Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DETERGENT COMPOSITIONS COMPRISING A MANNANASE AND A CLAY
1o Field of the Invention
The present invention relates to laundry detergent and/or fabric care
compositions comprising a mannanase and a clay.
_Backgiround of the invention
Performance of a detergent product is judged by a number of factors, including
the ability to remove soils, and the ability to prevent the redeposition of
the
2o soils, or the breakdown products of the soils on the articles in the wash.
Therefore, detergent compositions include nowadays a complex combination of
active ingredients which fulfil certain specific needs. In particular, current
detergent formulations generally include surfactants and detergent enzymes
providing cleaning and fabric care benefits. Moreover, the need for detergent
compositions which exhibit not only good cleaning properties, but also fabric
softening performance and other fabric care benefits, is well-established in
the
art.
Softening clays are commonly used in current laundry detergent and fabric care
3o compositions to provide soft feeling. In EP-A-177 165, the use of softening
clay
together with cellulase in detergent compositions has been disclosed. EP-A-495
258 describes detergent compositions comprising a surfactant, a builder
system, a softening clay and a cellulase.
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
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additives. The term "gum denotes a group of industrially useful
polysaccharides
(long chain polymer) or their derivatives that hydrate in hot or cold water to
from
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
~o also Carbohydrate Chemistry for Food Scientists (Eagan Press - 1997) by R.
L.
Whistler and J.N. BeMiller, Chap 4, pp63-89 and Direct Food Additives in Fruit
Processing by P. Laslo, Bioprincipies and Applications, Vol1, Chapter II,
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 tetragonoloba. 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
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
3o 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
galactomanann is the main storage carbohydrate, comprising up to 20% of the
total dry weight in some cases. Galactomannan has a a-galactose 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.
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However, clay is not compatible with some hydrocolloid gums contained in those
food and cosmetic stains. It is recognised in the art that guar gum induces
precipitation of clay due to its potential to coagulate slimes or clay
particles (see
Industrial Gum, second editions, R.L. Whistler pp 307, Academic Press, 1973,
1SBN, 0-12-74-6252-x).
Accordingly, it is an object of the present invention to provide laundry
detergent
and/or fabric care compositions exhibiting an optimum softening performance
and providing an optimum stain removal and cleaning performance, especially on
~o cosmetic and food stains.
The above objectives have been met by formulating laundry detergent and/or
fabric care compositions comprising a mannanase enzyme and a clay as a
softening agent.
It has been further found that the performance of the detergent compositions
of
the present invention is enhanced by the addition of a laundry detergent
and/or
fabric care ingredient selected from a builder, a cellulase and/or a cationic
surfactant.
Mannanases have been ident~ed in several Bacillus organisms. For example,
Talbot et al., Appl. Environ. Microbiol., vol. 56, No. 11, pp. 3505-3510
(1990)
describes a ~-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 (i-mannanase
derived from Bacillus subtilisis having a MW of 38 kDa, an optimum activity at
pH
5.0 / 55°C and a pl of 4.8. J0304706 discloses a ~i-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
3o alkaline, thermostable (i-mannase, which hydrolyses ~i-1,4-D-
mannopyranoside
bonds of e.g. mannans and produces manno:oligo: saccharides. J63036774
relates to a Bacillus micro-organism FERM P-8856 which produces a-
mannanase and ~-mannosidase, at an alkaline pH. A purified mannanase from
Bacillus amyloliquefaciens and its method of preparation useful in the
bleaching
of pulp and paper, is disclosed in W097111164. W091I18974 describes an
hemicelluiase such as a glucanase, xylanase or mannanase, active at extreme
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pH and temperature and the production thereof. W094/25576 describes an
enzyme exhibiting a mannanase activity derived from Aspergiiius aculeatus CBS
101.43, that might be used for various purposes for which degradation or
modification of plant or algae cell wall material is desired. WO93/24622
discloses
a mannanase isolated from Trichoderma raeesie for bleaching lignocellulosic
pulps.
However, the synergistic combination of a mannanase and a clay for optimum
softening performance and optimum stain removal and cleaning performance,
o especi2~lly on cosmetic and food stains in a laundry detergent and/or fabric
care
composition, has never been previously recognised.
Summary of the invention
The present invention relates to laundry detergent and/or fabric care
compositions comprising a mannanase and clay for providing an optimum
softening performance and providing an optimum stain removal and cleaning
performance, especially on cosmetic and food stains.
Detailed description of the invention
It is known in the art that clay is not compatible with some the hydrocolloid
gums
contained in for example food and cosmetics stains. It is has been recognised
that guar gum induces precipitation of clay due to its potential to coagulate
slimes
or clay particles {see Industrial Gum, second editions, R.L. Whistler pp 307,
Academic Press, 1973, ISBN, 0-12-74-6252-x).
3o Without wishing to be bound by theory, it has been found that such
flocculated
clay have a reduced softening effect. Moreover, it has been observed that clay
and soil from the wash load redeposit on the guar gum residues and cause
staining. It is been surprisingly found that the mannanase enzyme hydrolyse
hydrocolloid gums, in particular guar gums. This enzyme hydrolysis results in
the
absence of guar gum in the wash. Therefore, there is no clay flocculation and
the clay retains its full fabric care potential. Moreover, there is no guar
gum
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residues remaining on the fabric and therefore no redeposition of clay and
soil
with the following wash load and therefore no staining.
5 The mannanase enzyme
An essential element of the laundry detergent and/or fabric care compositions
of
the present invention is a mannanase enzyme.
1o 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 laundry detergent and/or fabric care 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
2o mannan endo-1,4-beta-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
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
3o mannose; glucomannans are polysaccharides having a backbone or more or less
regularly alternating ~-1,4 linked mannose and glucose; galactomannans and
galactoglucomannans are mannans and glucomannans with a-1,6 linked
galactose sidebranches. These compounds may be acetylated.
s5 The degradation of galactomannans and galactoglucomannans is
facilitated by full or partial removal of the galactose sidebranches. Further
the
degradation of the acetylated mannans, glucomannans, galactomannans and
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galactoglucomannans is facilitated by full or partial deacetylation. Acetyl
groups
can be removed by alkali or by mannan acetylesterases. The oligomers which
are released from the mannanases or by a combination of mannanases and a-
galactosidase and/or mannan acetyl esterases can be further degraded to
release free maltose by (i-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)
describes a beta-mannanase derived from Bacillus stearothermophilus in dimer
o form having molecular weight of 162 kDa and an optimum pH of 5.5-7.5.
Mendoza et al., World J. Microbiol. Biotech., Vol. 10, No. 5, pp. 551-555
(1994)
describes a beta-mannanase derived from Bacillus subtilis having a molecular
weight of 38 kDa, an optimum activity at pH 5.0 and 55C and a pl of 4.8. JP-
0304706 discloses a beta-mannanase derived from Bacillus sp., having a
~5 molecular weight of 373 kDa measured by gel filtration, an optimum pH of 8-
10
and a pl of 5.3-5.4. JP-63056289 describes the production of an alkaline,
thermostable beta-mannanase which hydrolyses beta-1,4-D-mannopyranoside
bonds of e.g. mannans and produces manno-oligosaccharides. JP-63036774
relates to the Bacillus microorganism FERM P-8856 which produces beta-
2o mannanse and beta-mannosidase at an alkaline pH. JP-08051975 discloses
alkaline beta-mannanases from aikalophilic Bacillus sp. AM-001. A purified
mannanase from Bacillus amyloliquefaciens useful in the bleaching of pulp and
paper and a method of preparation thereof is disclosed in WO 97/11164. WO
91/18974 describes a hemicellulase such as a glucanase, xylanase or
25 mannanase active at an extreme pH and temperature. WO 94/25576 discloses
an enzyme from Aspergillus aculeatus, CBS 101.43, exhibiting mannanase
activity which may be useful for degradation or mod~cation of plant or algae
cell
wall material. WO 93/24622 discloses a mannanase isolated from Trichoderma
reseei useful for bleaching lignocellulosic pulps. An hemicellulase capable of
3o degrading mannan-containing hemicellulose is described in W091/18974 and a
purled mannanase from Bacillus amyloliquefaciens is described in
W097/11164.
In particular, this mannanase enzyme will be an alkaline mannanase as defined
35 below, most preferably, a mannanase originating from a bacterial source.
Especially, the laundry detergent composition of the present invention will
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comprise an alkaline mannanase selected from the mannanase from the strain
Bacillus agaradherens and/or Bacillus subtilises strain 168, gene yght.
The term "alkaline mannanase enzyme" is meant to encompass enzyme having
an enzymatic activity of at least 10%, preferably at least 25%, more
preferably at
least 40% of its maximum activity at a given pH ranging from 7 to 12,
preferably
7.5 to 10.5.
Most preferably, the laundry detergent and/or fabric care compositions of the
present invention will comprise the alkaline mannanase from Bacillus
agaradher~ens. Said mannanase is
i) a polypeptide produced by Bacillus agaradhenens, NCIMB 40482, or
ii) a polypeptide comprising an amino acid sequence as shown in positions
32-343 of SEQ ID N0:2 or
~ 5 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);
(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
inventors according to the Budapest Treaty on the International Recognition of
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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 enryme is the mannanase from the Bacillus subtilisis
strain 168, which mannanase:
i) is encoded by the coding part of the DNA sequence shown in SED ID No. 5
or an analogue of said sequence and/or
o ii) a polypeptide comprising an amino acid sequence as shown SEQ lD 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
~5 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
20 (a) polynucleotide molecules encoding a polypeptide having mannanase
activity and comprising a sequence of nucleotides as shown in SEQ ID
N0:5
(b) species homologs of (a);
(c) polynucleotide molecules that encode a polypeptide having mannanase
25 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
(e) degenerate nucleotide sequences of (a), (b), (c) or (d).
30 DEFINITIONS
Prior to discussing this invention in further detail, the following terms will
first be
defined
The term "ortholog" (or "species homoiog") denotes a polypeptide or protein
obtained from one species that has homology to an analogous polypeptide or
35 protein from a different species.
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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
o 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
~5 an extra chromosomal entity, the replication of which is independent of
chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one
which, when introduced into a host cell, is integrated into the host cell
genome
and replicated together with the chromosomes) into which it has been
integrated.
2o 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.
25 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
molecules are those that are separated from their natural environment and
3o include cDNA and genomic clones. Isolated DNA molecules of the present
invention are free of other genes with which they are ordinarily associated,
but
may include naturally occurring 5' and 3' untranslated regions such as
promoters
and terminators. The identification of associated regions will be evident to
one of
ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-
78,
35 1985).
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The term "an isolated polynucieotide" may alternatively be termed "a cloned
poiynucleotide". When applied to a protein/polypeptide, the term "isolated"
indicates that the protein is found in a condition other than its native
environment.
In a preferred form, the isolated protein is substantially free of other
proteins,
5 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
preferably greater than 95% pure, and even more preferably greater than 99%
o 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
polypeptide than the polypeptide of the invention) which originate from the
~5 homologous cell where the polypeptide of the invention is originally
obtained
from. The term "obtained from" as used herein in connection with a speck
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 have been
inserted.
2o 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.
The term "polynucleotide" denotes a single- or double- stranded polymer of
25 deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
Polynucieotides 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
3o 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
35 reference polynucleotide molecule that encodes a polypeptide). Degenerate
codons contain different triplets of
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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.
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
o 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:
~5 The disclosed sequence information herein relating to a polynucleotide
sequence
encoding a mannanase of the invention can be used as a tool to identify other
homologous mannanases. For instance, polymerase chain reaction (PCR) can
be used to amplify sequences encoding other homologous mannanases from a
variety of microbial sources, in particular of different Bacillus species.
ASSAY FOR ACTIVITY TEST
A polypeptide of the invention having mannanase activity may be tested for
mannanase activity according to standard test procedures known in the art,
such
as by applying a solution to be tested to 4 mm diameter holes punched out in
agar plates containing 0.2% AZCL galactomannan (carob), i.e. substrate for the
assay of endo-1,4-beta-D-mannanase available as CatNo.l- AZGMA from the
company Megazyme for US$110.00 per 3 grams (Megazyme's Internet address:
http://www.megazyme.com/Purchase/index.html).
3o POLYNUCLEOTIDES:
An isolated polynucleotide of the invention will hybridize to similar sized
regions
of SEQ ID No. 1, or a sequence complementary thereto, under at least 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
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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 chloride/Sodium 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
~o 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
~5 (medium/high stringency), even more preferably at least 70°C (high
stringency},
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
2o 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.
Polynucleotides encoding polypeptides having mannanase activity of the
invention are then identified and isolated by, for example, hybridization or
PCR.
25 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
30 can be cloned using information and compositions provided by the present
invention in combination with conventional cloning techniques. For example, a
DNA sequence of the present invention can be cloned using chromosomal DNA
obtained from a cell type that expresses the protein. Suitable sources of DNA
can be identified by probing Northern blots with probes designed from the
35 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
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13
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),
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
~o B.agaradherens, NCIMB 40482, expressed and purified as described in
Materials
and Methods and Example 1, or by an activity test relating to a polypeptide
having mannanase activity.
The mannanase encoding part of the DNA sequence cloned into plasmid
pSJ1678 present in Escherichia coli DSM 12180 andlor an analogue DNA
~5 sequence of the invention may be cloned from a strain of the bacterial
species
Bacillus agaradherens, preferably the strain NCIMB 40482, producing the
enzyme with mannan degrading activity, or another or related organism as
described herein.
