Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W O 92/08779 PC~r/US91/08041
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2U~62~6
LIQUID DETERGENT COMPOSITION CONTAINING
LIPASE AND PROTEASE
Technical Field
The present invention relates to liquid detergent compositions which
contain an enzyme system. The enzyme system is a combination of a
modified protease and a lipase.
Background
It is well known in the art that detergent compositions may
advantageously comprise enzyme systems. Such enzyme systems include
cellulase, protease, lipase and amylases. The present invention is
specifically aiming at providing liquid detergent compositions in which
the enzyme system comprises a mixture of protease and lipase.
Formulating such a combination in a granular detergent raises no
specific issue, since both enzymes can be physically separated. On the
contrary, formulating such a combination in a liquid detergent raises a
specific technical issue in that the protease is likely to take as a
substrate any protein present in the detergent composition.
W O 92/08779 z PCT/US91/08041
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Specifically, it has been observed that lipases which may also be
present in the detergent composition are particularly subject to such
proteolytic degradation; as a consequence, the residual activity of the
lipase in the detergent composition will rapidly diminish with the
storage time of the detergent composition, so that it was up to now
impossible to formulate liquid detergent compositions comprising at the
same time a lipase and a protease, said detergent compositions being
sufficiently stable for a commercial exploitation.
It is thus an object of the present invention to provide a liquid
detergent composition comprising an enzyme system comprising a lipase
and a protease, wherein said enzyme system is stable; by stable, it is
meant that the proteolytic degradation of the lipase is substantially
reduced.
It has now been found that this object can be met by using anv
lipase, or mixtures thereof, together with a bacterial serine protease
wherein the methionine adjacent to the serine of the active site has
been replaced by another amino acid, or mixtures of such proteases.
Indeed, it has been discovered that this specific combination would
provide an enzyme system comprising a protease and a lipase, which would
be stable in a liquid detergent composition.
This solution has the advantage of being simple because it onlv
requires ingredients which are commercially available; indeed, several
modified bacterial serine proteases suitable for the purpose of this
invention are commercially available, as well as several lipases
suitable for use in a detergent comp~sition. Furthermore, the detergent
compositions according to the invention require no addition of specific
lipase stabilizers, and are therefore particularly attractive in terms
of product cost and environmental compatibility.
Modified bacterial serine proteases including proteases suitable for
use in the compositions according to the invention are disclosed for
instance in EP-A-0 328 229 as well as their use in detergent compositions.
This patent application describes among others a modified bacterial serine
protease which is commercially available from GIST-BROCADES under the name
MAXAPEM 15R.
Biotechnology Newswatch, published March, 1988, page 6, and
EP A-O 258 268 describe a lipase enzyme which is commercially available from
NOVO NORDISK A/S under the trade name LIPOLASER. This European patent
application mentions that LIPOLASER can be combined with proteases to form a
granular enzymatic detergent additive.
EP-A-O 381 262 describes detergent compositions comprising a protease
and a lipase, preferably LIPOLASER, together with a stabilizing system. The
proteases disclosed in this reference include bacterial proteases.
Summary of the invention
Accordingly, the present invention is a liquid detergent composition
comprising from about 5% to about 60% by weight of an organic surface active
agent selected from nonionic, anionic, cationic, and zwitterionic surface
active agents and mixtures thereof, and an enzyme system comprising a,lipase
derived from Humico 1a 7anuginosa a bacterial serine protease derived from
Baci11us subti1is selected from the group consisting of a Baci11us subti1is
which has been modified by replacing the methionine at position 197 in its
amino acid sequence with cysteine or a Baci11us subti1is which has been
modified by replacing the methionine at position 216 in its amino acid
sequence with cysteine and wherein said lipase is present in an amount
sufficient to provide from 0.1 to 10,000 Lipolytic Units per gram and
wherein said protease is present in the amount of from 0.005 to 10 mg of
active protease per gram of finished product, and from 0.01% to 5% by
weight of the composition of an enzyme stabilization system selected from
the group consisting of boric acid, 1,2-propanediol, carboxylic acids, and
mixtures thereof and wherein said composition has a pH of from 7.0 to 8.5.
