Note: Descriptions are shown in the official language in which they were submitted.
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ANIONIC-RICH HIGH-pH LIOUID DETERGENT COMPOSITIONS
CONTAINING SUBTILISIN MUTANTS
BACKGROUND AND PRIOR ART
This invention relates to high-anionic, high-pH liquid
detergent compositions containing mutant protease enzymes
which provide enhanced stability.
The modification of subtilisin proteases by substitution at
an amino acid site is known in the art. US-A-4 760 025,
assigned to Genencor, for example, claims subtilisin mutants
with amino acid substitutions at amino acid sites 32, 155,
104, 222, 166, 64, 33, 169, 217 or 157 which are different
from subtilisins naturally produced by B. amyloliquefaciens.
These amino acid substitutions are said to lead to increased
oxidation stability of the protease.
WO 87/04461, assigned to Amgen, discloses the substitution in
Bacillus subtilisins of alternative amino acids (i.e. serin~,
valine, threonine, cysteine, glutamine and isoleucine) for
ASN, GLY or ASN-GLY sequences (specifically at position 218).
These mutations are said to increase the stability of the
enzyme at high temperatures or over a broader pH range than
the wild type enzyme. WO 88/08033, also to Amgen, claims
mutations which modify calcium-binding capacity (to replace
an amino acid with a negatively charged residue such as ASP
or GLU) and optionally a deletion and/or replacement of
either residue of ASN-GLY sequences which results in better
pH and thermal stability and higher specific activities. The
reference claims that sites 41, 75, 76, 77, 78, 79, 80, 81,
208, and 214 may be replaced by a negatively charged amino
acid and ASN may be replaced by SER, VAL, THR, CYS, GLU, or
ILE in ASN-GLY sequences.
EP-A-342 177 (Procter & Gamble) discloses compositions
comprising a protease with a specific mutation and having a
pH between 7.0 and 9Ø
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These references do not disclose anionic-rich, high-pH
detergent compositions comprising the subtilisin mutants of
the subject invention or the advantages provided by the use
of these mutants in these detergent compositions.
WO 89/09819 (corresponding to US-A-4 980 288), assigned to
Genex, discloses the subtilisin mutants which are used in the
liquid detergent compositions of the invention. Although the
use of mutants in washing preparations is disclosed (Claims 6
and 7), there is no teaching of the use of these mutants in
anionic-rich, high-pH compositions. In particular, there is
no disclosure of the use of these mutants in specific
detergent compositions and no teaching or disclosure that the
mutant enzymes will have enhanced stability in these
specifically defined compositions.
SUMMARY OF THE INVENTION
The subject invention provides liquid detergent compositions
comprising:
(1) from 5% to 65% by weight anionic surfactant or
anionic surfactant and one or more detergent-actives wherein
the ratio of anionic to non-anionic is greater than 1:1;
(2) from 0% to 50% by weight builder;
(3) a mutant subtilisin protease added in sufficient
quantity to have an activity level of 0.01 to 100,000 GU/g
having substitutions in 1 or more amino acid residues
compared to wild type subtilisin or commercially available
subtilisin; and
(4) remainder water and minor ingredients.
The pH of these compositions ranyes from 9 to 12, preferably
from 9.5 to 11.
According to the invention, when certain modified mutant
subtilisin proteases are used in the above-identified
anionic~rich, high-pH detergent compositions of the
invention, enhanced stability is observed.
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DETAILED DESCRIPTION OF THE INVENTION
Deteraent-Active
The compositions of the invention comprise from about 5% to
about 65~ by weight of anionic surfactant or anionic
surfactant and one or more detergent-actives wherein the
ratio of anionic to non-anionic is greater than l:1.
Preferably, the compositions of the invention may comprise
from 5-25% anionic and preferably from 10-2~% anionic; and
from 5-15% preferably from 7-10~ nonionic surfactant.
The detergent-active material other than anionic surfactant
may be an alkali metal or alkanolamine soap or a 10 to 24
carbon atom fatty acid, including polymerized fatty acids, or
a nonionic, cationic, zwitterionic or amphoteric synthetic
detergent material, or mixtures of any of these.
