Title of Invention


Abstract (57) Abstract: Enzymes produced by mutating the genes for a number of subtilases and expressing the mutated genes in suitable hosts are presented. The enzymes exhibit improved stability and/or Improved wash performance in any detergent in comparison to their wild type parent enzymes . The enzymes are well-suited for use in any detergent and for some in especially liquid or solid shaped detergent compositions. PRICE: THIRTY RUPEES
Full Text

xne present invention relates to a process for preparing a subtilase variant.
This invention relates to novel mutant enzymes or enzyme variants useful in formulating detergent compositions and ex¬hibiting improved storage stability while retaining or improving their wash performance; cleaning and detergent compositions containing said enzymes; mutated genes coding for the expression of said enzymes when inserted into a suitable host cell or organism; and such host cells transformed therewith and capable of expressing said enzyme variants.

Cn the detergent industry enzymes have for more than 30 years seen implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, is well as other enzymes, or mixtures thereof. Commercially lost important are proteases.
Though proteases have been used in the detergent industry for ore than 30 years, much remains unknown as to details of how hese enzymes interact with substrates and/other substances resent in e.g. detergent compositions. Some factors related to pecific residues and influencing certain properties, such as xidative and thermal stability in general have been lucidated, but much remains to be found out. Also, it is still ot exactly known which physical or chemical characteristics re responsible for a good washing performance or stability of protease in a specific detergent composition.
currently used proteases have for the most part been found isolating proteases from nature and testing them in deter-int formulations.

An increasing number of commercially used protease are protein engineered variants of the corresponding naturally occurring wildtype protease, e.g. DURAZYM" (Novo Nordisk A/.S), REIJVSE" (Novo Nordisk A/S) , MAXAPEM* (Gist-Brocades N. V. ) , PUI^AFECT' 5 (Genencor International, Inc.)-
Therefore, an object of the present invention, is to provide improved protein engineered protease variants, especially for use in the detergent industry.
10 Enzymes cleaving the amide linkages i, ii protein substrates are classified as proteases, or (interch.uiqeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. V/. H. Freeman and Company, San Francisco, Chapter 3) . fiacterj.a of the Ba.ci,lUis species secrete two extracellular species of protease, a
15 neutral, or metalloprotease, and an alkaline protease which is functionally a serine endopeptidase and visually referred to as subtilisin. Secretion of these proteases has been linked to the bacterial growth cycle, with greatest expression of protease during the stationary phase, when sporulation also occurs.
0 Joliffe et al. (1980) J. Bacteriol 141 1199-1208, have suggested that Bacillus proteases function in cell wall turnover.
A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 "Principles of Biochemistry," Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272).
The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Daltons range. They are inhibited by diisoiaro-pylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkcjl.ine pr-otease, covering a sub-group, reflects the high pH optimum of some! of

the serine proteases, from pH 9.0 to 11. o (for review, see Priest (1977) Bacteriological Rev. 41 711-753).

In the context of this application substrate should be interpreted in its broadest form as comprising a compound containing at least one peptide bond susceptible to hydrolysis by a subtilisin protease.
Also the expression "product" should in the context ot: this invention be interpreted to include tho products of a hydrolysis reaction involving a subtilisin protease. A product may be the substrate in a subsequent hydrolysis reaction.
One subgroup of the subtilases, I-Sl, comprise::-, the "classical" subtilisins, such as subtilisin 168, subtilisin EJPN' , subtilisin Carlsberg (ALCALASE*, NOVO NORDISK A/53) , and subtilisin DY.
A further subgroup of the subtilases I-S2, is recognised by Siezen et al. (supra) . Sub-group I--S2 protea;-.es are described as highly alkaline subtilisins and comprise enzymes such as subtilisin PB92 (MAXACAL®, Gist-Eirocades NV) , subtilisin 309 (SAVINASE*, NOVO NORDISK A/S), sul:)t i 1 is in 117 (ESPERASK*, NOVO NORDISK A/S), and alkaline elastase YaB.
In the context of this invention, a subtilase variant or mutated subtilase means a subtilase that has been produced by an organism which is expressing a mutant gene derived from, a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene i from which said mutated subtilisin protease is produced when expressed in a suitable host.
Random and site-directed mutations of the subtilase gene have both arisen from knowledge of the physical and chemical properties of the enzyme and contrUnited inlCrmation relating to subtilase's catalytic activity, substrate specificity,

Soc- Lond.A. 317 415-423; Hwang and Warshel (1987) Biochem. 26 2669-2673; Rao et al.. (1987) Nature 328 551-554.
More recent publications covering this area are Carler et a1. (1989) Proteins 6 240-248 relating to design of variants that cleave a specific target sequence in a substrate (positions 24

Especially site-directed mutagenesis of the subtilisin genes has attracted much attention, and various mutations are described in the following patent applications and patents:
EP publ. no. 130756 (GENENTECH)(corresponding to US Reissue Patent No, 34,606 (GENENCOR)) relating to site specific or randomly generated mutations in "carbonyl hydrolases" and

the filing of the application, and therefore evident to select, this application does not contribute much to solving the problem of deciding where to introduce mutations in order to obtain enzymes with desired properties.
EP publ. no. 214435 (HENKEL) relating to cloning and expression

In International patent publication No. WO 8 7/04 4 61 (AMGEN) it is proposed to reduce the number of Asn-Gly sequences present in the parent enzyme in order to obtain mutated enzymes exhibiting improved pH and heat stabilities, in the application emphasis is put on removing, mutating, or modifying the ^^'Asn and the ^^^Asn residues in subtilisin BPN' . Mo examples are provided for any deletions or for modifying the Gly-residues.
International patent publication No. WO fr7/05050 (GENEX) discloses random mutation and subsequent screening of a Lai'ge number of mutants of subtilisin BPM' for impir)ved properties.

In EP Publication no. 251 446 (GENENCOR) it .:i_s described how lomology considerations at both primary and tertiary structural levels may be applied to identify equivalent • amino acid residues whether conserved or not. This information together with the inventors knowledge of the tertiary structure of subtilisin BPN' lead the inventors to select a number of positions susceptible to mutation with an expectation of

mutations are exemplified to support these suggestions, in addition to single mutations in these positions the inventors also performed a number of multiple mutations. Further the inventors identify and amino acid
residues within the segments 97-103, 126-129, 213-215, and 152-L72 as having interest, but mutations in any of these positions ire not exemplified.
Ispecially of interest for the purpose of the present invention ;he inventors of EP 2 51 44 6 suggest to

subtilisin BPN', type I-Sl), specifically they suggest to introduce Glu or Arg for the original Lys. It appears that the Glu variant was produced and it was found that it was highly susceptible to autolytic degradation (cf. pages 48, 121, 123 (Table XXI includes an obvious error, but indicates a reduction in autolysis halftime from 86 to 13 minutes) and Fig. 32).
EP publ. no. 260105 (GENENCOR) describes modification of certain properties in enzymes containing a catalytic triad by selecting an amino acid residue within about 15 A from the catalytic triad and replace the sele,cted ammo acid residue ith another residue. Enzymes of the subtilase type desci-ibed Ln the present specification are specificaJ i.y mentioned as belonging to the class of enzymes cont:;sining a -'atalytic triad. In subtilisins positions 222 and 217 are indicated as preferred ositions for replacement.
SO, it has been shown by Thomas, Russell, and Fersht (1985) ature 318 375-376 that exchange of '^Asp into '^Ser in ubtilisin BPN' changes the pH dependency of the enzyme.
n a subsequent article (1987) J. Mol. Biol. 193 803-813, the ame authors also discuss the substitution of ^'"^Ser in place of Glu.
oth these mutations are within a distance at about 15A from e active ^His.
Nature 328 496-500 (1987) Russel and Fersht discuss the sults of their experiments and present rules for changing pH-tivity profiles by mutating an enzyme to obtain changes in rface charge.
88/08028 (Genex) and WO 88/08033 (Amgen) both relate to edifications of amino acid residues in the calcium binding
tes of subtilisin BPN'. The enzyme is said to be stabilized substituting more negatively charged residues for the
iginal ones.

