.
3o One category of oxygen bleaching agent that can be used encompasses
percarboxylic acid bleaching agents and salts thereof. Suitable examples of
this
class of agents include magnesium monoperoxyphthalate hexahydrate, the
magnesium salt of meta-chloro perbenzoic acid, 4-nonylamino-4-
oxoperoxybutyric acid and diperoxydodecanedioic acid. Such bleaching agents
as are disclosed in U.S. Patent 4,483,781, U.S. Patent Application 740,446,
European Patent Application 0,133,354 and U.S. Patent 4,412,934. Highly
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58
preferred bleaching agents also include 8-nonylamino-6-oxoperoxycaproic acid
as described in U.S. Patent 4,634,551.
Another category of bleaching agents that can be used encompasses the
halogen bleaching agents. Examples of hypohalite bleaching agents, for
example, include trichloro isocyanuric acid and the sodium and potassium
dichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides. Such
materials are normally added at 0.5-10% by weight of the finished product,
preferably 1-5% by weight.
o The hydrogen peroxide releasing agents can be used in combination with
bleach activators such as tetraacetylethylenediamine (TAED),
nonanoyloxybenzene-sulfonate (NOES, described in US 4,412,934), 3,5,-
trimethylhexanoloxybenzenesulfonate (ISONOBS, described in EP 120,591) or
pentaacetylglucose (PAG)or Phenolsulfonate ester of N-nonanoyl-6-
~ 5 aminocaproic acid (NACA-OBS, described in W094/28106), which are
perhydrolyzed to form a peracid as the active bleaching species, leading to
improved bleaching effect. Also suitable activators are acylated citrate
esters
such as disclosed in Co-pending European Patent Application No. 91870207.7
and unsymetrical acyclic imide bleach activator of the following formula as
2o disclosed in the Procter 8~ Gamble co-pending patent applications US serial
No.
60/022,786 (filed July 30, 1996) and No. 60/028,122 (filed October 15, 1996)
O O
Ri
N R3
R2
wherein R1 is a C7-C13 linear or branched chain saturated or unsaturated alkyl
2s group, R2 is a C1-Cg, linear or branched chain saturated or unsaturated
alkyl
group and R3 is a C1-C4 linear or branched chain saturated or unsaturated
alkyl
group.
Useful bleaching agents, including peroxyacids and bleaching systems
3o comprising bleach activators and peroxygen bleaching compounds for use in
detergent compositions according to the invention are described in our co-
pending applications USSN 08/136,626, PCT/US95/07823, W095/27772,
W095/27773, W095/27774 and W095/27775.
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59
The hydrogen peroxide may also be present by adding an enzymatic
system {i.e. an enzyme and a substrate therefore) which is capable of
generating
hydrogen peroxide at the beginning or during the washing and/or rinsing
process.
Such enzymatic systems are disclosed in EP Patent Application 91202655.6 filed
October 9, 1991.
Metal-containing catalysts for use in bleach compositions, include cobalt-
containing catalysts such as Pentaamine acetate cobalt(III) salts and
1o manganese-containing catalysts such as those described in EPA 549 271; EPA
549 272; EPA 458 397; US 5,246,621; EPA 458 398; US 5,194,416 and US
5,114,611. Bleaching composition comprising a peroxy compound, a
manganese-containing bleach catalyst and a chelating agent is described in the
patent application No 9487020fi.3.
Bleaching agents other than oxygen bleaching agents are also known in
the art and can be utilized herein. One type of non-oxygen bleaching agent of
particular interest includes photoactivated bleaching agents such as the
sulfonated zinc and/or aluminum phthalocyanines. These materials can be
2o deposited upon the substrate during the washing process. Upon irradiation
with
light, in the presence of oxygen, such as by hanging clothes out to dry in the
daylight, the sulfonated zinc phthalocyanine is activated and, consequently,
the
substrate is bleached. Preferred zinc phthalocyanine and a photoactivated
bleaching process are described in U.S. Patent 4,033,718. Typically, detergent
compositions will contain about 0.025% to about 1.25%, by weight, of
sulfonated
zinc phthalocyanine.
Builder system
3o The laundry detergent compositions of the present invention will preferably
comprise builder, more preferably an inorganic builder, most preferably
Zeolite A,
a layered silicate and/or Sodium tripolyphosphate. It has been surprisingly
found
that the laundry detergent composition of the present invention further
comprising
a builder, provide enhanced food stain/soil removal, dingy cleaning and
whiteness maintenance. Without wishing to be bound by theory, it is believed
that
the saccharide gums may entrap calcium and thereby limit the enzyme
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WO 99/09127 PCT/US98/11995
hydrolysis. Therefore, the use of builder is expected to remove the entrapped
calcium and favouring the action of the saccharide gums degrading enzyme.
Any conventional builder system is suitable for use herein including
5 aluminosilicate materials, silicates, polycarboxylates, alkyl- or alkenyl-
succinic
acid and fatty acids, materials such as ethylenediamine tetraacetate,
diethylene
triamine pentamethyleneacetate, metal ion sequestrants such as
aminopolyphosphonates, particularly ethylenediamine tetramethylene
phosphonic acid and diethylene triamine pentamethylenephosphonic acid.
o Phosphate builders can also be used herein.
Suitable builders can be an inorganic ion exchange material, commonly an
inorganic hydrated aluminosilicate material, more particularly a hydrated
synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.
~5 Another suitable inorganic builder material is layered silicate, e.g. SKS-6
(Hoechst). SKS-6 is a crystalline layered silicate consisting of sodium
silicate
(Na2Si205).
Suitable polycarboxylates containing one carboxy group include lactic
acid, glycolic acid and ether derivatives thereof as disclosed in Belgian
Patent
2o Nos. 831,368, 821,369 and 821,370. Polycarboxylates containing two carboxy
groups include the water-soluble salts of succinic acid, malonic acid,
(ethylenedioxy) diacetic acid, malefic acid, diglycollic acid, tartaric acid,
tartronic
acid and fumaric acid, as well as the ether carboxylates described in German
Offenlegenschrift 2,446,686, and 2,446,687 and U.S. Patent No. 3,935,257 and
2s the sulfinyl carboxylates described in Belgian Patent No. 840,623.
Polycarboxylates containing three carboxy groups include, in particular, water-
solubie citrates, aconitrates and citraconates as well as succinate
derivatives
such as the carboxymethyloxysuccinates described in British Patent No.
1,379,241, lactoxysuccinates described in Netherlands Application 7205873, and
3o the oxypolycarboxylate materials such as 2-oxa-1,1,3-propane
tricarboxylates
described in British Patent No. 1,387,447.
Polycarboxylates containing four carboxy groups include oxydisuccinates
disclosed in British Patent No. 1,261,829, 1,1,2,2-ethane tetracarboxylates,
35 1,1,3,3-propane tetracarboxylates and 1,1,2, 3-propane tetracarboxylates.
Polycarboxylates containing sulfo substituents include the sulfosuccinate
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61
derivatives disclosed in British Patent Nos. 1,398,421 and 1,398,422 and in
U.S.
Patent No. 3,936,448, and the sulfonated pyrolysed citrates described in
British
Patent No. 1,082,179; while polycarboxylates containing phosphone substituents
are disclosed in British Patent No. 1,439,000.
Alicyclic and heterocyclic polycarboxylates include cyclopentane-
cis,cis,cis-tetracarboxylates, cyclopentadienide pentacarboxylates, 2,3,4,5-
tetrahydro-furan - cis, cis, cis-tetracarboxylates, 2,5-tetrahydro-furan -cis -
dicarboxylates, 2,2,5,5-tetrahydrofuran - tetracarboxylates, 1,2,3,4,5,6-
hexane -
hexacar-boxylates and and carboxymethyl derivatives of polyhydric alcohols
such as sorbitol, mannitol and xylitol. Aromatic poly-carboxylates include
mellitic
acid, pyromellitic acid and the phthalic acid derivatives disclosed in British
Patent
No. 1,425, 343.
Of the above, the preferred polycarboxylates are hydroxycarboxylates
~5 containing up to three carboxy groups per molecule, more particularly
citrates.
Preferred builder systems for use in the present compositions include a
mixture of a water-insoluble aluminosilicate builder such as zeolite A or of a
layered silicate (SKS-6), and a water-soluble carboxylate chelating agent such
as
2o citric acid. Other preferred builder systems include a mixture of a water-
insoluble
aluminosilicate builder such as zeolite A, and a watersoluble carboxylate
chelating agent such as citric acid. Preferred builder systems for use in
liquid
detergent compositions of the present invention are soaps and
polycarboxylates.
25 Other builder materials that can form part of the builder system for use in
granular compositions include inorganic materials such as alkali metal
carbonates, bicarbonates, silicates, and organic materials such as the organic
phosphonates, amino polyalkylene phosphonates and amino polycarboxylates.
Other suitable water-soluble organic salts are the homo- or co-polymeric
3o acids or their salts, in which the polycarboxylic acid comprises at least
two
carboxyl radicals separated from each other by not more than two carbon atoms.
Polymers of this type are disclosed in GB-A-1,596,756. Examples of such salts
are polyacrylates of MW 2000-5000 and their copolymers with malefic anhydride,
such copolymers having a molecular weight of from 20,000 to 70,000, especially
35 about 40,000.
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62
Detergency builder salts are normally included in amounts of from 5% to
80% by weight of the composition preferably from 10% to 70% and most usually
from 30% to 60% by weight.
Other surfactant system
The laundry detergent compositions of the present invention may also
contain cationic, ampholytic, zwitterionic, and semi-polar surfactants, as
well as
the nonionic and/or anionic surfactants other than those already described
1 o herein.
Ampholytic surfactants are also suitable for use in the laundry detergent
compositions of the present invention. These surfactants can be broadly
described as aliphatic derivatives of secondary or tertiary amines, or
aliphatic
~5 derivatives of heterocyclic secondary and tertiary amines in which the
aliphatic
radical can be straight- or branched-chain. One of the aliphatic substituents
contains at least about 8 carbon atoms, typically from about 8 to about 18
carbon
atoms, and at least one contains an anionic water-solubilizing group, e.g.
carboxy, sulfonate, sulfate. See U.S. Patent No. 3,929,678 to Laughlin et al.,
2o issued December 30, 1975 at column 19, lines 18-35, for examples of
ampholytic
surfactants.
When included therein, the laundry detergent compositions of the present
invention typically comprise from 0.2% to about 15%, preferably from about 1
to about 10% by weight of such ampholytic surtactants.
Zwitterionic surfactants are also suitable for use in laundry detergent
compositions. These surfactants can be broadly described as derivatives of
secondary and tertiary amines, derivatives of heterocyclic secondary and
tertiary
amines, or derivatives of quaternary ammonium, quaternary phosphonium or
3o tertiary sulfonium compounds. See U.S. Patent No. 3,929,678 to Laughlin et
al.,
issued December 30, 1975 at column 19, line 38 through column 22, line 48, for
examples of zwitterionic surfactants.
When included therein, the laundry detergent compositions of the present
invention typically comprise from 0.2% to about 15%, preferably from about 1
to about 10% by weight of such zwitterionic surfactants.
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63
Semi-polar nonionic surfactants are a special category of nonionic
surfactants which include water-soluble amine oxides containing one alkyl
moiety
of from about 10 to about 18 carbon atoms and 2 moieties selected from the
group consisting of alkyl groups and hydroxyalkyl groups containing from about
1
to about 3 carbon atoms; water-soluble phosphine oxides containing one alkyl
moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from
the group consisting of alkyl groups and hydroxyalkyl groups containing from
about 1 to about 3 carbon atoms; and water-soluble sulfoxides containing one
alkyl moiety of from about 10 to about 18 carbon atoms and a moiety selected
o from the group consisting of alkyl and hydroxyalkyl moieties of from about 1
to
about 3 carbon atoms.
