Note: Descriptions are shown in the official language in which they were submitted.
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 29
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CA 02633668 2008-06-17
WO 2007/087318 PCT/US2007/001802
DETERGENT COMPOSITIONS
FIELD OF THE INVENTION
The present invention relates to detergent compositions, particularly laundry
detergents,
comprising lipolytic enzymes.
BACKGROUND OF THE INVENTION
Improved removal of greasy soils is a constant aim for detergent
manufacturers,
especially in the laundry context. In spite of the use of many effective
surfactants and
combinations of surfactants, especially when used at low water temperatures,
many surfactant-
based products still do not achieve complete removal of greasy/oily soils.
Lipase enzymes have
been used in detergents since the late 1980s for removal of fatty soils by
breakdown of fatty
soils into tri-glycerides.
Until relatively recently, the main commercially available lipase enzymes,
such as
Lipolase (trade name, Novozymes) worked particularly effectively at the lower
moisture levels
of the drying phase of the wash process. These enzymes tended to produce
significant cleaning
only in the second wash step with fat breakdown significant only on soils
remaining on
laundered clothes during the drying stage, the broken down fats then being
removed in the next
washing step. However, more recently, higher efficiency lipases have been
developed that also
work effectively during the wash phase of the cleaning process, so that as
well as cleaning in the
second washing step, a significant improvement in cleaning effect due to
lipase enzyme can be
found in the first wash-cycle. Examples of such enzymes are as described in US
6,939,702 B1,
W000/60063 and Research Disclosure IP6553D. Such enzymes are referred to below
as first
wash lipases.
In addition, consumers prefer that articles, such as garments, be as clean as
possible.
Such consumers typically associate the odor of a cleaned or treated article
with the degree of
cleanliness of such article. Thus, the effectiveness of a cleaning and/or
treatment composition,
from a consumer's perspective, is typically directly linked with the odor that
such composition
imparts to an article that is cleaned or treated with such composition.
Applicants recognized that
certain materials, such as esterases and lipases, can generate objectionable
fatty acid odors,
particularly short-chain fatty acid odors such as the odor of butyric acid.
However, such
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materials can be particularly effective cleaning agents. Unfortunately,
consumers typically
associate the odors resulting from the use of such agents with a lack of
cleanliness. Examples of
reduced odour variants with a C-terminal extension are shared in W002/062973,
but these lipase
variants do not demonstrate the strong wash performance of the first wash
lipases such as those
from W000/60063 including the variant sold under the tradename Lipex .
Thus, there remains a need for a detergent compositions comprising lipqlytic
enzymes
for excellent greasy/oily soils removal while not generating any objectionable
fatty acid odors.
SUMMARY OF THE INVENTION
The present invention relates to detergent compositions comprising a detergent
ingredient and a lipase variant with reduced potential for odor generation,
without the
attachment of a C-terminal extension. The lipase variant is obtained by
introducing mutations in
one or more regions identified in the parent lipase.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the alignment of lipases_
t -
SEQUENCE LISTINGS
SEQ ID NO: 1 shows the DNA sequence encoding lipase from Thermomyces
lanvginosus.
SEQ ID NO: 2 shows the amino acid sequence of a lipase from Thermomyces
lanoginosus.
SEQ ID NO: 3 shows the amino acid sequence of a lipase from Absidia reflexa.
SEQ ID NO: 4 shows the amino acid sequence of a lipase from Absidia
corymbifera.
SEQ ID NO: 5 shows the amino acid sequence of a lipase from Rhizomucor miehei.
SEQ ID NO: 6 shows the amino acid sequence of a lipase from Rhizopus oryzae.
SEQ ID NO: 7 shows the amino acid sequence of a lipase from Aspergillus niger.
SEQ ID NO: 8 shows the amino acid sequence of a lipase from Aspergillus
tubingensis.
SEQ ID NO: 9 shows the amino acid sequence of a lipase from Fusarium
oxysporrum.
SEQ ID NO: 10 shows the amino acid sequence of a lipase from Fusarium
heterosporum.
SEQ ID NO: 11 shows the amino acid sequence of a lipase from Aspergillus
oryzae.
SEQ ID NO: 12 shows the amino acid sequence of a lipase from Penicillium
camemberti.
SEQ ID NO: 13 shows the amino acid sequence of a lipase from
Aspergillusfoetidus.
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SEQ ID NO: 14 shows the amino acid sequence of alipase from Aspergillus niger.
SEQ ID NO: 15 shows the amino acid sequence of a lipase from Aspergillus
oryzae.
SEQ ID NO: 16 shows the amino acid sequence of a lipase from Landerina
penisapora.
DETAILED DESCRIPTION OF THE INVENTION
LIPASE VARIANTS
Parent lipase
The parent lipase may be a fungal lipase with an amino acid sequence having at
least 50
% homology as defined in the section "Homology and aligment" to the sequence
of the T.
lanuginosus lipase shown in SEQ ID NO: 2.
The parent lipase may be a yeast polypeptide such as a Candida, Kluyveromyces,
Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or more
preferably a
filamentous fungal polypeptide such as an Acremonium, Aspergillus,
Aureobasidium,
Cryptococcus, Filobasidium, Fusarium, Humicola, Magnaporthe, Mucor,
Myceliophthora,
Neocallimastix, Neurospora, Paccilomyces, Penicilliurn, Piromyces,
Schizophyllum,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma
polypeptide.
In a preferred aspect, the parent lipase is a Saccharomyces carlsbergensis,
Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,
Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis
polypeptide
having lipase activity.
In another preferred aspect, the parent lipase is an Aspergillus aculeatus,
Aspergillus
awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus,
Aspergillus
nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus turbigensis,
Fusarium bactridioides,
Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium
graminearum,
Fusarium' graminum, Fusarium heterosporum, Fusarium negundi, Fusarium
oxysporum,
Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusariurn
sarcochroum,
Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium
trichothecioides, Fusarium venenatum, Humicola insolens, Thermomyces
lanoginosus
(synonym: Humicola lanuginose), Mucor miehei, Myceliophthora thermophila,
Neurospora
crassa, Penicillium purpurogenum, Triehoderma harzianum, Trichoderma koningii,
Trichoderma
longibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.
In another preferred aspect, the parent lipase is a Thermomyces lipase.
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In a more preferred aspect, the parent lipase is a Thermomyces lanuginosus
lipase. In an
even more preferred embodiment the parent lipase is the lipase of SEQ ID NO:
2.
Identification of regions and substitutions.
The positions referred to in Region I through Region IV below are the
positions of the amino
acid residues in SEQ ID NO:2. To find the corresponding (or homologous)
positions in a
different lipase, the procedure described in "Homology and alignment" is used.
Substitutions in Re ig on I
Region I consists.of amino acid residues surrounding the N-terminal residue
El. In this region it
is preferred to substitute an amino acid of the parent lipase with a more
positive amino, acid.
