Note: Descriptions are shown in the official language in which they were submitted.
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DETERGENT COMPOSITIONS COMPRISING A MANNANASE
AND PERCARBONATE
1 o Field of the Invention
The present invention relates to detergent compositions comprising a
mannanase and percarbonate.
Background of the invention
Perborate is well-known in the art as a laundry or dish additive that provide
available oxygen via a hydrogen peroxide release mechanism. Perborate is
2o broadly used in laundry or dish detergent due to its high performance and
its
attractive cost.
However it has been recognised in the art that the side product formed during
the
release of hydrogen peroxide from perborate, i.e. meta borate derivatives,
complex with sugar polymers such as starch and leads to cleaning negative (EP-
A-736 085). It has been surprisingly found that mannose polymers such as guar
gum also cross-link with perborate thereby rendering the food or cosmetic
stains
even harder to remove by perborate-containing detergents. It has additionally
been surprisingly found that the borate cross-finking with guar gum reduces
the
3o activity of the enzyme on such gum/borate complex substrates.
Indeed, food and cosmetic stains/soils represent the majority of consumer
relevant stains/soils and often comprise food additives such as thickener
stabiliser agents. Indeed, hydrocolloids gums and emulsifiers are commonly
used
food additives. The term "gum" denotes a group of industrially useful
polysaccharides (long chain polymer) or their derivatives that hydrate in hot
or
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cold water to from 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
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
1o Processing by P. Laslo, Bioprinciples and Applications, Vol1, Chapter fl,
pp313-
325 {1996) Technomie publishing. Some of these gums such as guar gum
(E412), locust bean (E410) are widely used alone or in combinations in many
food applications (Gums in ECT 4th Ed., Vol. 12 pp842-862, J. Baird, Kelco
division of Merck).
The guar gum used in these food and cosmetic stains is obtained from the seed
endosperm of the leguminous plant Cyamopsis tefragonoloba. The guar gum
(also called guaran) extracted from the dicotyledonous seed is composed of a 1-
4, b-D-mannopyranosyl unit backbone and is used as a thickening agent in
2o dressing and frozen products and cosmetics (H.-D. Belitz, Food Chemistry pp
243, English version of the second edition, Springer-verlag, 1987, ISBN 0-387-
15043-9 (US)) & (Carbohydrate Chemistry for Food Scientists, R.L. Wilstler,
eagan press, 1997, ISBN 0-913250-92-9) & (Industrial Gum, second editions,
R.L. Whistler pp 308, Academic Press, 1973, ISBN, 0-12-74-6252-x). The locus
bean gum (also called carob bean gum or St Jon's bread) is also used in the
food industry and is extracted from the seed of an evergreen cultivated in the
Mediterranean area. The locus bean gum probably differs from the structure of
guar gum only in smaller number of D-galactosyl side chains and have the same
1-4, b-D-mannopyranosyl backbone. In leguminous seeds, water-soluble
3o galactomanann is the main storage carbohydrate, comprising up to 20% of the
total dry weight in some cases. Galactomannan has a a-gaiactose linked to O-6
of mannose residues and it can also be acetylated to various degree on O-2 and
O-3 of the mannose residues.
Additionally, the action of percarbonate on bleacheable stains is broadly
known
in the art. The active ingredient released from percarbonate, i.e. H202, is
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identical to the active ingredient released from perborate, but without the
release of borate material. It has now been surprisingly discovered that the
combined use of percarbonate and mannanase, especially at specific levels,
provides a synergetic removal of difficult stains such as food and cosmetic
stains comprising mannans, in particular at low temperatures.
It has further been found that the ternary system consisting of percarbonatel
mannanaselprotease at specific levels provides an even better result on said
stains.
Mannanases have been identified in several Bacillus organisms. For example,
Talbot et al., Appl. Environ. Microbiol., vol. 56, No. 11, pp. 3505-3510
(1990)
describes a ~3-mannanase derived from Bacillus stearothermophilus in dimer
form
having a MW of 162 kDa and an optimum pH of 5.5-7.5. Mendoza et al., World J.
Micobio. Boitech., vol. 10, no. 5, pp. 551-555 (1994) describes a ~3-mannanase
derived from Bacillus subtilises having a MW of 38 kDa, an optimum activity at
pH
5.0 / 55°C and a pl of 4.8. J0304706 discloses a ~3-mannanase derived
from
Bacillus sp. having a MW of 37+/- 3kDa measured by gel filtration, an optimum
pH of 8-10 and a pl of 5.3-5.4. J63056289 describes the production of an
2o alkaline, thermostable ~i-mannase, which hydrolyses ~i-1,4-D-
mannopyranoside
bonds of e.g. mannans and produces manno:oligoaaccharides. J63036774
relates to a Bacillus micro-organism FERM P-8856 which produces ~3-
mannanase and ~-mannosidase, at an alkaline pH. A purified mannanase from
Bacillus amylolipuefaciens and its method of preparation useful in the
bleaching
of pulp and paper, is disclosed in W097/11164. W091/18974 describes an
hemicellulase such as a glucanase, xylanase or mannanase, active at extreme
pH and temperature and the production thereof. W094/25576 describes an
enzyme exhibiting a mannanase activity derived from Aspergillus aculeafus CBS
101.43, that might be used for various purposes for which degradation or
3o modification of plant or algae cell wall material is desired. W093/24622
discloses
a mannanase isolated from Trichoderma reesie for bleaching lignocellulosic
pulps.
However, the synergistic combination of a mannanase and percarbonate, for
superior cleaning performance in a detergent composition, i.e., superior stain
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removal, especially on mannans-containing cosmetic and food stains, dingy
cleaning and whiteness maintenance, has never been previously recognised.
Additionally, the synergistic combination of a mannanase and percarbonate and
protease, for superior cleaning performance in a detergent composition, i.e.,
superior stain removal, dingy cleaning and whiteness maintenance, has never
been previously recognised.
Summaryr of the invention
The present invention relates to detergent compositions comprising a
mannanase and percarbonate. These compositions provide superior cleaning
performance, i.e. superior stain removal, especially on mannans-containing
~5 cosmetic and food stains, dingy cleaning and whiteness maintenance.
Detailed desc~intion of the invention
2o An essential element of the detergent composition of the present invention
is a
mannanase enzyme. The use of mannanase provides significant stain removal
benefits on stains such as cosmetic and food stains containing hydrocolloid
gums
such as Guar Gum.
2s We have surprisingly found that the combination of perborate and mannanase
provide limited performance benefits. It is believed that this limited
performance
is due to the presence of the meta borate that can cross-link with the
hydrocolloid
gum contained in the stains. In contrast, it has been found that the use of
percarbonate, i.e., a material that provides available oxygen without forming
3o meta borate derivatives, provides in combination with the mannanase of the
present invention, an outstanding synergistic cleaning.
Therefore, the present invention relates to the use of the mannanase enzyme in
combination with percarbonate. This combination provides outstanding
35 synergistic whitening and/or stain removal benefits on food and cosmetic
stains
that contain mannanase sensitive hydrocolloid gums such as Guar gum or Locus
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bean gum. Without wishing to be bound by theory, it is believed the
percarbonate
bleaching agent does not complex with the gums present in the stains and
therefore facilitate the access of the enzyme and/or of the bleaching agent to
said stains. Moreover, it is believed that this synergistic effect is due to
a) the use
5 of percarbonate that bleach the chromophore of the gum containing stains
without cross linking and b) the action of the mannanase on the hydrocolloid
polymer to form more soluble small residues and providing provide high removal
of the hydrocolloid residues known to have a high affinity for the cotton
surface of
the garments. Additionally, it is believed that the synergy is due to the
absence of
o borate ions that are known to act as crosslinking agent with hydrated guar
gum
(see Industrial Gum, second editions, R.L. Whistler pp 317, Academic Press,
1973, ISBN, 0-12-74-6252-x).
~ 5 The mannanase enzyme
An essential element of the detergent compositions of the present invention is
a
mannanase enzyme.
2o Encompassed in the present invention are the following three mannans-
degrading enzymes : EC 3.2.1.25 : (i-mannosidase, EC 3.2.1.78 : Endo-1,4-~i-
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).
More preferably, the 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-
3o mannosidase and having the alternative names beta-mannanase and endo-1,4-
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
which degrade mannans and denote enzymes which are capable of cleaving
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polyose chains contaning mannose units, i.e. are capable of cleaving
glycosidic
bonds in mannans, glucomannans, galactomannans and galactogluco-mannans.
Mannans are polysaccharides having a backbone composed of ~i-1,4- linked
mannose; glucomannans are polysaccharides having a backbone or more or less
regularly alternating (i-1,4 linked mannose and glucose; galactomannans and
galactoglucomannans are mannans and glucomannans with a-1,6 linked
galaetose sidebranches. These compounds may be acetylated.
The degradation of galactomannans and galactoglucomannans is facilitated by
~o full or partial removal of the galactose sidebranches. Further the
degradation of
the acetylated mannans, glucomannans, galactomannans and galactogluco-
mannans 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-
~5 galactosidase and/or mannan acetyl esterases can be further degraded to
release free maltose by ~3-mannosidase and/or ~i-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)
2o describes a beta-mannanase derived from Bacillus stearothermophilus in
dimer
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-
25 0304706 discloses a beta-mannanase derived from Bacillus sp., having a
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
3o relates to the Bacillus microorganism FERM P-8856 which produces beta-
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
35 91/18974 describes a hemicellulase such as a glucanase, xylanase or
mannanase active at an extreme pH and temperature. WO 94125576 discloses
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an enzyme from Aspergillus aculeatus, CBS 101.43, exhibiting mannanase
activity which may be useful for degradation or modification of plant or algae
cell
wall material. WO 93/24622 discloses a mannanase isolated from Trichodenna
reseei useful for bleaching lignocellulosic pulps. An hemicellulase capable of
degrading mannan-containing hemicellulose is described in W091/18974 and a
purified mannanase from Bacillus amyloliquefaciens is described in
W097/11164.
In particular, this mannanase enzyme will be an alkaline mannanase as defined
below, most preferably, a mannanase originating from a bacterial source.