Semi-polar nonionic detergent surfactants include the amine oxide surfactants
having the formula
0
T
R3(OR4)xN(R5)2
wherein R3 is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixtures therof
2o containing from about 8 to about 22 carbon atoms; R'f is an alkylene or
hydroxyalkylene group containing from about 2 to about 3 carbon atoms ar
mixtures thereof; x is from 0 to about 3; and each R5 is an alkyl or
hydroxyalkyl
group containing from about 1 to about 3 carbon atoms or a polyethylene oxide
group containing from about 1 to about 3 ethylene oxide groups. The R5 groups
can be attached to each other, e.g., through an oxygen or nitrogen atom, to
form
a ring structure.
These amine oxide surfactants in particular include C1p-C1g alkyl
dimethyl amine oxides and Cg-C12 alkoxy ethyl dihydroxy ethyl amine oxides.
When included therein, the cleaning compositions of the present invention
3o typically comprise from 0.2% to about 15%, preferably from about 1 % to
about
10% by weight of such semi-polar nonionic surfactants.
The laundry detergent composition of the present invention may further
comprise a cosurfactant selected from the group of primary or tertiary amines.
Suitable primary amines for use herein include amines according to the formula
R1 NH2 wherein R1 is a Cg-C12, preferably Cg-C10 alkyl chain or R4X(CH2)n, X
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64
is -O-,-C(O)NH- or -NH-, R4 is a Cg-C12 alkyl chain n is between 1 to 5,
preferably 3. R1 alkyl chains may be straight or branched and may be
interrupted with up to 12, preferably less than 5 ethylene oxide moieties.
Preferred amines according to the formula herein above are n-alkyl amines.
Suitable amines for use herein may be selected from 1-hexylamine, 1-
octylamine, 1-decylamine and laurylamine. Other preferred primary amines
include CS-C10 oxypropylamine, octyloxypropylamine, 2-ethylhexyl-
oxypropylamine, lauryl amido propylamine and amido propylamine.
Suitable tertiary amines for use herein include tertiary amines having the
formula R1 R2R3N wherein R1 and R2 are C1-Cg alkylchains or
Rs
-C CHz-CH-O~H
~5 Rg is either a Cg-C12, preferably Cg-C10 alkyl chain, or R3 is R4X(CH2)n,
whereby X is -O-, -C(O)NH- or -NH-,R4 is a C4-C12, n is between 1 to 5,
preferably 2-3. R5 is H or C1-C2 alkyl and x is between 1 to 6 .
R3 and R4 may be linear or branched ; R3 alkyl chains may be interrupted with
up to 12, preferably less than 5, ethylene oxide moieties.
Preferred tertiary amines are R1 R2RgN where R1 is a C6-C12 alkyl chain,
R2 and R3 are C1-C3 alkyl or
Rs
I
-C CHz-CH-O~H
where R5 is H or CH3 and x = 1-2.
Also preferred are the amidoamines of the formula:
0
I I
R1-C-NH-( CH2 )-N-( R2 )
n a
3o wherein R1 is Cg-C12 alkyl; n is 2-4,
preferably n is 3; R2 and Rg is C1-C4
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Most preferred amines of the present invention include 1-octylamine, 1-
hexylamine, 1-decylamine, 1-dodecylamine,CB-10oxypropylamine, N coco 1-
3diaminopropane, coconutalkyldimethylamine, lauryldimethylamine, lauryl bis-
(hydroxyethyl)amine, coco bis{hydroxyehtyl)amine, lauryl amine 2 moles
5 propoxylated, octyl amine 2 moles propoxylated, lauryl amidopropyl-
dimethylamine, C8-10 amidopropyldimethylamine and C10 amidopropyl-
dimethylamine.
The most preferred amines for use in the compositions herein are 1-hexylamine,
1-octylamine, 1-decylamine, 1-dodecylamine. Especially desirable are n
1o dodecyldimethylamine and bishydroxyethylcoconutalkylamine and oleylamine 7
times ethoxylated, lauryl amido propylamine and cocoamido propylamine.
Color care and fabric care benefits
Technologies which provide a type of color care benefit can also be
included. Examples of these technologies are metallo catalysts for color
2o maintenance. Such metallo catalysts are described in co-pending European
Patent Application No. 92870181.2. Dye fixing agents, polyolefin dispersion
for
anti-wrinkles and improved water absorbancy, perfume and amino-functional
polymer for color care treatment and perfume substantivity are further
examples
of color care / fabric care technologies and are described in the co-pending
Patent Application No. 96870140.9, filed November 07, 1996.
Fabric softening agents can also be incorporated into laundry detergent
compositions in accordance with the present invention. These agents may be
inorganic or organic in type. Inorganic softening agents are exemplified by
the
3o smectite clays disclosed in GB-A-1 400 898 and in USP 5,019,292. Organic
fabric softening agents include the water insoluble tertiary amines as
disclosed in
GB-A1 514 276 and EP-BO 011 340 and their combination with mono C12-C14
quaternary ammonium salts are disclosed in EP-B-0 026 527 and EP-B-0 026
528 and di-long-chain amides as disclosed in EP-B-0 242 919. Other useful
organic ingredients of fabric softening systems include high molecular weight
polyethylene oxide materials as disclosed in EP-A-0 299 575 and 0 313 146.
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66
Levels of smectite clay are normally in the range from 2% to 20%, more
preferably from 5% to 15% by weight, with the material being added as a dry
mixed component to the remainder of the formulation. Organic fabric softening
agents such as the water-insoluble tertiary amines or dilong chain amide
materials are incorporated at levels of from 0.5% to 5% by weight, normally
from
1 % to 3% by weight whilst the high molecular weight polyethylene oxide
materials and the water soluble cationic materials are added at levels of from
0.1 % to 2%, normally from 0.15% to 1.5% by weight. These materials are
1o normally added to the spray dried portion of the composition, although in
some
instances it may be more convenient to add them as a dry mixed particulate, or
spray them as molten liquid on to other solid components of the composition.
Chelating Agents
The laundry detergent compositions herein may also optionally contain
one or more iron andlor manganese chelating agents. Such chelating agents
2o can be selected from the group consisting of amino carboxylates, amino
phosphonates, polyfunctionally-substituted aromatic chelating agents and
mixtures therein, all as hereinafter defined. Without intending to be bound by
theory, it is believed that the benefit of these materials is due in part to
their
exceptional ability to remove iron and manganese ions from washing solutions
by
formation of soluble chelates.
Amino carboxylates useful as optional chelating agents include
ethylenediaminetetracetates, N-hydroxyethylethylenediaminetriacetates, nitrilo-
triacetates, ethylenediamine tetraproprionates, triethylenetetraamine-
3o hexacetates, diethylenetriaminepentaacetates, and ethanoldiglycines, alkali
metal, ammonium, and substituted ammonium salts therein and mixtures therein.
Amino phosphonates are also suitable for use as chelating agents in the
compositions of the invention when at lease low levels of total phosphorus are
permitted in detergent compositions, and include ethylenediaminetetrakis
(methylenephosphonates) as DEQUEST. Preferably, these amino phosphonates
do not contain alkyl or alkenyl groups with more than about 6 carbon atoms.
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Polyfunctionally-substituted aromatic chelating agents are also useful in
the compositions herein. See U.S. Patent 3,812,044, issued May 21, 1974, to
Connor et al. Preferred compounds of this type in acid form are
dihydroxydisulfobenzenes such as 1,2-dihydroxy-3,5-disulfobenzene.
A preferred biodegradable chelator for use herein is ethylenediamine
disuccinate ("EDDS"), especially the [S,S] isomer as described in U.S. Patent
4,704,233, November 3, 1987, to Hartman and Perkins.
o The compositions herein may also contain water-soluble methyl glycine
diacetic acid (MGDA) salts (or acid form) as a chelant or co-builder useful
with,
for example, insoluble builders such as zeolites, layered silicates and the
like.
If utilized, these chelating agents will generally comprise from about 0.1
~5 to about 15% by weight of the detergent compositions herein. More
preferably, if
utilized, the chelating agents will comprise from about 0.1 % to about 3.0% by
weight of such compositions.
Suds suppressor
Another optional ingredient is a suds suppressor, exemplified by silicones,
and silica-silicone mixtures. Silicones can be generally represented by
alkylated
polysiloxane materials while silica is normally used in finely divided forms
exemplified by silica aerogels and xerogels and hydrophobic silicas of various
types. These materials can be incorporated as particulates in which the suds
suppressor is advantageously releasably incorporated in a water-soluble or
water-dispersible, substantially non-surface-active detergent impermeable
carrier. Alternatively the suds suppressor can be dissolved or dispersed in a
liquid carrier and applied by spraying on to one or more of the other
components.
3o A preferred silicone suds controlling agent is disclosed in Bartollota et
al.
U.S. Patent 3 933 672. Other particularly useful suds suppressors are the self-
emulsifying silicone suds suppressors, described in German Patent Application
DTOS 2 646 126 published April 28, 1977. An example of such a compound is
DC-544, commercially available from Dow Corning, which is a siloxane-glycol
copolymer. Especially preferred suds controlling agent are the suds suppressor
system comprising a mixture of silicone oils and 2-alkyl-alcanols. Suitable 2-
alkyl-
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alkanols are 2-butyl-octanol which are commercially available under the trade
name Isofol 12 R.
Such suds suppressor system are described in Co-pending European Patent
application N 92870174.7 filed 10 November, 1992.
Especially preferred silicone suds controlling agents are described in Co-
pending European Patent application N°92201649.8. Said compositions can
comprise a silicone/silica mixture in combination with fumed nonporous silica
such as AerosilR.
1o The suds suppressers described above are normally employed at levels of
from 0.001 % to 2% by weight of the composition, preferably from 0.01 % to 1 %
by
weight.
Others
Other components used in laundry detergent compositions may be
employed, such as soil-suspending agents, soil-release agents, optical
brighteners, abrasives, bactericides, tarnish inhibitors, coloring agents,
and/or
encapsulated or non-encapsulated perfumes.
Especially suitable encapsulating materials are water soluble capsules
which consist of a matrix of polysaccharide and polyhydroxy compounds such as
described in GB 1,464,616. Other suitable water soluble encapsulating
materials
comprise dextrins derived from ungelatinized starch acid-esters of substituted
dicarboxylic acids such as described in US 3,455,838. These acid-ester
dextrins
are,preferably, prepared from such starches as waxy maize, waxy sorghum,
sago, tapioca and potato. Suitable examples of said encapsulating materials
include N-Lok manufactured by National Starch. The N-Lok encapsulating
material consists of a modified maize starch and glucose. The starch is
modified
3o by adding monofunctional substituted groups such as octenyl succinic acid
anhydride.
Antiredeposition and soil suspension agents suitable herein include
cellulose derivatives such as methylcellulose, carboxymethylcellulose and
hydroxyethyfcellulose, and home- or co-polymeric polycarboxylic acids or their
salts. Polymers of this type include the polyacrylates and malefic anhydride-
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69
acrylic acid copolymers previously mentioned as builders, as well as
copolymers
of malefic anhydride with ethylene, methylvinyl ether or methacrylic acid, the
malefic anhydride constituting at least 20 mole percent of the copolymer.
These
materials are normally used at levels of from 0.5% to 10% by weight, more
preferably from 0.75% to 8%, most preferably from 1 % to 6% by weight of the
composition.
Preferred optical brighteners are anionic in character, examples of which
are disodium 4,4'-bis-(2-diethanolamino-4-anilino -s- triazin-6-
ylamino)stilbene-
0 2:2' disulphonate, disodium 4, - 4'-bis-(2-morpholino-4-anilino-s-triazin-6-
ylamino-
stilbene-2:2' - disulphonate, disodium 4,4' - bis-(2,4-dianilino-s-triazin-6-
ylamino)stilbene-2:2' - disulphonate, monosodium 4',4" -bis-(2,4-dianilino-s-
tri-
azin-6 ylamino)stilbene-2-sulphonate, disodium 4,4' -bis-(2-anilino-4-(N-
methyl-N-
2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2' - disulphonate, di-
sodium
~5 4,4' -bis-(4-phenyl-2,1,3-triazol-2-yl)-stilbene-2,2' disulphonate, di-so-
diem
4,4'bis(2-anilino-4-(1-methyl-2-hydroxyethylamino)-s-triazin-6- ylami-
no)stilbene
2,2'disulphonate, sodium 2(stilbyl-4"-(naphtho-1',2':4,5)-1,2,3 - triazole-2"
sulphonate and 4,4'-bis(2-sulphostyryl)biphenyl. Highly preferred brighteners
are
the specific brighteners of co-pending European Patent application No.