Alternatively, the analogous sequence may be constructed on the basis of the
2o DNA sequence obtainable from the plasmid present in Escherichia coli DSM
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
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
25 intended for production of the enzyme, or by introduction of nucleotide
substitutions which may give rise to a different amino acid sequence (i.e. a
variant of the mannan degrading enzyme of the invention).
POLYPEPTIDES:
3o 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
35 used herein to denote pofypeptides having 70%, preferably at least 80%,
more
preferably at least 85%, and even more preferably at least 90%, sequence
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14
identity to the sequence shown in amino acids nos. 32-343 of SEQ ID N0:2 or
their orthologs or paralogs. Such polypeptides will more preferably be at
least
95% identical, and most preferably 98% or more identical to the sequence shown
in amino acids nos. 32-343 of SEQ ID N0:2 or its orthologs or paralogs.
Percent
sequence identity is determined by conventional methods, by means of computer
programs known in the art such as GAP provided in the GCG program package
(Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics
Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 ) as
disclosed in Needleman, S.B. and Wunsch, C.D., (1970), Journal of Molecular
~o 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
using GAP with the following settings for DNA sequence comparison: GAP
~5 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
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
2o 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 pu~~ication (an affinity tag),
such as
a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;
Nilsson et
25 al., Methods Enz~rmol. 198:3, 1991. See, in general Ford et al., Protein
Expression and Purification 2: 95-107, 1991, which is incorporated herein by
reference. DNAs encoding affinity tags are available from commercial suppliers
(e.g., Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, MA).
CA 02301200 2000-02-11
WO 99/09129 PCT/US98/12015
However, even though the changes described above preferably are of a minor
nature, such changes may also be of a larger nature such as fusion of larger
polypeptides of up to 300 amino acids or more both as amino- or carboxyl=
terminal extensions to a Mannanase polypeptide of the invention.
5
Table 1
Conservative amino acid substitutions
Basic : arginine, lysine, histidine
Acidic : glutamic acid, aspartic acid
Polar : glutamine, asparagine
Hydrophobic : leucine, isoleucine, valine
Aromatic : phenylalanine, tryptophan, tyrosine
Small : glycine, alanine, serine, threonine,
methionine
~o 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
~5 substituted for amino acid residues. "Unnatural amino acids" have been
modified
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, thiazolidine carboxylic acid, dehydroproline, 3- and 4-
2o methylproline, and 3,3-dimethylproline.
Essential amino acids in the mannanase polypeptides of the present invention
can be identified according to procedures known in the art, such as site-
directed
mutagenesis or alanine-scanning mutagenesis {Cunningham and Wells, Science
244: 1081-1085, 1989). In the latter technique, single alanine mutations are
introduced at every residue in the molecule, and the resultant mutant
molecules
are tested for biological activity (i.e mannanase activity) to identify amino
acid
residues that are critical to the activity of the molecule. See also, Hilton
et al., J.
Biol. Chem. 271:4699-4708, 1996. The active site of the enzyme or other
biological interaction can also be determined by physical analysis of
structure, as
3o determined by such techniques as nuclear magnetic resonance,
crystallography,
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16
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
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
0 241:53-57, 1988), Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156,
1989), W095/17413, or WO 95/22625. Briefly,. these authors disclose methods
for simultaneously randomizing two or more positions in a polypeptide, or
recombination/shuffling of different mutations (W095/17413, W095/22625),
followed by selecting for functional a polypeptide, and then sequencing the
~s mutagenized polypeptides 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,
20 1988).
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
25 sequenced using modem 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
3o 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
35 proteins, fragments thereof and fusion proteins, can be produced in
genetically
engineered host cells according to conventional techniques. Suitable host
cells
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17
are those cell types that can be transformed or transfected with exogenous DNA
and grown in culture, and include bacteria, fungal cells, and cultured higher
eukaryotic cells. Bacterial cells, particularly cultured cells of gram-
positive
organisms, are preferred. Gram-positive cells from the genus of Bacillus are
especially preferred, such as from the group consisting of Bacillus subtilis,
Bacillus lentus, Bacillus br~evis, Bacillus stearothennophilus, Bacillus
alkalophilus,
Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus
lautus,
Bacillus thuringiensis, Bacillus licheniformis, and Bacillus agaradhen3ns, in
particular Bacillus agaradherens.
o Techniques for manipulating cloned DNA molecules and introducing exogenous
DNA into a variety of host cells are disclosed by Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, 1989; Ausubel et al. (eds.), Current Protocols in
Molecular Bioloav, John Wiley and Sons, Inc., NY, 1987; and "Bacillus subtilis
is 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
including a transcription promoter and terminator within an expression vector.
2o The vector will also commonly contain one or more selectable markers and
one
or more origins of replication, although those skilled in the art will
recognize that
within certain systems selectable markers may be provided on separate vectors,
and replication of the exogenous DNA may be provided by integration into the
host cell genome. Selection of promoters, terminators, selectable markers,
25 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
3o 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,
35 Washington D.C.; and Cutting, S. M.(eds.) "Molecular Biological Methods for
Bacillus", John Wiley and Sons, 1990, for further description of suitable
secretory
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18
signal sequences especially for secretion in a Bacillus host cell. The
secretory
signal sequence is joined to the DNA sequence in the correct reading frame.
Secretory signal sequences are commonly positioned 5' to the DNA sequence
encoding the polypeptide of interest, although certain signal sequences may be
positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al.,
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,
o 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
exogenously added DNA by, for example, drug selection or deficiency in an
~5 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
2o be purified from the growth media. Preferably the expression host cells are
removed from the media before purification of the polypeptide (e.g. by
centrifugation).
When the expressed recombinant polypeptide is not secreted from the host cell,
the host cell are preferably disrupted and the polypeptide released into an
25 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.
3o 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 pur~cation methods and media.
35 Ammonium sulfate precipitation and acid or chaotrope extraction may be used
for
fractionation of samples. Exemplary purification steps may include
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19
hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid
chromatography. Suitable anion exchange media include derivatized dextrans,
agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI,
DEAE,
QAE and Q derivatives are preferred, with DEAE Fast-Flow Sepharose
(Pharmacia, Piscataway, NJ) being particularly preferred. Exemplary
chromatographic media include those media derivatized with phenyl, butyl, or
octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl fi50
(Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or
polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
~o 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
that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl
~5 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
other solid media are well-known and widely used in the art, and are available
2o from commercial suppliers.
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
Chromatography: Principles 8~ Methods, Pharmacia LKB Biotechnology, Uppsala,
Sweden, 1988.
25 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.
3o 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,
35 t preparing a genomic library from a Bacillus strain, especially the strain
8.
agaradherens, NCIMB 40482;
CA 02301200 2000-02-11
WO 99/09129 PCT/US98/12015
t 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
t identifying a clone from said Bacillus agaradher~ens NCIMB 40482 genomic
5 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
0 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
as alkalophilic species of Bacillus.
~5 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 ftom the plasmid present in
2o Escherichia coli DSM 12180.
Accordingly, the polynucleotide molecule of the invention may be isolated from
Escherichia coli, DSM 12180, in which the plasmid obtained by cloning such as
described above is deposited. Also, the present invention relates to an
isolated
substantially pure biological culture of the strain Escherichia coli, DSM
12180.
In the present context, the term "enzyme preparation" is intended to mean
either a conventional enzymatic fermentation product, possibly isolated and
purified, from a single species of a microorganism, such preparation usually
comprising a number of difFerent enzymatic activities; or a mixture of
monocomponent enzymes, preferably enzymes derived from bacterial or fungal
species by using conventional recombinant techniques, which enzymes have
been fermented and possibly isolated and purified separately and which may
originate from different species, preferably fungal or bacterial species; or
the
fermentation product of a microorganism which acts as a host cell for
expression
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
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21
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 .
conventional fermentation techniques, e.g. culturing in shake flasks or
fermentors
with agitation to ensure sufficient aeration on a growth medium inducing
o 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
~5 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:
2o 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
25 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
ion exchange chromatography, e.g. on DEAE-Sephadex. Immunochemical
3o 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., supra, Chapters 3 and 4), or by
rocket
immunoelectrophoresis (N. Axelsen et al., Chapter 2).
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WO 99/09129 PCTNS98/12015
22
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,
more preferably a strain of Bacillus agaradherens, especially the strain
Bacillus
agaradhenens, 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.
~o DETERMINATION OF CATALYTIC ACTIVITY (ManU) OF MANNANASE
Colorimetric Assay:Substrate: 0.2% AZCL-Galactomannan (Megazyme,
Australia) from carob in 0.1 M Glycin buffer, pH10Ø The assay is carried out
in
an Eppendorf Micro tube 1.5 ml on a thermomixer with stirring and temperature
control of 40°C. Incubation of 0.750 ml substrate with 0.05 ml enzyme
for 20 min,
~5 stop by centrifugation for 4 minutes at 15000 rpm. The color of the
supernatant is
measured at 600 nm in a 1 cm cuvette. One ManU (Mannanase units) gives 0.24
abs in 1 cm.
OBTENTION OF THE BACILLUS AGARADHERENS MANNANASE NCIMB
20 40482
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.,
25 Jensen, B. R., Sjwholm, C. (1990) Cloning of aldB, which encodes alpha-
acetolactate decarboxylase, an exoenzyme from Bacillus brevis. J. Bacteriol.,
172, 4315-4321 ), were prepared for and transformed by electroporation using a
Gene PuIserTM electroporator from BIO-RAD as described by the supplier.
B.subtilis PL2306. This strain is the B.subtilis DN1885 with disrupted apr and
npr
3o genes (Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen, B. R., Sjmholm,
C.
(1990) Cloning of aldB, which encodes alpha-acetolactate decarboxylase, an
exoenzyme from Bacillus brevis. J. Bacteriol., 172, 4315-4321) 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
35 ( Eds. A.L. Sonenshein, J.A. Hoch and Richard Losick (1993) Bacillus
subtilis
and other Gram-Positive Bacteria, American Society for microbiology, p.618).
CA 02301200 2000-02-11
WO 99/09129 PCT/US98/12015
23
Competent cells were prepared and transformed as described by Yasbin, R.E.,
~Ison, G.A. and Young, F.E. (1975) Transformation and transfection in
lysogenic
strains of Bacillus subtilis: evidence for selective induction of prophage in
competent cells. J. Bacteriol,121:296-304.
s
Plasmids
pSJ1678 (as described in detail in WO 94/19454 which is hereby incorporated by
reference in its entirety).
pMOL944: This plasmid is a pUB110 derivative essentially containing elements
o 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.licheniformis ATCC14580. The signal peptide contains a Sacll site making it
convenient to clone the DNA encoding the mature part of a protein in-fusion
with
the signal peptide. This results in the expression of a Pre-protein which is
~5 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:
The pUB110 plasmid (McKenzie, T. et al., 1986, Plasmid 15:93-103) was
2o digested with the unique restriction enzyme Ncil. A PCR fragment amplified
from
the amyl promoter encoded on the plasmid pDN1981 (P.L. Jargensen 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:
25 # LWN5494 5'-
GTCGCCGGGGCGGCCGCTATCAATTGGTAACTGTATCTCAGC -3'
# LWN5495 5'-
GTCGCCCGGGAGCTCTGATCAGGTACCAAGCTTGTCGACCTGCAGAA
TGAGGCAGCAAGAAGAT-3'
The primer #LWN5494 inserts a Notl site in the piasmid.
The plasmid pSJ2624 was then digested with Sacl and Notl and a new PCR
fragment ampl~ed 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.
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WO 99/09129 PCTNS98/12015
24
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'
o 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
entirety) was digested with Pstl and Bcll and inserted to give the plasmid
~5 pMOL944. The two primers used for PCR amplification have the following
sequence:
#LWN7864 5' -AACAGCTGATCACGACTGATCTI~'TAGCTTGGCAC-3'
#LWN7901 5' -AACTGCAGCCGCGGCACATCATAATGGGACAAATGGG -3'
The primer #LWN7901 inserts a Sacll site in the plasmid.
2o Cloning of the mannanase gene from Bacillus a~aradher~ens
Genomic DNA preparation:
Strain Bacillus agaradher~ens NCIMB 40482 was propagated in liquid medium as
described in W094/01532. After 16 hours incubation at 30°C and 300 rpm,
the
cells were harvested, and genomic DNA isolated by the method described by Pit-
25 cher 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:
Genomic DNA was partially digested with restriction enzyme Sau3A, and size
3o 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.
Anal. Biochem., 112, 295-298).
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Isolated DNA fragments were ligated to BamHl digested pSJ1678 plasmid DNA,
and the ligation mixture was used to transform E. coli SJ2.
Identification of positive clones:
A DNA library in E. coli, constructed as described above, was screened on LB
5 agar plates containing 0.2% AZCL-galactomannan (Megazyme) and 9 Ng/ml
Chloramphenicol and incubated overnight at 37oC. Clones expressing
mannanase activity appeared with blue diffusion halos. Plasmid DNA from one of
these clone was isolated by Qiagen plasmid spin preps on 1 ml of overnight
culture broth (cells incubated at 37°C in TY with 9 Ng/ml
Chloramphenicol and
1o 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.
~5 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.subfilis:
2o The mannanase encoding DNA sequence of the invention was PCR amplified
using the PCR primer set consisting of these two oligo nucleotides:
Mannanase.upper.Sacll
5'-CAT TCT GCA GCC GCG GCA GCA AGT ACA GGC TTT TAT GTT GAT GG-
3'
25 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.