Examples of the anionic synthetic detergents are salts
(including sodium, potassium, ammonium and substituted
ammonium salts) such as mono-, di- and triethanolamine salts
of 9 to 20 carbon alkylbenzenesulphonates, 8 to 22 carbon
primary or secondary alkanesulphonates, 8 to 24 carbon
olefinsulphonates, sulphonated polycarboxylic acids prepared
by sulphonation of the pyrolyzed product of alkaline earth
metal citrates, e.g., as described in GB-A-1 082 179, 8 to 22
carbon alkylsulphates, 8 to 2~ carbon alkylpolyglycol-ether-
sulphates, -carboxylates and -phosphates (containing up to 10
moles of ethylene oxide); further examples are described in
"Surface Active Agents and Detergents" (Vol. I and II) by
Schwartz, Perry and Berch. Any suitable anionic may be used
and the examples are not intended to be limiting in any way.
Examples of nonionic synthetic detergents which may be used
with the invention are the condensation products of ethylene
oxide, propylene oxide and/or butylene oxide with 8 to 18
carbon alkylphenols, 8 to 18 carbon primary or secondary
aliphatic alcohols, 8 to 18 carbon fatty acid amides; further
4 C 6132 (R)
examples of nonionics include tertiary amine oxides with 8 to
18 carbon alkyl chain and two 1 to 3 carbon alkyl chains. The
above reference also describes further examples of nonionics.
The average number of moles of ethylene oxide and/or
propylene oxide present in the above nonionics varies from
1-30; mixtures of various nonionics, including mixtures of
nonionics with a lower and a higher degree of alkoxylation,
may also be used.
Examples of cationic detergents which may be used are the
quaternary ammonium compounds such as alkyldimethyl ammonium
halogenides.
Examples of amphoteric or zwitterionic detergents which may
be used with the invention are N-alkylamino acids,
sulphobetaines, condensation products of fatty acids with
protein hydrolysates; but, owing to their relatively high
costs, they are usually used in combination with an anionic
or a nonionic detergent. Mixtures of the various types of
active detergents may also be used, and preference is given
to mixtures of an anionic and a nonionic detergent active.
Soaps (in the form of their sodium, potassium and substituted
ammonium salts) of fatty acids may also be used, preferably
in conjunction with an anionic and/or nonionic synthetic
detergent.
Builders
Builders which can be used according to this invention
include conventional alkaline detergency builders, inorganic
or organic, which can be used at levels from 0% to about 50%
by weight of the composition, preferably from 1% to about 20
by weight, most preferably from 2% to about 8%.
Examples of suitable inorganic alkaline detergency builders
are water-soluble alkalimetal phosphates, polyphosphates,
borates, silicates and also carbonates. Specific examples of
such salts are sodium and potassium triphosphates,
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pyrophosphates, orthophosphates, hexametaphosphates,
tetraborates, silicates and carbonates.
Examples of suitable organic alkaline detergency builder
salts are: (1) water-soluble amino polycarboxylates, e.g.
sodium and potassium ethylenediaminetetraacetates,
nitrilotriacetates and N-(2 hydroxyethyl)-nitrilodiacetates;
(2) water-soluble salts of phytic acid, e.g. sodium and
potassium phytates (see US-A-2 379 942), (3) water-soluble
polyphosphonates, including specifically, sodium, potassium
and lithium salts of ethane-l-hydroxy- 1,l-diphosphonic acid;
sodium, potassium and lithium salts of methylene diphosphonic
acid; sodium, potassium and lithium salts of ethylene
diphosphonic acid; and sodium, potassium and lithium salts of
ethane-1,1,2- triphosphonic acid. Other examples include the
alkali metal salts of ethane-2-carboxy-1,1-diphosphonic acid
hydroxymethanediphosphonic acid, carboxyldiphosphonic acid,
ethane-1-hydroxy-1,1,2-triphosphonic acid, ethane-2-hydroxy-
1,1,2-triphosphonic acid, propane- 1,1,3,3-tetraphosphonic
acid, propane-1,1,2,3- tetraphosphonic acid, and propane-
l,2,2,3- tetraphosphonic acid; (4) water-soluble salts of
polycarboxylate polymers and co-polymers as described in
US-A-3 308 067.
In addition, polycarboxylate builders can be used
satisfactorily, including water-soluble salts of mellitic
acid, citric acid, and carboxymethyloxysuccinic acid and
salts of polymers of itaconic acid and maleic acid. Certain
zeolites or aluminosilicates can be used. One such !
aluminosilicate which is useful in the compositions of the
invention is an amorphous water-insoluble hydrated compound
of the formula Nax(yAlO2.SiO2), wherein x is a number from 1.0
to 1.2 and y is 1, said amorphous material being further
characterized by an Mg++ exchange capacity of from about 50
mg eq. CaCO3/g and a particle diameter of from about 0.01
micron to about 5 microns. This ion-exchange builder is more
fully described in GB-A-l 470 250.