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 subtilises strain 168, gene yght
~ 5 The term "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.
2o Most preferably, the (detergent composition of the present invention will
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
25 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
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 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 subtilises
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 polypeptide 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) polynucieotide 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;
(d) molecules complementary to (a), (b) or (c); and
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(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
o The term "ortholog" (or "species homolog") denotes a polypeptide or protein
obtained from one species that has homology to an analogous polypeptide or
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
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
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
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
3o and replicated together with the chromosomes) into which it has been
integrated.
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.
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The term "isolated", when applied to a polynucleotide molecule, denotes that
the polynucleotide has been removed from its natural genetic milieu and is
thus
free of other extraneous or unwanted coding sequences, and is in a form
suitable
for use within genetically engineered protein production systems. Such
isolated
s 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
0 ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-
78,
1985).
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.
~5 In a preferred form, the isolated protein is substantially free of other
proteins,
particularly other homologous proteins (i.e. "homologous impurities" (see
below)).
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
2o preferably greater than 95% pure, and even more preferably greater than 99%
pure, as determined by SDS-PAGE.
The term "isolated protein/polypeptide may alternatively be termed "purified
protein/polypeptide".
The term "homologous impurities" means any impurity (e.g., another
2s pofypeptide than the polypeptide of the invention) which originates from
the
homologous cell where the polypeptide of the invention is originally obtained
from. The term "obtained from" as used herein in connection with a specific
microbial source, means that the polynucleotide and/or polypeptide produced by
the specific source, or by a cell in which a gene from the source has been
3o inserted.
The term "operably linked", when referring to DNA segments, denotes that the
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.
35 The term "polynucleotide" denotes a single- or double- stranded polymer of
deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
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Polynucleotides include RNA and DNA, and may be isolated from natural
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
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
o 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 polymerase 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.
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
3o 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
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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
company Megazyme for US$110.00 per 3 grams (Megazyme's Internet address:
http://www.megazyme.com/Purchase/index.html).
POLYNUCLEOTIDES:
An isolated polynucleotide of the invention will hybridize to similar sized
regions
0 of SEQ ID No. 1, or a sequence complementary thereto, under at least 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 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 chloridelSodium 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),
3o and even more preferably at least 75°C (very high stringency).
Molecules to which the oligonucleotide probe hybridizes under these conditions
are detected using a x-ray film.
As previously noted, the isolated polynucleotides 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 further 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
1o 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 encoding an
polypeptide
~5 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 (Mullis, U.S. Patent 4,683,202),
2o 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 as described in Materials
25 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 NC1MB 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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14
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
s variant of the mannan degrading enzyme of the invention).
POLYPEPTIDES:
o The sequence of amino acids nos. 32-343 of SEQ ID NO: 2 is a mature
mannanase sequence.
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
~5 used herein to denote polypeptides having 70%, preferably at least 80%,
more
preferably at least 85%, and even more preferably at least 90%, sequence
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
2o 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
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
2s 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.
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
3o 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 arches 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
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all species preferably are at least 95%, even more preferably at least 98%,
homologous to Bacillus agaradherens based on aligned 16S rDNA sequences.
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
5 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, 1985;
Nilsson et
o al., Methods Enzymol. 198:3, 1991. See, in general Ford et al., Protein
Exaression 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).
However, even though the changes described above preferably are of a minor
~5 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.
Table 1
2o 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 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 been
modified
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16
after protein synthesis, and/or have a chemical structure in their 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, thiazofidine carboxylic acid, dehydroproline, 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
o 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 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.,
FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also
be
2o inferred from analysis of homologies with polypeptides 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 86:2152-2156,
1989), W095I17413, 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 for functional a polypeptide, and then sequencing the
3o mutagenized poiypeptides to determine the spectrum of 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).
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17
. Mutagenesis/shuffling methods as disclosed above can be combined with high-
throughput, automated screening methods to detect activity of cloned,
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
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
~o 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
~5 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
20 organisms, are preferred. Gram-positive cells from the genus of Bacillus
are
especially preferred, such as from the group consisting of Bacillus subfilis,
Bacillus lentus, Bacillus br~evis, Bacillus stearothermophilus, Bacillus
alkalophilus,
Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus
lautus,
Bacillus thuringiensis, Bacillus licheniformis, and Bacillus agaradherens, in
2s 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
3o Molecular Bioloay, John Wiley and Sons, Inc., NY, 1987; and "Bacillus
subtilis
and Other Gram-Positive Bacteria", Sonensheim et al., 1993, American Society
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
s5 including a transcription promoter and terminator within an expression
vector.
The vector will also commonly contain one or more selectable markers and one
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18
or more origins of replication, although those skilled in the art will
recognize that
within certain systems selectable markers may be provided on 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
o 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
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
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
2o 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.,
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 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
3o exogenously added DNA by, for example, drug selection or deficiency in an
essential nutrient which is complemented by the selectable 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
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19
removed from the media before purification of the polypeptide (e.g. by
centrifugation).
When the expressed recombinant pofypeptide 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
lysozyme digestion or by forcing the cells through high pressure. See (Robert
K.
o Scobes, Protein Purification, Second edition, Springer-Verlag) for further
description of such cell disruption techniques.
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.
s Ammonium sulfate precipitation and acid or chaotrope extraction may be used
for
fractionation of samples. Exemplary purification steps may include
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,
2o QAE and Q derivatives are preferred, with DEAE Fast-Flow Sepharose
(Pharmacia, Piscataway, NJ) being particularly preferred. Exemplary
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
2s polyacrylic resins, such as Arnberchrom CG 71 (Toso Haas) and the like.
Suitable solid supports include glass beads, silica-based resins, cellulosic
resins,
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
3o that allow attachment of proteins by amino groups, carboxyl groups,
sulfhydryl
groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling
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
35 other solid media are well-known and widely used in the art, and are
available
from commercial suppliers.
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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, Affini
Chromatographw. Principles 8~ Methods, Pharmacia LKB Biotechnology, Uppsaia,
Sweden, 1988.
5 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
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,
15 ~ preparing a genomic library from a Bacillus strain, especially the strain
8.
agaradherens, NCIMB 40482;
~ plating such a library on suitable substrate plates;
t identifying a clone comprising a polynucleotide 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.
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 Bacillus
such
3o 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 prepared on
the basis of the DNA sequence obtainable from the plasmid present in
Escherichia coli DSM 12180.
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21
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
1o microorganism which acts as a host cell for expression 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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22
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. 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., su ra, Chapters 3 and 4), or by
rocket
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 Giycin buffer, pH10Ø 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 nrn 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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23
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., Sjreholm, C. (1990) Cloning of aldB, which encodes alpha-
acetolactate decarboxyiase, 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
o genes (Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen, B. R., Sjraholm,
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 gene, resulting
in
cellulase negative cells. The disruption was performed essentially as
described in
~5 ( 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, 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
2o competent cells. J. Bacteriol,121:296-304.
Plasmids
pSJ1678 (as described in detail in WO 94/19454 which is hereby incorporated by
reference in its entirety).
25 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
B.lichenifonnis 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
3o 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 engineering
techniques which are briefly described in the following.
Construction of pMOL944:
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24
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'-GTCGCCGGGGCGGCCGCTATCAATTGGTAACTGTATCT
CAGC -3'
# LWN5495 5'-GTCGCCCGGGAGCTCTGATCAGGTACCAAGCTTGTCGAC
o CTGCAGAATGAGGCAGCAAGAAGAT-3'
The primer #LWN5494 inserts a Notl site in the plasmid.
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 promoter
but
in the opposite direction. The two primers used for PCR amplification have the
following sequences:
#LWN5938 5'-
GTCGGCGGCCGCTGATCACGTACCAAGCTTGTCGACCTGCAGAATG
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
alkaline amylase SP722 (disclosed in the International Patent Application
published as W095/26397 which is hereby incorporated by reference in its
3o 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:
#LWN7864 5' -AACAGCTGATCACGACTGATCTTTTAGCTTGGCAC-3'
#LWN7901 5' -AACTGCAGCCGCGGCACATCATAATGGGACAAATGGG -3'
The primer #LWN7901 inserts a Sacl I site in the plasmid.
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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
5 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).
Genomic library construction:
o 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
method for the recovery of DNA fragments from agarose and acrylamide gels.
15 Anal. Biochem., 112, 295-298).
Isolated DNA fragments were iigated to BamHl digested pSJ1678 plasmid DNA,
and the ligation mixture was used to transform E. coli SJ2.
Identification of positive clones:
A DNA library in E. coli, constructed as described above, was screened on LB
2o agar plates containing 0.2% AZCL-galactomannan (Megazyme) and 9 Nglml
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
culture broth (cells incubated at 37°C in TY with 9 Nglml
Chloramphenicol and
25 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, USA), fluorescent
labelled terminators and appropriate oligonucleotides as primers.
3o Analysis of the sequence data was pertormed 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
No.2.
Subcloning and expression of mannanase in B.subtilis:
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26
The mannanase encoding DNA sequence of the invention was PCR amplified
using the PCR primer set consisting of these two oligo nucleotides:
Mannanase. upper.Sacl I
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'
Restriction sites Sacll and Notll are underlined.
o 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
~ 5 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-NI
aliquots of the
2o 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
25 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
with Sacll and Notl, electrophoresed in 0.8% low gelling temperature agarose
30 (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-Noti digested and purified pMOL944. The ligation was performed
overnight at 16°C using 0.5Ng of each DNA fragment, 1 U of T4 DNA
ligase and
3s T4 ligase buffer (Boehringer Mannheim, Germany).
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27
The ligation mixture was used to transform competent B.subtilis PL2306. The
transformed cells were plated onto LBPG-10 pg/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.
s 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 plasmid
o 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
~5 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 M
25 potassium phosphate, pH 7.0
BPX media is described in EP 0 506 780 (WO 91109129).