Chromosomal DNA isolated from B.agaradher~ens NCIMB 40482 as described
3o above was used as template in a PCR reaction using Amplitaq DNA Polymerase
(Perkin Elmer) according to manufacturers instructions. The PCR reaction was
set up in PCR buffer (10 mM Tris-HCI, pH 8.3, 50 mM KCI, 1.5 mM MgCl2, 0.01
(w/v) gelatin) containing 200 NM of each dNTP, 2.5 units of AmpIiTaq
polymerase (Perkin-Elmer, Cetus, USA) and 100 pmol of each primer.
The PCR reaction was performed using a DNA thermal cycler (Landgraf,
Germany). One incubation at 94°C for 1 min followed by thirty cycles
of PCR
CA 02301200 2000-02-11
WO 99/09129 PCT/US98/12015
26
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
amplification product was analysed by electrophoresis in 0.7 % agarose gels
(NuSieve, FMC). The appearance of a DNA fragment size 1.4 kb indicated
s proper amplification of the gene segment.
Subcloning of PCR fragment.
Fortyfive-NI aliquots of the PCR products generated as described above were
purified using QIAquick PCR purification kit (Qiagen, USA) according to the
manufacturer's instructions. The purified DNA was eluted in 50 NI of 10mM Tris
1o HCI, pH 8.5.
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
(SeaPlaque GTG, FMC) gels, the relevant fragments were excised from the gels,
and purled using QIAquick Gel extraction Kit (Qiagen, USA) according to the
~s manufacturer's instructions. The isolated PCR DNA fragment was then ligated
to
the Sacll-Notl digested and purified pMOL944. The ligation was performed
overnight at 16°C using 0.5~rg of each DNA fragment, 1 U of T4 DNA
ligase and
T4 ligase buffer (Boehringer Mannheim, Germany).
The ligation mixture was used to transform competent B.subtiJis PL2306. The
2o transformed cells were plated onto LBPG-10 Ng/ml of Kanamycin plates. After
18
hours incubation at 37°C colonies were seen on plates. Several clones
were
analysed by isolating plasmid DNA from overnight culture broth.
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
25 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
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
3o appended SEQ ID N0:3. The derived mature protein is shown in SEQ ID N0:4.
It
will appear that the 3' end of the mannanse encoded by the sequence of SEQ ID
N0:1 was changed to the one shown in SEQ ID N0:3 due to the design of the
lower primer used in the PCR. The resulting amino acid sequence is shown in
SEQ ID N0:4 and it is apparent that the C terminus of the SEQ ID N0:2
35 (SHHVREIGVQFSAADNSSGQTALYVDNVTLR) is changed to the C terminus of
SEQ ID N0:4 (IIMLGK).
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Media:
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
potassium phosphate, pH 7.0
BPX media is described in EP 0 506 780 {V110 91/09129).
o Expression, purification and characterisation of mannanase from Bacillus
a4aradherens
The clone MB 594 obtained as described above under Materials and Methods
was grown in 25 x 200m1 BPX media with 10 Nglml of Kanamycin in 500m1 two
baffled shakeflasks for 5 days at 37°C at 300 rpm.
~5 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
anionic agent (A130) was added during agitation for flocculation. The
flocculated
material was separated by centrifugation using a Sorval RC 3B centrifuge at
9000 rpm for 20 min at 6°C. The supernatant was clarified using Whatman
glass
2o 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.
25 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.
Determination of kinetic constants:
Substrate: Locust bean gum (carob) and reducing sugar analysis (PHBAH).
3o Locust bean gum from Sigma (G-0753).
Kinetic determination using different concentrations of locust bean gum and
incubation for 20 min at 40°C at pH 10 gave
Kcat: 467 per sec.
Km: 0.08 gram per I
35 MW:38kDa
pl (isoelectric point): 4.2
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The temperature optimum of the mannanase was found to be 60°C.
The pH activity profile showed maximum activity between pH 8 and 10.
DSC differential scanning calometry gives 77°C as melting point at pH
7.5 in Tris
buffer indicating that this enzyme is very thermostable.
Detergent compatibility using 0.2% AZCL-Galactomannan from carob as
substrate and incubation as described above at 40°C shows excellent
compatibility with conventional liquid detergents and good compatibility with
conventional powder detergents.
o OBTENTION OF THE BACILLUS SUBTILISIS MANNANASE 168
The Bacillus subfilisis ~3-mannanase was characterised and purified as follows
The Bacillus subtilis genome was searched for homology with a known Bacillus
sp ~i-Mannanase gene sequence (Mendoza et al., Biochemica et Bio~~hysica
Acta 1243:552-554, 1995). The coding region of ydhT, whose product was
~5 unknown, showed a 58% similarity to the known Bacillus (i-Mannanase. The
following oligonucleotides were designed to amplify the sequences coding for
the
mature portion of the putative (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
GGC G-3'. Total genomic DNA from Bacillus subtilis strain 1A95 was used as a
2o template to amplify the ydhT mature region using the aforementioned
primers.
PCR is performed using the GENE-AMP PCR Kit with AMPLITAQ DNA
Polymerase (Perkin Elmer, Applied Biosystems, Foster City, CA). An initial
melting period at 95°C for 5 min was followed by 25 cycles of the
following
program: melting at 95°C for 1 min, annealing at 55°C for 2 min,
and extension at
25 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).
The ydhT mature region amplified from Bacillus subtilis strain 1A95 was
inserted
into the expression vector pPG1524 (previously described) as follows. The
3o amplified 1028bp fragment was digested with Mfe I and BamH 1. 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
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
35 cultured for DNA preparations. The DNA was then characterized by
restriction
analysis. Plasmid pPG3200 contains the mature region of the ydhT gene.
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29
Plasmid pPG3200 was then used to transform competent Bacillus subtilis strain
PG 632 (Saunders et al., 1992).
Seven kanamycin resistant Bacillus subtilis clones and one PG 632 control
clone
were picked and grown in 20m1 of 20/20/5 media ( 20g/I tryptone, 20g/I yeast
extract, 5g/I NaCI ) supplemented with 1ml 25% maltrin, 1201 10mM MnCl2, and
20p1 of 50 mg/ml kanamycin: Clones were grown overnight in 250m1 baffled
flasks shaking at 250 rpm at 37°C for expression of the protein. Cells
were spun
out at 14,OOOrpm for 15 minutes. One ~I of each supernatant was diluted in
99w1
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
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,
~s San Diego, Ca) to confirm expected protein size of 38kDa. Samples were
prepared as follows. A 5001 sample of ydhT clone 7 and PG 632 supernatants
were precipitated with 55.5.1 100% Trichloroacetic acid (Sigma), washed with
1001 5% Trichloroacetic, resuspended in 501 of Tris-glycine SDS sample
buffer(Novex) and boiled for five minutes. One ~.I of each sample was
2o electrophoresed on the gel at 30 mA for 90minutes. A large band of protein
was
observed to run at 38kDa for ydhT clone 7.
A 10 I fermentation of Bacillus subtilis ydhT clone 7 was performed in a
B.Braun
Biostat C fermentator. Fermentation conditions were as follows. Cells were
grown
for 18h in a rich media similar to 20/20/5 at 37°C. At the end of the
fermentation
25 run, the cells were removed and the supernatant concentrated to 1 liter
using a
tangential flow filtration system. The final yield of ~i-Mannanase in the
concentrated supernatant was determined to be 3 g/l.
The purification of the ~-Mannanase from the fermentation supernatant was
performed as follows: 500m1 of supernatant was centrifuged at 10,000 rpm for
10
3o min at 4°C. The centrifuged supernatant was then dialyzed overnight
at 4°C in
two 4 I changes of 10 mM potassium phosphate {pH 7.2) through Spectrapor
12,000-14,000 mol.wt. cutoff membrane (Spectrum). The dialyzed supernatant
was centrifuged at 10,000 rpm for 10 min at 4°C. A 200 ml Q Sepharose
fast flow
(Pharmacia) anion exchange column was equilibrated with 1 liter of 10 mM
35 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)
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were collected. The two fractions were assayed as before, except that the
samples were diluted with 199 ~I of 50 mM sodium acetate (pH 6.0), and they
showed Absorbance of .38 and .52 respectively. Two pl of each sample was
added to 8pl of Tris-glycine SDS sample buffer (Novex, CA) and boiled for 5
min.
5 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
present in each sample and comprised greater than 95% of the total protein. A
BCA protein assay (Pierce) was performed on both samples according to the
manufacturers instructions, using bovine serum albumin as standard. Samples A
and B contained 1.3 mg/ml and 1.6 mg/ml of ~i-Mannanase respectively. The
identity of the protein was confirmed by ion spray mass spectrometry and amino
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-~i-Mannanase Beta-Mannazyme
~5 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 ~i-Mannanase was found to
be pH 6.0-6.5. Temperature activity profiles were performed in 50mM citrate
2o 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 p,mollmin~mg ~-Mannanase using endo-1,4-(i-
Mannanase Beta-Mannazyme Tabs (Megazyme, Ireland) according to the
25 manufacturers directions. The nucleotide and amino acid sequences of the
Bacillus subtilisis ~i-mannanase are shown in SEQ. ID. No. 5 and 6.
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
3o 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
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
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31
introduced into the enzyme thus creating an enzyme hybrid. In this context,
the
term "cellulose-binding domain" is intended to be understood as defined by
Peter
Tomme et al. "Cellulose-Binding Domains: 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
class~ies more than 120 cellulose- binding domains into 10 families (I-X), and
demonstrates that CBDs are found in various enzymes such as cellulases,
xylanases, mannanases, arabinofuranosidases, acetyl esterases and chitinases.
CBDs have also been found in algae, e.g. the red alga Porphyra purpurea as a
o non-hydrolytic polysaccharide-binding protein, see Tomme et al., op.cit.
However, most of the CBDs are from cellulases and xylanases, CBDs are found
at the N and C termini of proteins or are internal. Enzyme hybrids are known
in
the art, see e.g. WO 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
~5 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
express the fused gene. Enzyme hybrids may be described by the following
formula:
CBD-MR-X
2o wherein CBD is the N-terminal or the C-terminal region of an amino acid
sequence corresponding to at least the cellulose- binding domain; MR is the
middle region (the linker), and may be a bond, or a short linking group
preferably
of from about 2 to about 100 carbon atoms, more preferably of from 2 to 40
carbon atoms; or is preferably from about 2 to to about 100 amino acids, more
25 preferably of from 2 to 40 amino acids; and X is an N-terminal or C-
terminal
region of the enzyme of the invention.
The above-mentioned enzymes may be of any suitable origin, such as vegetable,
animal, bacterial, fungal and yeast origin. Origin can further be mesophilic
or
3o extremophilic (psychrophilic, psychrotrophic, thermophilic, barophilic,
alkalophilic,
acidophilic, halophilic, etc.). Purified or non-purified forms of these
enzymes may
be used. Nowadays, it is common practice to modify wild-type enzymes via
protein / genetic engineering techniques in order to optimise their
performance
efficiency in the cleaning compositions of the invention. For example, the
variants
35 may be designed such that the compatibility of the enzyme to commonly
encountered ingredients of such compositions is increased. Alternatively, the
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32
variant may be designed such that the optimal pH, bleach or chelant stability,
catalytic activity and the like, of the enzyme variant is tailored to suit the
particular cleaning application.
In particular, attention should be focused on amino acids sensitive to
oxidation in
the case of bleach stability and on surface charges for the surfactant
compatibility. The isoelectric point of such enzymes may be modified by the
substitution of some charged amino acids, e.g. an increase in isoelectric
point
may help to improve compatibility with anionic surfactants. The stability of
the
1o enzymes may be further enhanced by the creation of e.g. additional salt
bridges
and enforcing metal binding sites to increase chelant stability.
The softening clay
A second essential component of the present laundry detergent and/or fabric
care compositions is a softening clay. Any clay used in the art or mixtures
thereof
can be used in the present invention. Preferred examples have been disclosed
in
GB 1.400.898 or US 5.019.292.
Included among such clays are various heat-treated kaolins and various multi-
layer smectites or bentonites also called montmorillonite. As known from the
art,
preferred smectite clays exhibit a cation-exchange capacity of at least 50 meq
per 100 grams of clay, which corresponds to a layer charge of 0.2 to 0.6.
Further
preferred are clays which have a particle size in the 5-50 micrometer range.
Additionally preferred smectite clays are hectorite clays of the general
formula
f(M93-x~ix) Si4-yMeIIlyplO(~H2-zFz)j-(x+Y) x+ Mn+
n
wherein y=0; or, if y = 0, Melll is AI, Fe, or B; Mn+ is a monovalent (n=1) or
divalent (n=2) metal ion, for example selected from Na, K, Mg, Ca, Sr. The
value
of (x+y) is the layer charge of the hectorite clay. The hectorite clays
suitable for
the detergent compositions of the present invention have a layer charge
distribution such that at least 50% is in the range of from 0.23 to 0.31.
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Preferred are hectorite clays of natural origin having a layer charge
distribution
such that at least 65% is in the range of from 0.23 to 0.31.
Specific non-limiting examples of fabric softening smectite clay minerals are
- Sodium Montmoriilonite : Borck (R), Volclay BC (R), Gelwhite GP (R), Thixo-
Jel
(R), Ben-A-Gel (R)-
- Sodium Hectorite : Veegum F (R) and Laponite SP (R)
- Sodium Saponite : Barasym NAS 100 (R)
- Calcium Montmorillonite : Soft Clark (R), Gelwhite L (R), Imvite K (R), CSM-
Clay
o (R) from Kimoulos
- Lithium Hectorite : Barasym LIH 200 R
The amount of softening clay useful in the present invention depends upon the
form of the laundry detergent and/or fabric care composition. In general, it
can
~5 range from lower limits of 0.1 %, 3% or 4% to upper limits of 50%, 25% or
15%.