Detailed description of the invention
The enzyme system according to the present invention comprises a
lipase and a protease. Any lipase suitable for use in a detergent
composition can be used in the compositions according to the invention, as
described for instance in EP 0 381 262 or EP 271 152. The preferred lipase
to be used in the compositions according to the present invention
C
f
W O 92/08779 P ~ /US91/08041
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~ is a lipase derived from Humicola lanu~inosa, as described in EP-A-0 258
068 to NOVO INDUSTRI A/S. This patent application describes how to
obtain said specific lipase, but said specific lipase is also
commercially available from NOVO NORDISK A/S under the trade name
LIPOLASER. Other commercially available lipases suitable for use herein
are Amono-P Lipase R, Amono-B Lipase R, Amono CES Lipase R, Amono AKG
Lipase R, all from Amono Pharmaceuticals, Japan; Toyo Jozo Co, Japan and
US biochemical Corp. USA as well as Diosynth Co, NL also commercialize
suitable lipases for use in the compositions according to the present
invention.
The compositions according to the present invention typically
comprise from 0.l to l0000 Lipolytic Units per gram of finished
product, preferably from l0 to 2500 Lipolytic Units per gram of finished
product. Lipolytic units are defined for instance in EP 0 258 268, page
5 line 38..
The proteases to be used according to the present invention are
modified bacterial serine proteases. All native bacterial serine
proteases are characterized in that the active site invariably comprises
a triade of amino acids which are serine, histidine and aspartic acid.
These amino acids are positioned in the native form of the enzyme in
such a way that they catalyse the cleavage of internal peptide bonds of
proteins. Another common point between these bacterial serine prote~ses
is that there always is a methionine adjacent to the serine of .he
active site, in the native sequence. The bacterial serine proteaseS
suitable for use according to the present invention are those wherein
the methionine adjacent to the serine of the active site has been
substituted by another amino acid. The serine of the active site can
also be defined as the serine which is homologuous to the serine in
position 221 in the amino acid sequence of the bacterial subtilisin
protease produced by Bacillus Subtilis; said sequence is listed herein
after in figure l.
W O 92/08779 5 P ~ /US91/08041
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In the sequence of this bacterial subtilisin protease produced by
Bacillus Subtilis, the methionine is immediately after the serine in
position 221 and therefore it is the methionine in position 222 which
needs to be substituted by another amino acid. It is possible that, in
the sequence of other bacterial serine proteases, this methionine would
not be immediately following the serine of the active site; in such a
case, it is the methionine homologuous to the methionine in position 222
in the sequence of this bacterial subtilisin protease produced by
Bacillus Subtilis which needs to be substituted by another amino acid.
It is to be understood that the present invention does not reside in
these modified proteases per se, rather in ehe particular applica~ion of
these modified proteases to liquid detergent compositions also
comprising a lipase; it is therefore not the aim of the present
description to specify how these modified proteases can be obtained;
This modification can be done bv site-directed mutagenesis or any other
genetic engeneering technique well known in the art for this purpose:
for instance, EP-A-0 328 229, to GIST-BROCADES N.V. describes how to
obtain such proteases. Another suitable method is described in EP 130
756, which also describes a mofified bacterial serine protease suitable
for use in the compositions according .o the invention.
Furthermore, some modified bacterial serine proteases suitable for
use in the compositions according to the invention are commerciall.
available, such as DURAZY~ R from NOVO, which is the methionine moaified
version of SAVINASE R; another e~ample of available modified protease is
.YAXAPEM 15 from GIST-BROCADES, ~hich is ~he modified verSion of .~.CA'
wherein the methionine in position 216 has been subs.i.uted. Also
available are experimental samples of modified OPTICLE~ R and
OPTIMASER, from SOLVAY enzymefi; both are modified in that the methionine
in position 222 is substituted by a c~steine. Preferred modified
bacterial serine protease according to the present invention are .~AXAPEM
15R from GIST BROCADES and DURAZYM R from NOVO.