Expression, purification and characterisation of mannanase from Bacillus
ayaradherens
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 ml 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
35 anionic agent (A130) was added during agitation for flocculation. The
flocculated
material was separated by centrifugation using a Sorval RC 3B centrifuge at
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28
9000 rpm for 20 min at 6°C. The supernatant was clarified using Whatman
glass
filters GF/D 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 translated
DNA sequence, is shown in SEQ ID No.2.
o 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
~ 5 Kcat: 467 per sec.
K,": 0.08 gram per I
MW: 38kDa
pl {isoelectric point): 4.2
The temperature optimum of the mannanase was found to be 60°C.
2o 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
25 compatibility with conventional liquid detergents and good compatibility
with
conventional powder detergents.
OBTENTION OF THE BACILLUS SUBTILISIS MANNANASE 168
The Bacillus subtilisis ~i-mannanase was characterised and purified as follows
so The Bacillus subtilis genome was searched for homology with a known
Bacillus
sp ~i-Mannanase gene sequence (Mendoza et al., Biochemica et Bioph sy ica
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
35 mature portion of the putative ~i-Mannanase: 5'-GCT CAA TTG GCG CAT ACT
GTG TCG CCT GTG-3' and 5'-GAC GGA TCC CGG ATT CAC TCA ACG ATT
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29
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
s 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 10 min to
complete extension. The PCR products were purified using QIAquick PCR
purification kit (Qiagen, Chatsworth, CA).
o 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 restriction products
were purified using QIAquick PCR purification kit (Qiagen, Chatsworth, CA).
The
~5 two fragments were ligated using T4 DNA ligase (13 hr, 16°C) and
used to
transform competent E. coli strain DH5-a. Ampicilin resistant colonies were
cultured for DNA preparations. The DNA was then characterized by restriction
analysis. Plasmid pPG3200 contains the mature region of the ydhT gene.
Plasmid pPG3200 was then used to transform competent Bacillus subtilis strain
2o 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/I yeast
extract, 5g/I NaCI ) supplemented with 1 ml 25% maltrin, 120.1 1 OmM MnCl2,
and
201 of 50 mg/ml kanamycin. Clones were grown overnight in 250m1 baffled
2s flasks shaking at 250 rpm at 37°C for expression of the protein.
Cells were spun
out at 14,OOOrpm for ~15 minutes. One wl 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-~i-Mannanase Beta-Mannazyme Tabs (Megazyme, Ireland) according
to the manufacturers instructions. Absorbance was read at 590 nm on a
3o 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 were
prepared as follows. A 500p.1 sample of ydhT clone 7 and PG 632 supernatants
3s were precipitated with 55.51 100% Trichloroacetic acid (Sigma), washed with
1001 5% Trichloroacetic, resuspended in 501 of Tris-glycine SDS sample
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buffer(Novex) and boiled for five minutes. One pl of each sample was
eiectrophoresed 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
s 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 ~3-Mannanase in the
concentrated supernatant was determined to be 3 g/I.
o 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 ! changes of 10 mM potassium phosphate (pH 7.2) through Spectrapor
12,000-14,000 mol.wt. cutoff membrane (Spectrum). The dialyzed supernatant
s 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 ml (sample B)
were collected. The two fractions were assayed as before, except that the
2o samples were diluted with 199 pl 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 8~1 of Tris-glycine SDS sample buffer (Novex, CA) and boiled for 5
min.
The resulting samples were electrophoresed on a 10-20% Tris-Glycine gel
(Novex, Ca) at 30 mA for 90minutes. A major band corresponding to 38kDa was
25 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 ~-Mannanase respectively. The
identity of the protein was confirmed by ion spray mass spectrometry and amino
3o terminal amino acid sequence analysis.
The purified ~i-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 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 mM CAPS buffer was
employed. The optimum pH for the Bacillus subtilis ~-Mannanase was found to
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31
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 ~i-1,4-Galactomannan was
determined to be 160,000 pmol/min~mg ~i-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. 5 and 6.
o The mannanase is incorporated into the compositions of the invention
preferably
at a level of from 0.0001 % to 2%, more preferably from 0.0005% to 0.1 %, most
preferred from 0.001 % to 0.02% pure enzyme by weight of the composition.
The enzyme of the invention, in addition to the enzyme core comprising the
~5 catalytically domain, also comprise a cellulose binding domain (CBD), the
cellulose binding domain and enzyme core (the 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
2o term "cellulose-binding domain" is intended to be understood as defined by
Peter
Tomme et al. "Cellulose-Binding Domains: 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
25 demonstrates that CBDs are found in various enzymes such as cellulases,
xylanases, mannanases, 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
3o at the N and C termini of proteins or are internal. Enzyme hybrids are
known in
the art, see e.g. WO 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 cellulose- binding domain ligated, with or without a linker,
to a
DNA sequence encoding the mannanase enzyme and growing the host cell to
35 express the fused gene. Enzyme hybrids may be described by the following
formula:
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32
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
s of from about 2 to about 100 carbon atoms, more preferably of from 2 to 40
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.
o The above-mentioned enzymes may be of any suitable origin, such as
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
~5 protein I genetic engineering techniques in order to optimise 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,
2o 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
25 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
enzymes may be further enhanced by the creation of e.g. additional salt
bridges
and enforcing metal binding sites to increase chelant stability.
The percarbonate compound
The detergent compositions herein typically contain from 0.1 % to 50%,
preferably from 0.5% to 35% by weight, most preferably from 1 % to 25% by
weight of an alkali metal percarbonate bleach in the form of particles having
a
mean size from 250 to 900 micrometers, preferably 500 to 700 micrometers.
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Laundry additives typically contain from 20% to 80% of said percarbonate
particles.
Preferred detergent compositions according to the present invention comprise a
level of mannanase (pure enzyme by weight of total composition) of from
0.0001 % to 2% and a level of percarbonate of from 0.1 % to 50% by weight of
the total composition, preferably a mannanase level of from 0.0005% to 0.5%
and a percarbonate level of from 0.5 % to 35%, more preferably a level of
mannanase of from 0.001 % to 0.1 % and a percarbonate level of from 1 % to 25%
The alkali metal percarbonate bleach is usually in the form of the sodium
salt.
Sodium percarbonate is an addition compound having a formula corresponding
to 2Na2C03 3H202. To enhance storage stability the percarbonate bleach
can be coated with e.g. a further mixed salt of an alkali metal sulphate and
carbonate. Such coatings together with coating processes have previously
been described in GB-1,466,799, granted to Interox on 9th March 1977. The
weight ratio of the mixed salt coating material to percarbonate lies in the
range
from 1:2000 to 1:4, more preferably from 1:99 to 1:9, and most preferably from
1:49 to 1:19. Preferably, the mixed salt is of sodium sulphate and sodium
2o carbonate which has the general formula Na2S04.n.Na2C03 wherein n is from
0.1 to 3, preferably n is from 0.3 to 1.0 and most preferably n is from 0.2 to
0.5.
Suitable percarbonate for the purpose of the present invention is the sodium
percarbonate described in W097/35591 being characterised by an intrinsic
mean particle size of 500 to 100 mu m, not more than 20% below 350 mu m
and a moisture pick-up of no more than 30g per 1000g sample at 80% relative
humidity and 32°C in 24h or characterised by a mean particle size of
500 to
1200 mu m and a 7-days aged heat emission at 40°C of below 3 mu Wlg in
16h. Also suitable is the sodium percarbonate manufactured from hydrogen
3o peroxide and sodium carbonate in aqueous medium without the use of chloride
salting-out agent as described in W097/35806.
Other suitable coating materials are sodium silicate, of Si02:Na20 ratio from
1.6:1 to 2.8:1, and magnesium silicate.
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Commercially available carbonate/sulphate coated percarbonate bleach may
include a low level of a heavy metal sequestrant such as EDTA, 1-
hydroxyethylidene 1,1-diphosphonic acid (HEDP) or an aminophosphonate,
that is incorporated during the manufacturing process. Preferred heavy metal
s sequestrants for incorporation as described herein above include the organic
phosphonates and amino alkylene poly(alkylene phosphonates) such as the
alkali metal ethane 1-hydroxy diphosphonates, the nitrilo trimethylene
phsphonates, the ethylene diamine tetra methylene phosphonates and the
diethylene triamine penta methylene phosphonates.
Determent components
The detergent compositions of the invention must contain at least one
additional
detergent component. The precise nature of these additional component, 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 detergent compositions according to the invention can be liquid, paste,
gels,
bars, tablets, spray, foam, powder or granular. Granular compositions can also
2o be in "compact" form and the liquid compositions can also be in a
"concentrated"
form.
In a preferred embodiment, the present invention relates to a laundry
composition comprising a mannanase and percarbonate. In a second
embodiment, the present invention relates to dishwashing compositions.
The compositions of the invention may for example, be formulated as hand and
machine dishwashing compositions, hand and machine laundry detergent
compositions including laundry additive compositions and compositions suitable
3o for use in the soaking and/or pretreatment of stained fabrics, rinse added
fabric
softener compositions.
When formulated as compositions for use in manual dishwashing methods the
compositions of the invention preferably contain a surfactant and preferably
other
detergent compounds selected from organic polymeric compounds, suds
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enhancing agents, group II metal ions, solvents, hydrotropes and additional
enzymes.
When formulated as compositions suitable for use in a laundry machine washing
5 method, the compositions of the invention preferably contain both a
surfactant
and a builder compound and additionally one or more detergent 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
1o compositions can also contain softening agents, as additional detergent
components. Such compositions containing mannanase and percarbonate can
provide fabric cleaning, stain removal, whiteness maintenance, color
appearance, dye transfer inhibition and sanitisation when formulated as
laundry
detergent compositions.
The compositions of the invention can also be used as detergent additive
products in solid or liquid form. Such additive products are intended to
supplement or boost the performance of conventional detergent compositions
and can be added at any stage of the cleaning process.
If needed the density of the laundry detergent compositions herein ranges from
400 to 1200 g/litre, preferably 500 to 950 g/litre of composition measured at
20°C.
The "compact" form of the compositions herein is best reflected by density
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 the
total
3o 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.
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
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36
conventional liquid detergents. Typically the water content of the
concentrated
liquid detergent is preferably less than 40%, more preferably less than 30%,
most
preferably less than 20% by weight of the detergent composition.