20 95201943.8.
Other useful polymeric materials are the polyethylene glycols, particularly
those of molecular weight 1000-10000, more particularly 2000 to 8000 and most
preferably about 4000. These are used at levels of from 0.20% to 5% more
25 preferably from 0.25% to 2.5% by weight. These polymers and the previously
mentioned homo- or co-polymeric polycarboxylate salts are valuable for
improving whiteness maintenance, fabric ash deposition, and cleaning
performance on clay, proteinaceous and oxidizable soils in the presence of
transition metal impurities.
Soil release agents useful in compositions of the present invention are
conventionally copolymers or terpolymers of terephthalic acid with ethylene
glycol
and/or propylene glycol units in various arrangements. Examples of such
polymers are disclosed in the commonly assigned US Patent Nos. 4116885 and
4711730 and European Published Patent Application No. 0 272 033. A particular
preferred polymer in accordance with EP-A-0 272 033 has the formula
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(CH3(PEG)43)0.75(POH)p.25[T-PO}2.g(T-PEG)p,4]T(PO
H)0.25((PEG)43CH3)0.75
5 where PEG is -(OC2H4)O-,PO is (OC3Hg0) and T is {pcOCgH4C0).
Also very useful are modified polyesters as random copolymers of
dimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and 1-2
propane diol, the end groups consisting primarily of sulphobenzoate and
o secondarily of mono esters of ethylene glycol and/or propane-diol. The
target is
to obtain a polymer capped at both end by sulphobenzoate groups, "primarily",
in
the present context most of said copolymers herein will be end-capped by
sulphobenzoate groups. However, some copolymers will be less than fully
capped, and therefore their end groups may consist of monoester of ethylene
15 glycol and/or propane 1-2 diol, thereof consist "secondarily" of such
species.
The selected polyesters herein contain about 46% by weight of dimethyl
terephthalic acid, about 16% by weight of propane -1.2 diol, about 10% by
weight
ethylene glycol about 13% by weight of dimethyl sulfobenzoic acid and about
15% by weight of sulfoisophthalic acid, and have a molecular weight of about
20 3.000. The polyesters and their method of preparation are described in
detail in
EPA 311 342.
It is well-known in the art that free chlorine in tap water rapidly
deactivates
the enzymes comprised in detergent compositions. Therefore, using chlorine
25 scavenger such as perborate, ammonium sulfate, sodium sulphite or
polyethyleneimine at a level above 0.1 % by weight of total composition, in
the
formulas will provide improved through the wash stability of the detergent
enzymes. Compositions comprising chlorine scavenger are described in the
European patent application 92870018.6 filed January 31, 1992.
Alkoxylated polycarboxylates such as those prepared from polyacrylates
are useful herein to provide additional grease removal performance. Such
materials are described in WO 91/08281 and PCT 90/01815 at p. 4 et seq.,
incorporated herein by reference. Chemically, these materials comprise
polyacrylates having one ethoxy side-chain per every 7-8 acrylate units. The
side-chains are of the formula -(CH2CH20)m(CH2)~CHg wherein m is 2-3 and n
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71
is 6-12. The side-chains are ester-linked to the polyacrylate "backbone" to
provide a "comb" polymer type structure. The molecular weight can vary, but is
typically in the range of about 2000 to about 50,000. Such alkoxylated
polycarboxyiates can comprise from about 0.05% to about 10%, by weight, of the
compositions herein.
Dispersants
The laundry detergent compositions of the present invention can also
o contain dispersants : Suitable water-soluble organic salts are the homo- or
co-
polymeric acids or their salts, in which the polycarboxylic acid comprises at
least
two carboxyl radicals separated from each other by not more than two carbon
atoms. Polymers of this type are disclosed in GB-A-1,596,758. Examples of
such salts are polyacrylates of MW 2000-5000 and their copolymers with malefic
~5 anhydride, such copolymers having a molecular weight of from 1,000 to
100,000.
Especially, copolymer of acrylate and methylacrylate such as the 480N
having a molecular weight of 4000, at a level from 0.5-20% by weight of
composition can be added in the laundry detergent compositions of the present
invention.
The compositions of the invention may contain a lime soap peptiser
compound, which has preferably a lime soap dispersing power (LSDP), as
defined hereinafter of no more than 8, preferably no more than 7, most
preferably
no more than 6. The lime soap peptiser compound is preferably present at a
level
from 0% to 20% by weight.
A numerical measure of the effectiveness of a lime soap peptiser is given
by the lime soap dispersant power (LSDP) which is determined using the lime
soap dispersant test as described in an article by H.C. Borghetty and C.A.
3o Bergman, J, Am. Oil. Chem. Soc., volume 27, pages 88-90, (1950). This lime
soap dispersion test method is widely used by practitioners in this art field
being
referred to, for example, in the following review articles; W.N. Linfield,
Surfactant
science Series, Volume 7, page 3; W.N. Linfield, Tenside surf. det., volume
27,
pages 159-163, (1990); and M.K. Nagarajan, W.F. Masler, Cosmetics and
Toiletries, volume 104, pages 71-73, (1989). The LSDP is the % weight ratio of
dispersing agent to sodium oleate required to disperse the lime soap deposits
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formed by 0.0258 of sodium oleate in 30m1 of water of 333ppm CaCo3
(Ca:Mg=3:2) equivalent hardness.
Surfactants having good lime soap peptiser capability will include certain
amine oxides, betaines, sulfobetaines, alkyl ethoxysulfates and ethoxylated
alcohols.
Exemplary surfactants having a LSDP of no more than 8 for use in accord
with the present invention include C1g-C1g dimethyl amine oxide, C12-C18 alkyl
1o ethoxysulfates with an average degree of ethoxylation of from 1-5,
particularly
C12-C15 alkyl ethoxysulfate surfactant with a degree of ethoxylation of amount
3
(LSDP=4), and the C14-C15 ethoxylated alcohols with an average degree of
ethoxylation of either 12 (LSDP=6) or 30, sold under the tradenames Lutensol
A012 and Lutensol A030 respectively, by BASF GmbH.
Polymeric lime soap peptisers suitable for use herein are described in the
article by M.K. Nagarajan, W.F. Masler, to be found in Cosmetics and
Toiletries,
volume 104, pages 71-73, (1989).
2o Hydrophobic bleaches such as 4-[N-octanoyl-6-aminohexanoyl]benzene
sulfonate, 4-[N-nonanoyl-6-aminohexanoyl]benzene sulfonate, 4-[N-decanoyl-6-
aminohexanoyl]benzene sulfonate and mixtures thereof; and nonanoyloxy
benzene sulfonate together with hydrophilic / hydrophobic bleach formulations
can also be used as lime soap peptisers compounds.
Dye transfer inhibition
The laundry detergent compositions of the present invention can also
include compounds for inhibiting dye transfer from one fabric to another of
3o solubilized and suspended dyes encountered during fabric laundering
operations
involving colored fabrics.
Polymeric dye transfer inhibiting agents
The laundry detergent compositions according to the present invention
also comprise from 0.001 % to 10 %, preferably from 0.01 % to 2%, more
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preferably from 0.05% to 1 % by weight of polymeric dye transfer inhibiting
agents. Said polymeric dye transfer inhibiting agents are normally
incorporated
into laundry detergent compositions in order to inhibit the transfer of dyes
from
colored fabrics onto fabrics washed therewith. These polymers have the ability
to
complex or adsorb the fugitive dyes washed out of dyed fabrics before the dyes
have the opportunity to become attached to other articles in the wash.
Especially suitable polymeric dye transfer inhibiting agents are polyamine N-
oxide
polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole,
polyvinylpyrrolidone polymers, polyvinyloxazolidones and polyvinylimidazoies
or
o mixtures thereof.
Addition of such polymers also enhances the performance of the enzymes
according the invention.
a) Polyamine N-oxide polymers
~5 The polyamine N-oxide polymers suitable for use contain units having the
following structure formula
P
I
20 (I) Ax
R
wherein P is a polymerisable unit, whereto the R-N-O group can be attached to
25 or wherein the R-N-O group forms part of the polymerisable unit or a
combination of both.
O O O
II II II
3o A is NC, CO, C, -O-,-S-, -N- ; x is O or 1;
R are aliphatic, ethoxylated aliphatics, aromatic, heterocyclic or
alicyclic groups or any combination thereof whereto the nitrogen of the
N-O group can be attached or wherein the nitrogen of the N-O group is
part of these groups.
The N-O group can be represented by the following general structures
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O O
(R1 )x -N- (R2)y =N- (R1 )x
(R3)z
wherein R1, R2, and R3 are aliphatic groups, aromatic, heterocyclic or
alicyclic
groups or combinations thereof, x or/and y or/and z is 0 or 1 and
o wherein the nitrogen of the N-O group can be attached or wherein the
nitrogen of the N-O group forms part of these groups.
The N-O group can be part of the polymerisable unit (P) or can be
attached to the polymeric backbone or a combination of both.
~5 Suitable polyamine N-oxides wherein the N-O group forms part of the
polymerisable unit comprise polyamine N-oxides wherein R is selected from
aliphatic, aromatic, alicyclic or heterocyclic groups.
One Glass of said polyamine N-oxides comprises the group of polyamine N
oxides wherein the nitrogen of the N-O group forms part of the R-group.
2o Preferred polyamine N-oxides are those wherein R is a heterocyclic group
such
as pyrridine, pyrrole, imidazole, pyrrolidine, piperidine, quinoline, acridine
and
derivatives thereof.
Another Glass of said polyamine N-oxides comprises the group of polyamine N-
oxides wherein the nitrogen of the N-O group is attached to the R-group.
Other suitable polyamine N-oxides are the polyamine oxides whereto the
N-O group is attached to the polymerisable unit.
Preferred Glass of these polyamine N-oxides are the polyamine N-oxides having
the general formula (I) wherein R is an aromatic, heterocyclic or alicyclic
groups
3o wherein the nitrogen of the N-0 functional group is part of said R group.
Examples of these classes are polyamine oxides wherein R is a heterocyclic
compound such as pyrridine, pyrrole, imidazole and derivatives thereof.
Another preferred Glass of polyamine N-oxides are the polyamine oxides having
the general formula (I) wherein R are aromatic, heterocyclic or aiicyclic
groups
wherein the nitrogen of the N-0 functional group is attached to said R groups.
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Examples of these classes are polyamine oxides wherein R groups can be
aromatic such as phenyl.
Any polymer backbone can be used as long as the amine oxide polymer
5 formed is water-soluble and has dye transfer inhibiting properties. Examples
of
suitable polymeric backbones are polyvinyls, polyalkylenes, polyesters,
polyethers, polyamide, polyimides, polyacrylates and mixtures thereof.
The amine N-oxide polymers of the present invention typically have a ratio
0 of amine to the amine N-oxide of 10:1 to 1:1000000. However the amount of
amine oxide groups present in the polyamine oxide polymer can be varied by
appropriate copolymerization or by appropriate degree of N-oxidation.
Preferably,
the ratio of amine to amine N-oxide is from 2:3 to 1:1000000. More preferably
from 1:4 to 1:1000000, most preferably from 1:7 to 1:1000000. The polymers of
~5 the present invention actually encompass random or block copolymers where
one monomer type is an amine N-oxide and the other monomer type is either an
amine N-oxide or not. The amine oxide unit of the polyamine N-oxides has a PKa
< 10, preferably PKa < 7, more preferred PKa < 6.
The polyamine oxides can be obtained in almost any degree of polymerisation.
2o The degree of polymerisation is not critical provided the material has the
desired
water-solubility and dye-suspending power.