Amino acid residues corresponding to the following positions are comprised by
Region I: 2 to
I1 and 223-239. The following positions are of particular interest: 4, 8, 11,
223, 227, 229, 231,
233, 234, 236. In particular the following substitutions have been identified:
X4V, X227G,
X23IR and X233R.
In a preferred embodiment the parent lipase has at least 80%, such as 85% or
90%, such
as at least 95% or 96% or 97% or 98% or 99%, identity to SEQ ID NO:2 . In a
most preferred
embodiment the parent lipase is identical to SEQ ID NO: 2.
Substitutions in Re ig o n II
Region II consists of amino acid residues in contact with substrate on one
side of the acyl chain
and one side of the alcohol part. In this region it is preferred to substitute
an amino acid of the
parent lipase with a more positive amino acid or with a less hydrophobic amino
acid. Amino
acid residues corresponding to the following positions are comprised by Region
II: 202 to 211
and 249 to 269. The following positions are of particular. interest : 202,
210, 211, 253, 254, 255,
256. In particular the following substitutions have been identified: X202G,
X210K, X255Y/V
and X256K/R.
In a preferred embodiment the parent lipase has at least 80%, such as 85% or
90%, such
as at least 95% or 96% or 97% or 98% or 99%, identity to SEQ ID NO:2. In a
most preferred
embodiment the parent lipase is identical to SEQ ID NO: 2.
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Substitutions in Region III
Region III consists of amino acid residues that form a flexible structure and
thus allowing the
substrate to get into the active site. In this region it is preferred to
substitute an amino acid of the
parent lipase with a more positive amino acid or a less hydrophobic amino
acid. Amino acid
5 residues corresponding to the following positions are comprised by Region
III: 82 to 102. The
following positions are of particular interest: 83, 86, 87, 90, 91, 95, 96,
99. In particular the
following substitutions have been identified: X83T, X86V and X90A/R.
In a preferred embodiment the parent lipase has at least 80%, such as 85% or
90%, such
as at least 95% or 96% or 97% or 98% or 99%, identity to SEQ ID NO:2 . In a
most preferred
embodiment the parent lipase is identical to SEQ ID NO: 2.
Substitutions in Region IV
Region IV consists of amino acid residues that bind electrostatically to a
surface. In this region it
is preferred to substitute an amino acid of the parent lipase with a more
positive amino acid.
Amino acid residues corresponding to the following positions are comprised by
Region IV: 27
and 54 to 62. The following positions are of particular interest: 27, 56, 57,
58, 60. In particular
the following substitutions have been identified: X27R, X58N/AG/T1P and
X60V/S/G/N/R/KI.A/L.
In a preferred embodiment the parent lipase has at least 80%, such as 85% or
90%, such
as at least 95% or 96% or 97% or 98% or 99%, identity to SEQ ID NO:2 . In a
most preferred
embodiment the parent lipase is identical to SEQ ID NO: 2.
Amino acids at other positions
The parent lipase may optionally comprise substitution of other amino acids,
particularly less
than 10 or less than 5 such substitutions. Examples are substitutions
corresponding to one or
more of the positions 24, 46, 74, 81, 83, 127, 131, 137, 147, 150, 203, 206,
211, 263, 264, 265,
267 and 269 of the parent lipase. In a particular embodiment there is a
substitution in at least one
of the positions corresponding to position 81, 147, 150 and 249. In a
preferred embodiment the
at least one substitution is selected from the group consisting of X81Q/E,
X1471V1/Y, X150G and
X249R/I/L.
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Further substitutions may, e.g., be made according to principles known in the
art, e.g.
substitutions described in WO 92/05249, WO 94125577, WO 95/22615, WO 97/04079
and WO
97/07202.
Parent lipase variants
In one aspect, said variant, when compared to said parent, comprising a total
of at least
three substitutions, said substitutions being selected from one or more of the
following groups of
substitutions:
a) at least two substitutions in Region I,
b) at least one substitution in Region 11,
c) at least one substitution in Region III, and/or
d) at least one substitution in Region IV.
The variant may comprise substitutions, compared to the variant's parent,
corresponding
to those substitutions listed below in Table 1.
Region I Region II Region III Region IV Outside regions
X4V + X227G + X210K + X83T + X58A + X60S X150G
X231 R+ X233R X256K X86V
X227G + X231R X256K X86V X58N + X60S X150G
+ X233R
X231R + X233R X255Y
X231 R + X233R X202G
X227G + X231R X256K X86V
+ X233R
X4V + X231R + X58N + X60S
X233R
X231R + X233R X90R X58N + X60S
X231R + X233R X255V X90A
X227G + X231R X256K X86V X58N + X60S XISOG
+ X233R
X231R + X233R X211L X58N + X60S X147M
Table 1: Some particular variants.
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In a further particular embodiment the parent lipase is identical to SEQ ID
NO:2, and
the variants of Table I will thus be:
Region I Region II Region III Region IV Outside regions
Q4V + L227G + E210K + S83T + 186V S58A + V60S A150G
T231R + N233R P256K
L227G + T231R P256K 186V S58N + V60S A150G
+ N233R
T231R + N233R 1255Y
T231R + N233R 1202G
L227G + T231R + P256K 186V
N233R
Q4V + T231R + S58N + V60S
N233R
T231R + N233R 190R S58N + V60S
T231R + N233R 1255V 190A
L227G + T231R + P256K 186V 858N + V60S A1506
N233R
T231R + N233R F211L S58N + V60S L147M
Table 2: Some particular variants of SEQ ID NO:2
Nomenclature for amino acid modifications
In describing lipase variants according to the invention, the following
nomenclature is used for
ease of reference: Original amino acid(s):position(s):substituted amino
acid(s)
According to this nomenclature, for instance the substitution of glutamic acid
for
glycine in position 195 is shown as G195E. A deletion of glycine in the same
position is shown
as G195*, and insertion of an additional amino acid residue such as lysine is
shown as G195GK.
Where a specific lipase contains a"deletion" in comparison with other lipases
and an insertion is
made in such a position this is indicated as *36D for insertion of an aspartic
acid in position 36.
Multiple mutations are separated by pluses, i.e.: R170Y+G195E, representing
mutations in
positions 170 and. 195 substituting tyrosine and glutamic acid for arginine
and glycine,
respectively.
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8
X231 indicates the amino acid in a parent polypeptide corresponding to
position 231,
when applying the described alignment procedure. X231R indicates that the
amino acid is
replaced with R. For SEQ ID NO:2 X is T, and X231R thus indicates a
substitution of T in
position 231 with R. Where the amino acid in a position (e.g. 231) may be
substituted by another
amino acid selected from a group of amino acids, e.g. the group consisting of
R and P and Y, this
will be indicated by X231R/P/Y.
In all cases, the accepted IUPAC single letter or triple letter amino acid
abbreviation is
employed.
Amino acid grouping
In this specification, amino acids are classified as negatively charged,
positively charged or
electrically neutral according to their electric charge at pH 10. Thus,
negative amino acids are E,
D, C (cysteine) and Y, particularly E and D. Positive amino acids are R, K and
H, particularly R
and K. Neutral amino acids are G, A, V, L, I, P, F, W, S, T, M, N, Q and C
when forming part of a
disulfide bridge. A substitution with another amino acid in the same group
(negative, positive or
neutral) is termed a conservative substitution.