Detergent components
2o The laundry detergent and/or fabric care 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 laundry detergent and/or fabric care compositions of the present invention
preferably further comprise a laundry detergent and/or fabric care ingredient
selected from a cellulase, a builder selected from zeolite, sodium
tripolyphosphate and/or layered silicate, a cationic surfactant and/or
mixtures
3o thereof.
The laundry detergent and/or fabric care compositions according to the
invention
can be liquid, paste, gels, bars, tablets, spray, foam, powder or granular.
Granular compositions can also be in "compact" form and the liquid
compositions
can also be in a "concentrated" form.
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34
The compositions of the invention may for example, be formulated as hand and
machine laundry detergent compositions including laundry additive compositions
and compositions suitable for use in the soaking and/or pretreatment of
stained
fabrics, rinse added fabric softener compositions.
When formulated as compositions suitable for use in a laundry machine washing
method, the compositions of the invention preferably contain both a surfactant
system that may contain anionic, cationic, nonionic, zwitterionic or mixture
of
surfactants and a builder system that may contain phosphate based builder, non
o phosphate based inorganic zeolite, layered silicate, organic builder such as
citrate 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 compositions can also
~5 contain softening agents different from those inorganic clay claimed in
this
present invention, as additional detergent components. Such compositions
containing a mannananase and a clay can provide fabric cleaning, stain
removal,
softening, color appearance when formulated as laundry detergent compositions.
2o 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.
25 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
3o 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 composition, preferably not exceeding 10%, most preferably not
35 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-
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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
5 the present invention will contain a lower amount of water, compared to
conventional liquid detergents. Typically the water content of the
concentrated
liquid detergent is preferably less than 40%, more preferably less than 30%,
most
preferably less than 20% by weight of the detergent composition.
o Suitable detergent compounds for use herein are selected from the group
consisting of the below described compounds.
Surfactant system
The laundry detergent and/or fabric care compositions according to the present
invention can further comprise a surfactant system wherein the surfactant can
be
selected from nonionic and/or anionic cationic and/or and/or ampholytic and/or
zwitterionic and/or semi-polar surfactants. The laundry detergent and/or
fabric
2o care compositions of the present invention will preferably further comprise
a
cationic surfactant. It has been surprisingly found that said compositions
further
comprising a cationic surfactant, provide improved cleaning and softening
performance.
The other 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 laundry detergent and/or fabric care compositions
in accord with the invention.
3o The surfactant is preferably formulated to be compatible with enzyme
components present in the composition. In liquid or gel compositions the
surfactant is most preferably formulated such that it promotes, or at least
does
not degrade, the stability of any enzyme in these compositions.
Polyethylene, polypropylene, and polybutylene oxide condensates of alkyl
phenols are suitable for use as the nonionic surfactant of the surfactant
systems
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36
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
mole of alkyl phenol. Commercially available nonionic surfactants of this type
include IgepaITM CO-630, marketed by the GAF Corporation; and TritonTM X-
~0 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).
The condensation products of primary and secondary aliphatic alcohols with
from
~5 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
2o 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
preferably from 2 to 5 moles of ethylene oxide per mole of alcohol are present
in
said condensation products. Examples of commercially available nonionic
25 surfactants of this type include TergitoITM 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
oxide with a narrow molecular weight distribution), both marketed by Union
Carbide Corporation; NeodoITM 45-9 (the condensation product of C14-C15
30 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
ethylene oxide), NeodoITM 45-5 (the condensation product of C14-C15 linear
alcohol with 5 moles of ethylene oxide) marketed by Shell Chemical Company,
35 KyroTM EOB (the condensation product of C13-C15 alcohol with 9 moles
ethylene oxide), marketed by The Procter & Gamble Company, and Genapol LA
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37
030 or 050 (the condensation product of C12-C14 alcohol with 3 or 5 moles of
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,
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
~o 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
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
group is attached at the 2-, 3-, 4-, etc. positions thus giving a glucose or
~5 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 prefer-ed alkylpolyglycosides have the formula
2o R20(CnH2n0)t(glYcosyl)x
wherein R2 is selected from the group consisting of alkyl, alkylphenyl,
hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which the alkyl
groups
contain from about 10 to about 18, preferably from about 12 to about 14,
carbon
25 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
30 (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
35 the condensation of propylene oxide with propylene glycol are also suitable
for
use as the additional nonionic surfactant systems of the present invention.
The
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38
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
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.
The hydrophobic moiety of these products consists of the reaction product of
~5 ethylenediamine and excess propylene oxide, and generally has a molecular
weight of from about 2500 to about 3000. This hydrophobic moiety is condensed
with ethylene oxide to the extent that the condensation product contains from
about 40% to about 80% by weight of polyoxyethylene and has a molecular
weight of from about 5,000 to about 11,000. Examples of this type of nonionic
2o surfactant include certain of the commercially available TetronicTM
compounds,
marketed by BASF.
Preferred for use as the nonionic surfactant of the surfactant systems of the
present invention are polyethylene oxide condensates of alkyl phenols,
25 condensation products of primary and secondary aliphatic alcohols with from
about 1 to about 25 moles of ethylene oxide, alkylpolysaccharides, and
mixtures
thereof. Most preferred are Cg-C14 alkyl phenol ethoxylates having from 3 to
15
ethoxy groups and Cg-C1g alcohol ethoxylates (preferably C1p avg.) having from
2 to 10 ethoxy groups, and mixtures thereof.
Highly preferred nonionic surfactants are polyhydroxy fatty acid amide
surfactants of the formula.
R2-C-N-Z,
O R1
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39
wherein R1 is H, or R1 is C1~ 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
mixtures thereof, and Z is derived from a reducing sugar such as glucose,
fructose, maltose, lactose, in a reductive amination reaction.
1o Suitable anionic surfactants to be used are linear alkyl benzene sulfonate,
alkyl
ester sulfonate surfactants including linear esters of Cg-C2p carboxylic acids
(i.e., fatty acids) which are sulfonated with gaseous S03 according to 'The
Journal of the American Oil Chemists Society", 52 (1975), pp. 323-329.
Suitable
starting materials would include natural fatty substances as derived from
tallow,
palm oil, etc.
The preferred alkyl ester sulfonate surfactant, especially for laundry
applications,
comprise alkyl ester sutfonate surfactants of the structural formula:
O
II
2o R3 - CH - C - OR4
I
S03M
wherein R3 is a Cg-C2p hydrocarbyl, preferably an alkyl, or combination
thereof,
R4 is a C1-C6 hydrocarbyl, preferably an alkyl, or combination thereof, and M
is
a ration which forms a water soluble salt with the alkyl ester sulfonate.
Suitable
salt-forming rations include metals such as sodium, potassium, and lithium,
and
substituted or unsubstituted ammonium rations, such as monoethanolamine,
diethanolamine, and triethanolamine. Preferably, R3 is C1 p-C16 alkyl, and R4
is
methyl, ethyl or isopropyl. Especially preferred are the methyl ester
sulfonates
3o wherein R3 is C1p-C16 alkyl.
Other suitable anionic surfactants include the alkyl sulfate surfactants which
are
water soluble salts or acids of the formula ROS03M wherein R preferably is a
C1p-C24 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C1p-C2p
alkyl
component, more preferably a C12-C1g alkyl or hydroxyalkyl, and M is H or a
ration, e.g., an alkali metal ration (e.g. sodium, potassium, lithium), or
CA 02301200 2000-02-11
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~o
ammonium or substituted ammonium (e.g. methyl-, dimethyl-, and trimethyl
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
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
~o the cleaning compositions of the present invention. These can include salts
(including, for example, sodium, potassium, ammonium, and substituted
ammonium salts such as mono-, di- and triethanolamine salts) of soap, Cg-C22
primary of secondary alkanesulfonates, Cg-C24 olefinsulfonates, sulfonated
polycarboxylic acids prepared by sulfonation of~the pyrolyzed product of
alkaline
~5 earth metal citrates, e.g., as described in British patent specification
No.
1,082,179, Cg-C24 alkylpolyglycolethersulfates (containing up to 10 moles of
ethylene oxide); alkyl glycerol sulfonates, fatty acyl glycerol sulfonates,
fatty oleyl
glycerol sulfates, alkyl phenol ethylene oxide ether sulfates, paraffin
sulfonates,
alkyl phosphates, isethionates such as the acyl isethionates, N-acyl taurates,
2o alkyl succinamates and sulfosuccinates, monoesters of sulfosuccinates
{especially saturated and unsaturated C12-C1g monoesters) and diesters of
sulfosuccinates (especially saturated and unsaturated C6-C12 diesters), acyl
sarcosinates, sulfates of alkylpolysaccharides such as the sulfates of
alkylpolyglucoside {the nonionic nonsulfated compounds being described below),
25 branched primary alkyl sulfates, and alkyl polyethoxy carboxylates such as
those
of the formula RO(CH2CH20)k-CH2C00-M+ wherein R is a Cg-C22 alkyl, k is
an integer from 1 to 10, and M is a soluble salt-forming ration. 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
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).
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When included therein, the laundry laundry detergent and/or fabric care
compositions of the present 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
an unsubstituted C10-C24 alkyl or hydroxyalkyl group having a C10-C24 alkyl
component, preferably a C12-C20 alkyl or hydroxyalkyl, more preferably C12-
o 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 ration which can be, for example, a metal ration
(e.g.,
sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or
substituted-ammonium ration. Alkyl ethoxylated sulfates as well as alkyl
propoxylated sulfates are contemplated herein. Specific examples of
substituted
ammonium rations include methyl-, dimethyl, trimethyl-ammonium rations and
quaternary ammonium rations such as tetramethyl-ammonium and dimethyl
piperdinium rations and those derived from alkylamines such as ethylamine,
diethylamine, triethylamine, mixtures thereof, and the like. Exemplary
surfactants
2o are C12-C1g alkyl poiyethoxylate (1.0) sulfate (C12-C18E(1.0)M), C12-C1g
alkyl
polyethoxyiate (2.25) sulfate (C12-C18E{2.25)M), C12-C1g alkyl polyethoxylate
(3.0) sulfate (C12-C18E(3.0)M), and C12-C1g alkyl polyethoxyiate (4.0) sulfate
{C12-C18E(4.0)M), wherein M is conveniently selected from sodium and
potassium.
The laundry detergent and/or fabric care compositions of the present invention
may also contain ampholytic, zwitterionic, and semi-polar surfactants, as well
as
the nonionic and/or anionic surfactants other than those already described
herein.
Ampholytic surtactants are also suitable for use in the laundry detergent
andlor
fabric care 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
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42
about 18 carbon atoms, and at least one contains an anionic water-solubilizing
group, e.g. carboxy, sulfonate, sulfate. See U.S. Patent No. 3,929,678 to
Laughlin et al., issued December 30, 1975 at column 19, lines 18-35, for
examples of ampholytic surfactants.
When included therein, the laundry detergent and/or fabric care 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 laundry detergent and/or
~o fabric care 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
~5 through column 22, line 48, for examples of zwitterionic surfactants.
When included therein, the laundry detergent and/or fabric care 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.
2o Semi-polar nonionic surfactants are a special category of nonionic
surfactants
which include water-soluble amine oxides containing one alkyl moiety of from
about 10 to about 18 carbon atoms and 2 moieties selected from the group
consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to
about 3 carbon atoms; water-soluble phosphine oxides containing one alkyl
25 moiety of from about 10 to about 18 carbon atoms and 2 moieties selected
from
the group consisting of alkyl groups and hydroxyalkyl groups containing from
about 1 to about 3 carbon atoms; and water soluble sulfoxides containing one
alkyl moiety of from about 10 to about 18 carbon atoms and a moiety selected
from the group consisting of alkyl and hydroxyalkyl moieties of from about 1
to
3o about 3 carbon atoms.
Semi-polar nonionic detergent surfactants include the amine oxide surfactants
having the formula
0
T
35 R3(OR4)xN(R5)2
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43
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
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
s 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
o amine oxides and Cg-C12 alkoxy ethyl dihydroxy ethyl amine oxides.
When included therein, the cleaning compositions of the present invention
typically comprise from 0.2% to about 15%, preferably from about 1 % to about
10% by weight of such semi-polar nonionic surfactants.
15 The laundry detergent and/or fabric care composition of the present
invention
may further comprise a co-surfactant selected from the group of primary or
tertiary amines.
Suitable primary amines for use herein include amines according to the formula
R1 NH2 wherein R1 is a Cg-C12, preferably Cg-C10 alkyl chain or R4X(CH2)n, X
20 is -O-,-C(O)NH- or -NH-, R4 is a Cg-C12 alkyl chain n is between 1 to 5,
preferably 3: R1 alkyl chains may be straight or branched and may be
interrupted with up to 12, preferably less than 5 ethylene oxide moieties.
Preferred amines according to the formula herein above are n-alkyl amines.
Suitable amines for use herein may be selected from 1-hexylamine, 1-
2s 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
so R1 R2R3N wherein R1 and R2 are C1-Cg alkylchains or
Rs
I
-C CHz--CH-O ~H
R3 is either a Cg-C12~ preferably Cg-C10 alkyl chain, or R3 is R4X(CH2)n,
whereby X is -O-, -C(O)NH- or -NH-,R4 is a C4-C12, n is between 1 to 5,
preferably 2-3. R5 is H or C1-C2 alkyl and x is between 1 to 6 .