Suitable detergent compounds for use herein are selected from the group
consisting of the below described compounds.
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37
Surfactant system
The detergent compositions according to the present invention generally
comprise a surtactant system wherein the surfactant can be selected from
nonionic and/or anionic and/or cationic andlor ampholytic and/or zwitterionic
andlor semi-polar surfactants.
The surfactant is typically present at a level of from 0.1 % to 60% by weight.
More
preferred levels of incorporation are 1 % to 35% by weight, most preferably
from
1 % to 30% by weight of 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
~5 not degrade, the stability of any enzyme in these compositions.
Preferred surfactant systems to be used according to the present invention
comprise as a surfactant one or more of the nonionic and/or anionic
surfactants
described herein.
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
moles, more preferably from about 3 to about 15 moles, of ethylene oxide per
3o 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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38
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 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
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 Te~gitoITM 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 4,565,647,
3o 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 saccharide
containing
35 5 or 6 carbon atoms can be used, e.g., glucose, galactose and galactosyl
moieties can be substituted for the glucosyl moieties (optionally the
hydrophobic
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39
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
wherein R2 is selected from the group consisting of alkyl, alkylphenyl,
o 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
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-
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 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 content is about
50% of the total weight of the condensation product, which corresponds to
3o 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.
Also suitable for use as the nonionic surfactant of the nonionic surfactant
system
of the present invention, are the condensation products of ethylene oxide with
the product resulting from the reaction of propylene oxide and
ethylenediamine.
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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
5 about 40% to about 80% by weight of poiyoxyethylene 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
~5 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
surfactants of the formula.
2o
R2-C-N-Z,
O R1
25 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.
Suitable anionic surfactants to be used are linear alkyl benzene sulfonate,
alkyl
ester sulfonate surfactants including linear esters of Cg-C2p carboxylic acids
35 (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
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41
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 I
R3 - CH - C - OR4
o S03M
wherein R3 is a Cg-C20 hydrocarbyl, preferably an alkyl, or combination
thereof,
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
~5 substituted or unsubstituted ammonium cations, such as monoethanolamine,
diethanolamine, and triethanolamine. Preferably, R3 is C10-C1g alkyl, and R4
is
methyl, ethyl or isopropyl. Especially preferred are the methyl ester
sulfonates
wherein R3 is C10-C1g alkyl.
2o Other suitable anionic surtactants include the alkyl sulfate surfactants
which are
water soluble salts or acids of the formula ROS03M wherein R 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
25 ammonium or substituted ammonium (e.g. methyl-, dimethyl-, and trimethyi
ammonium cations and quaternary ammonium cations such as tetramethyl-
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
3o 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).
Other anionic surfactants useful for detersive purposes can also be included
in
the detergent compositions of the present invention. These can include salts
35 (including, for example, sodium, potassium, ammonium, and substituted
ammonium salts such as mono-, di- and triethanolamine salts) of soap, Cg-C22
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42
primary of secondary aikanesulfonates, 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 alkylpolygiycolethersulfates (containing up to 10 moles of
ethylene oxide); alkyl glycerol sulfonates, fatty acyl glycerol sulfonates,
fatty oleyl
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
o sulfosuccinates (especially saturated and unsaturated Cg-C12 diesters), acyl
sarcosinates, sulfates of alkylpolysaccharides such as the sulfates of
alkylpolyglucoside (the nonionic nonsulfated compounds being described below),
branched primary alkyl sulfates, and alkyl poiyethoxy carboxylates such as
those
of the formula RO(CH2CH20)k-CH2C00-M+ wherein R is a Cg-C22 alkyl, k is
~5 an integer from 1 to 10, and M is a soluble salt-forming cation. Resin
acids and
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.
I
2o and II by Schwartz, Perry and Berch). A variety of such 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
2s invention typically comprise from about 1 % to about 40%, preferably from
about
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
3o an unsubstituted C10-C24 alkyl or hydroxyalkyl group having a C1p-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 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.,
35 sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or
substituted-ammonium cation. Alkyl ethoxylated sulfates as well as alkyl
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43
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
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.
The 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 herein.
Cationic detersive surfactants suitable for use in the detergent compositions
of
the present invention are those having one long-chain hydrocarbyl group.
Examples of such cationic surfactants include the ammonium surfactants such as
alkyltrimethylammonium halogenides, and those surfactants having the formula
2o IR2(OR3)y)IR4(OR3)yJ2R5N+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(CHg)-, -CH2CH(CH20H)-, -CH2CH2CH2-, and mixtures
thereof; each R4 is selected from the group consisting of C1-C4 alkyl, C1-C4
hydroxyaikyl, benzyl ring structures formed by joining the two R4 groups, -
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
3o 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
anion.
Quaternary ammonium surfactant suitable for the present invention has the
formula (I):
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44
3,,~~'~4
O+ N,,,
~O 'R5 X-
Formula I
whereby R1 is a short chainlength alkyl (C6-C10) or alkylamidoalkyl of the
formula (II)
~~~~N~CH~,
II 2
O
Formula II
y is 2-4, preferably 3.
whereby R2 is H or a C1-C3 alkyl,
1o 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.
Rs
H
O~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
2o R1 is Cg, C1p or mixtures thereof, x=o,
R3, R4 = CHg and Rb = CH2CH20H.
Highly preferred cationic surfactants are the water-soluble quaternary
ammonium compounds useful in the present composition having the formula
R1 R2RgR4N+X- (i)
wherein R1 is Cg-C1g alkyl, each of R2, R3 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, R3 or R4 should be benzyl.
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The preferred alkyl chain length for R1 is C12-C15 particularly where the
alkyl
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 R2R3 and R4 are methyl and hydroxyethyl groups and the anion X
5 may be selected from halide, methosulphate, acetate and phosphate ions.
Examples of suitable quaternary ammonium compounds of formulae (i) for use
herein are
coconut trimethyl ammonium chloride or bromide;
coconut methyl dihydroxyethyl ammonium chloride or bromide;
o decyl triethyl ammonium chloride;
decyl dimethyl hydroxyethyl ammonium chloride or bromide;
C12-15 dimethyl hydroxyethyl ammonium chloride or bromide;
coconut dimethyl hydroxyethyl ammonium chloride or bromide;
myristyl trimethyl ammonium methyl sulphate;
~5 lauryl dimethyl benzyl ammonium chloride or bromide;
lauryl dimethyl (ethenoxy)4 ammonium chloride or bromide;
choline esters (compounds of formula (i) wherein R1 is
CH2-CH2-O-C-C12-14 alkyl and R2R3R4 are methyl).
II
20 O
di-alkyl imidazoiines [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
25 Application EP 000,224.
Typical cationic fabric softening components include the water-insoluble
quaternary-ammonium fabric softening actives or thei corresponding amine
precursor, the most commonly used having been di-long alkyl chain ammonium
30 chloride or methyl sulfate.
Preferred cationic softeners among these include the following:
1) ditallow dimethylammonium chloride (DTDMAC);
2) dihydrogenated tallow dimethylammonium chloride;
3) dihydrogenated tallow dimethylammonium methylsulfate;
35 4) distearyl dimethylammonium chloride;
5) dioleyl dimethylammonium chloride;
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46
6) dipaimityl 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) C12_1g alkyl dihydroxyethyl methylammonium chloride;
12) di(stearoyloxyethyl) dimethylammonium chloride (DSOEDMAC);
13) di(tallow-oxy-ethyl) dimethylammonium chloride;
14) ditallow imidazolinium methylsulfate;
0 15) 1-(2-tallowylamidoethyl)-2-tallowyl imidazolinium methylsulfate.
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
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
2o formula (I) or (II), below
R3 R2
_ + N-(CH,)n-CH -CHZ X
N (CH?~n Q~l' 1 X R 3 Q
T~ T?
or
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;
3o R3 is C1-C4 alkyl or C1-C4 hydroxyalkyl or H;
R4 is H or C1-C4 alkyl or C1-C4 hydroxyalkyl;
T1, T2, T3, T4, T5 are independently 011-022 alkyl or alkenyl;
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47
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.
s 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
branched. Tallow is a convenient and inexpensive source of long chain alkyl
and
alkenyl material. The compounds wherein T1, T2, T3, T4, T5 represents the
mixture of long chain materials typical for tallow are particularly preferred.
Specific examples of quaternary ammonium compounds suitable for use in the
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;
4) N,N-di(2-tallowyl-oxy-ethylcarbonyl-oxy-ethyl)-N,N-dimethyl ammonium
chloride;
5) N-(2-tallowyl-oxy-2-ethyl)-N-(2-tailowyl-oxy-2-oxo-ethyl)-N,N-dimethyl
ammonium chloride;
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.
When included therein, the 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.
Ampholytic surfactants are also suitable for use in the detergent compositions
of
the present invention. These surfactants can be broadly described as aliphatic
derivatives of secondary or tertiary amines, or aliphatic 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
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48
contains an anionic water-solubilizing group, e.g. carboxy, sulfonate,
sulfate. See
U.S. Patent No. 3,929,678 to Laughlin et al., issued December 30, 1975 at
column 19, lines 18-35, for examples of ampholytic surfactants.
When included therein, the 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 surfactants.
Zwitterionic surfactants are also suitable for use in 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 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 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.
Semi-polar nonionic surfactants are a special category of nonionic surfactants
2o 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
2s 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
from the group consisting of alkyl and hydroxyalkyl moieties of from about 1
to
about 3 carbon atoms.
3o Semi-polar nonionic detergent surfactants include the amine oxide
surfactants
having the formula
0
T
R3(OR4)xN(R5)2
3s wherein R3 is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixtures
therof
containing from about 8 to about 22 carbon atoms; R4 is an alkylene or
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49
hydroxyalkylene group containing from about 2 to about 3 carbon atoms or
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 C10-C1g alkyl dimethyl
amine oxides and Cg-C12 alkoxy ethyl dihydroxy ethyl amine oxides.