Typically, the average molecular weight is within the range of 500 to
1000,000; preferably from 1,000 to 50,000, more preferably from 2,000 to
30,000,
most preferably from 3,000 to 20,000.
b) Copolymers of N-vinylpyrrofidone and N-vinylimidazole
The N-vinylimidazole N-vinyipyrrolidone polymers used in the present
invention have an average molecular weight range from 5,000-1,000,000,
preferably from 5,000-200,000.
3o Highly preferred polymers for use in detergent compositions according to
the present invention comprise a polymer selected from N-vinylimidazole N-
vinylpyrrolidone copolymers wherein said polymer has an average molecular
weight range from 5,000 to 50,000 more preferably from 8,000 to 30,000, most
preferably from 10,000 to 20,000.
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The average molecular weight range was determined by light scattering as
described in Barth H.G. and Mays J.W. Chemical Analysis Vol 113,"Modern
Methods of Polymer Characterization".
Highly preferred N-vinylimidazole N-vinylpyrrolidone copolymers have an
average molecular weight range from 5,000 to 50,000; more preferably from
8,000 to 30,000; most preferably from 10,000 to 20,000.
The N-vinylimidazole N-vinylpyrrolidone copolymers characterized by
having said average molecular weight range provide excellent dye transfer
o inhibiting properties while not adversely affecting the cleaning performance
of
detergent compositions formulated therewith.
The N-vinylimidazole N-vinylpyrrolidone copolymer of the present invention has
a
molar ratio of N-vinylimidazole to N-vinylpyrrolidone from 1 to 0.2, more
preferably from 0.8 to 0.3, most preferably from 0.6 to 0.4 .
c) Polyvinylpyrrolidone
The detergent compositions of the present invention may also utilize
polyvinylpyrrolidone ("PVP") having an average molecular weight of from about
2,500 to about 400,000, preferably from about 5,000 to about 200,000, more
2o preferably from about 5,000 to about 50,000, and most preferably from about
5,000 to about 15,000. Suitable polyvinylpyrrolidones are commercially
available
from ISP Corporation, New York, NY and Montreal, Canada under the product
names PVP K-15 (viscosity molecular weight of 10,000), PVP K-30 (average
molecular weight of 40,000), PVP K-60 (average molecular weight of 160,000),
and PVP K-90 (average molecular weight of 360,000). Other suitable
polyvinylpyrrolidones which are commercially available from BASF Cooperation
include Sokalan HP 165 and Sokalan HP 12; pofyvinylpyrrolidones known to
persons skilled in the detergent field (see for example EP-A-262,897 and EP-A-
256,696).
d) Polyvinyloxazolidone
The detergent compositions of the present invention may also utilize
polyvinyloxazolidone as a polymeric dye transfer inhibiting agent. Said
polyvinyloxazolidones have an average molecular weight of from about 2,500 to
about 400,000, preferably from about 5,000 to about 200,000, more preferably
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from about 5,000 to about 50,000, and most preferably from about 5,000 to
about
15, 000.
e) Polyvinylimidazole
s The detergent compositions of the present invention may also utilize
polyvinylimidazole as polymeric dye transfer inhibiting agent. Said
polyvinylimidazoles have an average about 2,500 to about 400,000, preferably
from about 5,000 to about 200,000, more preferably from about 5,000 to about
50,000, and most preferably from about 5,000 to about 15,000.
f) Cross-linked polymers
Cross-linked polymers are polymers whose backbone are interconnected
to a certain degree; these links can be of chemical or physical nature,
possibly
with active groups n the backbone or on branches; cross-linked polymers have
been described in the Journal of Polymer Science, volume 22, pages 1035-1039.
In one embodiment, the cross-linked polymers are made in such a way
that they form a three-dimensional rigid structure, which can entrap dyes in
the
pores formed by the three-dimensional structure. In another embodiment, the
cross-linked polymers entrap the dyes by swelling. Such cross-linked polymers
2o are described in the co-pending patent application 94870213.9
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78
Method of washing
The compositions of the invention may be used in essentially any washing
or cleaning methods, including soaking methods, pretreatment methods and
methods with rinsing steps for which a separate rinse aid composition may be
added.
The process described herein comprises contacting fabrics with a
o laundering solution in the usual manner and exemplified hereunder.
The process of the invention is conveniently carried out in the course of
the cleaning process. The method of cleaning is preferably carried out at
5°C to
95°C, especially between 10°C and 60°C. The pH of the
treatment solution is
15 preferably from 7 to 11. Preferably, the laundry detergent composition of
the
present invention will have a pH in a 1 % solution in water, of between 7 and
9.5
(referred to as "alkaline" detergent).
The following examples are meant to exemplify compositions of the
2o present invention, but are not necessarily meant to limit or otherwise
define the
scope of the invention.
In the detergent compositions, the enzymes levels are expressed by pure
enzyme by weight of the total composition and unless otherwise specified, the
25 detergent ingredients are expressed by weight of the total compositions.
The
abbreviated component identifications therein have the following meanings:
LAS : Sodium linear C11-13 alkyl benzene sulphonate.
TAS : Sodium tallow alkyl sulphate.
CxyAS : Sodium C1x - C1y alkyl sulfate.
CxySAS : Sodium C1x - C1y secondary (2,3) alkyl sulfate.
CxyEz : C1x - C1y predominantly linear primary alcohol
condensed with an average of z moles of ethylene
oxide.
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CxyEzS : C1x - C1y sodium alkyl sulfate condensed
with an
average of z moles of ethylene oxide.
QAS : R2.N+(CH3)2(C2H40H) with R2 = C12-C14~
QAS 1 : R2.N+(CH3)2(C2H40H) with R2 = Cg-C11.
APA : Cg_10 amido propyl dimethyl amine.
Soap : Sodium linear alkyl carboxylate derived from
a 80/20
mixture of tallow and coconut fatty acids.
Nonionic : C13-C15 mixed ethoxylated/propoxylated fatty
alcohol
with an average degree of ethoxylation of 3.8
and an
average degree of propoxylation of 4.5.
Neodol 45-13 : C14-C15 linear primary alcohol ethoxylate,
sold by Shell
Chemical CO.
Quat : Quaternary surfactant selected from one or
more of the
following: lauryl trimethyl ammonium chloride,
myristyl
trimethyl ammonium chloride, palmityl trimethyl
ammonium chloride, coconut trimethylammonium
chloride, coconut trimethylammonium methylsulfate,
coconut dimethyl-monohydroxyethyl-ammonium
chloride,
coconut dimethyl-monohydroxyethylammonium
methylsulfate, steryl dimethyl-monohydroxy-
ethylammonium chloride, steryl dimethylmonohydroxy-
ethylammonium methylsulfate, di- C12-C14 alkyl
dimethyl ammonium chloride.
STS : Sodium toluene sulphonate.
CFAA : C12-C14 alkyl N-methyl glucamide.
TFAA : C 1 g-C 1 g alkyl N-methyl glucamide.
TPKFA : C 12-C 14 topped whole cut fatty acids.
DEQA : Di-(tallow-oxy-ethyl) dimethyl ammonium chloride.
DEQA (2) : Di-(soft-tallowyloxyethyl) hydroxyethyl methyl
ammonium
methylsulfate.
DTDMAMS : Ditalllow dimethyl ammonium methylsulfate.
SDASA : 1:2 ratio of stearyldimethyl amineariple-pressed
stearic
acid.
Silicate : Amorphous Sodium Silicate (Si02:Na20 ratio = 1.6-3.2).
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Zeolite A : Hydrated Sodium Aluminosilicate of formula
Nal2(A102Si02)12. 27H20 having a primary
particle
size in the range from 0.1 to 10 micrometers
(Weight
expressed on an anhydrous basis).
Na-SKS-6 : Crystalline layered silicate of formula
8-Na2Si205,
Citrate : Tri-sodium citrate dihydrate of activity
86.4% with a
particle size distribution between 425 and
850
micrometres.
Citric : Anhydrous citric acid.
Borate : Sodium borate
Carbonate : Anhydrous sodium carbonate with a particle
size
between 200 and 900 micrometres.
Bicarbonate : Anhydrous sodium hydrogen carbonate with
a particle
size distribution between 400 and 1200 micrometres.
Sulphate : Anhydrous sodium sulphate.
Mg Sulphate : Anhydrous magnesium sulfate.
STPP : Sodium tripolyphosphate.
TSPP : Tetrasodium pyrophosphate.
MA/AA : Random copolymer of 4:1 acrylate/maleate,
average
molecular weight about 70,000-80,000.
MAIAA 1 : Random copolymer of 6:4 acrylate/maleate,
average
molecular weight about 10,000.
AA : Sodium polyacrylate polymer of average
molecular
weight 4,500.
PB1 : Anhydrous sodium perborate monohydrate
of nominal
formula NaB02.H202.
PB4 : Sodium perborate tetrahydrate of nominal
formula
NaB02.3H20. H202.
Percarbonate : Anhydrous sodium percarbonate of nominal
formula
2Na2C03.3H202 .
NaDCC : Sodium dichloroisocyanurate.
TAED : Tetraacetylethylenediamine.
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NOBS : Nonanoyloxybenzene sulfonate in the form
of the sodium
salt.
NACA-OBS : (6-nonamidocaproyl) oxybenzene sulfonate.
DTPA : Diethylene triamine pentaacetic acid.
HEDP : 1,1-hydroxyethane diphosphonic acid.
DETPMP : Diethyltriamine penta (methylene) phosphonate,
marketed by Monsanto under the Trade name
bequest
2060.
EDDS : Ethylenediamine-N,N'-disuccinic acid, (S,S)
isomer in the
form of its sodium salt
Photoactivated : Sulfonated zinc phtalocyanine encapsulated
in dextrin
Bleach soluble polymer.
Photoactivated : Sulfonated alumino phtalocyanine encapsulated
in
Bleach 1 dextrin soluble polymer.
PAAC : Pentaamine acetate cobalt(///) salt.
Mannanase : Mannanase sold under the tradename Gamanase~
by
Novo Nordisk A/S and/or Galactomannase extracted
from the enzyme product sold under the tradenarne
Rohapec~ B1 L by Rohm.
Alkaline mannanase : Mannanase from Bacillus agardherens, NCIMB 40482.
Protease : Proteolytic enzyme sold under the tradename Savinase,
Alcalase, Durazym by Novo Nordisk A/S, Maxacal,
Maxapem sold by Gist-Brocades and proteases
described in patents W091/06637 and/or W095/10591
and/or EP 251 446.
Amylase : Amylolytic enzyme sold under the tradename Purafact
Ox AmR described in WO 94/18314, W096/05295 sold
by Genencor; Termamyl~, Fungamyl~ and Duramyl~,
all available from Novo Nordisk A/S and those described
in W095/26397.
Lipase : Lipolytic enzyme sold under the tradename Lipolase,
Lipolase Ultra by Novo Nordisk A/S and Lipomax by
Gist-Brocades.
Cellulase : Cellulytic enzyme sold under the tradename Carezyme,
Celluzyme and/or Endolase by Novo Nordisk A/S.
CMC : Sodium carboxymethyi cellulose.
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PVP : Polyvinyl polymer, with an average molecular
weight of
60, 000.
PVNO : Polyvinylpyridine-N-Oxide, with an average
molecular
weight of 50,000.
PVPVI : Copolymer of vinylimidazole and vinylpyrrolidone,
with an
average molecular weight of 20,000.
Brightener 1 : Disodium 4,4'-bis(2-sulphostyryl)biphenyl.
Brightener 2 : Disodium 4,4'-bis(4-anilino-6-morpholino-1.3.5-triazin-2-
yl) stilbene-2:2'-disulfonate.
Silicone antifoam: Polydimethylsiloxane foam controller with
siloxane-
oxyalkylene copolymer as dispersing agent
with a ratio of
said foam controller to said dispersing agent
of 10:1 to
100:1.
Suds Suppressor : 12% Silicone/silica, 18% stearyl alcoho1,70%
starch in
granular form.
Opacifier : Water based monostyrene latex mixture,
sold by BASF
Aktiengesellschaft under the tradename Lytron
621.
SRP 1 : Anionically end capped poly esters.
SRP 2 : Diethoxylated poly (1,2 propylene terephthalate)
short
block polymer.