In WO 89/06279 (NOVO NORDISK A/S) position 170 is indicated as interesting and it is suggested to replace the existing residue with Tyr. However, no data are given in respect of such a variant. In WO 91/00345 (NOVO NORDISK A/S) the same suggestion is made, and it is shown that the Tyr variant of position 170 in subtilisin 309 (type I-S2) exhibits an improved wash performance in detergents at a pH of about 8 (variant SCi03 in Tables III, IV, V, VI, VIII, X) ., The same substitution in combination with other substitutions in otlier positions also indicates an improved wash performance(S004, S011-S014, S022-S024, 8019, S020, S203, S225, S227 in the same Table and Table VII) all in accordance with the generic concept of said application.
[n EP 525 610 Al (SOLVAY) it is suggested to improve the stability of the enzyme (a type I-S2 subtilase closely related o subtilisin PB92) towards ionic tensides by decreasing the ydrophobicity in certain surface regions thereof. It is onsequently suggested to substitute Gin for the Arg in osition 164 (170 if using BPN' numbering). No variants omprising this substitution are dlscloBed in the application.
n WO 94/02618 (GIST-BROCADES N.V.) a numbc^r of position 164 170 if using BPN' numbering) variants oi: the I~S2 type ubtilisin PB92 are described. Examples are provided showing ubstitution of Met, Val, Tyr, lie, for the original Arg. Wash erformance testing in powder detergents of the variants ndicates a slight improvement. Especially for the lie variant ash performance tests on cacao an improvement of about 20-30% indicated. No stability data are provided.
WO 95/30011, WO 95/30010, and WO 95/29979 (PROCTER & GAMBLE MPANY) describe 6 regions, especially position 199-220 (BPN' mbering), in both Subtilisin BPN' and subtilisin 309, which e designed to change (i.e. decrease) the adsorbtion of the zyme to surface-bound soils. It ir. suggested that decreased sorbtion by an enzyme to a subst.rate results in better

detergent cleaning performance. No specific detergent wash performance data are provided for the suggesjted variants.
* WO 95/27049 (SOLVAY S.A.) descriebe a subtilisin 309 type protease with following mutations: N43R+N116R+N117R (BPN^ 5 numbering. Data indicate the corresponding variant is having improved stability, compared to wildtype.
INDUSTRIAL APPLICATIONS OF SUBTILASES Proteases such as subtilisins have found much utility in industry, particularly in detergent formulations, as th«?y are 10 useful for removing proteinaceous stains.
At present at least the following proteases are knoi^/n to be commercially available and many of them are marketed in large quantities in many countries of the v/orld.
Subtilisin BPN' or Novo, available from e.g. CIGI'IA, St. Louis, 15 U.S.A.
Subtilisin Carlsberg, marketed by NOVO NORDISK A/S (Denmark) as ALCALASE* and by Gist-Brocades N.V. (Holland) as MAXATASE';
Both of these belong to subtilase subgroup I-fil
Among the subtilase sub-group I-S2 the following are known to 0 be marketed.
A Bacillus lentus subtilisin, subtilisin J09, marketed by NOVO NORDISK A/S (Denmark) as SAVINASE*. A protein engineered variant of this enzyme is marketed as DURAZYM*.
Enzymes closely resembling SAVINASE", such as subtilisin PB92, MAXACAL* marketed by Gist-Brocades N.V. (a protein engineered variant of this enzyme is marketed as MAXAPEM") , OPTICLEAN" marketed by SOLVAY et Cie. and PURAFECT* marketed by GENENCOR International.

A Bacillus lentus subtilisin, subtilisin 14 7, marketed by NOVO NORDISK A/S (Denmark) as ESPERASE*;
To be effective, however, such enzymes must not only exhibit activity under washing conditions, but must also be compatible 5 with other detergent components during detergent production and storage.
For example, subtilisins may be used in combination with other enzymes active against other substrates, and the selected subtilisin should possess stability towards such enzymes, and
10 also the selected subtilisin preferably should not catalyze degradation of the other enzymes. Also, the c:hosen subtilisin should be resistant to the action from other components in the detergent formulation, such as bleaching agents, oxidizing agents, etc., in particular an enzyme to be ut;ed in a detergent
15 formulation should be stable with respect to the oxirlizing power, calcium binding properties, and pH conditions rendered by the non-enzymatic components in the detergent during storage and in the wash liquor during wash.
The ability of an enzyme to catalyze the degradation of various 20 naturally occurring substrates present on the objects to be cleaned during e.g. wash is often referred to as its washing ability, washability, detergency, or wash perfox-mance. Throughout this application the term wash performance will be used to encompass this property.
25 The ability of an enzyme to remain active in the presence of other components of a detergent composition prior to being put to use (normally by adding water in tlie washing process) is usually referred to as storage stability or shelf life. It is often measured as half-life, t1/2. We will u,se the expression
30 storage stability for this property tlirougliout: this application to encompass this property.
Naturally occurring subtilisins have been found to possess properties which are highly variable in relation to their

variations in parameters such as pH. Several of the above marketed detergent proteases, indeed, have a better performance than those marketed about 2 0 years ago, but for optimal performance each enzyme has its own 5 specific conditions regarding formulation and wash conditions, e.g. pH, temperature, ionic strength ( = 1),, active system (tensides, surfactants, bleaching agent, etc.), builders, etc.
As a consequence it is found that an enzyme possessing desirable properties at low pH and low I may be less attractive 10 at more alkaline conditions and high I, or an enzyme exhibiting fine properties at high pH and high I may be less attractive at low pH, low I conditions.
Also, it has been found that the storage stability differs between the enzymes, but it has further been found that a
15 specific enzyme exhibits large variations in storage stability in respect of different detergent formulations, dependent upon a number of parameters, such as pH, pi, bleach system, tensides, etc., and upon the physical state of the detergent compositions, which may be in powder, dust, or liquid Corm.
20 Furthermore it may be concentrated or dilute.
The advent and development of recombinant DHA techniques has had a profound influence in the field of protein chemistry.
Through the application of this technology it is possible now to construct enzymes having desired amino acid sequences, and 25 as indicated above a fair amount of research has been devoted to designing subtilisins with altered properties.
Among the proposals the technique of producing and screening a large number of mutated enzymes as described in EP publ. no. 130756 (GENENTECH) (US Reissue Patent No. 34,606 (GENENCOR)) 30 and International patent publ. no. WO B7/05050 (GENEX) correspond to a large extend to the classical method of isola¬ting native enzymes, submit them to classical mutagenesis programs (using radiation or chemical mutagen;;) and screen them