When included therein, the cleaning compositions of the present invention
o typically comprise from 0.2% to about 15%, preferably from about 1 % to
about
10% by weight of such semi-polar nonionic surfactants.
The detergent composition of the present invention may further comprise a
cosurfactant selected from the group of primary or tertiary amines.
~5 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
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.
2o 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 C8-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
R3 is either a Cg-C12, preferably Cg-C1p alkyl chain, or R3 is R4X(CH2)n,
3o 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 ; Rg alkyl chains may be interrupted with
up to 12, preferably less than 5, ethylene oxide moieties.
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Preferred tertiary amines are R1 R2R3N where R1 is a C6-C12 alkyl chain, R2
and R3 are C1-C3 alkyl or
Rs
-C CHZ-CH-O ~H
5 where R5 is H or CH3 and x = 1-2.
Also preferred are the amidoamines of the formula:
0
I I
Ri-C-NH-(CH2n N-(R2i
wherein R1 is Cg-C12 alkyl; n is 2-4,
o preferably n is 3; R2 and Rg is C1-C4
Most preferred amines of the present invention include 1-octylamine, 1-
hexylamine, 1-decylamine, 1-dodecyiamine,CB-l0oxypropylamine, N coco 1-
3diaminopropane, coconutalkyldimethylamine, lauryldimethylamine, lauryl
15 bis(hydroxyethyl)amine, coco bis(hydroxyehtyl)amine, lauryl amine 2 moles
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,
20 1-octylamine, 1-decylamine, 1-dodecylamine. Especially desirable are n
dodecyldimethylamine and bishydroxyethylcoconutalkylamine and oleylamine 7
times ethoxylated, lauryl amido propylamine and cocoamido propylamine.
25 Conventional detergent enzymes
The detergent compositions can in addition to mannanase enzyme and
percarbonate further comprise one or more enzymes which provide cleaning
performance, fabric care and/or sanitisation benefits. Preferably, the
detergent
3o composition of the present invention will comprise a protease. It has been
surprinsingly found that the compositions of the present invention further
comprising a protease enzyme, provide better whitening and stain removal
benefits.
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51
Suitable proteases are the proteases from the IUPAC classification EC 3.4.-.-,
preferably the Endo-serine protease from the IUPAC classification EC 3.4.21.-,
more preferably the Subtilisin proteases from the IUPAC classification EC
3.4.21.62. These EC 3.4.21.62 proteases are the subtilisins which are obtained
from particular strains of 8. subtilis and B. 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
o 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~, PROPERASE~
and MAXAPEM~ (protein engineered Maxacal) from Gist-Brocades. Proteolytic
enzymes also encompass modified bacterial serine proteases, such as those
~5 described in European Patent Application Seria! 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",
2o which is a variant of an 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
25 C, are also included herein.
A preferred protease referred to as "Protease D" is a carbonyl hydroiase
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 W095/10591 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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52
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
following residues : +33, +62, +67, +76, +100, +101, +103, +104, +107, +128,
+129, +130, +132, +135, +156, +158, +164, +166, +167, +170, +2pg, +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
o 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
92/03529 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.
Said enzymes include enzymes selected from celluiases, hemicellulases,
peroxidases, gluco-amylases, amylases, xylanases, lipases, phospholipases,
3o esterases, cutinases, pectinases, keratanases, reductases, oxidases,
phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases,
pentosanases, malanases, f3-glucanases, arabinosidases, hyaluronidase,
chondroitinase, laccase or mixtures thereof.
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A preferred combination is a 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.
The cellulases usable in the present invention include both bacterial or
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
cellulases
are disclosed in U.S. Patent 4,435,307, Barbesgoard et al, J61078384 and
W096/02653 which discloses fungal cellulase produced respectively from
o Humicola insolens, Trichoderma, Thielavia and Sporotrichum. EP 739 982
describes cellulases isolated from novel Bacillus species. Suitable cellulases
are
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
~5 insolens (Humicola grisea var. thermoidea), particularly the Humicola
strain DSM
1800.
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
20 1800, exhibiting cellulase activity; a preferred endoglucanase 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.
25 Examples of such cellulases are cellulases described in European patent
application No. 91202879.2, filed November 6, 1991 (Novo). Carezyme and
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.
3o Said cellulases are normally incorporated in the detergent composition at
levels
from 0.0001 % to 2% of pure enzyme by weight of the detergent composition.
Peroxidase enzymes are used in combination with oxygen sources, e.g.
percarbonate, perborate, persulfate, hydrogen peroxide, etc and with a
phenolic
35 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
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54
operations to other substrates in the wash solution. Peroxidase enzymes are
known in the art, and include, for example, horseradish peroxidase, ligninase
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. 96870013.8, filed
February 20, 1996. Also suitable is the laccase enzyme.
Enhancers are generally comprised at a level of from 0.1 % to 5% by weight of
total 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.
~5 Said peroxidases are normally incorporated in the detergent composition at
levels from 0.0001 % to 2% of pure enzyme by weight of the detergent
composition.
Other preferred enzymes that can be included in the detergent compositions of
2o the present invention include lipases. Suitable lipase enzymes for
detergent
usage include those produced by microorganisms of the Pseudomonas group,
such as Pseudomonas 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
25 Pseudomonas fluorescent lAM 1057. This lipase is available from Amano
Pharmaceutical Co. Ltd., Nagoya, Japan, under the trade name Lipase P
"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.,
3o Tagata, Japan; Chromobacter viscosum lipases from U.S. Biochemical Corp.,
U.S.A. and Disoynth Co., The Netherlands, and lipases ex Pseudomonas
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
3s present invention. Also suitables are the lipolytic enzymes described in EP
258
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068, WO 92/05249 and WO 95/22615 by Novo Nordisk and in WO 94/03578,
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
5 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
and WO 94/14964 {Uniiever).
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.
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
detergent compositions which incorporate mutant amylases. See also
15 W095/10603, Novo Nordisk A/S, published April 20, 1995. Other amylases
known for use in detergent compositions include both a- and ~-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
2o amylases are stability-enhanced amylases described in W094/18314, published
August 18, 1994 and W096/05295, Genencor, published February 22, 1996 and
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
2s (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
3o specific activity of Termamyl~ at a temperature range of 25°C to
55°C and at a
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
35 level are described in W095/35382.
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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.
The above-mentioned enzymes may be of any suitable origin, such as 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
o 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 detergent 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
~5 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
2o 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
enzymes may be further enhanced by the creation of e.g. additional salt
bridges
25 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.
3o 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 (prills, granulates,
stabilized liquids, etc., containing one enzyme ) or as mixtures of two or
more
enzymes (e.g. cogranulates ).
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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, Jufy 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, 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.
2o 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 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
(PCT/US97/16546) 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 detergent compositions
in
accordance with the present invention. These agents may be inorganic or
organic in type. Inorganic softening agents are exemplified by the 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
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58
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.
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
~o 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
normally added to the spray dried portion of the composition, although in some
~5 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.
Bleaching agent
2o In addition to the percarbonate, the composition of the present invention,
may
comprise optionally other bleaching agents such as bleach activators,
photoactivated bleach, enzyme generating bleach species and bleach catalysts.
Alternative source of available oxygen encompasses percarboxylic acid
2s 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 are disclosed in U.S. Patent
4,483,781, U.S. Patent Application 740,446, European Patent Application
30 0,133,354 and U.S. Patent 4,412,934. Highly preferred bleaching agents also
include 6-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
3s example, include trichloro isocyanuric acid and the sodium and potassium
dichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides. Such
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59
materials are normally added at 0.5-10% by weight of the finished product,
preferably 1-5% by weight.
The hydrogen peroxide releasing agents can be used in combination with bleach
activators such as tetraacetylethylenediamine (TAED), nonanoyloxybenzene-
sulfonate (NOBS, 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-
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
disclosed in the Procter & Gamble co-pending patent applications US serial No.
~5 60/022,786 {filed July 30, 1996) and No. 60/028,122 (filed October 15,
1996)
O O
R;
N R3
R2
wherein R1 is a C7-C13 linear or branched chain saturated or unsaturated alkyl
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
2o group.
Useful bleaching agents, including peroxyacids and bleaching systems
comprising bleach activators and peroxygen bleaching compounds for use in
detergent compositions according to the invention are described in our co-
25 pending applications USSN 08/136,626, PCT/US95/07823, W095/27772,
W095127773, W095/27774 and W095/27775.
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
3o peroxide at the beginning or during the washing andlor rinsing process.
Such
enzymatic systems are disclosed in EP Patent Application 91202655.6 filed
October 9, 1991.
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Metal-containing catalysts for use in bleach compositions, include cobalt-
containing catalysts such as Pentaamine acetate cobalt(III) salts and
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 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 94870206.3.
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 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
The compositions according to the present invention may further comprise a
2s builder system. Any conventional builder system is suitable for use herein
including 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
3o phosphonic acid and diethylene triamine pentamethylenephosphonic acid.
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
35 synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.
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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,
gfycolic acid and ether derivatives thereof as disclosed in Belgian Patent
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 the sulfinyl
carboxylates described in Belgian Patent No. 840,623. Polycarboxylates
containing three carboxy groups include, in particular, water-soluble
citrates,
aconitrates and citraconates as well as succinate derivatives such as the
carboxymethyloxysuccinates described in British Patent No. 1,379,241,
~5 lactoxysuccinates described in Netherlands Application 7205873, and 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
2o disclosed in British Patent No. 1,261,829, 1,1,2,2-ethane
tetracarboxylates,
1,1,3,3-propane tetracarboxylates and 1,1,2,3-propane tetracarboxylates.
Polycarboxylates containing sulfo substituents include the sulfosuccinate
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
25 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
30 - 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.
35 Of the above, the preferred polycarboxylates are hydroxycarboxylates
containing
up to three carboxy groups per molecule, more particularly citrates.
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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
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.
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 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 maieic anhydride,
such copolymers having a molecular weight of from 20,000 to 70,000, especially
2o about 40,000.
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.
Chelating Agents
The detergent compositions herein may also optionally contain one or more iron
3o and/or manganese chelating agents. Such chelating agents 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.