QEA : bis((C2H50)(C2H40)n)(CH3) -N+-C6H12-N+-(CH3)
bis({C2H50)-(C2H40))n, wherein n = from 20
to 30.
PEI : Polyethyleneimine with an average molecular
weight of
1800 and an average ethoxylation degree of
7
ethyleneoxy residues per nitrogen.
Polymer A : Modified polyamines of PEI (MW = 182) with
average
degree of ethoxylation = 15.
Polymer B : Modified polyamines of PEI (MW = 600) with
average
degree of ethoxylation = 20.
Polyamide- Polyamide-polyamines herein are commercially
polyamine marketed under the tradenames: Kymene~, Kymene
557H~, Kymene 557LX~, Reten~, and Cartaretin~.
SCS : Sodium cumene sulphonate.
HMWPEO : High molecular weight polyethylene oxide.
PEGx : Polyethylene glycol, of a molecular weight of x .
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PEO : Polyethylene oxide, with an average molecular weight of
5, 000.
TEPAE : Tetreaethylenepentaamine ethoxylate.
Example 1
The following high density laundry detergent compositions were prepared
according to the present invention
I II III IV V VI
LAS 8.0 8.0 8.0 2.0 6.0 6.0
TAS - 0.5 - 0.5 1.0 0.1
C46(S)AS 2.0 2.5 - - - -
C25AS - - - 7.0 4.5 5.5
C68AS 2.0 5.0 7.0 - - -
C25E5 - - ~ 3.4 10.0 4.6 4.6
C25E7 3.4 3.4 1.0 - - -
C25E3S - - - 2.0 5.0 4.5
~
QAS - 0.8 - - - -
QAS 1 - - - 0.8 0.5 1.0
Zeolite A 18.1 18.0 14.1 18.1 20.0 18.1
Citric - - - 2.5 - 2.5
Carbonate 13.0 13.0 27.0 10.0 10.0 13.0
Na-SKS-6 - - - 10.0 - 10.0
Silicate 1.4 1.4 3.0 0.3 0.5 0.3
Citrate - 1.0 - 3.0 - -
Sulfate 26.1 26.1 26.1 6.0 - -
Mg sulfate 0.3 - - 0.2 - 0.2
MA/AA 0.3 0.3 0.3 4.0 1.0 1.0
CMC 0.2 0.2 0.2 0.2 0.4 0.4
PB4 9.0 9.0 5.0 - - -
Percarbonate - - - - 18.0 18.0
TAED 1.5 0.4 1.5 - 3.9 4.2
NACA-OBS - 2.0 1.0 - - -
DETPMP 0.25 0.25 0.25 0.25 - -
SRP 1 - - - 0.2 - 0.2
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1 II III fV V VI
EDDS - 0.25 0.4 - 0.5 0.5
CFAA - 1.0 - 2.0 - -
HEDP 0.3 0.3 0.3 0.3 0.4 0.4
QEA _ _ - 0.2 - 0.5
Mannanase 0.005 0.002 0.0008 0.001 0.002 0.001
Protease 0.009 0.009 0.01 0.04 0.05 0.03
Amylase 0.002 0.002 0.002 0.006 0.008 0.008
Cellulase 0.0007 - - 0.0007 0.0007 0.0007
Lipase 0.006 - - 0.01 0.01 0.01
Photoactivated15 15 15 - 20 20
bleach (ppm)
PVNO/PVPVI - - - 0.1 - -
Brightener 0.09 0.09 0.09 - 0.09 0.09
1
Perfume 0.3 0.3 0.3 0.4 0.4 0.4
Silicone antifoam0.5 0.5 0.5 - 0.3 0.3
Density in 850 850 850 850 850 850
g/iitre
Miscellaneous Up to 100%
and minors
Example 2
The following granular laundry detergent compositions of particular utility
under
European machine wash conditions were prepared according to the present
invention
I II III IV V VI
LAS 5.5 7.5 5.0 5.0 6.0 7.0
TAS 1.25 1.9 - 0.8 0.4 0.3
C24AS/C25AS - 2.2 5.0 5.0 5.0 2.2
C25E3S - 0.8 1.0 1.5 3.0 1.0
C45E7 3.25 - - - - 3.0
TFAA - - 2.0 - - -
C25E5 - 5.5 - - - -
QAS 0.8 - - - - -
QAS 1 - 0.7 1.0 0.5 1.0 0.7
STPP 19.7 - - - - -
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I II III IV V Vl
Zeolite A - 19.5 25.0 19.5 20.0 17.0
NaSKS-6/citric - 10.6 - 10.6 - -
acid
(79:21 )
Na-SKS-6 - - 9.0 - 10.0 10.0
Carbonate 6.1 21.4 9.0 10.0 10.0 18.0
Bicarbonate - 2.0 7.0 5.0 - 2.0
Silicate 6.8 - - 0.3 0.5 -
Citrate - - 4.0 4.0 - -
Sulfate 39.8 - - 5.0 - 12.0
Mg sulfate - - 0.1 0.2 0.2 -
MA/AA 0.5 1.6 3.0 4.0 1.0 1.0
CMC 0.2 0.4 1.0 1.0 0.4 0.4
PB4 5.0 12.7 - - - -
Percarbonate - - - - 18.0 15.0
TAED 0.5 3.1 - - 5.0 -
NACA-OBS 1.0 3.5 - - - 2.5
DETPMP 0.25 0.2 0.3 0.4 - 0.2
HEDP - 0.3 - 0.3 0.3 0.3
Q EA - - 1.0 1.0 1.0 -
Mannanase 0.005 0.002 0.008 0.005 0.002 0.001
Protease 0.009 0.03 0.03 0.05 0.05 0.02
Lipase 0.003 0.003 0.006 0.006 0.006 0.004
Cellulase 0.0006 0.0006 0.0005 0.0005 0.0007 0.0007
Amylase 0.002 0.002 0.006 0.006 0.01 0.003
PVNO/PVPVI - - 0.2 0.2 - -
PVP 0.9 1.3 - - - 0.9
SRP 1 - - 0.2 0.2 0.2 -
Photoactivated 15 27 - - 20 20
bleach (ppm)
Photoactivated 15 - - - - -
bleach 1 (ppm)
Brightener 1 0.08 0.2 - - 0.09 0.15
Brightener 2 - 0.04 - - - -
Perfume 0.3 0.5 0.4 0.3 0.4 0.3
Silicone antifoam0.5 2.4 0.3 0.5 0.3 2.0
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I II III IV V VI
Density in g/litre 750 750 750 750 750 750
Miscellaneous and Up to 100%
minors
Example 3
The following of particular underEuropean
detergent compositions utility
machine wash conditions preparedaccording ent
were to invention
the
pres
I I! III IV V VI
Blown Powder
LAS 6.0 5.0 11.0 11.0 6.0 6.0
TAS 2.0 - - - 2.0 2.0
Zeolite A 24.0 - - - 20.0 20.0
STPP - 27.0 24.0 24.0 - -
Sulfate 4.0 6.0 13.0 13.0 - -
MA/AA 1.0 4.0 6.0 6.0 2.0 2.0
Silicate 1.0 7.0 3.0 3.0 3.0 3.0
CMC 1.0 1.0 0.5 0.5 0.6 0.6
Brightener 1 0.2 0.2 0.2 0.2 0.2 0.2
Silicone antifoam 1.0 1.0 1.0 1.0 0.3 0.3
DETPMP 0.4 0.4 0.2 0.2 0.4 0.4
Spray On
Brightener 0.02 - - - 0.02 0.02
C45E7 - - - - 5.0 5.0
C45E2 2.5 2.5 2.0 2.0 - -
C45E3 2.6 2.5 2.0 2.0 - -
Perfume 0.5 0.3 0.5 0.5 0.2 0.2
Silicone antifoam 0.3 0.3 0.3 0.3 - -
Dry additives
QEA - - - - 1.0 1.0
EDDS 0.3 - - - - -
Sulfate 2.0 3.0 5.0 5.0 10.0 10.0
Carbonate 6.0 13.0 15.0 15.0 14.0 14.0
Citric 2.5 - - - 2.0 2.0
QAS 1 0.5 - - - 0.5 0.5
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I II III IV V VI
Na-SKS-6 10.0 - - - - -
Percarbonate 18.5 - - - - -
PB4 - 18.0 10.0 10.0 21.5 21.5
TAED 2.0 2.0 - - 2.0 2.0
NACA-OBS 3.0 2.0 4.0 4.0 - -
Mannanase 0.005 0.002 0.0008 - 0.001 -
Alkaline mannanase- - - 0.001 - 0.002
Protease 0.03 0.03 0.03 0.03 0.03 0.03
Lipase 0.008 0.008 0.008 0.008 0.004 0.004
Amylase 0.003 0.003 0.003 0.003 0.006 0.006
Brightener 1 0.05 - - - 0.05 0.05
Miscellaneous Up to
and minors 100%
Example 4
The following granular detergent compositions were prepared according to the
present invention
I II III IV V Vl
Blown Powder
LAS 23.0 8.0 7.0 9.0 7.0 7.0
TAS - - - - 1.0 -
C45AS 6.0 6.0 5.0 8.0 - -
C45AES - 1.0 1.0 1.0 - -
C45E35 - - - - 2.0 4.0
Zeolite A 10.0 18.0 14.0 12.0 10.0 10.0
MA/AA - 0.5 - - - 2.0
MA/AA 1 7.0 - - - - -
AA - 3.0 3.0 2.0 3.0 3.0
Sulfate 5.0 6.3 14.3 11.0 15.0 19.3
Silicate 10.0 1.0 1.0 1.0 1.0 1.0
Carbonate 15.0 20.0 10.0 20.7 8.0 6.0
PEG 4000 0.4 1.5 1.5 1.0 1.0 1.0
DTPA - 0.9 0.5 - - 0.5
Brightener 2 0.3 0.2 0.3 - 0.1 0.3
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I ll 111 IV V VI
Spray On
C45E7 - 2.0 - - 2.0 2.0
C25E9 3.0 - - - - -
C23E9 - - 1.5 2.0 - 2.0
Perfume 0.3 0.3 0.3 2.0 0.3 0.3
Agglomerates
C45AS - 5.0 5.0 2.0 - 5.0
LAS - 2.0 2.0 - - 2.0
Zeolite A - 7.5 7.5 8.0 - 7.5
Carbonate - 4.0 4.0 5.0 - 4.0
PEG 4000 - 0.5 0.5 - - 0.5
Misc (Water etc.)- 2.0 2.0 2.0 - 2.0
Dry additives
QAS - - - - 1.0 -
Citric - _ _ - 2.0 -
PB4 - - - - 12.0 1.0
PB1 4.0 1.0 3.0 2.0 - -
Percarbonate - - - - 2.0 10.0
Carbonate - 5.3 1.8 - 4.0 4.0
NOBS 4.0 - 6.0 - - 0.6
Methyl cellulose0.2 - - - - -
Na-SKS-6 8.0 - - - - -
STS - - 2.0 - 1.0 -
Culmene sulfonic- 1.0 - - - 2.0
acid
Mannanase 0.005 0.002 0.001 0.008 0.001 0.001
Protease 0.02 0.02 0.02 0.01 0.02 0.02
Lipase 0.004 - 0.004 - 0.004 0.008
Amylase 0.003 - 0.002 - 0.003 -
Cellulase 0.0005 0.0005 0.0005 0.0007 0.0005 0.0005
PVPVI - - - - 0.5 0.1
PVP - - - - 0.5 -
PVNO - - 0.5 0.3 - -
Q EA - - - - 1.0 -
SRP 1 0.2 0.5 0.3 - 0.2 -
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I II III IV V VI
Silicone antifoam 0.2 0.4 0.2 0.4 0.1 -
Mg sulfate - - 0.2 - 0.2 -
Miscellaneous and minors Up to 100%
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Example 5
The following nil bleach-containing detergent compositions of particular use
in