6 C 6132 (R)
A second water-insoluble synthetic aluninosilicate ion-
exchange material useful herein is crystalline in nature and
has the formula Naz[(AlO2)y.(SiO2)]xH2O, wherein z and y are
integers of a least 6; the molar ratio of z to y is in the
range from 1.0 to about 0.5, and x is an integer from about
15 to about 264; said aluminosilicate ion-exchange material
having a particle size diameter from about 0.1 micron to
about lO0 microns; a calcium ion-exchange capacity on an
anhydrous basis of at least about 200 milligrams equivalent
of CaCO3 hardness per gram; and a calcium-exchange rate on an
anhydrous basis of at least about 2 grams/gallon/minute/gram.
These synthetic aluminosilicates are more fully described in
GB-A-1 429 143.
Mutant Subtilisin Protease
Proteins exist in a dynamic equilibrium between a folded,
ordered state and an unfolded, disordered state. This
equilibrium in part reflects the short range interactions
between the different segments of the polypeptide chain which
tend to stabilize the protein's structure, and, on the other
hand, those thermodynamic forces which tend to promote the
randomization of the molecule.
The largest class of naturally occurring proteins is made up
of enzymes. Each enzyme generally catalyses a different kind
of chemical reaction, and is usually highly specific in its
function. Enzymes have been studied to determine correlations
between the three-dimensional structure of the enzyme and its
activity or stability.
The amino acid sequence of an enzyrne determines the
characteristics of the enzyme, and the enzyme's amino acid
sequence is specified by the nucleotide sequenc~ of a gene
coding for the enzyme. A change of the amino acid sequence of
an enzyme may alter the enzyme's properties to varying
degrees, or may even inactivate the enzyme, depending on the
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7 C 6132 (R)
location, nature and/or magnitude of the change in the amino
acid sequence.
Although there may be slight variations in a distinct type of
naturally occurring enzyme within a given species or
organism, enzymes of a specific type produced by organisms of
the same species generally are substantially identical with
respect to substrate specificity, thermal stability, activity
levels under various conditions(e.g. temperature and pH),
oxidation stability, and the like. Such characteristics of a
naturally occurring or "wild-type" enzyme are not necessarily
optimized for utilization outside of the natural environment
of the enzyme. It may thus be desirable to alter a natural
characteristic of an enzyme to optimize a certain property of
the enzyme for a specific use, or for use in a specific
environment.
Amino acids are naturally occurring compounds that are the
building blocks of proteins. The natural amino acids are
usually abbreviated to either three letters or one letter.
The most common amino acids, and their symbols, are given in
Table 1. The amino acids are joined head to tail to form a
long main chain. Each kind of amino acid has a different side
group.
J
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Table 1. Amino acid names and abbreviations
________________________________________________________
Amino acid Three letter code Single letter code
_________________________________________________ ______
5 Alanine Ala A
Arginine Arg R
Aspartic acid Asp D
Asparagine Asn N
Cysteine Cys C
10 Glutamic acid Glu E
Glutamine Gln Q
Glycine Gly G
Histidine His H
Isoleucine Ile
15 Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
20 Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V
9 C 6132 (R)
All amino acids have the same atoms in the main chain and
differ only in the side chains. The main-chain atoms are a
nitrogen, two carbons, and one oxygen. The first atom is the
nitrogen, called N. The next atom is a carbon and is called
the alpha-carbon. Side groups are attached to this
alpha-carbon. The alpha-carbon is connected to the carbonyl
carbon which is called C. C is connected to the carbonyl
oxygen (called O) and to the N of the next residue. The side
group atoms are given names composed of the symbol for the
element (C, 0, N, S), a Greek letter (alpha, beta, gamma,
delta, epsilon, zeta and eta), and perhaps an Arabic numeral
if the side group is forked.
The subtilisin enzymes used in the detergent compositions of
this invention have been modified by mutating the various
nucleotide sequences that code for the enzymes. Use of the
modified subtilisin enzymes provides enhanced stability in
the compositions.
The subtilisin enzymes of this invention belong to a class of
enzymes known as proteases. A protease is a catalyst for the
cleavage of peptide bonds. An example of this cleavage is
given below:
Rl ~ 12
/ C~ \ / CQ
H N H
H
~H20 protease
I 1 f R2 o
~ C~ I Cl~ :~
H
; i . ,. .,~ '~ ' ` '~1
C 6132 (R)
One type of protease is a serine protease. A serine protease
will catalyse the hydrolysis of peptide bonds in which there
is an essential serine residue at the active site. Serine
proteases can be inhibited by phenylmethyl sulphonylfluoride
and by diisopropylfluoro phosphate.