The neutral amino acids may be divided into hydrophobic or non-polar (G, A, V,
L, 1, P, F, W and
C as part of a disulfide bridge) and hydrophilic or polar (S, T, M, N, Q).
Amino acid identity
The relatedness between two amino acid sequences or between two) nucleotide
sequences is described by the parameter "identity".
For purposes of the present invention, the alignment of two amino acid
sequences is
determined by using the Needle program from the EMBOSS package
(http://emboss.org)
version 2.8Ø The Needle program irnplements the global alignment algorithm
described in
Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453. The
substitution matrix
used is BLOSUM62, gap opening penalty is 10, and gap extension penalty is 0.5.
The degree of identity between an amino acid sequence of the present invention
("invention sequence"; e.g. amino acids I to 269 of SEQ ID NO:2) and a
different amino acid
sequence ("foreign sequence") is calculated as the number of exact matches in
an alignment of
the two sequences, divided by the length of the "invention sequence" or the
length of the
"foreign sequence", whichever is the shortest. The result is expressed in
percent identity.
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An exact match occurs when the "invention sequence" and the "foreign sequence"
have
identical amino acid residues in the same positions of the overlap. The length
of a sequence is
the number of amino acid residues in the sequence (e.g. the length of SEQ ID
NO:2 is 269).
The parent lipase has an amino acid identity of at least 50 % with the T.
lanuginosus
lipase (SEQ ID NO: 2), particularly at least 55 %, at least 60 %, at least 75
%, at least 85 % , at
least 90 %, more than 95 % or more than 98 %. In a particular embodiment the
parent lipase is
identical to the T. lanuginosus lipase (SEQ ID NO:2).
The above procedure may be used for calculation of identity as well as
homology and
for alignment. In the context of the present invention homology and alignment
has been
calculated as described below.
Homology and alignment
For purposes of the present invention, the degree of homology may be suitably
determined by means of computer programs known in the art, siuch 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)
(Needleman,
S.B. and Wunsch, C.D., (1970), Journal of Molecular Biology, 48, 443-45),
using GAP with the
following settings for polypeptide sequence comparison: GAP creation penalty
of 3.0 and GAP
extension penalty of 0.1.
In the present invention, corresponding (or homologous) positions in the
lipase
sequences of Absidia reflexa, Absidia corymbefera, Rhizmucor miehei, Rhizopus
delemar,
Aspergillus niger, Aspergillus tubigensis, Fusarium oxysporum, Fusarium
heterosporum,
Aspergillus oryzea, Penicilium camembertii, Aspergillus foetidus, Aspergillus
niger,
Thermomyces lanoginosus (synonym: Humicola lanuginose) and Landerina
penisapora are
defined by the alignment shown in Figure 1.
To find the homologous positions in lipase sequences not shown in the
alignment, the
sequence of interest is aligned to the sequences shown in Figure 1. The new
sequence is aligned
to the present alignment in Figure 1 by using the GAP alignment to the most
homologous
sequence found by the GAP program. GAP is 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) (Needleman, S.B. and Wunsch,
C.D., (1970),
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Journal of Molecular Biology, 48, 443-45). The following settings are used for
polypeptide se-
quence comparison: GAP creation penalty of 3.0 and GAP extension penalty of
0.1.
The parent lipase has a homology of at least 50 % with the T. lanuginosus
lipase (SEQ
ID NO: 2), particularly at least 55 %, at least 60 %, at least 75 %, at least
85 % , at least 90 %,
5 more than 95 % or more than 98 %. In a particular embodiment the parent
lipase is identical to
the T. lanuginosus lipase (SEQ ID NO:2).
Hybridization
The present invention also relates to isolated polypeptides having lipase
activity which are
10 encoded by polynucleotides which hybridize under very low stringency
conditions, preferably
low stringency conditions, more preferably medium stringency conditions, more
preferably
medium-high stringency conditions, even more preferably high stringency
conditions, and most
preferably very high stringency conditions with (i) nucleotides 178 to 660 of
SEQ ID NO: 1, (ii)
the cDNA sequence contained in nucleotides 178 to 660 of SEQ ID NO: 1, (iii) a
subsequence of
(i) or (ii), or (iv) a complementary strand of (i), (ii), or (iii) (J.
Sambrook, E.F. Fritsch, and T.
Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold
Spring Harbor, New
York). A subsequence of SEQ ID NO: 1 contains at least 100 contiguous
nucleotides or
preferably at least 200 contiguous nucleotides. Moreover, the subsequence may
encode a
polypeptide fragment which has lipase activity.
For long probes of at least 100 nucleotides in length, very low to very high
stringency
conditions are defined as prehybridization and hybridization at 42 C in 5X
SSPE, 0.3% SDS,
200 ug/mi sheared and denatured salmon sperm DNA, and either 25% formamide for
very low
and low stringencies, 35% formamide for medium and medium-high stringencies,
or 50%
formamide for high and very high stringencies, following standard Southern
blotting procedures
for 12 to 24 hours optimally.
For long probes of at least 100 nucleotides in length, the carrier material is
finally
washed three times each for 15 minutes using 2X SSC, 0.2% SDS preferably at
least at 45 C
(very low stringency), more preferably at least at 50 C (low stringency), more
preferably at least
at 55 C (medium stringency), more preferably at least at 60 C (medium-high
stringency), even
more preferably'at least at 65 C (high stringency), and most preferably at
least at 70 C (very
high stringency).
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DNA sequence, Expression vector, Host cell, Production of lipase
The invention provides a DNA sequence encoding the lipase of the invention, an
expression
vector harboring the DNA sequence, and a transformed host cell containing the
DNA sequence
or the expression vector. These may be obtained by methods known in the art.
The invention also provides a method of producing the lipase by culturing the
transformed host
cell under conditions conducive for the production of the lipase and
recovering the lipase from
the resulting broth_ The method may be practiced according to principles known
in the art.
Lipase activity
- Lipase activity on tributyrin at neutral pH (LU)
A substrate for lipase is prepared by emulsifying tributyrin (glycerin
tributyrate) using gum
Arabic as emulsifier. The hydrolysis of tributyrin at 30 C at pH 7 or 9 is
followed in a pH-stat
titration experiment. One unit of lipase activity (1 LU) equals the amount of
enzyme capable of
releasing 1 micro mol butyric acid/min at pT-17.
- Benefit Risk
The Benefit Risk factor describing the performance compared to the reduced
risk for odour
smell is defined as: BR = RPaVg / R. Lipase variants described herein may have
BRs greater than
1, greater than 1.1, or even greater than 1 to about 1000.
-Average Relative Performance
The procedure for calculating average relative performance (RPavg) is found in
Example 5 of
the present specification. Lipase variants described herein may have (RPavg)
of at least 0.8, at
least 1.1, at least 1.5, or even at least 2 to about 1000.