The compositions according to the present invention typically will
contain from 0.005 to 10 mg of active protease per gram of finished
product, preferably from 0.01 to 5.0 mg of active protease per gram of
W O 92/08779 P ~ /US91/08041
finished product2 ~19xtur~es of the modified bacterial serine protease
described herein above are also suitable for use in the compositions
according to the invention.
The rest of the liquid detergent composition according to the
present invention is made of conventional detergency ingredients, i.e.
water, surfactants, builders and others. The following description of
these ingredients is for the sake of completeness of the description and
is not to be construed as limiting the compositions of the present
invention to those conventional ingredients described.
The liquid detergent compositions herein comprises from 5% to 60% by
weight of the total liquid detergent composition, preferably from 10% by
weight to 40~ by weight of an organic surface-active agent selected from
nonionic, anionic, cationic and zwitterionic surface-active agents and
mixtures thereof.
Suitable anionic surface-active salts are selected from the group of
sulfonates and sulfates. The like anionic surfactants are well-known in
the detergent arts and have found wide applica.ion in com~ercial
detergents. Preferred anionic water-soluble sulfonate or sulfate salts
have in their molecular structure an alkvl radical containing from abou.
3 to about 22 carbon atoms.
Examples of such preferred anionic surfactan. salts are the react on
?roducts obtained by sulfating Cg-Clg fat,y alconols deri.~ed from e.g.
tallow oil, palm oil, palm kernel oil and coconut oil; alkylbenzene
sulfonates wherein the alkyl group contains from about 9 to abou~ 15
carbon atoms; sodium alkylglyceryl ether sulfonates; ether sulfates of
fatty alcohols derived from tallow and coconut oils; coconut fatty acid
monoglyceride sulfates and sulfonates; and water-soluble salts of
paraffin sulfonates having from about 8 to about 22 carbon atoms in the
alkyl chain. Sulfonated olefin surfactants as more fully descr bed in
e.g. ~.S. Patent Specification '.332.880 can aiso be used. The
W O 92/08779 7 P ~ /~S91/08041
2~9625~
neutralizing cation for the anionic synthetic sulfonaees and/or sulfates
is represented by conventional cations which are widely used in
detergené technology such as sodium, potassium or alkanolammonium.
A suitable anionic synthetic surfactant component herein is
represented by the water-soluble salts of an alkylbenzene sulfonic acid,
preferably sodium alkylbenzene sulfonates, preferably sodium
alkylbenzene sulfonates having from about 10 to 13 carbon atoms in the
alkyl group.Another preferred anionic surfactant component herein is
sodium alkyl sulfates having from about 10 to 15 carbon atoms in the
alkyl group.
The nonionic surfactants suitable for use herein include those
produced by condensing ethylene oxide with a hydrocarbon having a
reactive hydrogen atom, e.g., a hydroxyl, carboxyl, or amido group, in
the presence of an acidic or basic catalyst, and include compounds
having the general formula RA(CH2CH2O)nH wherein R represents the
hydrophobic moiety, A represents the group carrying the reactive
hydrogen atom and n represents the average number of ethylene oxide
moieties. R typically contains from about 8 to 22 carbon atoms They
can also be formed by the condensation of propylene oxide with a lower
molecular weight compound. n usually varies from about 2 to about 24.
A preferred class of nonionic ethoxylates is represented by the
condensation product of a fatty alcohol having from 12 to 15 carbon
atoms and from about 4 to 10 moles of ethvlene oxide per mole or fattv -
alcohol. Suitable species of this class of ethoxylates include : thecondensation product of C12-Cls oxo-alcohols and 3 to 9 moles of
ethylene oxide per mole of alcohol; the condensation product or narrow
cut C14-Cls oxo-alcohols and 3 to 9 moles of ethylene oxide per mole of
fatty(oxo)alcohol; the condensation product of a narrow cut C12-C13
fatty(oxo)alcohol and 6,5 moles of ethylene oxide per mole of fatty
alcohol; and the condensation products of a Clo-C14 coconut fatty
alcohol with a degree of ethoxylation (moles E0/mole fatty alcohol) in
the range from 4 to 8. The fatty oxo alcohols while mainly linear can
have, depending upon the processing conditions and raw material olefins,
W O 92/08779 2 0'9 6 2 S 6 8 P ~ /US91/08041
a certain degree of br~n~hine, particularly short chain such as methyl
branching. A degree of br~nching in the range from 15% to 50% (weight%)
is frequently found in commercial oxo alcohols.