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Amino carboxylates useful as optional chelating agents include
ethylenediaminetetracetates, N-hydroxyethylethyienediaminetriacetates, nitrilo-
triacetates, ethylenediamine tetraproprionates, triethylenetetraamine-
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. Preferred, these amino phosphonates
do not contain alkyl or alkenyl groups with more than about 6 carbon atoms.
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.
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 %
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.
3o 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
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64
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.
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-
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
~5 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.
The suds suppressors 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 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-
3o 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
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are,preferabiy, 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
5 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 hydroxyethyl-
cellulose, and homo- or co-polymeric poiycarboxylic acids or their salts.
Polymers
of this type include the polyacrylates and malefic anhydride-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
~5 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
2o disodium 4,4'-bis-(2-diethanolamino-4.-anilino -s- triazin-6-
ylamino)stilbene-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-
25 2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2' - disuiphonate, di-
sodium
4,4' -bis-(4-phenyl-2,1,3-triazol-2-yl)-stilbene-2,2' disulphonate, di-so-dium
4,4'bis(2-anilino-4-(1-methyl-2-hydroxyethylamino)-s-triazin-6- ylami-
no)stiibene-
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
3o the specific brighteners disclosed in EP 753 567.
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
s5 preferably from 0.25% to 2.5% by weight. These polymers and the previously
mentioned homo- or co-polymeric poiycarboxylate salts are valuable for
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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
(CH3(PEG)43)0.75(POH)0.25IT-PO)2.8(T-PEG)0.41T(PO
H)0.25((PEG)43CH3)0.75
~5 where PEG is -(OC2H4)O-,PO is (OC3H60) and T is (pcOC6H4C0).
Also very useful are modified polyesters as random copolymers of dimethyl
terephthalate, dimethyl suifoisophthalate, ethylene glycol and 1-2 propane
diol,
the end groups consisting primarily of sulphobenzoate and secondarily of mono
2o 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 glycol and/or propane 1-2 diol,
25 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
3o 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
35 scavenger such as perborate, ammonium sulfate, sodium sulphite or
polyethyleneimine at a level above 0.1 % by weight of total composition, in
the
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67
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 a~crylate units. The side-chains are of
the
1o formula -(CH2CH20)m(CH2)nCH3 wherein m is 2-3 and n 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 polycarboxylates can
comprise from about 0.05% to about 10%, by weight, of the compositions herein.
Dispersants
The detergent compositions of the present invention can also contain
dispersants
Suitable water-soluble organic salts are the homo- or co-polymeric acids or
their
2o 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 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 detergent compositions of the present invention.
The compositions of the invention may contain a lime soap peptiser compound,
3o 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
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68
dispersant test as described in an article by H.C. Borghetty and C.A. 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 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.
~5 Exemplary surfactants having a LSDP of no more than 8 for use in accord
with
the present invention include C16-C1g dimethyl amine oxide, C12-C1g alkyl
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
2o 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
25 104, pages 71-73, (1989).
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
3o benzene sulfonate together with hydrophilic / hydrophobic bleach
formulations
can also be used as lime soap peptisers compounds.
Dye transfer inhibition
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The detergent compositions of the present invention can also include compounds
for inhibiting dye transfer from one fabric to another of solubilized and
suspended
dyes encountered during fabric laundering operations involving colored
fabrics.
s Polymeric dye transfer inhibiting agents
The detergent compositions according to the present invention also comprise
from 0.001 % to 10 %, preferably from 0.01 % to 2%, more preferably from 0.05%
to 1 % by weight of polymeric dye transfer inhibiting agents. Said polymeric
dye
transfer inhibiting agents are normally incorporated into 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.
~5 Especially suitable polymeric dye transfer inhibiting agents are polyamine
N-oxide
polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole,
polyvinylpyrrolidone polymers, polyvinyloxazolidones and polyvinylimidazoles
or
mixtures thereof.
Addition of such polymers also enhances the performance of the enzymes
2o according the invention.
a) Polyamine N-oxide polymers
The poiyamine N-oxide polymers suitable for use contain units having the
following structure formula
25 P
I
(I) Ax
R
3o wherein P is a polymerisable unit, whereto the R-N-O group can be attached
to or
wherein the R-N-O group forms part of the polymerisable unit or a combination
of
both.
O O O
35 II II II
A is NC, CO, C, -O-,-S-, -N- ; x is O or 1;
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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.
5 The N-O group can be represented by the following general structures
O O
I I
(R1 )x -N- (R2)y =N- (R1 )x
10 I
(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 wherein the
~5 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.
2o 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 class 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.
25 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 class 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 class of these polyamine N-oxides are the polyamine N-oxides having
the general formula (I) wherein R is an aromatic, heterocyclic or alicyclic
groups
wherein the nitrogen of the N-0 functional group is part of said R group.
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Examples of these classes are polyamine oxides wherein R is a heterocyclic
compound such as pyrridine, pyrrole, imidazole and derivatives thereof.
Another preferred class of polyamine N-oxides are the polyamine oxides having
the general formula (I) wherein R are aromatic, heterocyclic or alicyclic
groups
wherein the nitrogen of the N-0 functional group is attached to said R groups.
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 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 of
~5 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
2o 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.
25 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-vinylpyrrolidone and N-vinylimidazole
The N-vinylimidazole N-vinylpyrrolidone polymers used in the present invention
have an average molecular weight range from 5,000-1,000,000, preferably from
5,000-200,000.
Highly preferred polymers for use in detergent compositions according to the
present invention comprise a polymer selected from N-vinylimidazole N-
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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.
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 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
2o 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
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
3o include Sokalan HP 165 and Sokalan HP 12; polyvinylpyrrolidones 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
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polyvinyloxazoiidones 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
from about 5,000 to about 50,000, and most preferably from about 5,000 to
about
15, 000.
e) Polyvinylimidazole
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
~5 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
2o 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 are
described in the co-pending patent application 94870213.9
Method of washins~
The compositions of the invention may be used in essentially any washing or
cleaning methods, including soaking methods, pretreatment methods and
3o methods with rinsing steps for which a separate rinse aid composition may
be
added.
The process described herein comprises contacting fabrics or dishware with a
cleaning solution in the usual manner and exemplified hereunder.
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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
preferably from 7 to 12.
A preferred machine dishwashing method comprises treating soiled articles with
an aqueous liquid having dissolved or dispensed therein an effective amount of
the machine diswashing or rinsing composition. A conventional effective amount
of the machine dishwashing composition means from 8-60 g of product dissolved
or dispersed in a wash volume from 3-10 litres. According to a manual
dishwashing method, soiled dishes are contacted with an effective amount of
the
diswashing composition, typically from 0.5-20g (per 25 dishes being treated).
Preferred manual dishwashing methods include the application of a concentrated
solution to the surfaces of the dishes or the soaking in large volume of
dilute
~5 solution of the detergent composition.
The following examples are meant to exemplify compositions of the 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
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 suiphonate.
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.
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(C2H4OH) with R2 = Cg-C11.
APA : Cg_1 p amido propyl dimethyl amine.
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Soap : Sodium linear alkyl carboxylate derived from
a 80/20
mixture of tallow and coconut fatty acids.
Nonionic : C13-C15 mixed ethoxylatedlpropoxylated 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.
STS : Sodium toluene sulphonate.
CFAA : C12-C14 alkyl N-methyl glucamide.
TFAA : C16-C1g alkyl N-methyl glucamide.
TPKFA : C12-C14 topped whole cut fatty acids.
Silicate : Amorphous Sodium Silicate (Si02:Na20 ratio
= 1.6-3.2).
Metasilicate : Sodium metasilicate (Si02:Na20 ratio = 1.0).
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 dehydrate 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.
MA/AA 1 : Random copolymer of 6:4 acrylate/maleate,
average
molecular weight about 10,000.
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AA : Sodium polyacrylate polymer of average molecular
weight 4,500.
PA30 : Polyacrylic acid of average molecular weight
of between
about 4,500 - 8,000.
480N : Random copolymer of 7:3 acrylate/methacrylate,
average molecular weight about 3,500.
Polygel/carbopol: High molecular weight crosslinked polyacrylates.
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.
NOES : 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
MnTACN : Manganese 1,4,7-trimethyl-1,4,7-triazacyclononane.
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(III) salt.
Paraffin : Paraffin oil sold under the tradename Winog
70 by
Wintershall.
NaBz : Sodium benzoate.
BzP : Benzoyl Peroxide.
Mannanase : Mannanase from Bacillus agaradherens, NCIMB
40482
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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 carboxymethyl cellulose.
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 vinyipyrrolidone, 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% Siliconelsilica, 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.
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QEA : bis((C2H50)(C2H4~)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.
SCS : Sodium cumene sulphonate.
HMWPEO : High molecular weight polyethylene oxide.
PEGx : Polyethylene glycol, of a molecular weight of x .
PEO : Polyethylene oxide, with an average molecular weight of
5,000.
TEPAE : Tetreaethylenepentaamine ethoxylate.
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79
BTA : Benzotriazole.
pH : Measured as a 1% solution in distilled water at 20°C.