the washing of colored clothing were prepared according to the present
invention
s
I II III IV V
Blown Powder
Zeolite A 15.0 15.0 15.0 - -
Sulfate - - 5.0 - -
LAS 3.0 3.0 3.0 - -
DETPMP 0.4 0.4 0.5 - -
CMC 0.4 0.4 0.4 - -
MA/AA 4.0 4.0 4.0 - -
Agglomerates
C45AS - - - 11.0 11.0
LAS 6.0 6.0 5.0 - -
TAS 3.0 3.0 2.0 - -
Silicate 4.0 4.0 4.0 - -
Zeolite A 10.0 10.0 15.0 13.0 13.0
CMC - - - 0.5 0.5
MA/AA - - - 2.0 2.0
Carbonate 9.0 9.0 7.0 7.0 7.0
Spray-on
Perfume 0.3 0.3 0.3 0.5 0.5
C45E7 4.0 4.0 4.0 4.0 4.0
C25E3 2.0 2.0 2.0 2.0 2.0
Dry additives
MA/AA - - - 3.0 3.0
Na-SKS-6 - - - 12.0 12.0
Citrate 10.0 10.0 - 8.0 8.0
Bicarbonate 7.0 7.0 3.0 5.0 5.0
Carbonate 8.0 8.0 5.0 7.0 7.0
PVPVIIPVNO 0.5 0.5 0.5 0.5 0.5
Mannanase 0.0008 - 0.0005 0.001 -
Alkaline mannanase- 0.0008 - - 0.002
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I II III IV V
Protease 0.03 0.03 0.02 0.05 0.05
Lipase 0.008 0.008 0.008 0.008 0.008
Amylase 0.01 0.01 0.01 0.01 0.01
Cellulase 0.001 0.001 0.001 0.001 0.001
Silicone antifoam 5.0 5.0 5.0 5.0 5.0
Sulfate - - 9.0 - -
Density (g/litre) 700 700 700 700 700
Miscellaneous and Up to 0%
minors 10
Example 6
The following detergent compositions were prepared according to the present
invention
I II III IV
Base granule
Zeolite A 30.0 22.0 24.0 10.0
Sulfate 10.0 5.0 10.0 7.0
MA/AA 3.0 - -
AA - 1.6 2.0 -
MA/AA 1 - 12.0 - 6.0
LAS 14.0 10.0 9.0 20.0
C45AS 8.0 7.0 9.0 7.0
C45AES - 1.0 1.0 -
Sificate - 1.0 0.5 10.0
Soap - 2.0 - -
Brightener 1 0.2 0.2 0.2 0.2
Carbonate 6.0 9.0 10.0 10.0
PEG 4000 - 1.0 1.5 -
DTPA - 0.4 - -
Spray On
C25E9 - - - 5.0
C45E7 1.0 1.0 - -
C23E9 - 1.0 2.5 -
Perfume 0.2 0.3 0.3 -
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I II III IV
Dry additives
Carbonate 5.0 10.0 18.0 8.0
PVPVI/PVNO 0.5 - 0.3 -
Mannanase 0.005 0.002 0.0008 0.001
Protease 0.03 0.03 0.03 0.02
Lipase 0.008 - - 0.008
Amylase 0.002 - - 0.002
Cellulase 0.0002 0.0005 0.0005 0.0002
NOBS - 4.0 - 4.5
PB1 1.0 5.0 1.5 6.0
Sulfate 4.0 5.0 - 5.0
SRP 1 - 0.4 - -
Suds suppressor - 0.5 0.5 -
Miscellaneous and minors Up to 100%
Example 7
The following granular preparedaccording
detergent compositions to the
were
present invention
I II III IV
Blown Powder
Zeolite A 20.0 - 15.0 15.0
STPP - 20.0 - -
Sulfate - - 5.0 5.0
Carbonate - - 5.0 5.0
TAS - - 1.0 1.0
LAS 6.0 6.0 6.0 6.0
C68AS 2.0 2.0 - -
Silicate 3.0 8.0 - -
MA/AA 4.0 2.0 2.0 2.0
CMC 0.6 0.6 0.2 0.2
Brightener 1 0.2 0.2 0.1 0.1
DETPMP 0.4 0.4 0.1 0.1
STS - - 1.0 1.0
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I II III IV
Spray On
C45E7 5.0 5.0 4.0 4.0
Silicone antifoam 0.3 0.3 0.1 0.1
Perfume 0.2 0.2 0.3 0.3
Dry additives
QEA - - 1.0 1.0
Carbonate 14.0 9.0 10.0 10.0
PB1 1.5 2.0 - -
PB4 18.5 13.0 13.0 13.0
TAED 2.0 2.0 2.0 2.0
QAS - - 1.0 1.0
Photoactivated bleach 15 ppm 15 ppm 15 ppm 15 ppm
Na-SKS-6 - - 3.0 3.0
Mannanase 0.005 0.002 0.0008 -
Alkaline mannanase - - - 0.001
Protease 0.03 0.03 0.007 0.007
Lipase 0.004 0.004 0.004 0.004
Amylase 0.006 0.006 0.003 0.003
Cellulase 0.0002 0.0002 0.0005 0.0005
Sulfate 10.0 20.0 5.0 5.0
Density (g/litre) 700 700 700 700
Miscellaneous and minors Up to 100%
Example 8
The following detergent to the present
compositions were prepared
according
invention
Blown Powder
Zeolite A 15.0 15.0 15.0
Sulfate - 5.0 -
LAS 3.0 3.0 3.0
QAS - 1.5 1.5
DETPMP 0.4 0.2 0.4
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I II III
EDDS - 0.4 0.2
CMC 0.4 0.4 0.4
MA/AA 4.0 2.0 2.0
Agglomerate
LAS 5.0 5.0 5.0
TAS 2.0 2.0 1.0
Silicate 3.0 3.0 4.0
Zeolite A 8.0 8.0 8.0
Carbonate 8.0 8.0 4.0
Spray On
Perfume 0.3 0.3 0.3
C45E7 2.0 2.0 2.0
C25E3 2.0 - -
Dry Additives
Citrate 5.0 - 2.0
Bicarbonate - 3.0 -
Carbonate 8.0 15.0 10.0
TAED 6.0 2.0 5.0
PB1 14.0 7.0 10.0
PEO - - 0.2
Bentonite clay - - 10.0
Mannanase 0.005 0.002 0.0008
Protease 0.03 0.03 0.03
Lipase 0.008 0.008 0.008
Cellulase 0.001 0.001 0.001
Amylase 0.01 0.01 0.01
Silicone antifoam 5.0 5.0 5.0
Sulfate - 3.0 -
Density (g/litre) 850 850 850
Miscellaneous and minors Up to 100%
Example 9
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The following detergent compositions were prepared according to the present
invention
I II III IV
LAS 18.0 14.0 24.0 20.0
QAS 0.7 1.0 - 0.7
TFAA - 1.0 - -
C23E56.5 - - 1.0 -
C45E7 - 1.0 - -
C45E3S 1.0 2.5 1.0 -
STPP 32.0 18.0 30,0 22.0
Silicate 9.0 5.0 9.0 8.0
Carbonate 11.0 7.5 10.0 5.0
Bicarbonate - 7.5 - -
PB1 3.0 1.0 - -
PB4 - 1.0 - -
NOBS 2.0 1.0 - -
DETPMP - 1.0 - -
DTPA 0.5 - 0.2 0.3
SRP 1 0.3 0.2 - 0.1
MA/AA 1.0 1.5 2.0 0.5
CMC 0.8 0.4 0.4 0.2
PEI - - 0.4 -
Sulfate 20.0 10.0 20.0 30.0
Mg sulfate 0.2 - 0.4 0.9
Mannanase 0.005 0.002 0.005 0.001
Protease 0.03 0.03 0.02 0.02
Amylase 0.008 0.007 - 0.004
Lipase 0.004 - 0.002 -
Cellulase 0.0003 - - 0.0001
Photoactivated bleach 30 ppm 20 ppm - 10 ppm
Perfume 0.3 0.3 0.1 0.2
Brightener 1/2 0.05 0.02 0.08 0.1
Miscellaneous and minors up to
100%
5
Example 10
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The following liquid detergent formulations were prepared according to the
present invention (Levels are given in parts per weight, enzyme are expressed
in
pure enzyme)
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II III IV V
LAS 11.5 8.8 - 3.9 -
C25E2.5S - 3.0 18.0 - 16.0
C45E2.25S 11.5 3.0 - 15.7 -
C23E9 - 2.7 1.8 2.0 1.0
C23E7 3.2 - - -
C FAA - - 5.2 - 3 .1
TPKFA 1.6 - 2.0 0.5 2.0
Citric (50%) 6.5 1.2 2.5 4.4 2.5
Ca formate 0.1 0.06 0.1 - -
Na formate 0.5 0.06 0.1 0.05 0.05
SCS 4.0 1.0 3.0 1.2 -
Borate 0.6 - 3.0 2.0 2.9
Na hydroxide 5.8 2.0 3.5 3.7 2.7
Ethanol 1.75 1.0 3.6 4.2 2.9
1,2 Propanediol 3.3 2.0 8.0 7.9 5.3
Monoethanolamine 3.0 1.5 1.3 2.5 0.8
TEPAE 1.6 - 1.3 1.2 1.2
Mannanase 0.005 0.001 0.002 0.0005 0.0002
Protease 0.03 0.01 0.03 0.02 0.02
Lipase - - 0.002 - -
Amylase - - - 0.002 -
Cellulase - - 0.0002 0.0005 0.0001
SRP 1 0.2 - 0.1 - -
DTPA - - 0.3 - -
PVNO - - 0.3 - 0.2
Brightener 1 0.2 0.07 0.1 - -
Silicone antifoam 0.04 0.02 0.1 0.1 0.1
Miscellaneous and
water
Example 11
The following liquid detergent formulations were prepared according to the
present invention (Levels are given in parts per weight, enzyme are expressed
in
pure enzyme)
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I II III IV
LAS 10.0 13.0 9.0 -
C25AS 4.0 1.0 2.0 10.0
C25E3S 1.0 - - 3.0
C25E7 6.0 8.0 13.0 2.5
TFAA - - - 4.5
APA - 1.4 - -
TPKFA 2.0 - 13.0 7.0
Citric 2.0 3.0 1.0 1.5
Dodecenyl / tetradecenyl 12.0 10.0 - -
succinic
acid
Rapeseed fatty acid 4.0 2.0 1.0 -
Ethanol 4.0 4.0 7.0 2.0
1,2 Propanediol 4.0 4.0 2.0 7.0
Monoethanolamine - - - 5.0
Triethanolamine - - 8.0 -
TEPAE 0.5 - 0.5 0.2
DETPMP 1.0 1.0 0.5 1.0
Mannanase 0.0002 0.0005 0.005 0.0005
Protease 0.02 0.02 0.01 0.008
Lipase - 0.002 - 0.002
Amylase 0.004 0.004 0.01 0.008
Cellulase - - - 0.002
SRP 2 0.3 - 0.3 0.1
Boric acid 0.1 0.2 1.0 2.0
Ca chloride - 0.02 - 0.01
Brightener 1 - 0.4 - -
Suds suppressor 0.1 0.3 - 0.1
Opacifier 0.5 0.4 - 0.3
NaOH up to pH 8.0 8.0 7.6 7.7
Miscellaneous and water
Example 12
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The following liquid detergent compositions were prepared according to the
present invention (Levels are given in parts per weight, enzyme are expressed
in
pure enzyme)
I II 111 IV V
LAS 25.0 - _ _ _
C25AS - 13.0 18.0 15.0 18.0
C25E3S - 2.0 2.0 4.0 2.0
C25E7 - - 4.0 4.0 4.0
TFAA - 6.0 8.0 8.0 8.0
APA 3.0 1.0 2.0 - 2.0
TPKFA - 15.0 11.0 11.0 11.0
Citric 1.0 1.0 1.0 1.0 1.0