Suitable cationic surfactants include quaternary ammonium compounds
of the formula RlR2R3R4N+ where Rl,R2 and R3 are methyl groups, and R4
is a C12 15 alkyl group, or where Rl is an ethyl or hydroxy ethyl group,
R2 and R3 are methyl groups and R4 is a C12 15 alkyl group.
Zwitterionic surfactants include derivatives of aliphatic
quaternary ammonium, phosphonium, and sulfonium compounds in which the
aliphatic moiety can be straight or branched chain and wherein one of
the aliphatic substituents contains from about 8 to about 24 carbon
atoms and another substituent contains, at least, an anionic water-
solubilizing group. Particularly preferred zwitterionic materials are
the ethoxylated ammonium sulfonates and sulfates disclosed in U.S.
Patents 3,925,262, Laughlin et al., issued December 9, 1975 and
3,929,678, Laughlin et al., issued December 30, 1975.
Semi-polar nonionic surfactants include water-soluble amine oxides
cont~i~ing one alkyl or hydroxy alkyl moiety of from about 8 to about 28
carbon atoms and two moieties selected from the group consisting of
alkyl groups and hydroxy alkyl groups, containing from 1 to about 3
carbon atoms which can optionally be joined into ring structures.
Also suitable are Poly hydroxy fatty acid amide surfactants of .he
formula R2-C-N-Z, wherein Rl is H,
O Rl
Cl 4hydrocarbyl, 2-hydroxy ethyl, 2-hydroxy propyl or a mixture thereof,
R2 is Cs 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, Rl is
methyl, R2 is a straight Cll 15 alkyl or alkenyl chain or mixtures
thereof, and Z is derived from a reducing sugar such as glucose,
fructose, maltose, lactose, in a reductive amination reaction.
W O 92/08779 9 P ~ /US91/08041
The compositions according to the present invention may further
comprise 8 builder system. Any conventional builder system is suitable,
but preferred is a mixture of citric acid and a substituted succinic
acid.
The citric acid builder employed in the practicé of this invention
will be present in the finished product in the form of any water-soluble
salt of citric acid. Such salts include, for example, sodium,
potassium, ammonium or alkanolammonium salts. In practice it is
convenient to use a citric acid monohydrate slurry as a starting
material, which will be neutralized in situ, so as to form the above
mentioned salts.
The substituted succinic acid builders herein are of the general
formula R-CH(COOH)CH2(COOH), i.e., derivatives of succinic acid, wherein
R is Clo-Cl6 alkyl or alkenyl, preferably Cl2-Cl4 alkenyl.
These substituted succinic acid builders are preferably in the
finished product in the form of their water-soluble salts, including the
sodium, potassium, ammonium and alkanolammonium salts (e.g., mono-, di-,
or tri-ethanolammonium).
As raw materials, it is preferred to use these succinic acid
derivatives in their diacid or anhydride form. The diacid will be
neutralized in situ, while the anhydride will undergo a
hydrolysis/neutralization process.
Specific examples of substituted succinic acid builders include :
lauryl succinic acid, myristyl succinic acid, palmityl succinic acid, 2-
dodecenyl succinic acid (preferred), 2-tetradecenyl succinic acid, and
the like.
A preferred builder system comprises from 4% to 12% by weight of
the total composition of the above substituted succinic acid builders,
and from 4% to 12% by weight of the total composition of citric acid.