DETERGENT INGREDIENTS
As used herein detergent compositions include articles and cleaning and
treatment compositions.
As used herein, the term "cleaning and/or treatment composition" includes,
unless otherwise
indicated, tablet, granular or powder-form all-purpose 'or "heavy-duty"
washing agents,
especially laundry detergents; liquid, gel or paste-form all-purpose washing
agents, especially
the so-called heavy-duty liquid types; liquid fine-fabric detergents; hand
dishwashing agents or
light duty dishwashing agents, especially those of the high-foaming type;
machine dishwashing
agents, including the various tablet, granular, liquid and rinse-aid types for
household and
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institutional use. The compositions can also be in unit dose packages,
including those known in
the art and those that are water soluble, water insoluble and/or water
permeable.
The detergent composition of the present invention can comprise one or more
lipase variant(s)
of the present invention. In addition to the lipase variant(s), the detergent
composition will
further comprise a detergent ingredient. The non-limiting list of detergent
ingredients illustrated
hereinafter are suitable for use in the instant compositions and may be
desirably incorporated in
certain embodiments of the invention, for example to assist or enhance
cleaning performance,
for treatment of the substrate to be cleaned, or to modify the aesthetics of
the cleaning
composition as is the case with colorants, dyes or the like. The precise
nature of these additional
components, and levels of incorporation thereof, will depend on the physical
form of the
composition and the nature of the cleaning operation for which it is to be
used. Suitable
detergent ingredients include, but are not limited to, surfactants, builders,
chelating agents, dye
transfer inhibiting agents, dispersants, enzymes, and enzyme stabilizers,
bleach activators,
hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric
dispersing
agents, brighteners, suds suppressors, dyes, anti-corrosion agents, tarnish
inhibitors, perfumes,
fabric softeners, carriers, hydrotropes, processing aids, solvents and/or
pigments.
Typical detergents would comprise by weight any combination of the following
ingredients: 5-
30% surfactant, preferably anionic surfactants such as linear
alkylbenzenesulfonate and alcohol
ethoxysulfate; 0.005-0.1% protease active protein, wherein the protease is
preferably selected.
from CoronaseTM, FN4 FNA, or SavinaseTM, 0.001-0.1% amylase active protein,
wherein the
amylase is preferably selected from TermamylTM NatalaseTM, StainzymeTM and
PurastarTM and
0.1-3% chelants, preferably diethylene triamine pentaacetic acid. For granular
and tablet
products, such typical detergents would additionally comprise by weight: 5-20%
bleach,
preferably sodium percarbonate; 1-4% bleach activator, preferably TAED and/or
0-30%
builder, preferably 5-30%, more preferably less than 10% builder, such as the
aluminosilicate
Zeolite A andlor tripolyphosphate.
Bleaching Agents - The detergent compositions of the present invention may
comprise
one or more bleaching agents.
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In general, when a bleaching agent is used, the compositions of the present
invention may
comprise from about 0.1% to about 50% or even from about 0.1% to about 25%
bleaching agent
by weight of the subject cleaning composition. Examples of suitable bleaching
agents include:
(1) sources of hydrogen peroxide, for example, inorganic perhydrate salts,
including
alkali metal salts such as sodium salts of perborate (usually mono- or tetra-
hydrate),
percarbonate, persulphate, perphosphate, persilicate salts and mixtures
thereof. In one aspect of
the invention the inorganic perhydrate salts are selected from the group
consisting of sodium
salts of perborate, percarbonate and mixtures thereof. soaps; and
(2) bleach activators having R-(C=O)-L wherein R is an alkyl group, optionally
branched, having, when the bleach activator is hydrophobic, from 6 to 14
carbon atoms, or from
8 to 12 carbon atoms and, when the bleach activator is hydrophilic, less than
6 carbon atoms or
even less than 4 carbon atoms; and L is leaving group. Examples of suitable
leaving groups are
benzoic acid and derivatives thereof - especially benzene sulphonate. Suitable
bleach activators
include dodecanoyl oxybenzene sulphonate, decanoyl oxybenzene sulphonate,
decanoyl
oxybenzoic acid or salts thereof, 3,5,5-trimethyl hexanoyloxybenzene
sulphonate, tetraacetyl
ethylene diamine (TAED) and nonanoyloxybenzene sulphonate (NOBS). Suitable
bleach
activators are also disclosed in WO 98/17767. While any suitable bleach
activator may be
employed, in one aspect of the invention the subject cleaning composition may
comprise NOBS,
TAED or mixtures thereof.
(3) Pre-formed peracids.
When present, the peracid and/or bleach activator is generally present in the
composition
in an amount of from about 0.1 to about 60 wt%, from about 0.5 to about 40 wt
% or even from
about 0.6 to about 10 wt% based on the composition. One or more hydrophobic
precursors
thereof may be used in combination with one or more hydrophilic peracid or
precursor thereof.
The amounts of hydrogen peroxide source and peracid or bleach activator may be
selected such that the molar ratio of available oxygen (from the peroxide
source) to peracid is
from 1:1 to 35:1, or even 2:1 to 10:1.
Surfactants - The detergent compositions according to the present invention
may
comprise a surfactant or surfactant system wherein the surfactant can be
selected from nonionic
surfactants, anionic surfactants, cationic surfactants, ampholytic
surfactants, zwitterionic
surfactants, semi-polar nonionic surfactants and mixtures thereof. When
present, surfactant is
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14
typically present at a level of from about 0.1% to about 60%, from about 0.1%
to about 40%,
from about 0.1% to about 12%, frorri about 1% to about 50% or even from about
5% to about
40% by weight of the subject composition.
When included therein the detergent will usually contain from about 1% to
about 40% of
an anionic surfactant such as linear alkylbenzenesulfonate, alpha-
olefinsulfonate, alkyl sulfate
(fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,
alpha-sulfo fatty acid
methyl ester, alkyl- or alkenylsuccinic acid or soap.
The detergent may optionally contain from about 0.2% to about 40% of a non-
ionic
surfactant such as alcohol ethoxylate, nonylphenol ethoxylate,
alkylpolyglycoside,
alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid
monoethanolamide, polyhydroxy alkyl fatty acid arriide, or N-acyl N-alkyl
derivatives of
glucosamine ("glucamides").
Builders - The detergent compositions of the present invention may comprise
one or
more detergent builders or builder systems. When a builder is used, the
subject composition will
typically comprise at least about 1%, from about 5% to about 60% or even from
about 10% to
about 40% builder by weight of the subject composition. Builders include, but
are not limited
to, the alkali metal, ammonium and alkanolammonium salts of polyphosphates,
alkali metal
silicates or layered silicates, alkaline earth and alkali metal carbonates,
aluminosilicate builders
and the various alkali metal, ammonium and substituted ammonium salts of
polyacetic acids
such as ethylenediamine tetraacetic acid and nitrilotriacetic acid, as well as
polycarboxylates
such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid,
polymaleic acid, benzene
1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and soluble salts
thereof.