for their properties. The difference lies in that these methods are more efficient through the knowledge of the presence of a large number of variant enzymes substituted in a specific position.
5 A subtilisin enzyme typically comprises about: 275 amino acid residues. Each residue is capable of being 1 out of 2 0 possible naturally occurring amino acids.
Therefore one very serious draw-back, in that procedure is the very large number of mutations generated that have to be 10 submitted to a number of preliminary screenings to determine their properties.
A procedure as outlined in these patent applications will consequently only be slightly better than the traditional random mutation procedures which have been known for years.
15 The other known techniques relate to changing specific properties, such as oxidation stability, thermal stability, Ca-stability, transesterification and hydrolysis rate (EP publ. no. 260105 (GENENCOR)), pH-activity profile (Thomas, Russell, and Fersht, supra), and substrate specificity (International
20 patent publ. no. WO 88/07578 (GENENTECH) ) . None of these publications relates to changing either the wash performance of enzymes or their storage stability.
In International Patent Application no. PCT/DK 88/00002 (NOVO NORDISK A/S) it is proposed to use the concept of homology 25 comparison to determine which amino acid positions should be selected for mutation and which amino acids should be substituted in these positions in order to obtain a desired change in wash performance.
By using such a procedure the task, of screening is reduced
30 drastically, since the nuraber of mutants generated is much
smaller, but with that procedure it is only foreseen that

enzymes exhibiting the combined useful properties of the parent enzyme and the enzyme used in the comparison may be obtained.
Thus, as indicated above no relationship has yet been identified between well defined properties ol: an enzyme such as 5 those mentioned above and the wasli performance and storage stability of an enzyme in various detergent: ('ompositions.
The problem seems to be that although much research has been directed at revealing the mechanism of enzymsi activity, still only little is known about the factors in structure and amino 10 acid residue combination that determine the properties, such as storage stability in detergents, of enzymes in relation to most of their characteristics, especially when the enzymes are present in complex mixtures.
Consequently there still exists a need for further improvement 15 and tailoring of enzymes to detergent systems, as well as a better understanding of the mechaniism of protease action and degradation in the practical use of cleaning or detergent compositions. Such an understanding c;ould result in rules which may be applied for selecting mutations that with a reasonable 20 degree of certainty will result in an enzyme exhibiting improved storage stability under specified conditions in a detergent composition.
It has now surprisingly been found that a subtilase variant 25 having improved storage stability and/or improved performance in detergents, can be obtained by substituting one or more amino acid residues situated in, or in the vicinity of a hydrophobic domain of the parent subtilase for an amino acid residue more hydrophobic than the original residue, said 30 hydrophobic domain comprising the residues corresponding to residues P129, P131, 1165, Y167, Y171 of BtS309 (in BASBPN numbering), and said residues in the vicinity thereof comprises residues corresponding to the residues E13 6, G159, S164, R170,

A194, and G195 of BLS309 (in BASBPN numbering), with the exception of the R170M, R170I and R170V variants of BABP92.
The present invention relates consequently in its first aspect to enzyrae variants exhibiting improved stability and/or 5 improved wash performance in detergent.
In its second aspect the invention relates to DNA constructs capable of expressing the enzymes of the first aspect, when inserted in a suitable manner into a host cell that subsequently is brought to express tfie subti. I. isin enzyme (s) of 10 the first aspect.
In a third aspect the invention relates to tiie production of the subtilisin enzymes of the invention by inserting a DNA construct according to the second aspect into a suitable host, cultivating the host to express the desired subtilase enzyrae, 15 and recovering the enzyme product.
The invention relates, in part, but is not limited to, mutants of the genes expressing the subtilase sub-group I-S2 enzymes and the ensuing enzyme variants, as indicated above. Other subtilase gene variants encompassed by the invention are 20 such as those of the subtilase subgroup I-Si, e.g. Subtilisin BPN', and Subtilisin Carlsberg genes and ensuing variant Subtilisin BPN', Proteinase K, and Subtilisin Carlsberg enzymes, which exhibit improved stability in concentrated liquid detergents.
25 Still further subtilase gene variants encompassed by the invention are such as Proteinase K and other genes and ensuing variant Proteinase K, and other subtilase enzymes, which exhibit improved stability in concentrated liquid detergents.
Other examples of parent subtilase enzymes thai; can be modified 30 in accordance with the invention are listed in Table I,

Further the invention relates to the use of the mutant enzymes in cleaning compositions and cleaning compositions comprising the mutant enzymes, especially detergent compositions comprising the mutant subtilisin enzymes. Specifically the 5 invention relates to concentrated liquid detergent compositions comprising such enzyme variants.
A = Ala = Alanine
10 V = Val = Valine
L = Leu = Leucine
I = lie = Isoleucine
P = Pro = Proline
F = Phe = Phenylalanine
15 W = Trp = Tryptophan
M = Met = Methionine
G = Gly = Glycine
S = Ser = Serine
T = Thr = Threonine
20 C = Cys = Cysteine
y = Tyr = Tyrosine
N = Asn = Asparagine
Q = Gin = Glutaraine
D = Asp = Aspartic Acid
25 E = Glu = Glutamic Acid
K = Lys = Lysine
R = Arg = Arginine
H = His = Histidine
X = Xaa = Any amino acid
A = Adenine
G = Guanine
C = Cytosine
T = Thymine (only in DNA)
35 U = Uracil (only in RNA)

In describing the various enzyme variants produced or con¬templated according to the invention, the following nomen¬clatures have been adapted for ease of reference:
5 Original amino acid(s) position(s) substituted amino acid(s)
According to this the substitution of Glutamic acid for glycine in position 195 is designated as:
Gly 195 Glu or G195E
a deletion of glycine in the same position is:
10 Gly 195 * or G195*
and insertion of an additional amino aci(i residue such as lysine is:
Gly 19 5 GlyLYS or G195GK
Where a deletion in comparison with the sequence used for the 15 numbering is indicated, an insertion in such a position is indicated as:
* 3 6 Asp or *3 6D for insertion of an aspartic acid in position 3 6
Multiple mutations are separated by pluses, i.e.: 20 Arg 170 Tyr + Gly 195 Glu or R170Y+G195E
representing mutations in positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respec¬tively.
25 In describing the variants in this application and in the appended claims use is made of the alignment of various subtilases in Siezen et al., Supra. In other publications relating to subtilases other aligmnents or the numbering of specific enzymes have been used. It is a routine matter for the
30 skilled person to establish the position of a specific residue in the numbering used here. Reference is also made to Fig. 1 showing an alignment of residues relevant for the present

invention from a large number of subtilases. Reference is also made to Table I of WO 91/00345 showing an aliqnment of residues relevant for the present invention from a number of subtilases.

ARB172 Kamekura and Seno, (1990) Biochem. Cell Biol. 68
352-359 (amino acid sequencing of mature protease residues 1-35; residue 14 not determined).

from B. lentus differs in having hf87S, S99D, SlOlR, S103A, V104I and G159S). BDSM48 Rettenmaier et al. (1990) PCT Patent Appl. WO 90/04022. Publ.April 19, 1990. 5 BYSYAB Kaneko et al. (19 89) J. Bacteriol. 171 5232-5236 (M28537) .
BSEPR Sloma et al. (1988) J,, Bacteriol. 170 5 557-5563
(M22407) . Bruckner (1990) Mol. Gen. Genet. 221 486-490 (X53307) .
10 BSBPF Sloma et al. (199 0) J... Mi^tejrio.U 172 14 7 0-1477
(M29 03 5; corrected). Wu et al... (1990) J. Biol.Chem.
265 6845-6850 (J05400; this seMjuence differs in having A169V and 586 lerit! C-terminal residues due to a frameshift).
15 BSISPl Koide et al. (1986) ,1. .McterLcLl:.... 167 110-116
BSIA50 Strongin et al. (1978) .L,,,J5ac;:tei:io]U 133 1401-1411
(amino acid sequencing of mature protease residues
1-54; residues 3. 39, 40. 45, 46, 4 9 and 5 0 not
20 determined) ,
BTFINI Chestukhina et al. (1985) Biokhiniiya 50 1724-1730
(amino acid sequencing of mature protease residues
1-14 from B. thurinqiensis variety Israeliensis,
and residues .1-16 and 223-24 3 from variety
25 finitimus) . Kunitate et__ai_;- (1989) Agric. Biol.
Chem. 53 3251-3256 (amino acid sequencing of mature
protease residues 6-20 from variety kurstaki.
BCESPR Chestukhina et al. (1985) Biokhimiya 50 1724-1730
30 (amino acid sequencing of mature residues 1-16 and
223-243) . NDAPII Tsujibo et al. (199 0) Agric. Biol. Chem. 5 4 2177-2179 (amino acid sequencing of mature residues 1-26) . 35 TVTHER Meloun et al. (198 5) FEES Lett. 18 3 19 5-2 00 (FIR A00973; amino acid sequencing of mature protease residues 1-274). EFCYLA Segarra et al. (1991) Infect. Imniun. 59 1239-U46.