A subtilisin is a serine protease produced by Gram positive
bacteria or by fungi. The amino acid sequences of seven
subtilisins are known. These include five subtilisins from
Bacillus strains (subtilisin BPN', subtilisin Carlsberg,
subtilisin DY, subtilisin amylosacchariticus, and
mesenticopeptidase). (Vasantha et al., "Gene for alkaline
protease and neutral protease from Bacillus amyloliguefaciens
contain a large open-reading frame between the regions coding
for signal sequence and mature protein, "J. Bacteriol.
159:811-819_(1984); Jacobs et al., "Cloning sequencing and
expression of subtilisin Carlsberg form Bacillus
licheniformis, "Nucleic Acids Res. 13:8013-8926 (1985);
Nedkov et al., "Determination of the complete amino acid
sequence of subtilisin DY and its comparison with the primary
structures of the subtilisin BPN', Carlsberg and
amylosacchariticus, "Biol. Chem. Hoppe-Seyler 366:421-430
(1985); Kurihara et al., "Subtilisin amylosacchariticus,"
J. Biol. Chem. 247:5619-5631 (1972); and Svendsen et al.,
"Complete amino acid sequence of alkaline mesenterico-
peptidase," FEBS LQ t. 196:228-232 (1986)j.
The amino acid sequence of the subtilisin thermitase from
Thermoactinomyces vulqaris is also known (Meloun et al.,
"Complete primary structure of thermitase from
Thermoactinomyces vulqaris and its structural features
related to the subtilisin-type proteases," FEBS Lett.
183:195-200 (1985)). The amino acid sequences from two fungal
proteinases are known: Proteinase K from Tritirachium album
(Jany et al., "proteinase K from Tritirachium album Limber,"
Biol. Chem. Hoppe-Seyler 366:~85-492 (1985)) and
thermomycolase from the thermophilic fungus, Malbranchea
11 C 6132 (R)
~ulchella (Gaucher et al., "Endopeptidases: Thermomycolin,"
Methods Enzymol. 45:415-433 (1976)).
These enzymes have been shown to be related to subtilisin
BPN', not only through their primary sequences and
enzymological properties, but also by comparison of x-ray
crystallographic data. (McPhalen et al., "Crystal and
molecular structure of the inhibitor eglin from leeches in
complex with subtilisin Carlsberg," FEBS Lett. 188:55-58
(1985) and Pahler et al., "Three-dimensional structure of
fungal proteinase K reveals similarity to bacterial
subtilisin-," EMBO J. 3:1311-1314 (1984).)
The mutated enzymes used in the compositions of the
invention may be introduced into any serine protease which
has at least 50% and preferably 80% amino acid sequence
homology with the sequence referenced above for subtilisin
BPN', subtilisin Carlsberg, subtilisin DY, subtilisin
amylosacchariticus, mesenticopeptidase, thermitase,
proteinase K, or thermomycolase, and therefore may be
considered homologous.
Thus, the mutated subtilisin enzymes used in the detergent
composition of this invention have at least one of the
specific amino acid position substitutions shown in Table 2.
In Table 2, the naturally occurrinq amino acid and position
number is given first with the arrow to the right indicating
the amino acid substitution. The mutations-were made using
subtilisin BPN'. However, as explained her~n, these
mutations can be introduced at analogous positions in other
serine proteases using oligonucleotide-directed mutagenesis.
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12 C 6132 (R)
Table 2
Mutations in subtilisin BPN'
1 Val8 -> Ile
2 Thr22 -> Cys, Ser87 -> Cys
3 Thr22 -> Lys, Asn76 -> Asp
4 Met50 -> Phe
5 Ser53 -> Thr
6 Ser63 -> Asp, Tyr217 -> Lys
7 Asn76 -> Asp
8 Ser78 -> Asp
9 TyrlO4 -> Val, Glyl28 -> Ser
10 Alall6 -> Glu
11 Leul26 -> Ile
12 Glyl31 -> Asp
13 Glyl66 -> Ser
14 Glyl69 -> Ala
15 Prol72 -> Asp
16 Prol72 -> Glu
17 Serl88 -> Pro
18 Gln206 -> Cys
19 Gln206 -> Tyr
20 Ala216 -> Cys, Gln206 -> Cys
21 Tyr217 -> Lys
22 Tyr217 -> Leu
23 Asn218 -> Asp
24 Gln206 -> Tyr
25 Ser248 -> Asp, Ser249 -> Arg
26 Thr254 -> Ala
27 Gln271 -> Glu
In general, stability of a mutated subtilisin in a given
composition is expressed as the half life of the enzyme in
hours at a given temperature, e.g. 37C.