As an alternative builder, the compositions according to the invention
may also contain a fatty acid. Preferred are oleic and palmitoleic
acid.
It is well known from the man skilled in the art that the pH of the
W O 92/08779 10 P ~ /US91/08041
composition may~sign~f~cQntly affect the enzyme system's performance.~_~
Accordingly, the compositions according to the invention preferably have
a pH adjusted in the range of from 6 to 10, preferably from 7.5 to 8Ø
The compositions according to the invention may also comprise an
enzyme stabilizing system. Indeed, the present invention provides a
system wherein the protease does not significantly attack the native
lipase, but the enzyme system or components thereof may still be subject
to unstability problem due to the other detergency ingredients.
Therefore, stabilizing agents may be needed, which are conventional and
well known in the art. A preferred enzyme stabilizing system is
selected from boric acid, 1,2-propanediol, carboxylic acids, and
mixtures thereof. These enzyme stabilizing systems are typically
present in amounts of from 0.01% to 5% by weight of the total
composition.
The compositions of the invention may also comprise other enzymes.
such as cellulases or amylases. Amylases, particularly, seem to be
stable in the presence of protease, and the compositions of the
invention therefore preferably comprise an amylase.
The compositions herein can contain a series of further optional
ingredients. Examples of the like additives include : suds regulants,
opacifiers, agents .to improve the ~chinP compatibility in relation to
enamel-coated surfaces, bactericides, dyes, perfumes, bleaches including
perborate and percarbonate, brighteners, soil release agents, softening
agents and the like.
The liquid compositions herein can contain further additives,
typically at levels of from 0.05 to 5%. These additives include
polyaminocarboxylates such as ethylenediaminotetracetic acid,
diethylenetriaminopentacetic acid, ethylenediamino disuccinic acid or
water-soluble alkali metals thereof. Other additives include organo-
phosphonic acids; particularly preferred are ethylenediamino
tetramethylenephosphonic acid, hexamethylenediamino
tetramethylenephosphonic acid, diethylenetriamino
pentamethylenephosphonic acid and aminotrimethylenephosphonic acid.
WO 92J08779 PCT/VS91/08041
20962~6
.~ 11
EXAMPLES
The following compositions according to the invention are made by mixing the
listed ingredients in the listed proportions.
1 2 3 4 5
- Linear alkyl benzene sulfonate 12 7 6 7 8
- Sodium C12 15 alkyl sulfate 2 2 3 3 2
- C14 15 alkyl 2.5 times ethoxylated 0 0 2 2 0
sulfate
- C12 glucose amide 0 0 6 6 0
- C12 lsalcohol 7 times ethoxylated 8 0 0 0 0
- C12 lsalcohol 5 times ethoxylated 0 8 0 0 8
- Oleic Acid 2 0 0 0 0
- Citric Acid 3 9 9 13 15
- C12 14 alkenyl substituted 10 5 5 7 6
succinic acid
-.Ethanol 4 4 3 4 5
- 1,2-propanediol 2 3 3 l 2
- NaOH 6 8 8 ll ll
- diethylene triamine 0.5 0.7 0.7
penta(methylene phosphonic acid)
- Amylase(143KNU/g) 0.1 0.1 0.05 0.2 0.1