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44
R3 and R4 may be linear or branched ; R3 alkyl chains may be interrupted with
up to 12, preferably less than 5, ethylene oxide moieties.
Preferred tertiary amines are R1 R2R3N where R1 is a C6-C12 alkyl chain, R2
and R3 are C1-C3 alkyl or
Rs
-( CHz-CH-O~H
where R5 is H or CH3 and x = 1-2.
o Also preferred are the amidoamines of the formula:
0
I I
Rl-C-NH-( CH2 n N-( R2 2
wherein R1 is Cg-C12 alkyl; n is 2-4,
preferably n is 3; R2 and R3 is C1-C4
~5 Most preferred amines of the present invention include 1-octylamine, 1-
hexylamine, 1-decylamine, 1-dodecylamine,CB-10oxypropylamine, N corn 1-
3diaminopropane, coconutalkyldimethylamine, lauryldimethylamine, lauryl
bis(hydroxyethyl)amine, coco bis(hydroxyehtyl)amine, lauryl amine 2 moles
propoxylated, octyl amine 2 moles propoxylated, lauryl amidopropyl-
20 dimethylamine, C8-10 amidopropyldimethylamine and C10 amidopropyldi-
methylamine.
The most preferred amines for use in the compositions herein are 1-hexylamine,
1-octylamine, 1-decylamine, 1-dodecylamine. Especially desirable are n-
dodecyldimethylamine and bishydroxyethylcoconutalkylamine and oleylamine 7
25 times ethoxylated, lauryl amido propylamine and cocoamido propylamine.
Bleaching agent
so The laundry detergent and/or fabric care compositions of the present
invention
can further comprise a bleaching agent such as hydrogen peroxide, PB1, PB4
and percarbonate with a particle size of 400-800 microns. These bleaching
agent components can include one or more oxygen bleaching agents and,
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WO 99/09129 PCT/US98/12015
depending upon the bleaching agent chosen, one or more bleach activators.
When present oxygen bleaching compounds will typically be present at levels of
from about 1 % to about 25%.
5 The bleaching agent component for use herein can be any of the bleaching
agents useful for laundry detergent and/or fabric care compositions including
oxygen bleaches as well as others known in the art. The bleaching agent
suitable
for the present invention can be an activated or non-activated bleaching
agent.
o One category of oxygen bleaching agent that can be used encompasses
percarboxylic acid bleaching agents and salts thereof. Suitable examples of
this
class of agents include magnesium monoperoxyphthalate hexahydrate, the
magnesium salt of meta-chloro perbenzoic acid, 4-nonylamino-4-
oxoperoxybutyric acid and diperoxydodecanedioic acid. Such bleaching agents
~5 are disclosed in U.S. Patent 4,483,781, U.S. Patent Application 740,446,
European Patent Application 0,133,354 and U.S. Patent 4,412,934. Highly
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
2o halogen bleaching agents. Examples of hypohalite bleaching agents, for
example, include trichloro isocyanuric acid and the sodium and potassium
dichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides. Such
materials are normally added at 0.5-10% by weight of .the finished product,
preferably 1-5% by weight.
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
3o pentaacetyiglucose (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
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WO 99/09129 PCTlUS98/12015
46
disclosed in the Procter & Gamble co-pending patent applications US serial No.
60/022,786 (filed July 30, 1996) and No. 60/028,122 (filed October 15, 1996)
O O
Rj
R3
2
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
group.
1o Useful bleaching agents, including peroxyacids and bleaching systems
comprising bleach activators and peroxygen bleaching compounds for use in
laundry detergent and/or fabric care compositions according to the invention
are
described in our co-pending applications USSN 08/136,626, PCT/US95/07823,
W095/27772, W095/27773, W095/27774 and W095/27775.
The hydrogen peroxide may also be present by adding an enzymatic system (i.e.
an enzyme and a substrate therefore) which is capable of generating hydrogen
peroxide at the beginning or during the washing and/or rinsing process. Such
enzymatic systems are disclosed in EP Patent Application 91202655.6 filed
2o October 9, 1991.
Metal-containing catalysts for use in bleach compositions, include cobalt-
containing catalysts such as Pentaamine acetate cobalt(///) 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
5,114,611. Bleaching composition comprising a peroxy compound, a
manganese-containing bleach catalyst and a chelating agent is described in the
patent application No 94870206.3.
so 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
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47
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, laundry detergent and/or
fabric
care compositions will contain about 0.025% to about 1.25%, by weight, of
sulfonated zinc phthalocyanine.
Builder system
The laundry detergent and/or fabric care compositions of the present invention
can further comprise a builder. The laundry detergent and/or fabric care
compositions of the present invention will preferably further comprise a
builder
~5 selected from zeolite, sodium tripolyphosphate and/or layered silicate. It
has
been surprisingly found that said compositions further comprising a builder
selected from zeolite, sodium tripolyphosphate and/or layered silicate,
provide
improved cleaning and softening performance.
2o 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
25 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
3o synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.
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,
35 glycolic acid and ether derivatives thereof as disclosed in Belgian Patent
Nos.
831,368, 821,369 and 821,370. Polycarboxylates containing two carboxy groups
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WO 99/09129 PCTNS98/12015
48
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,
lactoxysuccinates described in Netherlands Application 7205873, and the
0 oxypolycarboxylate materials such as 2-oxa-1,1,3-propane tricarboxylates
described in British Patent No. 1,387,447.
Polycarboxylates containing four carboxy groups include oxydisuccinates
disclosed in British Patent No. 1,261,829, 1,1,2,2-ethane tetracarboxylates,
~ s 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
Patent No. 1,082,179, while polycarboxylates containing phosphone substituents
2o are disclosed in British Patent No. 1,439,000.
Alicyclic and heterocyclic polycarboxylates include cyclopentane-cis,cis,cis-
tetracarboxylates, cyclopentadienide pentacarboxylates, 2,3,4,5-tetrahydro-
furan
- cis, cis, cis-tetracarboxylates, 2,5-tetrahydro-furan -cis - dicarboxylates,
2,2,5,5-
25 tetrahydrofuran - tetracarboxylates, 1,2,3,4,5,6-hexane -hexacar-boxylates
and
and carboxymethyl derivatives of polyhydric alcohols such as sorbitol,
mannitol
and xylitol. Aromatic poly-carboxylates include mellitic acid, pyromellitic
acid and
the phthalic acid derivatives disclosed in British Patent No. 1,425,343.
Of the above, the preferred polycarboxylates are hydroxycarboxylates
containing
3o up to three carboxy groups per molecule, more particularly citrates.
Preferred builder systems for use in the present compositions include a
mixture
of a water-insoluble aluminosilicate builder such as zeolite A or of a layered
silicate (SKS-6), and a water-soluble carboxylate chelating agent such as
citric
35 acid. Other preferred builder systems include a mixture of a water-
insoluble
aluminosilicate builder such as zeolite A, and a watersoluble carboxylate
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49
chelating agent such as citric acid. Preferred builder systems for use in
liquid
laundry detergent and/or fabric care compositions of the present invention are
soaps and polycarboxylates.
s 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
o their salts, in which the polycarboxylic acid comprises at least two
carboxyl
radicals separated from each other by not more than two carbon atoms.
Polymers of this type are disclosed in GB-A-1,596,756. Examples of such salts
are polyacrylates of MW 2000-5000 and their copolymers with malefic anhydride,
such copolymers having a molecular weight of from 20,000 to 70,000, especially
~5 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.
Conventional detergent enzymes
The laundry detergent and/or fabric care compositions can in addition to the
mannanase enzyme further comprise one or more enzymes which provide
cleaning performance, fabric care and/or sanitisation benefits. The laundry
detergent and/or fabric care compositions of the present invention will
preferably
further comprise a cellulase. It has been surprisingly found that said
compositions further comprising a cellulase, provide improved cleaning and
3o softening performance.
Said enzymes include enzymes selected from cellufases, hemicellulases,
peroxidases, proteases, gluco-amylases, amylases, xylanases, lipases,
phospholipases, esterases, cutinases, pectinases, keratanases, reductases,
oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases,
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pentosanases, malanases, (3-glucanases, arabinosidases, hyaluronidase,
chondroitinase, laccase or mixtures thereof.
A preferred combination is a laundry detergent and/or fabric care composition
5 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.
Suitable proteases are the subtilisins which are obtained from particular
strains of
o B. subtilis and B. lichenifonnis (subtilisin BPN and BPN'). One suitable
protease
is obtained from a strain of Bacillus, having maximum activity throughout the
pH
range of 8-12, developed and sold as ESPERASE~ by Novo Industries A/S of
Denmark, hereinafter "Novo". The preparation of this enzyme and analogous
enzymes is described in GB 1,243,784 to Novo. Other suitable proteases
~5 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 described in European
Patent
Application Serial Number 87 303761.8, filed April 28, 1987 (particularly
pages
20 17, 24 and 98), and which is called herein "Protease B", and in European
Patent
Application 199,404, Venegas, published October 29, 1986, which refers to a
modified bacterial serine protealytic enzyme which is called "Protease A"
herein.
Suitable is the protease called herein "Protease C", which is a variant of an
alkaline serine protease from Bacillus in which lysine replaced arginine at
25 position 27, tyrosine replaced valine at position 104, serine replaced
asparagine
at position 123, and alanine replaced threonine at position 274. Protease C is
described in EP 90915958:4, corresponding to WO 91/06637, Published May 16,
1991. Genetically modified variants, particularly of Protease C, are also
included
herein.
3o A preferred protease referred to as "Protease D" is a carbonyl hydrolase
variant
having an amino acid sequence not found in nature, which is derived from a
precursor carbonyl hydrolase by substituting a different amino acid for a
plurality
of amino acid residues at a position in said carbonyl hydrolase equivalent to
position +76, preferably also in combination with one or more amino acid
residue
35 positions equivalent to those selected from the group consisting of +gg,
+101,
+103, +104, +107, +123, +27, +105, +109, +126, +128, +135, +156, +166, +195,
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WO 99/09129 PCT/US98/12015
51
+197, +204, +206, +210, +216, +217, +218, +222, +260, +265, and/or +274
according to the numbering of Bacillus amyloliquefaciens subtilisin, as
described
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,
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, +209, +215,
+217, +218, and +222, where the numbered position corresponds to naturally
occurring subtilisin from Bacillus amyloliquefaciens or to equivalent amino
acid
residues in other carbonyl hydrolases or subtilisins, such as Bacillus lentus
subtilisin (co-pending patent application US Serial No. 60/048,550, filed June
04,
~ 5 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.
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 &
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 laundry detergent and/or
fabric
care compositions of the present invention a level of from 0.0001 % to 2%,
preferably from 0.001 % to 0.2%, more preferably from 0.005% to 0.1 % pure
3o enzyme by weight of the composition.
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
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52
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
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
o amino acids; and a -43kD endoglucanase derived from Humicola insolens, DSM
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,
~5 1994. Especially suitable cellulases are the cellulases having color care
benefits.
Examples of such cellulases are cellulases described in European patent
application No. 91202879.2, filed November 6, 1991 (Novo). Carezyme and
Celluzyme (Novo Nordisk A/S) are especially useful. See also W091I17244 and
W091/21801. Other suitable cellulases for fabric care and/or cleaning
properties
2o are described in W096/34092, W096/17994 and W095/24471.
Said cellulases are normally incorporated in the laundry detergent andlor
fabric
care composition at levels from 0.0001 % to 2% of pure enzyme by weight of the
laundry detergent and/or fabric care composition.
2s Peroxidase enzymes are used in combination with oxygen sources, e.g.
percarbonate, perborate, persulfate, hydrogen peroxide, etc and with a
phenolic
substrate as bleach enhancing molecule. They are used for "solution
bleaching",
i.e. to prevent transfer of dyes or pigments removed from substrates during
wash
operations to other substrates in the wash solution. Peroxidase enzymes are
3o known in the art, and include, for example, horseradish peroxidase,
ligninase and
haloperoxidase such as chloro- and bromo-peroxidase. Peroxidase-containing
laundry detergent and/or fabric care compositions are disclosed, for example,
in
PCT International Application WO 89/099813, W089/09813 and in European
Patent application EP No. 91202882.6, filed on November 6, 1991 and EP No.
35 96870013.8, filed February 20, 1996. Also suitable is the lactase enzyme.
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53
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.
Said peroxidases are normally incorporated in the laundry detergent and/or
fabric
care composition at levels from 0.0001% to 2% of pure enzyme by weight of the
laundry detergent and/or fabric care composition.
Other preferred enzymes that can be included in the laundry detergent and/or
fabric care compositions of the present invention include lipases. Suitable
lipase
enzymes for detergent usage include those produced by microorganisms of the
~5 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 Pseudomonas fluorescent IAM 1057. This lipase is available from
Amano Pharmaceutical Co. Ltd., Nagoya, Japan, under the trade name Lipase P
20 "Amano," hereinafter referred to as "Amano-P". Other suitable commercial
lipases include Amano-CES, lipases ex Chromobacter viscosum, e.g.