Table 3 shows the strain designation of the host cell
secreting the mutated subtilisin enzymes.
13 C 6132 (R)
Table 3
Mutated Subtilisin BPN' Enzymes
Strain Mutation
5 GX7130 Wild type
GX7174 VAL8->ILE
GX7175 GLY169->ALA
GX7181 ASN218->ASP
THR22->CYS
SER87->CYS
GX7186 ASN218->SER
THR22->CYS
SER87->CYS
GLY169->ALA
GX7195 TYR217->LYS
GX7199 THR22~>CYS
SER87->CYS
GLY169->ALA
PR0172->ASP
GX8303 MET50->PHE
GX8309 SER248->ASP
SER249->ARG
GX8314 GLN206->CYS
GX8321 THR22->CYS
SER87->CYS
GLY169->ALA
MET50->PHE
TYR217->LYS
ASN218->SER
GX8324 THR22-~CYS
SER87-,CYS
GLY169->ALA
MET50->PHE
TYR217->LYS
ASN218-~SER
GLN206->CYS
GX8330 TYR217->LEU
14 C 6132 (R)
GX8336 GLN206->TYR
GX8350 MET50->PHE
GLY169->ALA
GLN206->CYS
TYR217->LYS
ASN218~>SER
ASN76->ASP
GX8352 SER63->ASP
TYR217-->LYS
GX8354 GLN271->GLU
GX8363 THR22->LYS
ASN76->ASP
GX8372 MET50->PHE
GLY169->ALA
GLN206->CYS
TYR217->LYS
ASN76->ASP
SER78->ASP
ASN218->SER
GX8376 TYR104->VAL
GLY128->SER
GX7148 GLY131->ASP
GX7150 ASN218->SER
GX7164 ASN218->ASP
GX7178 SER188->PRO
GX7188 ALA116->GLU
GX7189 LEUI.26->ILE
GX8301 ASN218->SER
GhY166->SER
GX8305 SER53->THR
GX8306 ASN218->SER
THR254->ALA
GX8315 ASN218->SER
GLYl31->ASP
THR254->ALA
GX7159 THR22->CYS
SER87->CYS
C 6132 (R)
GX8307 . GLN206->CYS
SER87->CYS
ALA216->CYS
GX7172 PR~172->ASP
GX8312 PRO172->GLU
GX8347 ASN76->ASP
GX8364 SER78->ASP
GX8373 ASN218->ASP
MET50->PHE
GLY169->ALA
GLN206->CYS
TYR217->LYS
ASN76->ASP
SER78->ASP
GX8397 MET50->PHE
ASN76->ASP
GLY169->ALA
GLN206->CYS
ASN218->SER
GX8398 MET50->PHE
ASN76->ASP
GLN206->CYS
TYR217->LYS
ASN218->SER
GX8399 MET50->PHE
ASN76->ASP
ASN218->SER
GLN206->CYS
The subtilisin enzyme mutations, shown in Tables 2 and 3, can
be made on other proteases which are closely related,
subtilisin Carlsberg for example. Closeness of relation is
measured by comparison of amino acid sequences. There are
many methods of aligning protein sequences, but the
differences are only manifested when the degree of
relatedness is quite small. The methods described in Atlas of
Protein Sequence and Structure, Margaret 0. Dayhoff editor,
16 C 6132 (R)
Vol. 5, Supplement 2, 1976, National Biomedical Research
Foundation, Georgetown University Medical Center, Washington,
D.C., p. 3 ff., entitled SEARCH and ALIGN, define
relatedness. As is well known in the art, related proteins
can differ in number of amino acids as well as identity of
each amino acid along the chain. That is, there can be
deletions or insertions when two structures are aligned for
maximum identity. For example, subtilisin Carlsberg has only
274 amino acids, while subtilisin BPN' has 275 amino acids.
Aligning the two sequences shows that Carlsberg has no
residue corresponding to ASN56 of subtilisin BPN'. Thus the
amino acid sequence of Carlsberg would appear very different
from BPN' unless a gap is recorded at location 56. Therefore
an analogous substitution of position 218 of BPN' may be made
at location 218 of subtilisin Carlsberg, provided that the
residues in Carlsberg are numbered by homology to BPN'.