Chelating Agents - The detergent compositions herein may contain a chelating
agent.
Suitable chelating agents include copper, iron and/or manganese chelating
agents and mixtures
thereof. When a chelating agent is used, the subject composition may comprise
from about
0.005% to about 15% or even from about 3.0% to about 10% chelating agent by
weight of the
subject composition.
Brighteners - The detergent compositions of the present invention can also
contain
additional components that may alter appearance of articles being cleaned,
such as fluorescent
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brighteners. These brighteners absorb in the UV-range and emit in the visible.
Suitable
fluorescent brightener levels include lower levels of from about 0.01, from
about 0.05, from
about 0.1 or even from about 0.2 wt % to upper levels of 0.5 or even 0.75 wt
%.
5 Dispersants - The compositions of the present invention can also contain
dispersants.
Suitable water-soluble organic materials include 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.
10 Enzymes - In addition to the lipase variant(s) of the present invention the
detergent
composition can comprise one or more further enzymes which provide cleaning
performance
and/or fabric care benefits such as a protease, another lipase, a cutinase, an
amylase, a
carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a
galactanase, a xylanase, an
oxidase, e.g., a laccase, and/or a peroxidase.
15 In general the properties of the chosen enzyme(s) should be compatible with
the selected
detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-
enzymatic ingredients,
etc.), and the enzyme(s) should be present in effective amounts.
Suitable proteases include those of animal, vegetable or microbial origin.
Microbial
origin is preferred. Chemically modified or protein engineered mutants are
included. The
protease may be a serine protease or a metallo protease, preferably an
alkaline microbial
protease or a trypsin-like protease. Examples of alkaline proteases are
subtilisins, especially
those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg,
subtilisin 309, subtilisin
147 and subtilisin 168 (described in WO 89/06279), SEQ ID no 4 and SEQ ID no 7
in WO
05/103244. Other suitable serin proteases include those from Micrococcineae
spp especially
Cellulonas spp and variants thereof as disclosured in W02005052146. Examples
of trypsin-like
proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium
protease described in
WO 89106270 and WO 94/25583.
Examples of useful proteases are the variants described in WO 92/19729, WO
98/20115,
WO 98/20116, and WO 98/34946, especially the variants with substitutions in
one or more of
the following positions: 27, 36, 57, 68, 76, 87, 97, 101, 104, 106, 120, 123,
167, 170, 194, 206,
218, 222, 224, 235, 245, 252 and 274, and amongst other variants with the
following mutations:
(K27R, V 104Y, N123S, T124A), (N76D, S 103A, V1041), or (S 101 G, S 103A,
V1041, G159D,
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16
A232V, Q236H, Q245R, N248D, N252K). Other examples of useful proteases are the
variants
described in WO 05/052146 especially the variants with substitutions in one or
more of the
following positions: 14, 16, 35, 65, 75, 76, 79, 123, 127, 159 and 179
Preferred commercially available protease enzymes include AlcalaseT"',
SavinaseTM,
PrimaseTM, DuralaseTM, EsperaseTM, CoronaseTM, PolarzymeTM and KannaseTM
(Novozymes
A/S), MaxataseTM, MaxacalTM, MaxapemTM, ProperaseTM, PurafectTM, Purafect
PrimeTM, Purafect
OxPTM, FN2, FN3 and FN4 (Genencor International Inc.).
Lipases include those of bacterial or fungal origin. Chemically modified or
protein
engineered mutants are included. Examples of useful lipases include lipases
from Humicola
(synonym Thermomyces), e.g. from H. lanuginosa (synonymous T. lanuginosus) as
described in
EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a
Pseudomonas
lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P.
cepacia (EP 331 376),
P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO
95/06720 and
WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B.
subtilis
(Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360), B.
stearothermophilus
(JP 64/744992) or B. pumilus (WO 91/16422).
Other examples are lipase variants such as those described in WO 92/05249, WO
94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96100292, WO 95/30744, WO
94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.
Other commecially available lipase enzymes include LipolaseTM, Lipolase
UltraT"' and
LipexTM (Novozymes A/S).
Suitable amylases (oc and/or (3) include those of bacterial or fungal origin.
Chemically
modified or protein engineered mutants are included. Amylases include, for
example, a-
amylases obtained from Bacillus, e.g. a special strain of B. licheniformis,
described in more
detail in GB 1,296,839.
Examples of useful amylases are the variants described in WO 94/02597, WO
94/18314,
WO 96/23873, and WO 97/43424, especially the variants with substitutions in
one or more of
the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188,
190, 197, 202, 208,
209, 243, 264, 304, 305, 391, 408, and 444.
Commercially available amylases are DuramylTM, TermamylTM, StainzymeTM
Stainzyme UltraTM, FungamylTM and BANTM (Novozymes A/S), RapidaseTM and
PurastarTM
(from Genencor International Inc.).
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Suitable cellulases include those of bacterial or fungal origin. Chemically
modified or
protein engineered mutants are included. Suitable cellulases include
cellulases from the genera
Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the
fungal cellulases
produced from Humicola insolens, Myceliophthora thermophila and Fusarium
oxysporum
disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO
89/09259.
Especially suitable cellulases are the alkaline or neutral cellulases having
colour care
benefits. Examples of such cellulases are cellulases described in EP 0 495
257, EP 0 531 372,
WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants
such as
those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US
5,763,254,
WO 95/24471, WO 98/12307 and PCTIDK98/00299.
Commercially available cellulases include RenozymeTm , CellucleanTM, Endolase,
TM
CelluzymeTM, and CarezymeTM (Novozymes A/S), Clazinase"M, and Puradax HATM
(Genencor
International Inc.), and KAC-500(B)TM (Kao Corporation).
Peroxidases/Oxi dases:
Suitable peroxidases/oxidases include those of plant, bacterial or fungal
origin.
Chemically modified or protein engineered mutants are included. Examples of
useful
peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and
variants thereof as
those described in WO 93/24618, WO 95/10602, and WO 98/15257.
Commercially available peroxidases include GuardzymeTM (Novozymes A/S).
When present in a cleaning composition, the aforementioned enzymes may be
present at levels
from about 0.00001% to about 2%, from about 0.0001% to about 1% or even from
about
0.001% to about 0.5% enzyme protein by weight of the composition.
Enzyme Stabilizers - Enzymes for use in detergents can be stabilized by
various
techniques. The enzymes employed herein can be stabilized by the presence of
water-soluble
sources of calcium and/or magnesium ions in the finished compositions that
provide such ions to
the enzymes. Further conventional stabilizing agents, e.g., a polyol such as
propylene glycol or
glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid
derivative, e.g., an
aromatic borate ester, or a phenyl boronic acid derivative such as 4-
formylphenyl boronic acid,
may also be used and the composition may be formulated as described in e.g. WO
92/19709 and
WO 92/19708.
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Solvents - Suitable solvents include water and other solvents such as
lipophilic fluids.