SEEPIP Schnell et al. (1991) personal communication
(Siezen et al. (supra)) . SPSCPA Chen et al. (199 0) J, Biol. Chem. 265 3161-3167
(J05224). 5 DNEBPR Kortt et al. (1991) Abstracts 5th Protein Society
Symposium, June 22-26, Baltimore, cibstract S76. LLSKll Vos et al. (1989) J. Biol. Chem. 2 64 13 579-13585
(J04962). Kok et al.,,, (19 88) Appl. Environ.
Microbiol. 54 231-238 (M24767; the sequence from
10 strain Wg2 differs in 44 positions, including 18
differences in the protease doiiifiin, and a deletion
of residues 1617-1676) . Kiwak::! e.t al.^ (1989)
Mol .Microbiol. . 3 359™-36g (X14:r!0; the sequence
from strain NCD0763 differs in 4 6 positions,
15 including 22 in the protease domain, and a deletion
of residues 1617-1676) . XCEXPR Liu et al. (1990) Mol. Gen. Genet. 220 433-440. SMEXSP Yanagida et al. (1986) J. Bacteriol. 166 937-994
(M13469) . 20 TAAQUA Terada et al. (1990) J. Biol. Chem. 265 6576-6581
(J054I4). TRT41A McHale et al. (1990) Abstracts 5th Eur. Congr.
Biotechn. Christiansen ,Munck and Villadsen (eds),
Munksgaard Int. Publishers, Copenhagen. 25 VAPROA Deane et al. (1989) Gene 7 6 281-2 88 (M2 54 99).
SRESPD Lavrenova et al. (1984) Biochemistry USSR. 49 447-
454 (amino acid sequencing of residues 1-23;
residues 13, 18 and 19 not determined).
AVPRCA Maldener et al (1991) Mol. Gen. Genet. 225 113-120
30 (the published sequence has 28 uncertain residues
near position 200-210 due to a frameshift reading
error). TAPROK Gunkel and Gassen (19 89) Eur. J. Biochem. 17 9 18 5-
194 (X14688/XI4689) . Jany et al. (1986) J,._ Biol.
35 Chem.Hoppe-Seyler 3 67 87(PIR A2 4 54 1; amino acid
sequencing; mature protease differs in having
S745G, SILST204-208DSL and VNLL2 64-2 67FNI.) .

TAPROR Samal et al. (1990) Mo.l, Microbiol. 4 17{19-1792
(X56116). TAPROT Samal et al. (1989) Gene 85 329- !3:].
AOALPR Tatsumi et al. (1989) Mol. Gen. Geiiet,^. 219 3 3-38.
5 Cheevadhanarah et al. (1991) EMBL Data Library
(X54726). MPTKMY Gaucher and Stevenson (1976) Methods Enzymol. 45
415-433 (amino acid sequencing of residues 1-28,and
hexapeptide LSGTSM with active site serine). 10 ACALPR Isogai et al. (1991) Agric. Biol. Chem. 55 471-477.
Stepanov et al. (1986) Int. J. Biochem. 18 369-375
(amino acid sequencing of residucis 1-27: the mature
protease differs in having H13[l]Q, R13[2]N and
S13[6]A). 15 KLKEXl Tanguy-Rougeau, WesolowGki~I.,ouvel and Fukuhara
(1988) FEBS lett. 234 464-470 (X07038)., SCKEX2 Mizuno et al. (1988) Ei i ochem . B i.ophys . Res . Commun.
156 246-254(M24201).
SCPRBl Moehle et al. (1987) M.fiI,=.._£,eJJ..,, Biol. 7 4 390-4399
20 (M18097) .
YLXYPR2 Davidow et al. (198 7) J. Bacterid. 169 4621-4629
(M17741) . Matoba et al. (1988) Mol. Cell Biol. 8
4904-4916 (M23353).
CEBL14 Peters and Rose (1991) The Worm Breeder's Gazette
25 11 28.
DMFURl Roebroek et al. (1991) FEBS Lett. 289 133-137
(X59384) .
DMFUR2 Roebroek et al. (1992) 267 17208-17215.
CMCUCU Kaneda et al. (1984) J. Biochem. 95 825-829 (amino
30 acid sequencing of octapeptide NIISGTSM with active
site serine). HSFURI van den Ouweland et al. (1990) Nucl. Acids Res. 18
664 (X04329) (the sequence of mouse furin differs
in 51 positions, including five in the catalytic
55 domain: A15E, Y21F, S223F, A232V and N258[2]D).
Misumi et al.(1990) Nucl. Acids Res. 18 6719
(X55660: the sequence of rat furin differs in 49

positions, including three in the catalytic domain:
A15E, Y21F, H24R).
HSIPC2 Smeekens and Steiner (1990) J,_ Biol. Chem. 265
2997-3000 (J05252), Seidah et al. (1990) DNA Cell
5 Biol. 9 415-424 (the sequence of mouse pituitary
PC2 protease differs in 23 positions,including
seven in the protease domain: 141', S42[2]Y, E45D,
N76S, D133E, V134L and G239[1]D).
MMPPC3 Smeekens et al. (1991) i:;i:x)c.^ Nd r J ,, Acad. Sci . USA
10 88 340-344 (M58507). Seidah et', a.i,_ (1990) DNA Cell
Biol. 9 415-424 (M55668/M55669; partial sequence). HSTPP Tomkinson and Jonsson (1991) Biochemistry 30 168-
174 (J05299) .
15 In the drawings, Fig. 1 shows an alignment of a number of the subtilases mentioned in Table T;
Fig. 2 is a 3-dimensional representation of subtilisin 309 showing the location of the hydrophobic domain and some of the amino acid residues in the vicinity thereof to be substituted 20 according to the invention.
It has surprisingly been found that the storage stability and/or improved performance in detergents of subtilases generally is improved when amino acid residues situated in, or 25 in the vicinity of a hydrophobic domain comprising the residues P129, P131, 1165, Y167, Y171 of subtilisin 309 are substituted for a more hydrophobic residue. The residue:;^ in question are especially E136, G159, S164, R170, A:i94, and i:;i95.