Chromobacfer viscosum var, lipolyticum NRRLB 3673 from Toyo Jozo Co.,
Tagata, Japan; Chromobacfer viscosum lipases from U.S. Biochemical Corp.,
U.S.A. and Disoynth Co., The Netherlands, and lipases ex Pseudomonas
25 gladioli. Especially suitable lipases are lipases such as M1 LipaseR and
LipomaxR (Gist-Brocades) and LipolaseR and Lipolase UItraR(Novo) which have
found to be very effective when used in combination with the compositions of
the
present invention. Also suitables are the lipolytic enzymes described in EP
258
068, WO 92/05249 and WO 95/22615 by Novo Nordisk and in WO 94/03578,
3o WO 95/35381 and WO 96100292 by Unilever.
Also suitable are cutinases [EC 3.1.1.50] which can be considered as a special
kind of lipase, namely lipases which do not require interfacial activation.
Addition
of cutinases to detergent compositions have been described in e.g. WO-A
88/09367 (Genencor); WO 90/09446 (Plant Genetic System) and WO 94/14963
3s and WO 94/14964 (Unilever).
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The lipases andlor cutinases are normally incorporated in the laundry
detergent
and/or fabric care composition at levels from 0.0001 % to 2% of pure enzyme by
weight of the laundry detergent and/or fabric care composition.
s Amylases (a and/or a) 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
W095110603, Novo Nordisk A/S, published April 20, 1995. Other amylases
known for use in detergent compositions include both a- and ~i-amylases. a-
Amylases are known in the art and include those disclosed in US Pat. no.
5,003,257; EP 252,666; W0/91/00353; FR.2,676,456; EP 285,123; EP 525,610;
EP 368,341; and British Patent specification no. 1,296,839 (Novo). Other
suitable
amylases are stability-enhanced amylases described in W094/18314, published
August 18, 1994 and W096/05295, Genencor, published February 22, 1996 and
~5 amylase variants having additional modification in the immediate parent
available
from Novo Nordisk A/S, disclosed in WO 95/10603, published April 95. Also
suitable are amylases described in EP 277 216, W095/26397 and W096/23873
(all by Novo Nordisk).
Examples of commercial a-amylases products are Purafect Ox Am~ from
2o Genencor and Termamyl~, Ban~ ,Fungamyl~ and Duramyi~, all available from
Novo Nordisk A/S Denmark. W095/26397 describes other suitable amylases : a
amylases characterised by having a specific activity at least 25% higher than
the
specific activity of Termamyl~ at a temperature range of 25°C to
55°C and at a
pH value in the range of 8 to 10, measured by the Phadebas~ a-amylase activity
2s assay. Suitable are variants of the above enzymes, described in W096/23873
(Novo Nordisk). Other amylolytic enzymes with improved properties with respect
to the activity level and the combination of thermostability and a higher
activity
level are described in W095/35382.
The amylolytic enzymes are incorporated in the laundry detergent and/or fabric
3o care 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,
3s animal, bacterial, fungal and yeast origin. Origin can further be
mesophilic or
extremophilic (psychrophilic, psychrotrophic, thermophilic, barophilic,
alkalophilic,
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acidophilic, halophilic, etc.). Purified or non-purified forms of these
enzymes may
be used. Nowadays, it is common practice to modify wild-type enzymes via
protein I genetic engineering techniques in order to optimise their
performance
efficiency in the detergent compositions of the invention. For example, the
5 variants may be designed such that the compatibility of the enzyme to
commonly
encountered ingredients of such compositions is increased. Alternatively, the
variant may be designed such that the optimal pH, bleach or chelant stability,
catalytic activity and the like, of the enzyme variant is tailored to suit the
particular cleaning application.
In particular, attention should be focused on amino acids sensitive to
oxidation in
the case of bleach stability and on surface charges for the surfactant
compatibility. The isoelectric point of such enzymes may be modified by the
substitution of some charged amino acids, e.g. an increase in isoelectric
point
may help to improve compatibility with anionic surfactants. The stability of
the
enzymes may be further enhanced by the creation of e.g. additional salt
bridges
and enforcing calcium binding sites to increase chelant stability. Special
attention
must be paid to the cellulases as most of the cellulases have separate binding
domains (CBD). Properties of such enzymes can be altered by modifications in
2o these domains.
Said enzymes are normally incorporated in the laundry detergent andlor fabric
care composition at levels from 0.0001 % to 2% of pure enzyme by weight of the
laundry detergent and/or fabric care 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 ).
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
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56
et al, July 18, 1978, and in U.S. 4,507,219, Hughes, March 26, 1985. Enzyme
materials useful for liquid detergent formulations, and their incorporation
into
such formulations, are disclosed in U.S. 4,261,868, Hora et al, April 14,
1981.
Enzymes for use in detergents can be stabilised by various techniques. Enzyme
stabilisation techniques are disclosed and exemplified in U.S. 3,600,319,
August
17, 1971, Gedge et al, EP 199,405 and EP 200,586, 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.
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
2o 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 laundry detergent and/or
fabric care compositions in accordance with the present invention. These
agents
may be inorganic or organic in type. Organic fabric softening agents include
the
water insoluble tertiary amines as disclosed in GB-A1 514 276 and EP-BO 011
340 and their combination with mono C12-C14 quaternary ammonium salts are
3o 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.
Organic fabric softening agents such as the water-insoluble tertiary amines or
dilong chain amide materials are incorporated at levels of from 0.5% to 5% by
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57
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 instances it may be more convenient to add them as a dry
mixed particulate, or spray them as molten liquid on to other solid components
of
the composition.
Chelating Agents
The laundry detergent and/or fabric care compositions herein may also
optionally
contain one or more iron 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
2o formation of soluble chelates.
Amino carboxylates useful as optional chelating agents include
ethylenediaminetetracetates, N-hydroxyethylethylenediaminetriacetates, 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. Preferably, 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
3s dihydroxydisulfobenzenes such as 1,2-dihydroxy-3,5-disulfobenzene.
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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.
ff utilized, these chelating agents will generally comprise from about 0.1 %
to
1o about 15% by weight of the laundry detergent and/or fabric care
compositions
herein. More preferably, if utilized, the chelating agents will comprise from
about
0.1 % to about 3.0% by weight of such compositions.
Suds suppressor
Another optional ingredient is a suds suppressor, exemplified by silicones,
and
silica-silicone mixtures. Silicones can be generally represented by alkylated
polysiloxane materials while silica is normally used in finely divided forms
exemplified by silica aerogels and xerogels and hydrophobic silicas of various
2o types. These materials can be incorporated as particulates in which the
suds
suppressor is advantageously releasably incorporated in a water-soluble or
water-dispersible, substantially non-surface-active detergent impermeable
carrier. Alternatively the suds suppressor can be dissolved or dispersed in a
liquid carrier and applied by spraying on to one or more of the other
components.
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
3o copolymer. Especially preferred suds controlling agent are the suds
suppressor
system comprising a mixture of silicone oils and 2-alkyl-alcan8ls. 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
application N 92870174.7 filed 10 November, 1992.
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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 laundry detergent and/or fabric care compositions
may be employed, such as soil-suspending agents, soil-release agents, optical
brighteners, abrasives, bactericides, tarnish inhibitors, coloring agents,
and/or
encapsulated or non-encapsulated perfumes.
Especially suitable encapsulating materials are water soluble capsules which
consist of a matrix of polysaccharide and polyhydroxy compounds such as
described in GB 1,464,616. Other suitable water soluble encapsulating
materials
2o comprise dextrins derived from ungelatinized starch acid-esters of
substituted
dicarboxylic acids such as described in US 3,455,838. These acid-ester
dextrins
are,preferably, prepared from such starches as waxy maize, waxy sorghum,
sago, tapioca and potato. Suitable examples of said encapsulating materials
include N-Lok manufactured by National Starch. The N-Lok encapsulating
material consists of a modified maize starch and glucose. The starch is
modified
by adding monofunctional substituted groups such as octenyl succinic acid
anhydride.
Antiredeposition and soil suspension agents suitable herein include cellulose
so derivatives such as methylcellulose, carboxymethylcellulose and
hydroxyethyl-
cellulose, and homo- or co-polymeric polycarboxylic 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
materials are normally used at levels of from 0.5% to 10% by weight, more
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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
5 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-
0 2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2' - disulphonate, 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-hyd~oxyethylamino)-s-triazin-6- ylami-
no)stilbene-
2,2'disulphonate, sodium 2(stilbyl-4"-(naphtho-1',2':4,5)-1,2,3 - triazole-2"-
sulphonate and 4,4'-bis(2-sulphostyryl)biphenyl. Highly preferred brighteners
are
~5 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
2o preferably from 0.25% to 2.5% by weight. These polymers and the previously
mentioned homo- or co-polymeric polycarboxylate salts are valuable for
improving whiteness maintenance, fabric ash deposition, and cleaning
performance on clay, proteinaceous and oxidizable soils in the presence of
transition metal impurities.
Soil release agents useful in compositions of the present invention are
conventionally copolymers or terpolymers of terephthalic acid with ethylene
glycol
and/or propylene glycol units in various arrangements. Examples of such
polymers are disclosed in the commonly assigned US Patent Nos. 4116885 and
4711730 and European Published Patent Application No. 0 272 033. A particular
preferred polymer in accordance with EP-A-0 272 033 has the formula
(CH3(PEG)43)0.75(POH)0.25IT-PO)2.8(T-PEG)0.41T(PO
H)0.25((PEG)43CH3)0.75
where PEG is -(OC2H4)O-,PO is (OC3Hg0) and T is (pcOCgH4C0).
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Also very useful are modified polyesters as random copolymers of dimethyl
terephthalate, dimethyl sulfoisophthalate, ethylene glycol and 1-2 propane
diol,
the end groups consisting primarily of sulphobenzoate and secondarily of mono
esters of ethylene glycol and/or propane-diol. The target is to obtain a
polymer
capped at both end by sulphobenzoate groups, "primarily", in the present
context
most of said copolymers herein will be end-capped by sulphobenzoate groups.
However, some copolymers will be less than fully capped, and therefore their
end
groups may consist of monoester of ethylene glycol and/or propane 1-2 diol,
o 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
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
2o scavenger such as perborate, ammonium sulfate, sodium sulphite or
polyethyleneimine at a level above 0.1 % by weight of total composition, in
the
formulas will provide improved through the wash stability of the detergent
enzymes. Compositions comprising chlorine scavenger are described in the
European patent application 92870018.6 filed January 31, 1992.
Alkoxylated polycarboxylates such as those prepared from polyacrylates are
useful herein to provide additional grease removal performance. Such materials
are described in WO 91/08281 and PCT 90/01815 at p. 4 et seq., incorporated
herein by reference. Chemically, these materials comprise polyacrylates having
one ethoxy side-chain per every 7-8 acrylate units. The side-chains are of the
formula -{CH2CH20)m(CH2)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.
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Dfspersants
The laundry detergent and/or fabric care composition of the present invention
can also contain dispersants : Suitable water-soluble organic salts are the
homo-
or co-polymeric acids or their salts, in which the polycarboxylic acid
comprises at
least two carboxyl radicals separated from each other by not more than two
carbon atoms. Polymers of this type are disclosed in GB-A-1,596,756. 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 laundry detergent and/or fabric care compositions of the
present
invention.
The compositions of the invention may contain a lime soap peptiser compound,
which has preferably a lime soap dispersing power (LSDP), as defined
hereinafter of no more than 8, preferably no more than 7, most preferably no
more than 6. The lime soap peptiser compound is preferably present at a level
2o from 0% to 20% by weight.
A numerical measure of the effectiveness of a lime soap peptiser is given by
the
lime soap dispersant power (LSDP) which is determined using the lime soap
dispersant test as described in an article by H.C. Borghetty and C.A. 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.
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63
Exemplary surfactants having a LSDP of no more than 8 for use in accord with
the present invention include C1g-C1g dimethyl amine oxide, C12-C1g alkyl
ethoxysulfates with an average degree of ethoxylation of from 1-5,
particularly
C12-C15 alkyl ethoxysulfate surtactant with a degree of ethoxylation of amount
3
(LSDP=4), and the C14-C15 ethoxylated alcohols with an average degree of
ethoxylation of either 12 (LSDP=6) or 30, sold under the tradenames Lutensol
A012 and Lutensol A030 respectively, by BASF GmbH.
o Polymeric lime soap peptisers suitable for use herein are described in the
article
by M.K. Nagarajan, W.F. Masler, to be found in Cosmetics and Toiletries,
volume
104, pages 71-73, (1989).
Hydrophobic bleaches such as 4-[N-octanoyl-6-aminohexanoyl]benzene
~5 sulfonate, 4-[N-nonanoyl-6-aminohexanoyl]benzene sulfonate, 4-(N-decanoyl-6-
aminohexanoyl]benzene sulfonate and mixtures thereof; and nonanoyloxy
benzene sulfonate together with hydrophilic / hydrophobic bleach formulations
can also be used as lime soap peptisers compounds.
2o Dye transfer inhibition
The laundry detergent and/or fabric care 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
25 operations involving colored fabrics.
Polymeric dye transfer inhibiting agents
The laundry detergent and/or fabric care compositions according to the present
3o 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 laundry detergent and/or fabric care compositions in order to inhibit the
transfer of dyes from colored fabrics onto fabrics washed therewith. These
35 polymers have the ability to complex or adsorb the fugitive dyes washed out
of
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64
dyed fabrics before the dyes have the opportunity to become attached to other
articles in the wash.
Especially suitable polymeric dye transfer inhibiting agents are polyamine N-
oxide
polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole,
polyvinylpyrrolidone polymers, polyvinyloxazoiidones and polyvinylimidazoles
or
mixtures thereof.