Examples of suitable lipophilic fluids include siloxanes, other silicones,
hydrocarbons, glycol
ethers, glycerine derivatives such as glycerine ethers, perfluorinated amines,
perfluorinated and
hydrofluoroether solvents, low-volatility nonfluorinated organic solvents,
diol solvents, other
environmentally-friendly solvents and mixtures thereof.
WASHING METHOD
The present invention includes a method for cleaning and /or treating a situs
inter alia a surface
or fabric. Such method includes the steps of contacting an embodiment of
Applicants' cleaning
composition, in neat form or diluted in a wash liquor, with at least a portion
of a surface or
fabric then optionally rinsing such surface or fabric. The surface or fabric
may be subjected to a
washing step prior to the aforementioned rinsing step. For purposes of the
present invention,
washing includes but is not limited to, scrubbing, and mechanical agitation.
As will be
appreciated by one skilled in the art, the cleaning compositions of the
present invention are
ideally suited for use in laundry applications. Accordingly, the present
invention includes a
method for laundering a fabric. The method comprises the steps of contacting a
fabric to be
laundered with a said cleaning laundry solution comprising at least one
embodiment of
Applicants' cleaning composition, cleaning additive or mixture thereof. The
fabric may
comprise most any fabric capable of being laundered in normal consumer use
conditions. The
solution preferably has a pH of from about 8 to about 10.5. The compositions
may be employed
at concentrations of from about 100 ppm, preferably 500ppm to about 15,000 ppm
in solution.
The water temperatures typically range from about 5 C to about 90 C. The
invention may be
particularly beneficial at low water ternperatures such as below 30 C or below
25 or 20 C. The
water to fabric ratio is typically from about 1:1 to about 30:1.
LIPASE VARIANTS EXAMPLES
Chemicals used as buffers and substrates were commercial products of at least
reagent grade.
- Media and Solutions: LAS (Surfac PST"') and Zeolite A (Wessalith PTM). Other
ingredients
used are standard laboratory reagents.
- Materials: EMl'A221 from EMPA St. Gallen, Lerchfeldstrasse 5, CH-9014 St.
Gallen,
Switzerland
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Example 1: Production of enzyme
A plasmid containing the gerie encoding the lipase is constructed and
transformed into a suitable
host cell using standard methods of the art.
Fermentation is carried out as a fed-batch fermentation using a constant
medium
temperature of 34 C and a start volume of 1.2 liter. The initial pH of the
medium is set to 6.5.
Once the pH has increased to 7.0 this value is maintained through addition of
10% H3P04. The
level of dissolved oxygen in the medium is controlled by varying the agitation
rate and using a
fixed aeration rate of 1.0 liter air per liter medium per minute. The feed
addition rate is
maintained at a constant level during the entire fed-batch phase.
The batch medium contained maltose syrup as carbon source, urea and yeast
extract as nitrogen
source and a mixture of trace metals and salts. The feed added continuously
during the fed-batch
phase contains maltose syrup as carbon source whereas yeast extract and urea
is added in order
to assure a sufficient supply of nitrogen.
Purification of the lipase may be done by use of standard methods known in the
art, e_g.
by. filtering the fermentation supernatant and subsequent hydrophobic
chromatography and
anion exchange, e.g. as described in EP 0 851 913, Example 3.
Example 2: AMSA - Automated Mechanical Stress Assay - for calculation of
Relative
Performance (RP).
The enzyme variants of the present application are tested using the Automatic
Mechanical Stress Assay (AMSA). With the AMSA test the wash performance of a
large
quantity of small volume enzyme-detergent solutions can be examined. The AMSA
plate has a
number of slots for test solutions and a lid firmly squeezing the textile
swatch to be washed
against all the slot openings. During the washing time, the plate, test
solutions, textile and lid are
vigorously shaken to bring the test solution in contact with the textile and
apply mechanical
stress. For further description see WO 02/42740 especially the paragraph
"Special method
embodiments" at page 23-24. The containers, which contain the detergent test
solution, consist
of cylindrical holes (6 mm diameter, 10 mm depth) in a metal plate. The
stained fabric (test
material) lies on the top of the metal plate and is used as a lid and seal on
the containers.
Another metal plate lies on the top of the stained fabric to avoid any
spillage from each
container. The two metal plates together with the stained fabric are vibrated
up and down at a
frequency of 30 Hz with an amplitude of 2 mm.
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The assay is conducted under the experimental conditions specified below:
0.5g/ILAS
Test solution 0.52 g/1 Na2CO3
1.07 g/1 Zeolite A
0.52 g/I Tri sodium Citrate
Test solution volume 160 micro I
pH As is (;:--9.9)
Wash time 20 minutes
Temperature 30 C
15 dH
Water hardness Ratio of CaZ"/Me/NaHCO3
4:1:7.5
0.125, 0.25, 0.50, 1.0 mg enzyme
Enzyme concentration in test solution
protein/Iiter (mg ep / 1)
Performance: After washing the
textile pieces is immediately flushed
in tap water and air-dried at 85C in 5
Drying min
Odor: After washing the textile
pieces is immediately flushed in tap
water and dried at room temperature
(20 C) for 2 hours
Cream turmeric swatch as described
Test material below (EMPA221 used as cotton
textile)
Table 3
5 Cream-turmeric swatches were prepared by mixing 5 g of turmeric (Santa
Maria,
Denmark) with 100 g cream (38% fat, Arla, Denmark) at 50 C, the mixture is
left at this
temperature for about 20 minutes and filtered (50 C) to remove any undissolved
particles. The
mixture is cooled to 20 C) woven cotton swatches, EMPA221, were immersed in
the cream-
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turmeric mixture and afterwards allowed to dry at room temperature over night
and frozen until
use. The preparation of cream-turmeric swatches is disclosed in the patent
application PA 2005
00775, filed 27 May 2005.
The performance of the enzyme variant is measured as the brightness of the
colour of
the textile samples washed with that specific enzyme variant. Brightness can
also be expressed
as the intensity of the light reflected from the textile sample when luminated
with white light.
When the textile is stained the intensity of the reflected light is lower,
than that of a clean textile.
Therefore the intensity of the reflected light can be used to measure wash
performance of an
enzyme variant.
Color measurements are made with a professional flatbed scanner (PFU
DL2400pro),
which is used to capture an image of the washed textile samples. The scans are
made with a
resolution of 200 dpi and with an output color depth of 24 bits. In order to
get accurate results,
the scanner is frequently calibrated with a Kodak reflective IT8 target.
To extract a value for the light intensity from the scanned images, a special
designed
software application is used (Novozymes Color Vector Analyzer). The program
retrieves the 24
bit pixel values from the image and converts them into values for red, green
and blue (RGB).
The intensity value (Int) is calculated by adding the RGB values together as
vectors and then
taking the length of the resulting vector:
Int=~r2 +gZ+b2
The wash performance (P) of the variants is calculated in accordance with the
formula:
P = Int(v) - Int(r) where
Int(v) is the light intensity value of textile surface washed with the tested
enzyme and Int(r) is
the light intensity value of textile surface washed without the tested enzyme.