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 5.0 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
Mannanase 0.001 0.002 0.05 0.001 0.002 0.003
Percarbonate 9.0 9.0 5.0 5.0 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
EDDS - 0.25 0.4 - 0.5 0.5
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CFAA - 1.0 - 2.0 - -
HEDP 0.3 0.3 0.3 0.3 0.4 0.4
QEA - - - 0.2 - 0.5
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/litre
Miscellaneous Up to 100%
and minors
Exam~~fe 2
s 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 - _ _ _ _
Zeolite A - 19.5 20.0 14.5 20.0 17.0
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II ~l~ ~V V V~
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
Mannanase 0.001 0.002 0.02 0.001 0.002 0.02
Percarbonate 5.0 12.7 5.0 5.0 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 -
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
Density in g/litre750 750 750 750 750 750
Miscellaneous Up to 100%
and minors
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Example 3
The following detergentcompositionsof particularutilityunder European
machine wash conditions according
were prepared to the
present
invention
I II III IV
Blown Powder
LAS 6.0 5.0 11.0 6.0
TAS 2.0 - - 2.0
Zeolite A 24.0 - - 20.0
STPP - 27.0 24.0 -
Sulfate 4.0 6.0 13.0 -
MA/AA 1.0 4.0 6.0 2.0
Silicate 1.0 7.0 3.0 3.0
CMC 1.0 1.0 0.5 0.6
Brightener 1 0.2 0.2 0.2 0.2
Silicone antifoam 1.0 1.0 1.0 0.3
DETPMP 0.4 0.4 0.2 0.4
Spray On
Brightener 0.02 - - 0.02
C45E7 - - - 5.0
C45E2 2.5 2.5 2.0 -
C45E3 2.6 2.5 2.0 -
Perfume 0.5 0.3 0.5 0.2
Silicone antifoam 0.3 0.3 0.3 -
Dry additives
Q EA - - - 1.0
EDDS 0.3 - - -
Sulfate 2.0 3.0 5.0 10.0
Carbonate 6.0 13.0 15.0 14.0
Citric 2.5 - - 2.0
QAS 1 0.5 - - 0.5
Na-SKS-6 10.0 - - -
Percarbonate 18.5 18.0 10.0 21.5
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I II III IV
Mannanase 0.001 0.002 0.02 0.02
TAED 2.0 2.0 - 2.0
NACA-OBS 3.0 2.0 4.0 -
Protease 0.03 0.03 0.03 0.03
Lipase 0.008 0.008 0.008 0.004
Amylase 0.003 0.003 0.003 0.006
Brightener 1 0.05 - - 0.05
Miscellaneous and Up to 100%
minors
Example 4
The following granular detergent compositions were prepared according to the
present invention
I II III IV V VI
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
MAIAA - 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
Spray On
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I II III IV V VI
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 -
Mannanase 0.001 0.02 0.001 0.02 0.001 0.01
Percarbonate 4.0 1.0 3.0 2.0 14.0 11.0
Carbonate - 5.3 1.8 - 4.0 4.0
NOES 4.0 - 6.0 - - 0.6
Methyl cellulose0.2 - - - - -
Na-SKS-G 8.0 - - - - -
STS - - 2.0 - 1.0 -
Culmene sulfonic- 1.0 - - - 2.0
acid
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 -
Silicone antifoam0.2 0.4 0.2 0.4 0.1 -
Mg sulfate - - 0.2 - 0.2 -
Miscellaneous Up to
and minors 100%
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Example 5
The following detergent compositions were prepared according to the present
5 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 -
Silicate - 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 -
Dry additives
Carbonate 5.0 10.0 18.0 8.0
PVPVI/PVNO 0.5 - 0.3 -
Mannanase 0.001 0.001 0.02 0.02
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
NOES - 4.0 - 4.5
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1 II III IV
Percarbonate 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 6
The following granular detergent compositions were prepared according to the
present invention
Blown Powder
Zeolite A 20.0 - 15.0
STPP - 20.0 -
Sulfate - - 5.0
Carbonate - - 5.0
TAS - - 1.0
LAS 6.0 6.0 6.0
C68AS 2.0 2.0 -
Silicate 3.0 8.0 -
MA/AA 4.0 2.0 2.0
CMC 0.6 0.6 0.2
Brightener 1 0.2 0.2 0.1
DETPMP 0.4 0.4 0.1
STS - - 1.0
Spray On
C45E7 5.0 5.0 4.0
Silicone antifoam 0.3 0.3 0.1
Perfume 0.2 0.2 0.3
Dry additives
QEA - - 1.0
Carbonate 14.0 9.0 10.0
Percarbonate 20.0 15.0 13.0
TAED 2.0 2.0 2.0
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I II III
QAS - - 1.0
Photoactivated bleach 15 ppm 9 5 ppm 15 ppm
Na-SKS-6 - - 3.0
Mannanase 0.001 0.02 0.0015
Protease 0.03 0.03 0.007
Lipase 0.004 0.004 0.004
Amylase 0.006 0.006 0.003
Cellulase 0.0002 0.0002 0.0005
Sulfate 10.0 20.0 5.0
Density (g/litre) 700 700 700
Miscellaneous and minors Up to 100%
Example 7
The following detergent compositions to the present
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
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
Pertume 0.3 0.3 0.3
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I II III
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 fi.0 2.0 5.0
Percarbonate 14.0 7.0 10.0
PEO - - 0.2
Bentonite - - 10.0
clay
Mannanase 0.001 0.02 0.01
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
Sil icone antifoam5.0 5.0 5.0
Sulfate - 3.0 -
Density (g/litre) 850 850 850
Miscellaneousand minors Up to
100%
Example 8
The followingdetergent prepared to the present
compositions according
were
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 28.0 20.0
Silicate 9.0 5.0 9.0 8.0
Carbonate 11.0 7.5 10.0 5.0
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I II III IV
Bicarbonate - 7.5 - -
Percarbonate 3.0 2.0 2.0 2.0
NOES 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 0.001 0.001 0.02 0.03
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 bleach30 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 up to
minors 100%
Examale 9
s The following granular fabric detergent compositions which provide
"softening
through the wash" capability were prepared according to the present invention
I II
C45AS - 10.0
LAS 7.6 -
C68AS 1.3 -
C45E7 4.0 -
C25E3 - 5.p
Coco-alkyl-dimethyl hydroxy-1.4 1.0
ethyl ammonium chloride
Citrate 5.0 3.0
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I II
Na-SKS-6 - 11.0
Zeolite A 15.0 15.0
MA/AA 4.0 4.0
DETPMP 0.4 0.4
Percarbonate 15.0 15.0
TAED 5.0 5.0
Smectite clay 10.0 10.0
HMWPEO - 0.1
Mannanase 0.001 0.02
Protease 0.02 0.01
Lipase 0.02 0.01
Amylase 0.03 0.005
Cellulase 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 minors Up to 100%
Example 10
5 The following detergent additive compositions were prepared according to the
present invention
I II
~S - 5.0
STPP 30.0 -
Zeolite A - 35.0
Percarbonate 20.0 15.0
TAED 10.0 8.0
Mannanase 0.001 0.02
P rotease - 0. 3
Amylase - 0.06
Minors, water and miscellaneous Up to 100%
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Examiole 11
The following compact high density (0.96Kg/I) dishwashing detergent
compositions were prepared according to the present invention
I II 111 IV V VI VII VIII
STPP - - 54.3 51.4 51.4 - - 50.9
Citrate 35.0 17.0 - - - 46.1 40.2 -
Carbonate - 17.5 14.0 14.0 14.0 - 8.0 32.1
Bicarbonate - - - - - 25.4 - -
Silicate 32.0 14.8 14.8 10.0 10.0 1.0 25.0 3.1
Metasilicate- 2.5 - 9.0 9.0 - - -
Percarbonate10.5 9.7 7.8 7.8 7.8 6.7 11.8 4.8
Nonionic 1.5 2.0 1.5 1.7 1.5 2.6 1.9 5.3
TAED 5.2 2.4 - - - 2.2 - 1.4
HEDP - 1.0 - - - - - _
DETPMP - 0.6 - - - -
MnTACN - - - - - - 0.008 -
PAAC - - 0.008 0.01 0.007 - - -
BzP _ _ - - 1.4 - _ _
Paraffin 0.5 0.5 0.5 0.5 0.5 0.6 - -
Mannanase 0.001 0.02 0.015 0.02 0.001 0.001 0.02 0.02
Protease 0.072 0.072 0.029 0.05 0.046 0.026 0.059 0.06
3
Amylase 0.012 0.012 0.006 0.01 0.013 0.009 0.017 0.03
2
Lipase - 0.001 - 0.00 - - - -
5
BTA 0.3 0.3 0.3 0.3 0.3 - 0.3 0.3
MA/AA - _ _ - _ _ 4.2 -