Dodecenyl / tetradecenyl15.0 - - - -
succinic acid
Rapeseed fatty acid 1.0 - 3.5 - 3.5
Ethanol 7.0 2.0 3.0 2.0 3.0
1,2 Propanediol 6.0 8.0 10.0 13.0 10.0
Monoethanolamine - - 9.0 9.0 9.0
TEPAE - - 0.4 0.3 0.4
DETPMP 2.0 1.2 1.0 - 1.0
Mannanase 0.0001 0.0002 0.005 0.0005
Alkaline mannanase - - - - 0.005
Protease 0.08 0.02 0.01 0.02 0.01
Lipase - - 0.003 0.003 0.003
Amylase 0.004 0.01 0.01 0.01 0.01
Cellulase - - 0.004 0.003 0.004
SRP 2 - - 0.2 0.1 0.2
Boric acid 1.0 1.5 2.5 2.5 2.5
Bentonite clay 4.0 4.0 - - -
Brightener 1 0.1 0.2 0.3 - 0.3
Suds suppressor 0.4 - - - -
Opacifier 0.8 0.7 - - -
NaOH up to pH 8.0 7.5 8.0 8.2 8.0
Miscellaneous and
water
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Example 13
The following liquid detergent compositions were prepared according to the
present invention (Levels are given in parts by weight, enzyme are expressed
in
pure enzyme)
I II III
LAS 27.6 18.9 27.6
C45AS 13.8 5.9 13.8
C13E8 3.0 3.1 3.0
Oleic acid 3.4 2.5 3.4
Citric 5.4 5.4 5.4
Na hydroxide 0.4 3.6 0.4
Ca Formate 0.2 0.1 0.2
Na Formate - 0.5 -
Ethanol 7.0 - 7,0
Monoethanolamine 16.5 8.0 16.5
1,2 propanediol 5.9 5.5 5.9
Xylene sulfonic acid- 2.4 -
TEPAE 1.5 0.8 1.5
Protease 0.05 0.02 0.05
Mannanase 0.005 0.0002 -
Alkaline mannanase - - 0.001
PEG - 0.7 -
Brightener 2 0.4 0.1 0.4
Perfume 0.5 0.3 0.5
Water and Minors
Example 14
to
The following gel detergent compositions were prepared according to the
present
invention
I II III IV V
C12-15E2.5S 21 20.2 22.7 13.6 20.2
C12LAS - - - 9.1 -
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I II III IV ~/
C12-14 glucosamide 4.0 2.5 - - 2.5
C12-14E07 4.5 - - - -
C12-15E09 - 0.6 0.6 0.6 0.6
C8-10 amidopropylamine 1.3 - - - -
C10 amidopropylamine - 1.3 1.3 1.3 1.3
Citric 1.0 5.0 1.0 1.0 5.0
C12/14 fatty acid - 10.0 10.0 10.0 10.0
Palm kernal fatty acid 8.0 - - - -
Rapeseed fatty acid 8.0 - - - -
Mannase 0.0001 0.0002 0.005 0.0005 -
Alkaline Mannanase - - - - 0.0008
Protease 0.02 0.03 0.03 0.03 0.03
Lipase 0.001 0.002 0.003 0.002 0.002
Amylase 0.003 0.002 0.002 0.002 0.002
Cellulase 0.0007 0.0001 0.0001 0.0001 0.0001
Brightener 1 0.15 0.15 0.15 0.15 0.15
Polymer A 0.7 0.6 0.6 0.6 0.6
Polymer B - 1.2 1.2 1.2 1.2
Polyamine-polyamide 2.0 1.0 1.0 - 1.0
Polyethoxylated-Polyamines- 2.0 - - 2.0
Soil release agent - 0.1 0.1 0.1 0.1
Ethanol 0.7 0.5 0.5 0.5 0.5
1,2-propanediol 4.0 4.0 4.0 4.0 4.0
MonoEthanolAmine 0.7 0.5 0.5 0.5 0.5
NaOH 2.8 7.0 7.0 7.0 7.0
Boric acid 2.0 - - - -
Borax - 2.5 2.5 2.5 2.5
Suds supressor - 0.1 0.1 0.1 0.1
Polydimethyl siloxane 0.2 - - - -
Perfume 0.5 0.75 0.75 0.75 0.75
Dye - 0.04 0.04 0.04 0.04
Miscellaneous and water Up to 100%
Example 15
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The following gel detergent compositions were prepared according to the
present
invention
I II III IV
C12-15E2.5S 18.2 22.6 27.6 22.6
C12-15E09 0.6 0.6 0.6 0.6
C10 amidopropylamine 1.3 1.3 1.3 1.3
Citric 1.0 1.0 1.0 1.0
C 12/14 fatty acid 10.0 10.0 7.5 10.0
Quat 1.0 5.0 - -
Mannanase 0.005 0.001 0.002 0.0005
Protease 0.03 0.01 0.03 0.03
Lipase 0.002 0.002 0.002 0.002
Amylase 0.003 0.002 0.001 0.002
Cellulase 0.0001 0.0004 0.0001 0.0001
Brightener 1 0.15 0.15 0.15 0.15
Polymer A 0.6 0.3 0.6 0.6
Polymer B 1.2 0.6 1.2 1.2
Soil release agent 0.1 0.1 0.1 0.1
Ethanol 0.5 0.5 0.5 0.5
1,2-propanediol 4.0 4.0 4.0 4.0
MonoEthanolAmine 0.5 0.5 0.5 0.5
NaOH 7.0 7.0 7.0 7.0
Boric acid - - - -
Borax 2.5 2.5 2.5 -
Suds supressor 0.1 0.1 0.1 0.1
Perfume 0.75 0.75 0.75 0.75
Dye 0.04 0.04 0.04 0.04
Miscellaneous and Up to 0%
water 10
Example 16
The following granular fabric detergent compositions which provide "softening
through the wash" capability were prepared according to the present invention
0
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I II
C45AS - 10.0
LAS 7.6 -
C68AS 1.3 -
C45E7 4.0 -
C25E3 - 5.0
Coco-alkyl-dimethyl hydroxy-1.4 1.0
ethyl ammonium chloride
Citrate 5.0 3.0
Na-SKS-6 - 11.0
Zeolite A 15.0 15.0
MA/AA 4.0 4.0
DETPMP 0.4 0.4
PB1 15.0 -
Percarbonate - 15.0
TAED 5.0 5.0
Smectite clay 10.0 10.0
HMWPEO - 0.1
Mannanase 0.01 0.001
Protease 0.02 0.01
Lipase 0.02 0.01
Amylase 0.03 0.005
Celluiase 0.001 -
Silicate 3.0 5.0
Carbonate 10.0 10.0
Suds suppressor 1.0 4.0
CMC 0.2 0.1
Miscellaneous and water Up to
100%
Example 17
The following rinse added fabric softener compositions were prepared according
to the present invention
I II
DEQA (2) 20.0 20.0
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I II
Alkaline mannanase - 0.002
Mannanase 0.0008 -
Cellulase 0.001 0.001
HCL 0.03 0.03
Antifoam agent 0.01 0.01
Blue dye 25ppm 25ppm
CaCl2 0.20 0.20
Perfume 0.90 0.90
Miscellaneous and Up to 100%
water
Example 18
The following fabric softener and dryer added fabric conditioner compositions
were prepared according to the present invention
I II III IV V
DEQA 2.6 19.0 - - -
DEQA(2) _ _ _ _ 51.8
DTMAMS - - - 26.0 -
SDASA - - 70.0 42.0 40.2
Stearic acid of IV=00.3 - - - -
Neodol45-13 - - 13.0 - -
Hydrochloride acid 0.02 0.02 - - -
Ethanol - - 1.0 - -
Mannanase 0.0008 0.0002 0.0005 0.005 0.0002
Perfume 1.0 1.0 0.75 1.0 1.5
Glycoperse S-20 - - - - 15.4
Glycerol monostearate- - - 26.0 -
Digeranyl Succinate - - 0.38 - -
Silicone antifoam 0.01 0.01 - - -
Electrolyte - 0.1 - -
Clay - - - 3.0 -
Dye l0ppm 25ppm 0.01 - -
Water and minors 100% 100% - - -
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Example 19
The following laundry bar detergent compositions were prepared according to
the
present invention (Levels are given in parts per weight, enzyme are expressed
in
pure enzyme)
I II III VI V III VI V
LAS - - 19.0 15.0 21.0 6.75 8.8
C28AS 30.0 13.5 - - - 15.75 11.2 22.5
Na Laurate 2.5 9.0 - - - - - -
Zeolite A 2.0 1.25 - - - 1.25 1.25 1.25
Carbonate 20.0 3.0 13.0 8.0 10.0 15.0 15.0 10.0
Ca Carbonate27.5 39.0 35.0 - - 40.0 - 40.0
Sulfate 5.0 5.0 3.0 5.0 3.0 - - 5.0
TSPP 5.0 - - - - 5.0 2.5 -
STPP 5.0 15.0 10.0 - - 7.0 8.0 10.0
Bentonite - 10.0 - - 5.0 - - -
clay
DETPMP - 0.7 0.6 - 0.6 0.7 0.7 0.7
CMC - 1.0 1.0 1.0 1.0 - - 1.0
Talc - - 10.0 15.0 10.0 - - -
Silicate - - 4.0 5.0 3.0 - - -
PVNO 0.02 0.03 - 0.01 - 0.02 - -
MA/AA 0.4 1.0 - - 0.2 0.4 0.5 0.4
SRP 1 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3
Mannanase .0005 .0005.0008 .0005 .0002 .0002 0.001.0005
Amylase - - 0.01 - - - 0.002-
Protease - 0.004- 0.003 0.003 - - 0.003
Lipase - 0.002- 0.002 - - - -
Celluiase - .0003- - .0003 .0002 - -
PEO - 0.2 - 0.2 0.3 - - 0.3
Perfume 1.0 0.5 0.3 0.2 0.4 - - 0.4
Mg sulfate - - 3.0 3.0 3.0 - - -
Brightener 0.15 0.1 0.15 - - - - 0.1
Photoactivated- 15.0 15.0 15.0 15.0 - - 15.0
bleach (ppm)
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Example 20
The following detergent additive compositions were prepared according to the
present invention
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I II III IV
LAS - 5.0 5.0 5.0
STPP 30.0 - 20.0 20.0
Zeolite A - 35.0 20.0 20.0
PB1 20.0 15.0 - -
TAED 10.0 $.0 - -
Mannanase 0.005 0.0002 0.001 -
Alkaline mannanase - - - 0.005
Protease - 0.3 0.3 0.3
Amylase - 0.06 0.06 0.06
Minors, water and miscellaneous Up to 100%
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
APPLICANT:
NAME: The Procter & Gamble Company
STREET: One Procter 8~ Gamble Plaza
CITY: Cincinnati, OHIO
COUNTRY: USA
POSTAL CODE: 45202
TITLE OF INVENTION: Laundry detergent compositions comprising a
saccharide gum degrading enzyme.