A relative performance score is given as the result of the AMSA wash in
accordance
with the definition: Relative Performance scores (RP) are summing up the
performances (P) of
the tested enzyme variants against the reference enzyme: RP = P(test enzyme) /
P(reference
enzyme).
RPavg indicates the average relative performance compared to the reference
enzyme at all four
enzyme concentrations (0.125, 0.25, 0.5, 1.0 mg ep/I)
RPavg = avg(RP(0_125), RP(0.25) RP(0.5), RP(1.0))
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A variant is considered, to exhibit improved wash performance, if it performs
better than the
reference. In the context of the present invention the reference enzyme is the
lipase of SEQ ID
NO:2 with the substitutions T231R + N233R.
Example 3: GC - Gas Chromatograph - for calculation of risk factor.
The butyric acid release from the lipase washed swatches were measured by
Solid Phase
Micro Extraction Gas Chromatography (SPME-GC) using the following method. Four
textile
pieces (5 mm in diameter), washed in the specified solution in Table 3
containing 1 mg/1 lipase,
were transferred to a Gas Chromatograph (GC) vial. The samples were analysed
on a Varian
3800 GC equipped with a Stabilwax- DA w/Integra-Guard column (30m, 0.32 mm ID
and 0.25
micro-m df) and a Carboxen PDMS SPME fibre (75 miero-m). Each sample is
preincubated for
10 min at 40 C followed by 20 min sampling with the SPME fibre in the head-
space over the
textile pieces. The sample is subsequently injected onto the column (injector
temperature=250 C). Column flow = 2 ml Helium/min. Column oven temperature
gradient: 0
min = 40 C, 2 min = 40 C, 22 min = 240 C, 32 min = 240 C. The butyric acid is
detected by FID
detection and the amount of butyric acid is calculated based on a butyric acid
standard curve.
The Risk Performance Odour, R, of a lipase variant is the ratio between the
amount of
released butyric acid from the lipase variant washed swatch and the amount of
released butyric
acid from a swatch washed with the lipase of SEQ ID NO: 2 with the
substitutions T231R +
N233R (reference enzyme), after both values have been corrected for the amount
of released
butyric acid from a non-lipase washed swatch. The risk (R) of the variants is
calculated in
accordance with the below formula:
Odour =measured in micro g butyric acid developed at I mg enzyme protein / 1
corrected for blank
atest enzyme = Odour test enzyme - Blank
arcference enzyme = Odour reference enzyme - Blank
R = atest enzyme / Ureference enzyme
A variant is considered to exhibit reduced odor compared to the reference, if
the R factor is
lower than 1.
Example 4: Activity (LU) relative to absorbance at 280nm
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23
The activity of a lipase relative to the absorbance at 280 nm is determined by
the following
assay LU/A280:
The activity of the lipase is determined as described above in the section
Lipase activity.
The absorbance of the lipase at 280 nm is measured (A280) and the ratio
LU/A280 is calculated.
The relative LU/A280 is calculated as the LU/A280 of the variant divided by
the LU/A280 of a
reference enzyme. In the context of the present invention the reference enzyme
is the lipase of
SEQ ID NO:2 with the substitutions T231R + N233R.
Example 5: BR - Benefit Risk
The Benefit Risk factor describing the performance compared to the reduced
risk for
odour smell is thus defined as: BR = RPaõg / R
A variant is considered to exhibit improved wash performance and reduced odor,
if the BR
factor is higher than 1.
Applying the above methods the following results were obtained:
Average
Variant Mutations in SEQ ID NO: 2 RP BR LU/A280
(Rpavg)
I 1202G + T231R + N233R 0.84 1.41 not
determined
2 186V + L227G + T231R + N233R + 1.08 1.52 1700
P256K
3 Q4V + S58N + V60S + T23 3 R+ N233R 0.87 1.73 1950
4 S58N + V60S + 190R + T231R + N233R 1.06 1.27 2250
5 1255Y + T231R + N233R 1.19 1.17 3600
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24
6 190A + T231R + N233R + 1255V 1.13 1.14 2700
Referen T231R +N233R 1.00 1.00 3650
ce
7 G91A + E99K + T231R+N233R + 0.43 not 850
Q249R + 270H + 271T + 272P + 273S + determined
274S + 275G + 276R + 277G + 278G +
279H + 280R
8 G91A + E99K + T231R, N233R. + 0.13 not 500
Q249R + 270H + 271T + 272P + 273S + deterrnined
274S +275G+276R+277G+278G
Table 4
The reference lipase and variants 7 and 8 in Table 4 are described in WO
2000/060063.
DETERGENT EXAMPLES
Abbreviated component identifications for the examples are as follows:
LAS Sodium linear Cl 1-13 alkyl benzene sulphonate.
CxyAS Sodium Clx - C1}, alkyl sulfate.
CxyEzS Clx - Cly sodium alkyl sulfate condensed with an average of z
moles of ethylene oxide.
CxyEy Clx - Cly alcohol with an average of ethoxylation of z
QAS RZ.N+(CH3)2(CZH4OH) with R2 = C10-C12
Silicate Amorphous Sodium Silicate (SiOz:Na2O ratio = 1.6-3.2:1).
Zeolite A Hydrated Sodium Aluminosilicate of formula Na,a(A1OZSiO2)12.
27H20 having a primary particle size in the range from 0.1 to 10
micrometers (Weight expressed on an anhydrous basis).
(Na-)SKS-6 Crystalline layered silicate of formula 8-Na2Si2O5,
Citrate Tri-sodiurn citrate dihydrate.
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Citric Anhydrous citric acid.
Carbonate Anhydrous sodium carbonate.
Sulphate Anhydrous sodium sulphate.
MA/AA Random copolymer of 4:1 acrylate/maleate, average molecular
weight about 70,000-80,000.
AA polymer Sodium polyacrylate polymer of average molecular weight 4,500.
PB 1/ PB4 Anhydrous sodium perborate monohydrate / tetrahydrate.
PC3 Anhydrous sodium percarbonate [ 2.74 Na2CO3.3H202 ]
TAED Tetraacetyl ethylene diamine.
NOBS Nonanoyloxybenzene sulfonate in the form of the sodium salt.
DTPA Diethylene triamine pentaacetic acid.
HEDP Hydroxyethane di phosphonate
EDDS Na salt of Ethylenediamine-N,N'-disuccinic acid, (S,S) isomer
STPP Sodium tripolyphosphate
Protease Proteolytic enzyme sold under the tradename Savinase
Alcalase , Everlase , CoronaseO, Polarzyme , by Novozymes
A/S, Properase , Purafect , Purafect MAO and Purafect Ox
sold by Genencor and proteases described in patents WO 91106637
and/or WO 95/10591 and/or EP 0 251 446 such as FNA, FN3
and/or FN4. -
Amylase Amylolytic enzyme sold under the tradename Purastar > Purafect
Oxam sold by Genencor; Termamyl , FungamylO Duramyl ,
Stainzyme and Natalase sold by Novozymes A/S .