In general, one should not transfer mutations if either
subtilisin has a gap at, or immediately adjacent to, the site
of the mutation. Therefore, after aligning the amino acid
sequences, those mutations at, or next to, gaps should be
deleted from the list of desirable mutations and the mutation
is not made. One can use this reasoning to transfer all of
the thermostable mutations described herein to other
homologous serine proteases.
In brief, in order to introduce the mutation(s) for the
subtilisin, the gene coding for the desired subtilisin
material generally is first isolated from :s natural source
and cloned in a cloning vector. Alternatively, mRNA which is
transcribed from the gene of interest can be isolated from
the source cell and converted into cDNA by reverse
transcription for insertion into a cloning vector. A cloning
vector can be a phage or plasmid, and generally includes a
replicon for autonomous replication of the vector in a micro-
organism independent of the genome of the micro-organism. A
cloning vector advantageously includes one or more phenotypic
17 c 6132 (R)
markers, such as DNA coding for antibiotic resistance, to aid
in selection of micro-organisms transformed by the vector.
Procedures for insertion of DNA or cDNA into a vector for
cloning purposes are well known in the art. These procedures
generally include insertion of the gene coding for the
subtilisin material into an opened restriction endonuclease
site in the vector, and may involve addition of homopolymeric
tails of deoxynucleotides to the ends of the gene and linking
the gene to opened ends of a cloning vector having
complementary homopolymeric tails. A subtilisin gene can then
be mutated by oligonucleotide-directed mutagenesis.
Oligonucleotide-directed mutagenesis, also called site-
directed mutagenesis, is described in detail in Bryan et al.,
Proc. Natl. Acad. Sci. USA 83:3743-3745 (1986), incorporated
herein by reference.
The protease used in these compositions is used in an
amount sufficient to have an activity of 0.01 to lO0,000 GU/g
based on the final composition. A GU is a glycine unit, which
is the amount of proteolytic enzyme which under standard
incubation conditions produces an amount of terminal
NH2-groups equivalent to l microgramme/ml of glycine.
Water
Finally, except for the stabilizer and optional components
described below, water comprises the remainder of the
compositions. Generally, the amount of water will vary from
30-80~ of the composition although this will depend on the
amount of actives and the ingredients used.
Stabilizer
Another component which may be optionally used in the
compositions of the invention is a stabilizer or stabilizer
3S system. The improvements in stability of the invention can be
demonstrated in systems with or without enzyme stabilization
systems although it is preferred that such systems be used.
18 C 6132 (R)
When present, the stabilization system comprises from about
0.1 to about 15% of the composition.
The enzyme stabilization systems may comprise calcium ion,
boric acid, propylene glycol and/or short chain carboxylic
acids. The composition preferably contains from about 0.01 to
about 50, preferably from about 0.1 to about 30, more
preferably from about 1 to about 20 millimoles of calcium ion
per liter.
When calcium ion is used, the level of calcium ion should be
seleeted so that there is always some minimum level available
for the enzyme after allowing for complexation with builders,
etc. in the composition. Any water-soluble calcium salt can
be used as the source of calcium ion including calcium
chloride, calcium formate, calcium acetate, and calcium
propionate. A small amount of calcium ion, generally from
0.05 to about 2.5 millimoles per liter, is often also present
in the composition due to calcium in the enzyme slurry and
formula water.
Another enzyme stabilizer which may be used is propionic acid
or a propionie acid salt capable of forming propionic acid.
When used, the stabilizer may be used in an amount from about
0.1% to about 15% by weight of the composition.
Another preferred enzyme stabilizer is polyols containing
only carbon, hydrogen and oxygen atoms. They preferably
contain from 2 to 6 carbon atoms and from 2 to 6 hydroxy
groups. Examples include propylene glycol (especially 1,2
propanediol which is preferred), ethylene glycol, glycerol,
sorbitol, mannitol and glucose. The polyol generally
represents from about 0.5% to about 15%, preferably from
about 1.0% to about 8% by weight of the composition.
The composition herein may also optionally contain from about
0.25% to about 5%, most preferably from about 0.5% to about
19 C 6132 (R)
3% by weight of boric acid. The boric acid may be, but is
preferably not, formed by a compound capable of forming boric
acid in the composition. Boric acid is preferred, although
other compounds such as boric oxide, borax and other alkali
metal borates (e.g. sodium ortho-, meta-, and pyroborate and
sodium pentaborate) are suitable. Substituted boric acids
(e.g. phenylboronic acid, butane boronic acid and p-bromo
phenylboronic acid) can also be used instead of boric acid.