NUMBER OF SEQUENCES: 6
COMPUTER READABLE FORM:
MEDIUM TYPE: Diskette
COMPUTER: IBM PC compatible
OPERATING SYSTEM: PC-DOS/MS-DOS
2o SOFTWARE: Patentln Release # 1.0 Version 1.25 (EPO)
SEQ ID N0:1
SEQUENCE CHARACTERISITICS:
LENGTH: 1407 base pairs
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
3o MOLECULE TYPE: genomic DNA
ORIGINAL SOURCE
FEATURE:
NAME/KEY: CDS
LOCATION:1-1482
SEQUENCE DESCRIPTION: SEQ ID NO: 1
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ATGAAAAAAAAGTTATCACAGATTTATCATTTAATTATTTGCACACTTATAATA
AGTGTGGGAATAATGGGGATTACAACGTCCCCATCAGCAGCAAGTACAGGC
TTTTATGTTGATGGCAATACGTTATATGACGCAAATGGGCAGCCATTTGTCAT
GAGAGGTATTAACCATGGACATGCTTGGTATAAAGACACCGCTTCAACAGCT
ATTCCTGCCATTGCAGAGCAAGGCGCCAACACGATTCGTATTGTTT'fATCAG
ATGGCGGTCAATGGGAAAAAGACGACATTGACACCATTCGTGAAGTCATTG
AGCTTGCGGAGCAAAATAAAATGGTGGCTGTCGTTGAAGTTCATGATGCCA
CGGGTCGCGATTCGCGCAGTGATTTAAATCGAGCCGTTGATTATTGGATAG
1o AAATGAAAGATGCGCTTATCGGTAAAGAAGATACGGTTATTATTAACATTGCA
AACGAGTGGTATGGGAGTTGGGATGGCTCAGCTTGGGCCGATGGCTATATT
GATGTCATTCCGAAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTG
ATGCAGCAGGATGGGGGCAATATCCGCAATCTATTCATGATTACGGACAAG
ATGTGTTTAATGCAGATCCGTTA~AAAAATACGATGTTCTCCATCCATATGTAT
~5 GAGTATGCTGGTGGTGATGCTAACACTGTTAGATCAAATATTGATAGAGTCA
TAGATCAAGACCTTGCTCTCGTAATAGGTGAATTCGGTCATAGACATACTGA
TGGTGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACA
GGGTGGCTCGCTTGGTCTTGGAAAGGCAACAGTACCGAATGGGACTAT-TTA
GACCTTfCAGAAGACTGGGCTGGTCAACATTTAACTGATTGGGGGAATAGAA
2o TTGTCCACGGGGCCGATGGCTTACAGGAAACCTCCAAACCATCCACCGTAT
TTACAGATGATAACGGTGGTCACCCTGAACCGCCAACTGCTACTACCTTGTA
TGACTTTGAAGGAAGCACACAAGGGTGGCATGGAAGCAACGTGACCGGTG
GCCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAAGC
CGATGTAAATTTAACCTCAAATTCTTCACATGAACTGTATAGTGAACAAAGTC
25 GTAATCTACACGGATACTCTCAGCTCAACGCAACCGTTCGCCATGCCAATTG
GGGAAATCCCGGTAATGGCATGAATGCAAGACTTTACGTGAAAACGGGCTC
TGATTATACATGGCATAGCGGTCCTTTTACACGTATCAATAGCTCCAACTCA
GGAACAACGTTATCTTTTGATTTAAACAACATCGAAAATAGTCATCATGTTAG
GGAAATAGGCGTGCAATTTTCAGCGGCAGATAATAGCAGTGGTCAAACTGC
3o TCTATACGTTGATAACGTTACTTTAAGATAG
SEQ ID N0:2
35 SEQUENCE CHARACTERISITICS:
LENGTH: 493 amino acids
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TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: protein
SEQUENCE DESCRIPTION: SEQ ID NO: 2
MKKKLSQIYHLIICTLIISVGIMGITTSPSAASTGFYVDGNTLYDANGQPFVMRGIN
HGHAWYKDTASTAIPAIAEQGANTIRIVLSDGGQWEKDDIDTIREViELAEQNKM
VAWEVHDATGRDSRSDLNRAVDYWIEMKDALIGKEDTVIINIANEWYGSWDGS
~o AWADGYIDVIPKLRDAGLTHTLMVDAAGWGQYPQSIHDYGQDVFNADPLKNTM
FSIHMYEYAGGDANTVRSNIDRVIDQDLALVIGEFGHRHTDGDVDEDTILSYSEE
TGTGWLAWSWKGNSTEWDYLDLSEDWAGQHLTDWGNRIVHGADGLQETSKP
STVFTDDNGGHPEPPTATTLYDFEGSTQGWHGSNVTGGPWSVTEWGASGNY
SLKADVNLTSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLYV
~5 KTGSDYTWHSGPFTRINSSNSGTTLSFDLNNIENSHHVREIGVQFSAADNSSGQ
TALYVDNVTLR
SEQ ID N0:3
SEQUENCE CHARACTERISITICS:
LENGTH: 1407 base pairs
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: genomic DNA
SEQUENCE DESCRIPTION: SEQ ID NO: 3
ATG GTTATCACAGATTTATCATTTAATTATTTGCACACTTATAATA
AGTGTGGGAATAATGGGGATTACAACGTCCCCATCAGCAGCAAGTACAGGC
TTTTATGTTGATGGCAATACGTTATATGACGCAAATGGGCAGCCATTTGTCAT
GAGAGGTATTAACCATGGACATGCTTGGTATAAAGACACCGCTTCAACAGCT
ATTCCTGCCATTGCAGAGCAAGGCGCCAACACGATTCGTATTGTTTTATCAG
ATGGCGGTCAATGGGAAAAAGACGACATTGACACCATTCGTGAAGTCATTG
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AGCTTGCGGAGCAAAATAAAATGGTGGCTGTCGTTGAAGTTCATGATGCCA
CGGGTCGCGATTCGCGCAGTGATTTAAATCGAGCCGTTGATTATTGGATAG
AAATGAAAGATGCGCTTATCGGTAAAGAAGATACGGTTATTATTAACATTGCA
AACGAGTGGTATGGGAGTTGGGATGGCTCAGCTTGGGCCGATGGCTATATT
GATGTCATTCCGAAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTG
ATGCAGCAGGATGGGGGCAATATCCGCAATCTATTCATGATTACGGACAAG
ATGTGTTTAATGCAGATCCGTTAAAAAATACGATGTTCTCCATCCATATGTAT
GAGTATGCTGGTGGTGATGCTAACACTGTTAGATCAAATATTGATAGAGTCA
TAGATCAAGACCTTGCTCTCGTAATAGGTGAATTCGGTCATAGACATACTGA
1o TGGTGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACA
GGGTGGCTCGCTTGGTCTTGGAAAGGCAACAGTACCGAATGGGACTATTTA
GACCTTTCAGAAGACTGGGCTGGTCAACATTTAACTGATTGGGGGAATAGAA
TTGTCCACGGGGCCGATGGCTTACAGGAAACCTCCAAACCATCCACCGTAT
TTACAGATGATAACGGTGGTCACCCTGAACCGCCAACTGCTACTACCTTGTA
~5 TGACTTTGAAGGAAGCACACAAGGGTGGCATGGAAGCAACGTGACCGGTG
GCCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAAGC
CGATGTAAATTTAACCTCAAATTCTTCACATGAACTGTATAGTGAACAAAGTC
GTAATCTACACGGATACTCTCAGCTCAACGCAACCGTTCGCCATGCCAATTG
GGGAAATCCCGGTAATGGCATGAATGCAAGACTTTACGTGAAAACGGGCTC
2o TGATTATACATGGCATAGCGGTCCTTTTACACGTATCAATAGCTCCAACTCA
GGAACAACGTTATCTTTTGATTTAAACAACATCGAAAATATCATCATGTTAGG
GAAATAG
25 SEQ ID N0:4
SEQUENCE CHARACTERISITICS:
LENGTH: 468 amino acids
TYPE: amino acid
3o TOPOLOGY: linear
MOLECULE TYPE: protein
SEQUENCE DESCRIPTION: SEQ ID NO: 4
35 MKKKLSQIYHLIICTLIISVGIMGITTSPSAASTGFYVDGNTLYDANGQPFVMRGIN
HGHAWYKDTASTAIPAIAEQGANTIRIVLSDGGQWEKDDIDTIREVIELAEQNKM
CA 02301404 2000-02-11
WO 99!09127 PC'T/US98/11995
112
VAWEVHDATGRDSRSDLNRAVDYWIEMKDALIGKEDTVIINIANEWYGSWDGS
AWADGYIDVIPKLRDAGLTHTLMVDAAGWGQYPQSIHDYGQDVFNADPLKNTM
FSIHMYEYAGGDANTVRSNIDRVIDQDLALVIGEFGHRHTDGDVDEDTILSYSEE
TGTGWLAWSWKGNSTEWDYLDLSEDWAGQHLTDWGNRIVHGADGLQETSKP
STVFTDDNGGHPEPPTATTLYDFEGSTQGWHGSNVTGGPWSVTEWGASGNY
SLKADVNLTSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLW
KTGSDYTWHSGPFTRINSSNSGTTLSFDLNNIENIIMLGK
1o SEQ ID N0:5
SEQUENCE CHARACTERISITICS:
LENGTH: 1029 base pairs
TYPE: nucleic acid
~5 STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: genomic DNA
2o SEQUENCE DESCRIPTION SEQ ID No:5
5' AAT TGG CGC ATA CTG TGT CGC CTG TGA ATC CTA ATG CCC AGC
AGA CAA CAA AAA CAG TGA TGA ACT GGC TTG CGC ACC TGC CGA ACC
GAA CGG AAA ACA GAG TCC TTT CCG GAG CGT TCG GAG GTT ACA GCC
25 ATG ACA CAT TTT CTA TGG CTG AGG CTG ATA GAA TCC GAA GCG CCA
CCG GGC AAT CGC CTG CTA TTT ATG GCT GCG ATT ATG CCA GAG GAT
GGC TTG AAA CAG CAA ATA TTG AAG ATT CAA TAG ATG TAA GCT GCA
ACG GCG ATT TAA TGT CGT ATT GGA AAA ATG GCG GAA TTC CGC AAA
TCA GTT TGC ACC TGG CGA ACC CTG CTT TTC AGT CAG GGC ATT TTA
3o AAA CAC CGA TTA CAA ATG ATC AGT ATA AAA ACA TAT TAG ATT CAG
CAA CAG CGG AAG GGA AGC GGC TAA ATG CCA TGC TCA GCA AAA TTG
CTG ACG GAC TTC AAG AGT TGG AGA ACC AAG GTG TGC CTG TTC TGT
TCA GGC CGC TGC ATG AAA TGA ACG GCG AAT GGT TTT GGT GGG GAC
TCA CAT CAT ATA ACC AAA AGG ATA ATG AAA GAA TCT CTC TAT ATA
35 AAC AGC TCT ACA AGA AAA TCT ATC ATT ATA TGA CCG ACA CAA GAG
GAC TTG ATC ATT TGA TTT GGG TTT ACT CTC CCG ACG CCA ACC GAG
CA 02301404 2000-02-11
WO 99/09127 PCT/US98111995
113
ATT TTA AAA CTG ATT TTT ACC CGG GCG CGT CTT ACG TGG ATA TTG
TCG GAT TAG ATG CGT ATT TTC AAG ATG CCT ACT CGA TCA ATG GAT
ACG ATC AGC TAA CAG CGC TTA ATA AAC CAT TTG CTT TTA CAG AAG
TCG GCC CGC AAA CAG CAA ACG GCA GCT TCG ATT ACA GCC TGT TCA
TCA ATG CAA TAA AAC AAA AAT ATC CTA AAA CCA TTT ACT TTC TGG
CAT GGA ATG ATG AAT GGA GCG CAG CAG TAA ACA AGG GTG CTT CAG
CTT TAT ATC ATG ACA GCT GGA CAC TCA ACA AGG GAG AAA TAT GGA
ATG GTG ATT CTT TAA CGC CAA TCG TTG AGT GAA TCC GGG ATC 3'
SEQ ID N0:6
SEQUENCE CHARACTERISITICS:
LENGTH: 363 amino acids
TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: protein
2o SEQUENCE DESCRIPTION: SEQ ID NO: 6
ydhT 1
LFKKHTISLLIIFLLASAVLAKPIEAHTVSPVNPNAQQTTKTVMNWLAHL 50
ydhT 51
PNRTENRVLSGAFGGYSHDTFSMAEADRIRSATGQSPAIYGCDYARGWLE 100
ydhT 101
TANIEDSIDVSCNGDLMSYWKNGGIPQISLHLANPAFQSGHFKTPITNDQ 150
ydhT 151
YKNILDSATAEGKRLNAMLSKIADGLQELENQGVPVLFRPLHEMNGEWFW 200
3o ydhT 201
WGLTSYNQKDNERISLYKQLYKKIYHYMTDTRGLDHLIWVYSPDANRDFK 250
ydhT 251
TDFYPGASYVDIVGLDAYFQDAYSINGYDQLTALNKPFAFTEVGPQTANG 300
ydhT 301
SFDYSLFINAIKQKYPKTIYFLAWNDEWSAAVNKGASALYHDSWTLNKGE 350
ydhT 351
CA 02301404 2000-02-11
WO 99/09127 PCT/US98/11995
114
IWNGDSLTPIVE*. 363