480N 3.3 6.0 - - - - - p,g
Perfume 0.2 0.2 0.2 0.2 0.2 0.2 0.1 0.1
Sulphate 7.0 20.0 5.0 2.2 0.8 12.0 4.6 -
pH 10.8 11.0 10.8 11.3 11.3 9.6 10.8 10.9
Miscellaneous Up to 100%
and water
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Examele 12
The following granular dishwashing detergent compositions of bulk density
1.02KgIL were prepared according to the present invention
I II III IV V
STPP 30.0 30.0 33.0 34.2 31.1
Carbonate 30.5 30.5 31.0 30.0 39.4
Silicate 7.4 7.4 7.5 7.2 3.4
Metasilicate - - 4.5 5.1 -
Percarbonate 4.4 4.2 4.5 4.5 4.0
NADCC - - - - -
Nonionic 1.2 1.0 0.7 0.8 0.7
TAED ~ 1.0 - - - 0.8
PAAC - 0.004 0.004 0.004 -
BzP - - - 1.4 -
Paraffin 0.25 0.25 0.25 0.25 -
Mannanase 0.01 0.001 0.02 0.001 0.001
Protease 0.036 0.015 0.03 0.028 0.03
Amylase 0.003 0.003 0.01 0.006 0.01
Lipase 0.005 - 0.001 - -
BTA 0.15 0.15 0.15 0.15 -
Perfume 0.2 0.2 0.2 0.2 0.2
Sulphate 23.4 25.0 22.0 18.5 19.3
pH 10.8 10.8 11.3 11.3 11.5
Miscellaneous Up to
and water 100%
Example 13
o The following tablet detergent compositions were prepared according to the
present invention by compression of a granular dishwashing detergent
composition at a pressure of 13KN/cm2 using a standard 12 head rotary press:
I II III IV V VI
STPP - 48.8 49.2 38.0 - 46.8
Citrate 26.4 - - - 31.1 -
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I II III IV V VI
Carbonate - 5.0 14.0 15.4 14.4 23.0
Silicate 26.4 14.8 15.0 12.6 17.7 2.4
Mannanase 0.001 0.02 0.001 0.002 0.03 0.002
Protease 0.058 0.072 0.041 0.033 0.052 0.013
Amylase 0.01 0.03 0.012 0.007 0.016 0.002
Lipase 0.005 - - - - -
Percarbonate 8.5 7.7 12.2 10.6 15.7 14.4
Nonionic 1.5 2.0 1.5 1.65 0.8 6.3
PAAC - - 0.02 0.009 - -
MnTACN - - - - 0.007 -
TAED 4.3 2.5 - - 1.3 1.8
HEDP 0.7 - - 0.7 - 0.4
DETPMP 0.65 - - - - -
Paraffin 0.4 0.5 0.5 0.55 - -
BTA 0.2 0.3 0.3 0.3 - -
PA30 3.2 - - - - -
M~AA - - - - 4.5 0.55
Perfume - - 0.05 0.05 0.2 0.2
Sulphate 24.0 13.0 2.3 - 10.7 3.4
Weight of tablet25g 25g 20g 30g 18g 20g
pH 10.6 10.6 10.7 10.7 10.9 11.2
Miscellaneous water Up to 100%
and
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
APPLICANT:
NAME: The Procter ~ Gamble Company
STREET: One Procter & Gamble Plaza
CITY: Cincinnati, OHIO
COUNTRY: USA
POSTAL CODE: 45202
TITLE OF INVENTION: Detergent compositions comprising a mannanase and
percarbonate
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
5 GAGAGGTATTAACCATGGACATGCTTGGTATAAAGACACCGCTTCAACAGCT
ATTCCTGCCATTGCAGAGCAAGGCGCCAACACGATTCGTATTGTTTTATCAG
ATGGCGGTCAATGGGAAAAAGACGACATTGACACCATTCGTGAAGTCATTG
AGCTTGCGGAGCAAAATAAAATGGTGGCTGTCGTTGAAGTTCATGATGCCA
CGGGTCGCGATTCGCGCAGTGATTTAAATCGAGCCGTTGATTATTGGATAG
o AAATGAAAGATGCGCTTATCGGTAAAGAAGATACGGTTATTATTAACATTGCA
AACGAGTGGTATGGGAGTTGGGATGGCTCAGCTTGGGCCGATGGCTATATT
GATGTCATTCCGAAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTG
ATGCAGCAGGATGGGGGCAATATCCGCAATCTATTCATGATTACGGACAAG
ATGTGTTTAATGCAGATCCGTTAAAAAATACGATGTTCTCCATCCATATGTAT
15 GAGTATGCTGGTGGTGATGCTAACACTGTTAGATCAAATATTGATAGAGTCA
TAGATCAAGACCTTGCTCTCGTAATAGGTGAATTCGGTCATAGACATACTGA
TGGTGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACA
GGGTGGCTCGCTTGGTCTTGGAAAGGCAACAGTACCGAATGGGACTATTTA
GACCTTTCAGAAGACTGGGCTGGTCAACATTTAACTGATTGGGGGAATAGAA
2o TTGTCCACGGGGCCGATGGCTTACAGGAAACCTCCAAACCATCCACCGTAT
TTACAGATGATAACGGTGGTCACCCTGAACCGCCAACTGCTACTACCTTGTA
TGACTTTGAAGGAAGCACACAAGGGTGGCATGGAAGCAACGTGACCGGTG
GCCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAAGC
CGATGTAAATTTAACCTCAAATTCTTCACATGAACTGTATAGTGAACAAAGTC
25 GTAATCTACACGGATACTCTCAGCTCAACGCAACCGTTCGCCATGCCAATTG
GGGAAATCCCGGTAATGGCATGAATGCAAGACTTTACGTGAAAACGGGCTC
TGATTATACATGGCATAGCGGTCCTTTTACACGTATCAATAGCTCCAACTCA
GGAACAACGTTATCTTTTGATTTAAACAACATCGAAAATAGTCATCATGTTAG
GGAAATAGGCGTGCAATTTTCAGCGGCAGATAATAGCAGTGGTCAAACTGC
30 TCTATACGTTGATAACGTTACTTTAAGATAG
SEQ ID N0:2
35 SEQUENCE CHARACTERISITICS:
LENGTH: 493 amino acids
CA 02301156 2000-02-11
WO 99109130 PCT/US98/12023
96
TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: protein
SEQUENCE DESCRIPTION: SEQ ID NO: 2
MKKKLSQIYHLIICTLIISVGIMGITTSPSAASTGFYVDGNTLYDANGQPFVMRGIN
HGHAWYKDTASTAIPAIAEQGANTIRIVLSDGGQWEKDDIDTIREVIELAEQNKM
VAWEVHDATGRDSRSDLNRAVDYWIEMKDALIGKEDTVI INIANEWYGSWDGS
~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
TTTTATGTTGATGG CAATACGTTATATGAC G CAAATG GG CAG C CATTTGTCAT
GAGAGGTATTAACCATGGACATGCTTGGTATAAAGACACCGCTTCAACAGCT
ATTCCTGCCATTGCAGAGCAAGGCGCCAACACGATTCGTATTGTTTTATCAG
ATGGCGGTCAATGGGAAAAAGACGACATTGACACCATTCGTGAAGTCATTG
CA 02301156 2000-02-11
WO 99/09130 PCT/US98/12023
97
AGCTTGCGGAGCAAAATAAAATGGTGGCTGTCGTTGAAGTTCATGATGCCA
CGGGTCGCGATTCGCGCAGTGATT'TAAATCGAGCCGTTGATTATTGGATAG
AAATGAAAGATGCGCT'TATCGGTAAAGAAGATACGGTTATTATTAACATTGCA
AACGAGTGGTATGGGAGT'TGGGATGGCTCAGCTTGGGCCGATGGCTATATT
GATGTCATTCCGAAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTG
ATGCAGCAGGATGGGGGCAATATCCGCAATCTATTCATGATTACGGACAAG
ATGTGTTTAATGCAGATCCGTTAAAAAATACGATGTTCTCCATCCATATGTAT
GAGTATGCTGGTGGTGATGCTAACACTGTTAGATCAAATATTGATAGAGTCA
TAGATCAAGACCTTGCTCTCGTAATAGGTGAATTCGGTCATAGACATACTGA
o TGGTGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACA
GGGTGGCTCGCTTGGTCTTGGAAAGGCAACAGTACCGAATGGGACTATTTA
GACCTTTCAGAAGACTGGGCTGGTCAACATTTAACTGATTGGGGGAATAGAA
TTGTCCACGGGGCCGATGGCTTACAGGAAACCTCCAAACCATCCACCGTAT
TTACAGATGATAACGGTGGTCACCCTGAACCGCCAACTGCTACTACCTTGTA
TGACTTTGAAGGAAGCACACAAGGGTGGCATGGAAGCAACGTGACCGGTG
GCCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAAGC
CGATGTAAATTTAACCTCAAATTCTTCACATGAACTGTATAGTGAACAAAGTC
GTAATCTACACGGATACTCTCAGCTCAACGCAACCGTTCGCCATGCCAATTG
GGGAAATCCCGGTAATGGCATGAATGCAAGACTTTACGTGAAAACGGGCTC
TGATTATACATGGCATAGCGGTCCTTTTACACGTATCAATAGCTCCAACTCA
GGAACAACGTTATCTT'TTGATTTAAACAACATCGAAAATATCATCATGTTAGG
GAAATAG
SEQ ID N0:4
SEQUENCE CHARACTERISITICS:
LENGTH: 468 amino acids
TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: protein
SEQUENCE DESCRIPTION: SEQ ID NO: 4
MKKKLSQIYHLIICTLIISVGIMGIT'TSPSAASTGFYVDGNTLYDANGQPFVMRGIN
HGHAWYKDTASTAIPAIAEQGANTIRIVLSDGGQWEKDDIDTIREVIELAEQNKM
CA 02301156 2000-02-11
WO 99/09130 PCT/US98/12023
98
VAWEVHDATGRDSRSDLNRAVDYWIEMKDALIGKEDTVIINIANEWYGSWDGS
AWADGYIDVIPKLRDAGLTHTLMVDAAGWGQYPQSIHDYGQDVFNADPLKNTM
FSIHMYEYAGGDANTVRSNIDRVIDQDLALVIGEFGHRHTDGDVDEDTILSYSEE
TGTGWLAWSWKGNSTEWDYLDLSEDWAGQHLTDWGNRIVHGADGLQETSKP
STVFTDDNGGHPEPPTATTLYDFEGSTQGWHGSNVTGGPWSVTEWGASGNY
SLKADVNLTSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLYV
KTGSDYTWHSGPFTRINSSNSGTTLSFDLNNIENIIMLGK
1o SEQ ID N0:5
SEQUENCE CHARACTERISITICS:
LENGTH: 1029 base pairs
TYPE: nucleic acid
~5 STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: genomic DNA
20 SEQUENCE DESCRIPTION SEQ ID No:S
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 02301156 2000-02-11
WO 99/09130 PCT/US98/12023
99
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
~5 TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: protein
2o SEQUENCE DESCRIPTION: SEQ ID NO: 6
ydhT 1
LFKKHTISLLIIFLLASAVLAKPIEAHTVSPVNPNAQQTTKTVMNWLAHL 50
ydhT 51
25 PNRTENRVLSGAFGGYSHDTFSMAEADRIRSATGQSPAIYGCDYARGWLE 100
ydhT 101
TANIEDS1DVSCNGDLMSYWKNGGIPQISLHLANPAFQSGHFKTPITNDQ 150
ydhT 151
YKN1LDSATAEGKRLNAMLSKIADGLQELENQGVPVLFRPLHEMNGEWFW 200
3o ydhT 201
WGLTSYNQKDNERISLYKQLYKKIYHYMTDTRGLDHLIWVYSPDANRDFK 250
ydhT 251
TDFYPGASYVDIVGLDAYFQDAYSINGYDQLTALNKPFAFTEVGPQTANG 300
ydhT 301
35 SFDYSLFINAIKQKYPKTIYFLAWNDEWSAAVNKGASALYHDSWTLNKGE 350
ydhT 351
CA 02301156 2000-02-11
WO 99/09130 PCT/US9$/12023
100
IWNGDSLTPIVE*. 363