Addition of such polymers also enhances the performance of the enzymes
according the invention.
a) Polyamine N-oxide polymers
The polyamine N-oxide polymers suitable for use contain units having the
following structure formula
P
I
(I) Ax
I
R
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
II II li
A is NC; CO, C, -O-,-S-, -N- ; x is O or 1;
3o R are aliphatic, ethoxylated aliphatics, aromatic, heterocyclic or
alicyclic groups
or any combination thereof whereto the nitrogen of the N-O group can be
attached or wherein the nitrogen of the N-O group is part of these groups.
The N-O group can be represented by the following general structures
O O
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(R1)x -N- (R2)y =N- (R1)x
(R3)z
5
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
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.
Suitable polyamine N-oxides wherein the N-O group forms part of the
polymerisable unit comprise poiyamine 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.
Preferred poiyamine N-oxides are those wherein R is a heterocyclic group such
as pyrridine, pyrrole, imidazole, pyrrolidine, piperidine, quinoline, acridine
and
2o 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.
Examples of these classes are polyamine oxides wherein R is a heterocyclic
so 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.
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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
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,
o 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
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
~5 < 10, preferably PKa < 7, more preferred PKa < 6.
The polyamine oxides can be obtained in almost any degree of polymerisation.
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;
2o 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
25 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-
vinylpyrrolidone copolymers wherein said polymer has an average molecular
3o 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".
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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
o molar ratio of N-vinylimidazole to N-vinylpyrrolidone from 1 to 0.2, more
preferably from 0.8 to 0.3, most preferably from 0.6 to 0.4 .
c) Polyvinylpyrrolidone
The laundry detergent and/or fabric care compositions of the present invention
~5 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,
2o Canada under the product names PVP K-15 (viscosity molecular weight of
10,000), PVP K-30 (average molecular weight of 40,000), PVP K-60 (average
molecular weight of 160,000), and PVP K-90 {average molecular weight of
360,000). Other suitable polyvinylpyrrolidones which are commercially
available
from BASF Cooperation include Sokalan HP 165 and Sokalan HP 12;
25 polyvinylpyrrolidones known to persons skilled in the detergent field (see
for
example EP-A-262,897 and EP-A-256,696).
d) Polyvinyloxazolidone
The laundry detergent and/or fabric care compositions of the present invention
3o may also utilize polyvinyloxazolidone as a polymeric dye transfer
inhibiting agent.
Said polyvinyloxazolidones have an average molecular weight of from about
2,500 to about 400,000, preferably from about 5,000 to about 200,000, more
preferably from about 5,000 to about 50,000, and most preferably from about
5,000 to about 15,000.
e) Polyvinylimidazole
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The laundry detergent and/or fabric care compositions of the present invention
may also utilize polyvinylimidazole as polymeric dye transfer inhibiting
agent.
Said polyvinylimidazoles have an average about 2,500 to about 400,000,
preferably from about 5,000 to about 200,000, more preferably from about 5,000
to about 50,000, and most preferably from about 5,000 to about 15,000.
f) Cross-linked polymers
Cross-linked polymers are polymers whose backbone are interconnected to a
certain degree; these links can be of chemical or physical nature, possibly
with
active groups n the backbone or on branches; cross-linked polymers have been
described in the Journal of Polymer Science, volume 22, pages 1035-1039. In
one embodiment, the cross-linked polymers are made in such a way that they
form a three-dimensional rigid structure, which can entrap dyes in the pores
formed by the three-dimensional structure. In another embodiment, the cross-
~ 5 linked polymers entrap the dyes by swelling. Such cross-linked polymers
are
described in the co-pending patent application 94870213.9
2o Method of washins~
The compositions of the invention may be used in essentially any washing or
cleaning methods, including soaking methods, pretreatment methods and
methods with rinsing, steps for which a separate rinse aid composition may be
2s added.
The process described herein comprises contacting fabrics with a laundering
solution in the usual manner and exemplified hereunder. The process of the
invention is conveniently carried out in the course of the laundering process.
The
3o 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.
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
35 the invention. In the laundry detergent and/or fabric care compositions,
the
enzymes levels are expressed by pure enzyme by weight of the total composition
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69
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 sulphonate.
TAS : Sodium tallow alkyl sulphate.
CxyAS : Sodium C1x - C1y alkyl sulfate.
CxySAS : Sodium C1x - C1y secondary (2,3) alkyl sulfate.
CxyEz : C1x - C1y predominantly linear primary alcohol
condensed with an average of z moles of ethylene
oxide.
CxyEzS : C1x - C1y sodium alkyl sulfate condensed
with an
average of z moles of ethylene oxide.
QAS : R2.N+(CH3)2(C2H4OH) with R2 = C12-C14~
QAS 1 : R2.N+(CH3)2(C2H4OH) with R2 = Cg-C11.
APA : Cg-10 amido propyl dimethyl amine.
Soap : Sodium linear alkyl carboxylate derived
from a 80/20
mixture of tallow and coconut fatty acids.
Nonionic : C13-C15 mixed ethoxylated/propoxylated fatty
alcohol
with an average degree of ethoxylation of
3.8 and an
average degree of propoxylation of 4.5.
Neodol 45-13 : C14-C15 linear primary alcohol ethoxylate,
sold by Shell
Chemical CO.
STS : Sodium toluene sulphonate.
CFAA : C12-C14 alkyl N-methyl glucarnide.
TFAA : C16-C1g alkyl N-methyl glucarnide.
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
S-Na2Si205,
Citrate : Tri-sodium citrate dehydrate of activity
86.4% with a
particle size distribution between 425 and
850
micrometres.
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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.
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.
NOBS : Nonanoyloxybenzene sulfonate in the form
of the sodium
salt.
NACA-OBS : (6-nonamidocaproyl) oxybenzene sulfonate.
DTPA : Diethylene triamine pentaacetic acid.
HEDP : 1,1-hydroxyethane diphosphonic acid.
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DETPMP : Diethyltriamine yenta (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(II1) salt.
Paraffin : Paraffin oil sold under the tradename Winog
70 by
Wntershall.
NaBz : Sodium benzoate.
BzP : Benzoyl Peroxide.
Mannanase : Mannanase from Bacillus agaradherens, NCIMB
40482
Protease : Proteolytic enzyme sold under the tradename
Savinase,
Alcalase, Durazym by Novo Nordisk A/S, Maxacal,
Maxapem sold by Gist-Brocades and proteases
described in patents W091/06637 and/or W095/10591
and/or EP 251 446.
Amylase : Amylolytic enzyme sold under the tradename
Purafact
Ox AmR described in WO 94/18314, W096/05295
sold
by Genencor; Termamyl~, Fungamyl~ and Duramyl~,
all available from Novo Nordisk A/S and those
described
in W095/26397.
Lipase : Lipolytic enzyme sold under the tradename
Lipolase,
Lipolase Ultra by Novo Nordisk A/S and Lipomax
by
Gist-Brocades.
Cellulase : Cellulytic enzyme sold under the tradename
Carezyme,
Celluzyme and/or Endolase by Novo Nordisk A/S.
Clay : Smectite clay or bentonite clay.
CMC : Sodium carboxymethyl cellulose.
PVP : Polyvinyl polymer, with an average molecular
weight of
60,000.
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PVNO : Polyvinylpyridine-N-Oxide, with an average
molecular
weight of 50,000.
PVPVI : Copolymer of vinylimidazole and vinylpyrrolidone,
with an
average molecular weight of 20,000.
Brightener 1 : Disodium 4,4'-bis(2-sulphostyryl)biphenyl.
Brightener 2 : Disodium 4,4'-bis(4-anilino-6-morpholino-1.3.5-triazin-2-
yl) stilbene-2:2'-disulfonate.
Silicone antifoam: Polydimethylsiloxane foam controller with
siloxane-
oxyalkylene copolymer as dispersing agent
with a ratio of
said foam controller to said dispersing agent
of 10:1 to
100:1.
Suds Suppressor : 12% Silicone/silica, 18% stearyl alcoho1,70%
starch in
granular form.
Opacifier : Water based monostyrene latex mixture,
sold by BASF
Aktiengesellschaft under the tradename Lytron
621.
SRP 1 : Anionically end capped poly esters.
SRP 2 : Diethoxylated poly (1,2 propylene terephthalate)
short
block polymer.
QEA : bis((C2H50)(C2H40)n)(CH3) -N+-C6H12-N+-(CH3)
bis((C2H50)-(C2H40))n, wherein n = from 20
to 30.
PEI : Polyethyleneimine with an average molecular
weight of
1800 and an average ethoxylation degree of
7
ethyleneoxy residues per nitrogen.
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.
Example 1
The following detergent compositions were prepared according to the present
invention
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Blown Powder
Zeolite A 13.0 13.0 15.0
Sulfate - 3.0 -
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 1.0 1.0
Silicate 3.0 2.0 4.0
Zeolite A 8.0 8.0 8.0
Carbonate 7.0 4.0 4.0
Spray On
Perfume 0.3 0.3 0.3
C45E7 2.0 2.0 2.0
C25E3 2.0 - -
Dry Additives
Citrate 3.0 - 2.0
Bicarbonate - 3.0 -
Carbonate 8.0 15.0 10.0
TAED fi.0 2.0 5.0
PB1 9.0 7.0 10.0
PEO - - 0.2
Bentonite clay 10.0 10.0 10.0
Mannanase 0.001 0.001 0.02
Protease 0.03 0.03 0.03
Lipase 0.008 0.008 0.008
Cellulase 0.001 0.001 0.001
Amylase 0.01 0.01 0.01
Silicone antifoam 5.0 5.0 5.0
Sulfate - 3.0 -
Density (g/litre) 850 850 850
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Miscellaneous and minors Up to 100%