Further, said variant exhibits a particulary high improved stability in liquid detergents and in detergents in a shaped solid form.
Fig, 2 shows the hydrophobic domain in subtilisin 309 and 5 residues in the vicinity thereof a number of which are to be substituted in order to increase the hydropliobicity of the domain. This may be achieved by substituting hydrophobic residues for non-hydrophobic residues and/or by substituting residues to become even more hydrophobic than in the parent 10 enzyme.
The same principle applies to the corresponding domain in other subtilases, the identification of v/hich is witliin the skills of the average person working in this technical field. Graphic representations like the one in Fig. 2 can be. produced for 15 other subtilases to determine the target residues to be substituted according to the invention.
A number hereof is indicated in Table II below:

20 Consequently the invention relates to subtilase variants in which the amino acid sequence has been changed through mutating the gene of the subtilisin enzyme, which it is desired to modify (the parent enzyme or gene), in the codon responsible for the expression of the amino acid residue in positions 129,
25 131, 165, 167, 171, 136, 159, 164, 170, 194, and 195, which residues are more hydrophobic than the residue(s) in the parent enzyme, especially such hydrophobic residu(:;::; that comprise a relatively long hydrophobic side chain, siicli as lie, Leu, and

Val, whereby, when the mutated gene is expressed, the amino acid residue is substituted by a more hydrophobic residue, which increases the hydrophobicity of the domain as such.
Hydrophobic amino acid residues are generally the following: 5 Val (V), He (I), Leu (L) , Met (M) , Phe (F) , Pro (P) and Trp(W). Among these Val, He and Leu are preferred.
By looking at Table II and applying the principle of the invention a number of candidates for substitution becomes clear.
For both BASBPN and BLSCAR it seems appropriate to make substitutions in positions 129, 131, 136, 159, 164, 167, 170, 171 and 195. In BLS309 positions 136, 164, 167, and 170, 171 would be the first choices, and positions 159 and 195 also would be a second choice. In BLS147 positions 129, 131,, 136, 167, 170, 171 and 195 are the first choice, while positions 159 and 164 are second. Finally, in TVTHER positions 129, 131, 136, 167, 171 and 194 are the first choices, with 164 as a second one.
According to the invention it would entail an advantage to substitute the Gly residues in the hydrophobic domain to bulkier and more hydrophobic residues.
Such considerations apply for any hydrophilic or hydrophobic residue that may occupy any of the above mentioned position, meaning that any increase in hydrophobicity seems to be advantageous. This means that e.g, a very hydrophilic residue such as the charged residues Arg (R) , Ar.p (D) , Glu (E) or Lys (K) may be substituted by any residue that is less hydrophilic. Such less hydrophilic residues comprises the residues Gly (G), Cys (C), Ser (S), Ala (A), Thr (T), Tyr (Y), Gin (Q), His (H) or Asn (N). It also means that a Tyr(y) may be substituted by a more hydrophobic residue such as Phe(F), Lou(L), or He (I)..

Similar considerations can be applied to other sulstilases having a hydrophobic domain in this part of (he surface of the enzyme.
In the context of this invention a subtilase is defined in accordance with Siezen et al. supra. In a more narrow sense, applicable to many embodiments of the invention, the subtilases of interest are those belonging to the subgroups I-Sl and I-S2. In a more specific sense, many of the embodiments of the invention relate to serine proteases of gram-posit:ive bacteria which can be brought into substantially unambiguous homology in their primary structure, with the subtilases listed in Table I above.
The present invention also coniprLses any one or more substitutions in the above mentioned positions in combination with any other substitution, deletion or addition to the amino acid sequence of the parent enzyme. Especially combinations with other substitutions known to provide improved properties to the enzyme are envisaged.
Such combinations comprise the positions: 222 (improve oxidation stability), 218 (improves thermal stability), substitutions in the Ca-binding sites stabilising the enzyme, e.g. position 76, and many other apparent from the prior art.
Furthermore combinations with the variants mentioned in EP 4 05 901 are also contemplated specifically.
A: Single variants:
Subtilisin BPN', Subtilisin Carlsberg, Subtilisin 168, and Sub-
tilisin DY variants:
A129V, A129I, A129L, A129M, A129F
G131V, G131I, G131L, G131M, G131F
K136V, K136I, K136L, K136M, K136F,
S159V, S159I, S159L, S159M, S159F,
T164V, T164I, T164L, T164M, T164F,

Any of the above variants are contemplated to prove advantageous if combined with other variants in any of the positions;
27, 36, 57, 76, 97, 101, 104, 120, 123, 206, 218, 222, 224, 235 5 and 274.
Specifically the following BLS309 and BAPB92 variants are considered appropriate for combination:
K27R, *36D, S57P, N76D, G97N, SIOIG, V104A, V104N, V104Y, H120D, N123S, A194P, Q206E, N218S, M222S, M222A, T224S, K235L 10 and T274A.
Also such variants comprising any one or two of the substitutions X167V, X167M, X167F, X167L, X167I, X170V, X170M, X170F, X170L, and/or X170I in combination with any one or more of the other substitutions, deletions and/or insertions 15 mentioned above are advantageous.
Furthermore variants comprising any of the variants V104N+S101G, K27R+V104Y+N123S+T274A, or N76D+V104A or other combinations of these mutations (V104N, SIOIG, K27R, V104Y, N123S, T274A, N76D, V104A) , in combination wit:h any one or more 20 of the substitutions, deletions and/or insertions mentioned above are deemed to exhibit improved properties.

The present invention also comprises the use of the mutant enzymes of the invention in cleaning and detergent compositions and such compositions comprising the mutant subtilisin enzymes. Such cleaning and detergent compositions can in principle have 25 any physical form, but the subtilase variants are preferably incorporated in liquid detergent compositions or in detergent compositions in the form of bars, tablets, sticks and the like for direct application, wherein they exhibit improved enzyme stability or performance.
30 Among the liquid compositions of ttie present invention are aqueous liquid detergents having for example a homogeneous physical character, e.g. they can consist of a micellar solution of surfactants in a continuous aqueous phase, so-called isotropic liquids.

Alternatively, they can have a heterogeneous physical phase and they can be structured, for example they can consist of a dispersion of lamellar droplets in a continuous a(jueous fihase, for example comprising a deflocculating polymer having a hydrophilic backbone and at least one hydrophobic side chain, as described in EP-A-346 995 (Unilever) (incorporated herein by reference). These latter liquids are heterogeneous and may contain suspended solid particles such as particles of builder materials e.g. of the kinds mentioned below.
Concerning powder detergent compositions such compositions comprise in addition to any one or more of the subtilisin enzyme variants in accordance to any of the preceding aspects of the invention alone or in combination any of the usual components included in such compositions whii:::h are well-known to the person skilled in the art.
Such components comprise builders, such as phosphate or zeolite builders, surfactants, such as anionic, cat Ionic, non--ionic or zwitterionic type surfactants, polymers, such as acrylic or equivalent polymers, bleach systems, such as perborate- or amino-containing bleach precursors or activatoi-s, structurants, such as silicate structurants, alkali or acid to adjust pH, humectants, and/or neutral inorganic salts.
Furthermore, a number of other ingredients are normally present in the compositions of the invention, such as:
A. Cosurfactants
B. Tartrate Succinate Builder
C. Neutralization System
D. Suds Suppressor
E. Other Enzymes
F. Other Optional Components
The weight ratio of anionic surfactant to nonionic surfactant is preferably from 1:1 to 5:1. The compositions have a pH in a 10% by weight solution in water at 2G°C of from 7.0 to 9.0, a