- LipolaseR(lOOKLU/g 0.4 0.2 0.3 0.3 0.3
commercial solution)
- PEMlSR(50mg/g Commercial solution) 0.3 0 0 0 0.4
- DurazymR (39 mg/g Commercial solution) 0 0.2 0 0 0
- Opticlean M222CR (experimental sample) 0 0.1 0 0.4 0
- Optimase M222CR (experimental sample) 0 0 0.3 0 0
- CaC12 0.01 0 0.01 0.01 0.02
- Na metaborate 2.2 2 2 4 3
- TEA 0 0 0 0 0
- Sodium formate 0 0 0 0 0
- Fatty Acids 0 0 0 0 0
- Uater and Minors -------------Balance to 100~------
W O 92/08779 PC~r/US91/08041
2 0 9 6 2 ~ 6 12 ~~
EXAMPLES
The following compositions according to the invention are made by mixing the listed
ingredients in the listed proportions
6 7 8 9 10
- Linear alkyl benzene sulfonate 5 7 9 8 10
- Sodium C12 15 alkyl sulfate 5 2 1.75 0 3
- C14 15 alkyl 2.5 times ethoxylated 2 0 2 0 0
sulfate
- C12 glucose amide 6 0 7 0 0
- C12 15alcohol 7 times ethoxylated 0 0 0.5 0 11.6
- C12 15alcohol 5 times ethoxylated 0 8 0 8
- Oleic Acid 0 0 0 3.5 2.5
- Citric Acid 10 9 9.5 4
- C12 14 alkenyl substituted 11 ~ 11.5 0 0
succinic acid
- STPP 0 20 0 0 0
- Zeolite 0 0 0 26 0
- Ethanol 6 4 4 3 6
- 1,2-propanediol 3 2 2 2 1.5
- NaOH 9 9 9.8 9 3.5
- diethylene triamine 1.0 1.0 1.0 0.5 0.8
penta(methylene phosphonic acid)
- Amylase(143KNU/g) 0.2 0.1 0.2 0.05
- LipolaseR(100KLU/g 0.5 0.5 0.3 0.2 0.3
commercial solution)
- PEM15R(50mg/g Commercial solution) 0.4 0 0 C 0.2
- DurazymR (39 mg/g Commercial solution) 0 0 0.5 0 0.2
- Opticlean M222CR (experimental sample) 0 0 0 0.3 0
- Optimase M222CR (experimental sample) 0 0.5 0 0 0
- CaC12 0.01 0.01 0.02 0.02 0.01
- Na metaborate 4 2 4 3 0
- TEA 0 0 0 0 6
- Sodium formate 0 0 0 0
- Fatty Acids 0 0 0 0 12
- Water and Minors ---------------Balance to 100%--------
W O 92/08779 PC~r/US91/08041
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EXAMPLES
The following compositions according to the invention are made by mixing the listed
ingredients in the listed proportions
' 11 12 13 14 15
- Linear alkyl benzene sulfonate 5 7 9 8 10
- Sodium C12 15 alkyl sulfate 5 2 1.75 0 3
- C14 15 alkyl 2.5 times ethoxylated 2 0 2 0 0
sulfate
- C12 glucose amide 6 0 7 0 0
- C12 lsalcohol 7 times ethoxylated 0 0 0.5 0 11.6
- C12 lsalcohol 5 times ethoxylated 0 8 0 8
- Oleic Acid 0 0 0 3.5 2.5
- Citric Acid 10 9 9.5 4
- C12 14 alkenyl substituted 11 0 11.5 0 0
succinic acid
- Tartrate monosuccinate 0 15 0 17 20
- Diethoxylated poly (1,2 propylene
terephtalate) 1.0 0.5 0.7 0 0.5
- Ethanol 6 4 4 3 6
- 1,2-propanediol 3 2 2 2 1.5
- NaOH 9 9 9.8 9 3.5
- diethylene triamine 1.0 1.0 1.0 0.5 0.8
penta(methylene phosphonic acid)
- Amylase(143KNU/g) 0.2 0.1 0.2 0.05
- LipolaseR(lOOKLU/g 0.5 0.5 0.3 0.2 0.3
commercial solution)
- PEM15R(50mg/g Commercial solution) 0.4 0 0 0 0.2
- DurazymR (39 mg/g Commercial solution) 0 0 0.5 0 0.2
- Opticlean M222CR (experimental sample) 0 0 0 0.3 0
- Optimase M222CR (experimental sample) 0 0.5 0 0 0
- CaC12 0.01 0.01 0.02 0.02 0.01
- Na ~etaborate 4 2 4 3 0
- T~A 0 0 0 0 6
- Sodium formate 0 0 0 0
- Fatty Acids 0 0 0 0 12
- Water ~hd~Mi,~r5 ---------------Balance to 100~--------