One especially preferred stabilization system is a polyol in
combination with boric acid. Preferably, the weight ratio of
polyol to boric acid added is at least 1, more preferably at
least 1.3.
Optional Components
In addition to the ingredients described hereinbefore, the
preferred compositions herein frequently contain a series of
optional ingredients which are used for the known
functionality in conventional levels. While the inventive
compositions are premised on aqueous enzyme-containing
detergent compositions, it is frequently desirable to use a
phase regulant. This component, together with water~ then
constitutes the solvent matrix for the claimed liquid
compositions. Suitable phase regulants are well known in
liquid detergent technology and, for example, can be
represented by hydrotropes such as salts of alkylaryl-
sulfonates having up to 3 carbon atoms in the alkylgroup,
e.g. sodium, potassium, ammonium and ethanolamine salts of
xylene-, toluene-, ethyl benzene-, cumene-, and
isopropylbenzene sulfonic acids. Alcohols may also be used as
phase regulants. This phase regulant is frequently used in an
amount from about 0.5% to about 20%, the sum of phase
regulant and water is normally in the range from 35% to 65%.
The preferred compositions herein can contain a series of
further optional ingredients which are mostly used in
additive levels, usually below about 5%. Examples of the like
j i ~ i
C 6132 (R)
additives include: polyacids, suds regulants, opacifiers,
antioxidants, bactericides, dyes, perfumes, brighteners and
the like.
The beneficial utilization of the claimed compositions under
various usage conditions can require the utilization of a
suds regulant. While generally all detergent suds regulants
can be utilized, preferred for use herein are alkylated
polysiloxanes such as dimethylpolysiloxane, also frequently
termed silicones. The silicones are frequently used in a
level not exceeding 0.5%, most preferably between 0.01% and
0.2%.
It can also be desirable to utilize opacifiers inasmuch as
they contribute to create a uniform appearance of the
concentrated liquid detergent compositions. Examples of
suitable opacifiers include: polystyrene commercially known
as LYTRON 621 manufactured by MONSANTO CHEMICAL CORPORATION.
The opacifiers are frequently used in an amount from 0.3% to
1.5%.
The compositions herein can also contain known antioxidants
for their known utility, frequently radical scavengers, in
the art established levels, i.e. 0.001% to 0.25% (by
reference to total composition). These antioxidants are
frequently introduced in conjunction with fatty acids.
The compositions of the invention may also contain other
enzymes in addition to the proteases of the invention such as
lipases, amylases and cellulases. When present, these enzymes
may be used in an amount from about 0.01% to about 5% of the
compositions.
In a preferred embodiment of the invention, the formulation
contains ingredients in the following ratio:
21 C 6132 (R)
Inqredients % by Weiqht
Linear alkylbenzene sulphonate 8-12
Alcohol ethoxylate 6-10
Alcohol ethoxysulphate 4- 8
5 Builder 5-10
Sodium xylene sulphonate 1- 5
Monoethanolamine 1- 3
Triethanolamine 1- 3
Mutant protease enzyme *
10 Calcium chloride dihydrate 0- 0.1
Minor ingredients < 1.0
Water balance to 100
pH 9.0-12.0
* as required to provide activity of 0.01 to 100,000 GU/g,
based on final composition.
In an especially preferred embodiment of this aspect of the
invention, the mutant protease used in the above-formulated
composition is GX 8379.
In a second preferred embodiment of the i~vention, the
formulation contains ingredients in the following ratio:
25 Ingredients % by Weiqht
Linear alkylbenzene sulphonate 8-12
Alcohol ethoxylate 6-10
Alcohol ethoxysulphate 4- 8
Builder 3- 7
30 Sodium xylene sulphonate 1- 5
Triethanolamine 1- 5
Borax pentahydrate 1- 5
Propylene glycol 2- 6
Calcium chloride dihydrate 0.035
35 Mutant protease enzyme *
Minor ingredients < 1.0
Water balance to 100
22 C 6132 (R)
pH 9.0-12.0
* as required to provide activity of o.Ol to lOO,oO0 GU/g,
based on final composition.
In an especially preferred embodiment of this aspect of the
invention, the mutant protease used in the above-formulated
composition is GX 8379.
Product pH
The pH of the compositions of the invention is from about 9
to about 12, preferably 9.5 to 11, most preferably 9.5 to
10.5.