crirical Micelle Concentration of less than or equal to 200 ppm, and an air/water Interfacial Tension at the Critical Micelle Concentration of less than or equal to 3 2 dynes/cm at 35°C in distilled water. The compositions are preferably clear, homogeneous and phase stable, and have good cleaning performance and enzyme stability.
1. Anionic Surfactant
The compositions of the present invention contain from about 10% to about 50%, preferably from about 15% to about 50%, more preferably from about 2 0% to 4 0%, and most preferably from 2 0% to about 30%, by weight of a natural or synthetic anionic surfactant. Suitable natural or synthetic anionic surfactants ii^e e.g. soaps and such as disclosed in U.S. Patent 4,285,841, ind in U.S. Patent 3,929,678.
Jseful anionic surfactants include the water-soluble salts,
)articularly the alkali metal, ammonium and aLkylolammonium
e.g., monoethanolammonium or triethanolammonium) salts, of
»rganic sulfuric reaction product!-; having in their molecular
tructure an alkyl group containing ('rem about 10 to about 20
arbon atoms and a sulfonic acid or sulfuric ,::icid ester group.
Included in the term "alkyl" is tlie alkyl portion of aryl
roups.) Examples of this group of synthetic: surfactants are
he alkyl sulfates, especially those obtained by sulfating the
igher alcohols (Cg-C^g carbon atoms) such as those produced by
sducing the glycerides of tallow or coconut oil; and the
Lkylbenzene sulfonates in which the alkyl group contains from
Dout 9 to about 15 carbon atoms, in straight chain or branched
lain configuration, e.g., those of the type described in U. S.
itents 2,220,099 and 2,477,383. Especially vaJ.uable are linear
:raight chain alkylbenzene sulfonates in which the average
imber of carbon atoms in the alkyl group is from about 11 to

other anionic surfactants herein are the water-soluble salts of: paraffin sulfonates containing from 8 to about 24 (pre¬ferably about 12 to 18) carbon atoms; alkyl glyceryl ether sul¬fonates, especially those ethers of Cg-C,g alcohols (e.g., those derived from tallow and coconut oil) ; alkyl phenol etliylene oxide ether sulfates containing from 1 to ;:ibout 4 units of ethylene oxide per molecule and from 8 to 12 carbon atoms in the alkyl group; and alkyl ethylene oxide etJier sulfates containing 1 to 4 units of ethylene oxide per molecule and from 10 to 20 carbon atoms in the alkyl group.
Other useful anionic surfactants include the water-soluble salts of esters of a-sulfonated fatty acids containing from 6 to 20 carbon atoms in the fatty acid group and from 1 to 10 carbon atoms in the ester group; water-soluble salts of 2-acyloxy-alkane-1-sulfonic acids containing from 2 to 9 carbon atoms in the acyl group and from 9 to 2 3 carbon atoms in the alkane moiety; water-soluble salts of olefin sulfonates containing from 12 to 24 carbon atoms; and /]-alkyloxy alkane sulfonates containing from 1 to 3 carbon atoms in the alkyl group and from 8 to 2 0 carbon atoms in tlie alkane moiety.
Preferred anionic surfactants are soaps, l.he C^Q-C^JJ alkyl sulfates and alkyl ethoxy sulfates containing an average of up to 4 ethylene oxide units per mole of alkyl sulfate, C^^-C^-^ linear alkyl benzene sulfonates, and mixtures thereof.
2. Nonionic Surfactant
Another optional ingredient is from 2% to 14% preferably from 2% to 8%, most preferably from 3% to 5% by weight, of an optionally ethoxylated nonionic surfactant. The weight ratio of natural or synthetic anionic surfactant (on an acid basis) to nonionic surfactant is from 1:1 to 5:1 prefei-ably from 2:1 to 5:1, most preferably from 3:1 to 4 ; 1. This is to ensure the formation and adsorption of sufficient hai-dness surfactants at the air/water interface to provide good greasy/oily soil removal.

The optionally ethoxylated nonionic surfactant is of the formula R^COCjH^)^ OH, wherein R^ is a C,g-C^^ alkyl group or a Cg-C^2 alkyl phenyl group, n is from 3 to 9, and said nonionic surfactant has an HLB (Hydrophilic-Lipophilic Balance) of from 6 to 14, preferably from 10 to 13. These surfactants are more fully described in U.S. Patents 4, 285,a41, and 4,284,532, Particularly preferred are condensation products of C^2~^i5 alcohols with from 3 to 8 moles of ethylene oxide per mole of alcohol, e.g., C^^'^n alcohol condensed witli about 6.5 moles of ethylene oxide per mole of alcohol. Other nonionic surfactants to be mentioned are APG, EGE, and glucamide surfactants.
3. Detergency Builder
Among the usual detergent ingredients which may be present in usual amounts in the detergent compositions of this invention are the following: The compositions may be built or unbuilt, and may be of the zero-P type (i.e, not cont.aining any phos¬phorus containing builders). Thus, the composition may contain in the aggregate for example from 1-50%, e.g. at least about 5% and often up to about 3 5-4 0% by weight, of one or more organic and/or inorganic builders. Typical examples o:l: builders include those already mentioned above, and more broadly include alkali metal ortho, pyro, and tripolyphosphates, alkali metal carbon¬ates, either alone or in admixture with calcite, alkali metal citrates, alkali metal nitrilotriacetates, carboxymethyloxysuc-cinates, zeolites, polyacetalcarboxylates, and so on.
More specifically the compositions herein contain from 5% to 20%, preferably from 10% to 15%, by weight of a detergency builder which can be a fatty acid containing from 10 to 18 carbon atoms and/or a polycarboxylate, zeolite, polyphoshonate and/or polyphosphate a builder. Preferred aire from 0 to 10% (more preferably from 3% to 10%) by weight oi': saturated fatty acids containing from 12 to 14 carbon atoms, .iiong with from 0 to 10%, more preferably from 2% to 8%, most preferably from 2.% to 5%, by weight of a polycarboxylate builder, most preferably citric acid, in a weight ratio nf from ] -. ^ tn i!i_

since the proteolytic enzywes herein appear to provide optimum storage stability benefits versus other enzymes when the builder to water hardness ratio is close to one, the compositions preferably contain sufficient builder to sequester 5 from 2 to 10, preferably from 3 to 8, grains per gallon of hardness.
Suitable saturated fatty acids can be obtained from natural sources such as plant or animal esters (e.g., palm kernel oil, palm oil and coconut oil) or synthetically prepared (e.g., via
10 the oxidation of petroleum or by hydrogenation of carbon monoxide via the Fisher-Tropsch process). Exainples of suitable saturated fatty acids for use in the compositions' of this invention include capric, lauric, myristic, coconut and palm kernel fatty acid. Preferred are satvirated coconut fatty acids;
5 from 5:1 to 1:1 (preferably about 3:1) weight ratio mixtures of lauric and myristic acid; mixtures of the above with minor amounts (e.g., l%-30% of total fatty acid) of: oleic acid; and palm kernel fatty acid.
The compositions herein preferably also contain the ) polycarboxylate, polyphosphonate and polyphosphate builders described in U.S. Patent 4,284,532, Water-soluble polycarboxylate builders, particularly citrates, are preferred of this group. Suitable polycarboxylate builders include the various aminopolycarboxylates, cycloalkane polycarboxylates, ether polycarboxylates, alkyl polycarboxylates, epoxy polycarboxylates, tetrahydrofuran polycarboxylates, benzene polycarboxylates, and polyacetal polycarboxylates.
Examples of such polycarboxylate builders are sodium and potassium ethylenediaminetetraacetate; sodium and potassium nitrilotriacetate; the water-soluble salts of phytic acid, e.g., sodium and potassium phytates, disclosed in U.S. Patent 1,739,942, the polycarboxylate materials described in U.S. Patent 3,354,103; and the water-soluble salts of polycarboxylate polymers and copolymers described in U.S. Patent 3,308,067.