Example 2
The following liquid detergent compositions were prepared according to the
present invention (Levels are given in parts per weight, enzyme are expressed
in
pure enzyme)
I II III IV
LAS 25.0 - - -
C25AS - 13.0 16.0 13.0
C25E3S - 2.0 2.0 4.0
C25E7 - - 4.0 4.0
TFAA - 6.0 6.0 6.0
APA 3.0 1.0 2.0 -
TPKFA - 14.0 11.0 11.0
Citric 1.0 1.0 1.0 1.0
Dodecenyl / tetradecenyl 15.0 - - -
succinic
acid
Rapeseed fatty acid 1.0 - 3.5 -
Ethanol 7.0 2.0 3.0 2.0
1,2 Propanediol 6.0 8.0 10.0 13.0
Monoethanolamine - - ~ 9.0 9.0
TEPAE - - 0.4 0.3
DETPMP 2.0 1.2 1.0 -
Mannanase 0.001 0.002 0.02 0.001
Protease 0.08 0.02 0.01 0.02
Lipase - - 0.003 0.003
Amylase 0.004 0.01 0.01 0.01
Cellulase - - 0.004 0.003
SRP 2 - - 0.2 0.1
Boric acid 1.0 1.5 2.5 2.5
Bentonite clay 4.0 5.0 4.0 4.0
Brightener 1 0.1 0.2 0.3 -
Suds suppressor 0.4 - - -
Opacifier 0.8 0.7 - -
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I II III IV
NaOH up to pH 8.0 7.5 8.0 8.2
Miscellaneous and water
Example 3
5 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 - 5.0
Coco-alkyl-dimethyl hydroxy-1.4 1.0
ethyl ammonium chloride
Citrate 5.0 3.0
Na-SKS-6 - 11.0
Zeolite A 15.0 15.0
MA/AA 4.0 4.0
DETPMP 0.4 0.4
PB1 15.0 -
Percarbonate - 15.0
TAED 5.0 5,0
Smectite clay 10.0 10.0
HMWPEO - 0.1
Mannanase 0.001 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
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Miscellaneous and minors Up to 100%
Example 4
The following laundry bar detergent compositions were prepared according to
the
present invention (Levels are given in parts per weight, enzyme are expressed
in
pure enzyme)
I II III VI V III VI V
LAS - - 19.0 15.0 21.0 6.75 8.8 -
C28AS 26.0 13.5 - - - 15.75 11.2 22.5
Na Laurate 2.5 9.0 - - - - -
Zeolite A 2.0 1.25 - - - 1.25 1.25 1.25
Carbonate 20.0 3.0 13.0 8.0 10.0 15.0 13.0 8.0
Ca Carbonate 27.5 39.0 31.0 - - 36.0 - 36.0
Sulfate 5.0 5.0 3.0 5.0 3.0 - - 5.0
TSPP 5.0 - _ _ - 5.0 2.5
STPP 5.0 15.0 10.0 - - 7.0 8.0 10.0
Bentonite 4.0 10.0 4.0 4.0 5.0 4.0 4.0 4.0
clay
DETPMP - 0.7 0.6 - 0.6 0.7 0.7 0.7
CMC - 1.0 1.0 1.0 1.0 - - 1.0
Talc - - 10.0 11.0 10.0 - - -
Silicate - - 4.0 5.0 3.0 - - -
PVNO 0.02 0.03 - 0.01 - 0.02 - -
MA/AA 0.4 1.0 - - 0.2 0.4 0.5 0.4
SRP 1 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3
Mannanase 0.001 0.001 0.02 0.001 0.02 0.03 0.01 0.001
Amylase - - 0.01 - - - 0.002 -
Protease - 0.004 - 0.003 0.003 - - 0.003
Lipase - 0.002 - 0.002 - - - -
Cellulase - .0003 - - .0003 .0002 - -
PEO - 0.2 - 0.2 0.3 - - 0.3
Pertume 1.0 0.5 0.3 0.2 0.4 - - 0.4
Mg sulfate - - 3.0 3.0 3.0 - - -
Brightener 0.15 0.1 0.15 - - - - 0.1
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II III VI V III VI V
Photoactivated - 15.0 15.0 15.0 15.0 - - 15.0
bleach (ppm)
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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 a
clay
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
ao MOLECULE TYPE: gencmic DNA
ORIGINAL SOURCE
FEATURE:
NAME/KEY: CDS
LOCATION:1-1482
SEQUENCE DESCRIPTION: SEQ ID NO: 1
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WO 99/09129 PCT/US98/12015
79
ATGAAAAAAAAGTTATCACAGATTfATCATTTAATTATTTGCACACTTATAATA
AGTGTGGGAATAATGGGGATTACAACGTCCCCATCAGCAGCAAGTACAGGC
TTTTATGTTGATGGCAATACGTTATATGACGCAAATGGGCAGCCATTTGTCAT
GAGAGGTATTAACCATGGACATGCTTGGTATAAAGACACCGCTTCAACAGCT
ATTCCTGCCATTGCAGAGCAAGGCGCCAACACGATTCGTATTGTTTTATCAG
ATGGCGGTCAATGGGAAAAAGACGACATTGACACCATTCGTGAAGTCATTG
AGCTTGCGGAGCAAAATAAAATGGTGGCTGTCGTTGAAGTTCATGATGCCA
CGGGTCGCGATTCGCGCAGTGATTTAAATCGAGCCGTTGATTATTGGATAG
1 o AAATGAAAGATGCGCTTATCGGTAAAGAAGATACGGTTATTATTAACATTGCA
AACGAGTGGTATGGGAGTTGGGATGGCTCAGCTTGGGCCGATGGCTATATT
GATGTCATTCCGAAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTG
ATGCAGCAGGATGGGGGCAATATCCGCAATCTATTCATGATTACGGACAAG
ATGTGTTTAATGCAGATCCGTTAAAAAATACGATGTTCTCCATCCATATGTAT
GAGTATGCTGGTGGTGATGCTAACACTGTTAGATCAAATATTGATAGAGTCA
TAGATCAAGACCTTGCTCTCGTAATAGGTGAATTCGGTCATAGACATACTGA
TGGTGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACA
GGGTGGCTCGCTTGGTCTTGGAAAGGCAACAGTACCGAATGGGACTATTTA
GACCTTTCAGAAGACTGGGCTGGTCAACATTTAACTGATTGGGGGAATAGAA
2o TTGTCCACGGGGCCGATGGCTTACAGGAAACCTCCAAACCATCCACCGTAT
TTACAGATGATAACGGTGGTCACCCTGAACCGCCAACTGCTACTACCTTGTA
TGACTTTGAAGGAAGCACACAAGGGTGGCATGGAAGCAACGTGACCGGTG
GCCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAAGC
CGATGTAAATTTAACCTCAAATTCTTCACATGAACTGTATAGTGAACAAAGTC
GTAATCTACACGGATACTCTCAGCTCAACGCAACCGTTCGCCATGCCAATTG
GGGAAATCCCGGTAATGGCATGAATGCAAGACTTTACGTGAAAACGGGCTC
TGATTATACATGGCATAGCGGTCCTTTTACACGTATCAATAGCTCCAACTCA
GGAACAACGTTATCTTTTGATTTAAACAACATCGAAAATAGTCATCATGTTAG
GGAAATAGGCGTGCAATTTTCAGCGGCAGATAATAGCAGTGGTCAAACTGC
3o TCTATACGTTGATAACGTTACTTTAAGATAG
SEQ ID N0:2
SEQUENCE CHARACTERISITICS:
LENGTH: 493 amino acids
CA 02301200 2000-02-11
WO 99/09129 PCT1US98/12015
TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: protein
5 SEQUENCE DESCRIPTION: SEQ ID NO: 2
MKKKLSQIYHLIICTLIISVGIMGITTSPSAASTGFYVDGNTLYDANGQPFVMRGIN
HGHAWYKDTASTAIPAIAEQGANTIRIVLSDGGQWEKDDIDTIREVIELAEQNKM
VAWEVHDATGRDSRSDLNRAVDYWIEMKDALIGKEDTVIINIANEWYGSWDGS
1o AWADGYIDVIPKLRDAGLTHTLMVDAAGWGQYPQSIHDYGQDVFNADPLKNTM
FSIHMYEYAGGDANTVRSNIDRVIDQDLALVIGEFGHRHTDGDVDEDTILSYSEE
TGTGWLAWSWKGNSTEWDYLDLSEDWAGQHLTDWGNRIVHGADGLQETSKP
STVFTDDNGGHPEPPTATTLYDFEGSTQGWHGSNVTGGPWSVTEWGASGNY
SLKADVNLTSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLYV
15 KTGSDYTWHSGPFTRINSSNSGTTLSFDLNNIENSHHVREIGVQFSAADNSSGQ
TALYVDNVTLR
SEQ ID N0:3
SEQUENCE CHARACTERISIT1CS:
LENGTH: 1407 base pairs
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: genomic DNA
SEQUENCE DESCRIPTION: SEQ ID NO: 3
ATGAAAAAAAAGTTATCACAGATTTATCATTTAATTATTTGCACACTTATAATA
AGTGTGGGAATAATGGGGATTACAACGTCCCCATCAGCAGCAAGTACAGGC
TTTTATGTTGATGGCAATACGTTATATGACGCAAATGGGCAGCCATTTGTCAT
GAGAGGTATTAACCATGGACATGCTTGGTATAAAGACACCGCTTCAACAGCT
ATTCCTGCCATTGCAGAGCAAGGCGCCAACACGATTCGTATTGTTTTATCAG
ATGGCGGTCAATGGGAAAAAGACGACATTGACACCATTCGTGAAGTCATTG
CA 02301200 2000-02-11
WO 99/09129 PCT/US98/12015
81
AGCTTGCGGAGCAAAATAAAATGGTGGCTGTCGTTGAAGTTCATGATGCCA
CGGGTCGCGATTCGCGCAGTGATTTAAATCGAGCCGTTGATTATTGGATAG
AAATGAAAGATGCGCTTATCGGTAAAGAAGATACGGTTATTATTAACATTGCA
AACGAGTGGTATGGGAGTTGGGATGGCTCAGCTTGGGCCGATGGCTATATT
GATGTCATTCCGAAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTG
ATGCAGCAGGATGGGGGCAATATCCGCAATCTATTCATGATTACGGACAAG
ATGTGTTTAATGCAGATCCGTTAAAAAATACGATGTTCTCCATCCATATGTAT
GAGTATGCTGGTGGTGATGCTAACACTGTTAGATCAAATATTGATAGAGTCA
TAGATCAAGACCTTGCTCTCGTAATAGGTGAATTCGGTCATAGACATACTGA
1o TGGTGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACA
GGGTGGCTCGCTTGGTCTTGGAAAGGCAACAGTACCGAATGGGACTATTTA
GACCTTTCAGAAGACTGGGCTGGTCAACATTTAACTGATTGGGGGAATAGAA
TTGTCCACGGGGCCGATGGCTTACAGGAAACCTCCAAACCATCCACCGTAT
TTACAGATGATAACGGTGGTCACCCTGAACCGCCAACTGCTACTACCTTGTA
TGACTTTGAAGGAAGCACACAAGGGTGGCATGGAAGCAACGTGACCGGTG
GCCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAAGC
CGATGTAAA'tTfAACCTCAAATTCTTCACATGAACTGTATAGTGAACAAAGTC
GTAATCTACACGGATACTCTCAGCTCAACGCAACCGTTCGCCATGCCAATTG
GGGAAATCCCGGTAATGGCATGAATGCAAGACTTTACGTGAAAACGGGCTC
2o TGATTATACATGGCATAGCGGTCCTTTTACACGTATCAATAGCTCCAACTCA
GGAACAACGTTATCTTTTGATTTAAACAACATCGAAAATATCATCATGTTAGG
GAAATAG
SEQ ID N0:4
SEQUENCE CHARACTERISITICS:
LENGTH: 468 amino acids
TYPE: amino acid
3o TOPOLOGY: linear
MOLECULE TYPE: protein
SEQUENCE DESCRIPTION: SEQ ID NO: 4
MKKKLSQIYHLIICTLIISVGIMGITTSPSAASTGFYVDGNTLYDANGQPFVMRGIN
HGHAWYKDTASTAIPAIAEQGANTIRIVLSDGGQWEKDDIDTIREVIELAEQNKM
CA 02301200 2000-02-11
WO 99/09129 PCT/US98/12015
82
VAWEVHDATGRDSRSDLNRAVDYWIEMKDALIGKEDTVIINIANEWYGSWDGS
AWADGYIDVIPKLRDAGLTHTLMVDAAGWGQYPQSIHDYGQDVFNADPLKNTM
FS1HMYEYAGGDANTVRSNIDRVIDQDLALVIGEFGHRHTDGDVDEDTILSYSEE
TGTGWLAWSWKGNSTEWDYLDLSEDWAGQHLTDWGNRIVHGADGLQETSKP
STVFTDDNGGHPEPPTATTLYDFEGSTQGWHGSNVTGGPWSVTEWGASGNY
SLKADVNLTSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLYV
KTGSDYTWHSGPFTRINSSNSGTTLSFDLNNIENIIMLGK
SEQ ID N0:5
SEQUENCE CHARACTERISITICS:
LENGTH: 1029 base pairs
TYPE: nucleic acid
~5 STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: genomic DNA
2o SEQUENCE DESCRIPTION SEQ ID No:5
5' AAT TGG CGC ATA CTG TGT CGC CTG TGA ATC CTA ATG CCC AGC
AGA CAA CAA AAA CAG TGA TGA ACT GGC TTG CGC ACC TGC CGA ACC
GAA CGG AAA ACA GAG TCC TTT CCG GAG CGT TCG GAG GTT ACA GCC
25 ATG ACA CAT TTT CTA TGG CTG AGG CTG ATA GAA TCC GAA GCG CCA
CCG GGC AAT CGC CTG CTA TTT ATG GCT GCG ATT ATG CCA GAG GAT
GGC TTG AAA CAG CAA ATA TTG AAG ATT CAA TAG ATG TAA GCT GCA
ACG GCG ATT TAA TGT CGT ATT GGA AAA ATG GCG GAA TTC CGC AAA
TCA GTT TGC ACC TGG CGA ACC CTG CTT TTC AGT CAG GGC ATT TTA
3o AAA CAC CGA TTA CAA ATG ATC AGT ATA AAA ACA TAT TAG ATT CAG
CAA CAG CGG AAG GGA AGC GGC TAA ATG CCA TGC TCA GCA AAA TTG
CTG ACG GAC TTC AAG AGT TGG AGA ACC AAG GTG TGC CTG TTC TGT
TCA GGC CGC TGC ATG AAA TGA ACG GCG AAT GGT TTT GGT GGG GAC
TCA CAT CAT ATA ACC AAA AGG ATA ATG AAA GAA TCT CTC TAT ATA
35 AAC AGC TCT ACA AGA AAA TCT ATC ATT ATA TGA CCG ACA CAA GAG
GAC TTG ATC ATT TGA TTT GGG TTT ACT CTC CCG ACG CCA ACC GAG
CA 02301200 2000-02-11
WO 99/09129 PCT/US98/i2015
83
ATT TTA AAA CTG ATT TTT ACC CGG GCG CGT CTT ACG TGG ATA TTG
TCG GAT TAG ATG CGT ATT TTC AAG ATG CCT ACT CGA TCA ATG GAT
ACG ATC AGC TAA CAG CGC TTA ATA AAC CAT TTG CTT TTA CAG AAG
TCG GCC CGC AAA CAG CAA ACG GCA GCT TCG ATT ACA GCC TGT TCA
TCA ATG CAA TAA AAC AAA AAT ATC CTA AAA CCA TTT ACT TTC TGG
CAT GGA ATG ATG AAT GGA GCG CAG CAG TAA ACA AGG GTG CTT CAG
CTT TAT ATC ATG ACA GCT GGA CAC TCA ACA AGG GAG AAA TAT GGA
ATG GTG ATT CTT TAA CGC CAA TCG TTG AGT GAA TCC GGG ATC 3'
SEQ ID N0:6
SEQUENCE CHARACTERISITICS:
LENGTH: 363 amino acids
TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: protein
2o SEQUENCE DESCRIPTION: SEQ ID NO: 6
ydhT 1
LFKKHTISLLIIFLLASAVLAKPIEAHTVSPVNPNAQQTTKTVMNWLAHL 50
ydhT 51
PNRTENRVLSGAFGGYSHDTFSMAEADRIRSATGQSPAIYGCDYARGWLE 100
ydhT 101
TANIEDSIDVSCNGDLMSYWKNGGIPQISLHLANPAFQSGHFKTPITNDQ 150
ydhT 151
YKNILDSATAEGKRLNAMLSKIADGLQELENQGVPVLFRPLHEMNGEWFW 200
3o ydhT 201
WGLTSYNQKDNERISLYKQLYKKIYHYMTDTRGLDHLIWVYSPDANRDFK 250
ydhT 251
TDFYPGASYVDIVGLDAYFQDAYSINGYDQLTALNKPFAFTEVGPQTANG 300
ydhT 301
SFDYSLFINAIKQKYPKTIYFLAWNDEWSAAVNKGASALYHDSWTLNKGE 350
ydhT 351
CA 02301200 2000-02-11
WO 99/09IZ9 PCT/US98/12015
84
iWNGDSLTPIVE*. 363