The following examples are intended to illustrate the
invention and facilitate its understanding and are not meant
to limit the invention in any way.
EXAMPLE 1
The stability of various wild-type subtilisins were compared
to mutant subtilisin strain GX8397 (subtilisin with 5 amino
acid mutations) in the following formulations without
stabilizer and with builder:
Anionic-rich formulation A
Wt.%
Linear alkylbenzene sulphonate 10.0
Alcohol ethoxylate 8.0
30 Alcohol ethoxysulphate 6.0
Sodium citrate 7.0
Sodium xylene sulphonate 3.0
Monoethanolamine 2.0
Triethanolamine 2.0
35 Mutant protease enzyme *
Calcium chloride dihydrate0.035
Minor ingredients 0.5
23 C 6132 (R)
Water balance to 100
pH 10
* as required to provide activity of 0. 01 to 100,000 GU/g,
based on final composition.
Enzyme Half-life at 37C (hrs) % Improvement
Savinase (from Novo) llg
Alcalase (from Novo~ 49
Wild type BPN' (from Novo) 89
GX 8397 440 269*
*~elative to Savinase; relative to Alcalase, improvement was
797% and relative to BPN', the improvement was 394%.
Anionic-rich formulation B
Wt.%
Linear alkylbenzene sulphonate 10.0
Alcohol ethoxylate 8.0
20 Alcohol ethoxysulphate 6.0
Sodium citrate 5.0
Sodium xylene sulphonate 2.5
Triethanolamine 3.0
Borax pentahydrate 2.4
25 Propylene glycol 4.0
Calcium chloride dihydrate 0.035
Mutant protease en~yme *
Minor ingredients < 1.0
Water balance to 100
30 pH 9.8
* as required to provide activity of 0.01 to 100,000 GU/g,
based on final composition.
nzyme Half-life at 37C (hrs) % Im~rovement
Savinase (from Novo) 197
GX 8397 500 153
24 C 6132 (R)
As can be seen from the results above, the stability of the
mutant strain GX8397, measured as the half-life of the enzyme
at 37OC, was significantly greater in the anionic rich, high
pH compositions of the invention compared to the wild-type
and/or commercially available enzymes tested in the same
formulations.
EXAMPLE 2
The stability of wild-type BPN' was compared to mutant
subtilisins with 6 or fewer amino acid mutations in
formulation A.
No. of Amino Acid
Enzyme SubstitutionsHalf-life at 37C (hrs)
15 Wildtype BPN' 0 73
GX 8350 6 441
GX 8397 5 523
GX 8398 5 459
GX 8399 4 380
GX 7160 1 135
GX 7175 1 103
GX 7195 1 120
GX 8303 1 125
GX 8314 1 145
GX 8347 1 155
The results show that the stability of the mutant enzymes was
clearly superior to wild-type BPN' in the composition of the
invention. The example also shows that stability was
significantly improved even when the enzyme was mutated in as
few as 1 amino acid site.
EXAMPLE 3
The stability of Savinase enzyme was compared to GX 8350
(subtilisin with 6 amino acid mutations) in Formulation A
with and without builder (i.e. 7.0% sodium citrate).
' ' ~ i`l
25 C 6132 (R)
Half-life at 37C (hrs)
Formulation A Savinase GX 8350
with builder 123 441
wi-thout builder 246 410
The results show that the stability of GX 8350 is not signi-
ficantly affected by builder, while the presence of a builder
in formulation A has a major impact on Savinase stability.
EXAMPLE 4
The stability of various subtilisins were compared to GX 8350
(subtilisin with 6 amino acid substitutions) in Formulation A
with varying amounts of enzyme stabilizer.
Half-Life at 37C (hours)
No 1/2 Full
Enzymestabilizerstabilizerstabilizer
BPN' 77 190 427
Savinase119 350 732
Alcalase49 105 198
GX 8350 433 1022 2300
The stabilizer system used in the above examples was a
propylene glycol/borax stabilizer system. The 1/2 stabilizer
system comprises 2.12% propylene glycol and 1.33~ borax
(introduced as sodium borate tetrahydrate) and the full
stabilizer system comprises 4% propylene gly~ ll and 2.7%
borax (also introduced as sodium borate tetrahydrate). All
percentages were by weight.
These results show that the stability of enzymes tested is
improved with the use of stabilizer (although use of the
stabilizer is not required). The stability in formulation A
was much greater, with or without stabilizer, when GX8350
enzyme was used.