other useful detergency builders include the water-soluble salts of polymeric aliphatic polycarboxylic acids having the following structural and physical characteristics: (a) a minimum molecular weight of about 3 50 calculated as to the acid 5 form; (b) an equivalent weight of 50 to 80 calculated as to acid form; (3) at least 45 mole percent of the monomeric species having at least two carboxyl radicals separated from each other by not more than two cajrbon atoms: (d) the site of attachment of the polymer chain ot: any carboxyl-gontaining
10 radical being separated by not moire than three carbon atoms along the polymer chain from the site of: attachment of the next carboxyl-containing radical. Specific examples of such builders are the polymers and copolymers of itaconic acid, aconitic acid, maleic acid, mesaconic acid, fumaric acid, methylene
15 malonic acid, and citraconic acid.
Other suitable polycarboxylate builders include the water-soluble salts, especially the sodium and potassium salts, of mellitic acid, citric acid, pyromellitic acid, benzene pentacarboxylic acid, oxydiacetic acid, carboxymethyloxy-20 succinic acid, carboxymethyloxymalonic acid, cis-cyclohexane-hexacarboxylic acid, cis-cyclopentaiietetracarboxylic acid and oxydisuccinic acid.
Other polycarboxylates are the polyacetal carboxylates described in U.S. Patent 4,14 4,22 6, and U.S. Patent 4,14 6,49 5.
25 Other detergency builders include the zeolites, such as the aluminosilicate ion exchange material described in U.S. Patent 4,405,483.
Other preferred builders are those of the general formula R-CH(COOH) CHjCCOOH), i.e. derivatives of succinic acid, wherein 30 R is C^p-Cjp alkyl or alkenyl, preferably Cy^-C^^^ or wherein R may be substituted with hydroxyl, sulfo, sulfoxy or sulfone substituents. These succinate builders are preferably used in the form of their water soluble salts, including the sodium, potassium and alkanolammonium salts,. Specific examples of

succinate builders include: lauryl succinsite, inyristyl succinate, palmityl succinate, 2-dodecenyl succinate, and the like.
4. Proteolytic Enzyme
5 The enzymes of the invention can be used in weiLl-known standard amounts in detergent compositions. The amounts may range very widely, e.g. about 0.0002-0.1, e.g. about 0.005-0.05, Anson units per gram of the detergent composition. Expressed in al¬ternative units, the protease can be included in the composi-
10 tions in amounts in the order of from about 0.1 to 100 GU/mg (e.g. 1-50, especially 5-20 GU/mg) of the detergent formula¬tion, or any amount in a wide range centering at about 0.01-4, e.g. 0.1-0.4 KNPU per g of detergent formulation.
It may for example be suitable to use the present enzymes at 15 the rate of about 0.25 rag of enzyme protein per litre of wash liquor, corresponding to an enzyme (ictivity of the order of 0.08 KNPU per litre. Corresponding detergent formulations can contain the enzymes in for example an amount of the order of 0.1-0.4 KNPU/g.
20 Expressed differently the compositions of the present invention contain from about 0.01% to about 5%, preferably from about 0.1% to about 2%, by weight of the proteolytic enzymes of the invention.
The described proteolytic enzyme is preferably included in an 25 amount sufficient to provide an activity of from 0.05 to about 1.0, more preferably from about 0.1 to 0.75, most preferably from about 0.125 to about 0.5,mg of active enzyme per gram of composition.
The enzyme component may be added to the other components in 30 any convenient form, such as in the form of a solution, slurry, LDP slurry, or crystals.

3. tnzyroe Stabilization System
The liquid detergents according to the present invention may comprise An enzyme stabilization system, comprising calcium ion, boric acid, propylene glycol and/or short chain carboxylic 5 acids. The enzyme stabilization system comprises from about 0.5% to about 15% by weight of the composition.
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 20 millimoles of calcium ion per" liter. The level of
10 calcium ion should be selected 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, and calcium
15 acetate. A small amount of calcium ion, generally from about 0.05 to 0.4 millimoles per liter, is often also present in the composition due to calcium in the enzyme slurry and formula water. From about 0.03% to about 0.6% of calcium formate is preferred.
20 A second preferred enzyme stabilizer is polyols containing only carbon, hydrogen and oxygen atoms, Tlujy 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
25 glucose. The polyol generally represents from about 0.5% to 15%, preferably from about 1.5% to about 8%, by weight of the composition. Preferably, the weight ratio of polyol to any boric acid added is at least 1, more preferably at least 1.3.
The compositions preferably also contain the water-soluble, 30 short chain carboxylates described in U.S. Patent 4,318,818. The formates are preferred and can be used at levels of from about 0. 05% to about 5%, preferably from about 0.2% to about 2%, most preferably from 0.4% to 1.5%, by weight of the composition. Sodium formate is preferred.

The compositions herein also optionally contain from about 0.25% to about 5%, most preferably from about 0.5% to about 3%, by weight of boric acid. The boric acid may be, but is preferably not, formed by a compound capable of forming boric
5 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
10 phenylboronic acid) can also be used in place of boric acid.
6. Water
The liquid compositions of the present: invention may be aciueous liquids or non-aqueous liquids. WVieii the are aquous liquids, they contain from about 15% to about 60%, pre]:erably from about 15 25% to about 45%, by weight of water.
A. Cosurfactants
Optional cosurfactants for use with the above nonionic surfactants include amides of the formula

wherein R^ is an alkyl,hydroxyalkyl or alkenyl radical containing from 8 to 20 carbon atoms, and R^ and R^ are selected 25 from the group consisting of hydrogen, methyl, ethyl, propyl, isopropyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, and said radicals additionally containing up to 5 ethylene oxide units, provided at least one of R^ and R-^ contains a hydroxyl group.

Preferred araides are the c^.-c,^ fatty acid alkylol .amides in which each alkylol group contains: Vrom 1 to :! carbon atoms, and additionally can contain up to 2 ethyl i-ne oxido units. Particularly preferred are the 0^,-0,^, fatty acid sonoothiariol and diethanal amidos.

wherein X is a salt-forming cation; and iii) mixtures thereof.
The tartrate succinate compounds used herein are described in U.S. Patent 4,663,071.
5 C. Neutralization System
The present compositions can also opbionally contain from about 0 to about 0.04 moles, preferably from about 0.01 to 0.035 moles, more preferably from about 0.015 to about 0.03 moles, per 100 grams of composition of an alkanolamine selected from
10 the group consisting of monoethanolamine, diethanolamine, triethanolamine,and mixtures thereof. Low levels of the alkanolamines, particularly monoethanolamine, are preferred to enhance product stability, detergency performance, and odour. However, the amount of alkanolamine should be minimized for
15 best chlorine bleach compatibility.
In addition, the compositions contain sodium ions, and preferably potassium ions,at a level sufficient to neutralize the anionic species and provide the desired product pH.
D. Suds Suppressor
20 Another optional component for use in the liquid detergents herein is from 0 to about 1.5%, preferably from about 0.5% to about 1.0%, by weight of silicone based suds suppressor agent. Silicones are widely known and taught for use as highly effective suds controlling agents. For example, U.S. Patent
25 3,455,839 relates to compositions and processes for defoaming aqueous solutions by incorporating therein small amounts of polydimethylsiloxane fluids.
Useful suds controlling silicones are mixtures of silicone and silanated silica as described, for instance, in German Patent 30 Application DOS 2,124,526.