Lipase Any lipase variant 1 to 5 described in example 5 Table 4, and
combinations thereof.
Mannanase Mannaway sold by Novozymes
CMC or HEC Carboxymethyl or Hydroxyethyl or ester modified cellulose.
or EMC
SS Agglom_ Suds Suppressor agglomerate: 12% Silicone/silica, 18% stearyl
alcohol,70% starch in granular form.
TEPAE Tetreaethylenepentaamine ethoxylate.
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pH Measured as a 1% solution in distilled water at 20 C.
Examnle A
Bleaching detergent compositions having the forni of granular laundry
detergents are
exemplified by the following, formulations.
A B C D E F
LAS 20 22 20 15 20 20
QAS 0.7 1 1 0.6 0.0 0.7
C25E3S 0.9 0.0 0.9 0.0 0.0 0.9
C25E7 . 0.0 0.5 0.0 1 3 1
STPP 23 30 23 17 12 23
Zeolite A 0.0 0.0 0.0 0.0 10 0.0
Silicate 7 7 7 7 7 7
Carbonate 15 14 15 18 15 15
AA Polymer 1 0.0 1 1 1.5 1
CMC 1 1 1 1 1 1
Protease 32.89mg/g 0.1 0.07 0.1 0.1 0.1 0.1
Amylase 8.65mg/g 0.1 0.1 0.1 0.0 0.1 0.1
Lipase 18mg/g 0.03 0.07 0.3 0.1 0.07 0.1
Brightener -Tinopal AMS (Ciba) 0.06 0.0 0.06 0.18 0.06 0.06
Brightener -Tinopal CBS-X (Cib 0.1 0.06 0.1 0.0 0.1 0.1
DTPA 0.6 0.3 0.6 0.25 0.6 0.6
MgSO4 1 1 1 0.5 1 1
PC3 0.0 5.2 0.1 0.0 0.0 0.0
PB 1 4.4 0.0 3.85 2.09 0.78 3.63
NOBS 1.9 0.0 1.66 1.77 0.33 0.75
TAED 0.58 1.2 0.51 0.0 0.015 0.28
Balance Balance to Balance Balance Balance Balance
Sulphate/Moisture to 100% 100% to 100% to 100% to 100% to 100%
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Any of the compositions in Example A is used to launder fabrics at a
concentration of 600 -
10000 ppm in water, with typical median conditions of 2500ppm, 250C, and a
25:1 water:cloth
ratio. The typical pH is about 10 but can be can be adjusted by altering the
proportion of acid to
Na- satt form of alkylbenzenesulfonate.
Example B
Bleaching detergent compositions having the form of granular laundry
detergents are
exemplified by the following formulations.
A B C D
LAS 8 7.1 7 6.5
C25E3S 0 4.8 0 5.2
C68S 1 0 1 0
C25E7 2.2 0 3.2 0
QAS 0.75 0.94 0.98 0.98
(Na-)SKS-6 4.1 0 4.8 0
Zeolite A 20 0 17 0
Citric 3 5 3 4
Carbonate 15 20 14 20
Silicate 0.08 0 0.11 0
Soil release agent 0.75 0.72 0.71 0.72
MA/AA 1.1 3.7 1.0 3.7
CMC 0.15 1.4 0.2 1.4
Protease (56.00mg activelg) 0.37 0.4 0.4 0.4
Terrnamyi (21.55mg active/g) 0.3 0.3 0.3 0.3
Lipase (18.00rng active/g) 0.05 0.15 0.1 0.5
Arnylase (8.65mg active/g) 0.1 0.14 0.14 0.3
TAED 3.6 4.0 3.6 4.0
PC3 13 13.2 13 13.2
EDDS 0.2 0.2 0.2 0.2
HEDP 0.2 0.2 0.2 0.2
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MgSO4 0.42 0.42 0.42 0.42
Perfume 0.5 0.6 0.5 0.6
SS Agglom. 0.05 0.1 0.05 0.1
Soap 0.45 0.45 0.45 0.45
Sulphate 22 33 24 30
Balance to Balance to Balance to Balance to
Water & Miscellaneous 100% 100% 100% 100%
Any of the above compositions in Example B is used to launder fabrics at a
concentration
of 10,000 ppm in water, 20-90 OC, and a 5:1 water:cloth ratio. The typical pH
is about 10 but
can be can be adjusted by altering the proportion of acid to Na-salt form of
alkylbenzenesulfonate.
Example C
A B C D E F
(wt%) (wt%) (wt%) (wt%) (wt%) (wt%)
C25E1.8S 11 10 4 6.32 6.0 8.2
LAS 4 5.1 8 3.3 4.0 3.0
Sodium formate 1.6 0.09 1.2 0.04 1.6 1.2
Sodium hydroxide 2.3 3.8 1.7 1.9 2.3 1.7
Monoethanolamine 1.4 1.490 1.0 0.7 1.35 1.0
Diethylene glycol 5.5 0.0 4.1 0.0 5.500 4.1
C23E9 0.4 0.6 0.3 0.3 2 0.3
DTPA 0.15 0.15 0.11 0.07 0.15 0.11
Citric Acid 2.5 3.96 1.88 1.98 2.5 1.88
C,2_,4 dimethyl
Amine Oxide 0.3 0.73 0.23 0.37 0.3 0.225
C12_1 g Fatty Acid 0.8 1.9 0.6 0.99 0.8 0.6
Borax 1.43 1.5 1.1 0.75 1.43 1.07
Ethanol 1.54 1.77 1.15 0.89 1.54 1.15
~
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TEPAE 0.3 0.33 0.23 0.17 0.0 0.0
ethoxylated
hexamethylene
diamine2 0.8 0.81 0.6 0.4 0.0 0.0
1,2-Propanediol 0.0 6.6 0.0 3.3 0.0 0.0
Protease* 36.4 36.4 27.3 18.2 36.4 27.3
Mannanase * 1.1 1.1 0.8 0.6 1.1 0.8
Amylase* 7.3 7.3. 5.5 3.7 7.3 5.5
Lipase* 10 3.2 0.5 3.2 2.4 3.2
Water, perfume,
dyes & other
components Balance Balance Balance Balance Balance Balance
* Numbers quoted in mg enzyme/ 100g
as described in US 4,597,898.
2 available under the tradename LUTENSIT from BASF and such as those
described in WO
01/05874
All documents cited in the Detailed Description of the Invention are in
relevant part
incorporated herein by reference: the citation of any document is not to be
construed as an
admission that it is prior art with respect to the present invention. To the
extent that any
meaning or definition of a term in this document conflicts with any meaning or
definition of the
same term in a document incorporated by reference, the meaning or definition
assigned to that
term in this document shall govern.
While particular embodiments of the present invention have been illustrated
and
described, it would be obvious to those skilled in the art that various other
changes and
modifications can be made without departing from the spirit and scope of the
invention. It is
therefore intended to cover in the appended claims all such changes and
modifications that are
within the scope of this invention.
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 29
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