silicone defoamers and suds controlling agents have been successfully incorporated into granular detergent compositions by protecting them from detergent surfactants as in U.S. Patent 3,933,672, and in U.S. Patent 4,652,392.
5 A preferred silicone based suds suppressor for use herein is a suds suppressing amount of a suds controlling agent consisting essentially of:
(i) polydimethylsiloxane fluid having a viscosity of from about 20 cs. to about 1500 cs. at 25°C;
10 (ii) from about 5 to about 50 parts per 100 parts by weight of (i) of siloxane resin composed of (CH^)^ ^^^1/2 units and SiOj units in a ratio of from (CHj).j SiO^^^ units and to SiOj units of from about 0.6:1 to about 1.2:1; and
(iii) from about 1 to about 20 parts per 100 parts by weight 15 of (i) of a solid silica gel.
By "suds suppressing amount" is meant that t;he formulator of the composition can select an amount of this suds controlling agent that will control the suds to the extent desired. The amount of suds control will vary with the detergent surfactant 20 selected. For example, with high sudsing surfactants, relatively more of the suds controlling agent- is used to achieve the desired suds control than with low foaming surfactants.
E. Other Enzymes
25 The detergent compositions of the invention may also contain further enzymes.
For example, lipase can usefully be addeil ni the form of a
solution or a slurry of lipolytic en/.yme with carrier material
(e.g. as in EP Patent Publication No. 258,068 (Novo Mordisk
^o A/S) ^

The added amount of lipase can be chosen within wide limits, for example 50 to 30,000 LU/g per gram of the surfactant system or of the detergent composition, e.g. often at least 100 LU/g, very usefully at least 500 LU/g, sometimes preferably above 5 1000, above 2000 LU/g or above 4000 LU/g or more, thus very often within the range of 50-4000 LU/g, and possibly within the range of 200-1000 LU/g. In this specification, lipase units are defined as they are in EP Patent Publication No. 258,068.
The lipolytic enzyme can be chosen among a wide range of
10 lipases. In particular, the lipases descrilaed in for example the following patent specifications: EP Patent Publications Nos. 214,761 (Novo Nordisk A/S), 250,068, and especially lipases showing immunological crass r'eactivity with antisera raised against lipase from Thermomyces lanu'i inosus ATCC 22070,
15 EP Patent Publications Nos. 205,208 and 206, .'I'M,) , and especially lipases showing immunological cross-reactivity with antisera raised against lipase from Chromobacter viscosum var lipolyti-cum NRRL B-3673, or against lipase from ALcallgenes PL-679, ATCC 31371 and FERM-P 3783, also the lipases described in
20 specifications WO 87/00859 (Gist-Brocades) and EP Patent Publication No. 204,284 (Sapporo Breweries). Suitable, in particular, are for example the following commercially available lipase preparations: Lipolase* Novo Nordisk A/S, Amano lipases CE, P, B, AP, M-AP, AML, and CES, and Meito lipases
25 MY-3 0, OF, and PL, also Esterase* MM (Novo Nordisk A/S), Lipozym, SP225, SP285, (all Novo Nordisk A/S) Saiken lipase, Enzeco lipase, Toyo Jozo lipase and Diosynth lipase (Trade Marks), Lumafast* (Genencor Inc.), Lipomax* (Gist-Brocades N.V.), and lipases as described in WO 94/03578 (Unilever).
30 Amylase can for example be used when desired, in an amount in the range of about 1 to about 100 MU (maltose units) per gram of detergent composition (or 0.014-1.4, e.g. 0.07-0.7, KNU/g (Novo units)). Amylases suitable are for example Termarayl*, and BAN (Novo Nordisk A/S). Cellulase can for example be used when
35 desired, in an amount in the range of about 0.3 to about 35 CEVU units per gram of the detergent composition. Suitable

cellulases are for example Celluzyme*, and Carezyme' (NOVO NORDISK A/S).
Other enzymes contemplated to be used in the present invention are oxidases and peroxidases 5 F. Other Optional Components
Other optional components for use in the liquid detergents herein include soil removal agents, soil release polymers, antiredeposition agents such as tetraethylGne pentamine ethoxylate (from about 0,5% to 3%, preferably from about 1% to
10 about 3%, by weight), suds regulants, poly vinyl pyrolidone, carboxy methyl cellulose, clays, and hydrotropes such as sodivm cumene sulfonate, opacifiers, antioxidants, bactericides, dyes, perfumes, and brighteners known in the art. Such optional components generally represent less than about 15%, preferably
15 from about 0.5% to 10%, more preferably from about 1% to about 10%, by weight of the composition.
The compositions may contain from 0% to about 8%, preferably from 0% to about 5%, by weight of a C,2~^u ^IJ^^-'fiyl succinic acid or salt thereof. These materials are of the general formula R-20 CH(C00X)CH2(C00X) , wherein R is a C,2-C^^ alkenyl group and each X is H or a suitable cation, such as sodium, potassium, ammonium or alkanolammonium (e.g., mono-, di-, or tri-ethanolammonium).
Specific examples are 2-dodecenyl succinate (preferred) and 2-25 tetradecenyl succinate.
The compositions herein optionally contain from about 0.1% to about 1%, preferably from about 0.2% to about 0.6%, by weight of water-soluble salts of erthylenedi amine tetramethylenephosphonic acid, die thylenetriamine 30 pentamethylenephosphonic acid, ethylenediaraine tetraacetic acid (preferred), or diethylenetriamine pentaacetic acid (most preferred) to enhance cleaning performance when pretreating fabrics.

Furthermore, the detergent compositions may contain from 1-35% of a bleaching agent or a bleach precursor or a system compris¬ing bleaching agent and/or precursor with activator therefor.
Further optional ingredients are lather boosters, anti-cor-5 rosion agents, soil-suspending agents, sequestering agents, anti-soil redeposition agents, and so on.
The compositions herein preferably contain up to about 10% of ethanol.
G. Other Properties
10 The instant composition usually has a pl(, it\ a 10% by weight solution in water at 2 0°C, of from about 7.0 to 9.0, preferably from about 8.0 to about 8.5.
The instant compositions can also have a Critical Micelle Concentration (CMC) of less than or equal to 200 parts per
15 million (ppm), and an air/water Interfacial Tension above the CMC of less than or equal to 32, preferably less than or equal to about 30, dynes per centimetre at 3 5°C in distilled water. These measurements are described in "Measurement of Interliacial Tension and Surface Tension - General Review fi:>r Practical Man"
20 C. Weser, GIT Fachzeitschrift fur das Laboralr.orium, 24 (1980) 642-648 and 734-742, FIT Verlag Ernst Giebeler, Darmstadt, and "Interfacial Phenomena - Equilibrium and Dynamic Effects", C. A. Miller and P. Neogi, Chapter 1, pp. 29-:i6 (1985), Marcel Dekker, Inc. New York.
25 The compositions of the invention can be used for the washing of textile materials, especially, but without limitation cotton and polyester based textiles and mixtures thereof. For example washing processes carried out at temperatures of about 60-65°C or lower, e.g. about 30-35°C or lower, are particularly

lower or higher concentrations, if deBired, without limitation it can for example be stated that a iise-rate from about 1 to 10 g/1, e.g. from about 3-6 g/1, of tlie detergont formulation is suitable for use in the case when the forTiiulati ons are substan-5 tially as in the Examples.
In this aspect the invention is especially related to:
a) A detergent composition formulated as an aqueous
detergent liquid comprising anionic surfactant, nonionic
surfactant, humectant, organic acid, caustic alkali, with a pH
0 adjusted to a value between 9 and 10.
b) A detergent composition formulated as a non-aqueous
detergent liquid comprising a liquid nonionic surfactant
consisting essentially of linear alkoKylated primary alcohol,
triacetin, sodium triphosphate, caustic alkali, perborate
5 monohydrate bleach precursor, and tertiary amine bleach activator, with a pH adjusted to a value between about 9 and 10.
c) An enzymatic liquid detergent composition formulated to
give a wash liquor pH of 9 or less when used at a rate corre-
3 spending to 0.4-0.8 g/1 surfactant.
d) An enzymatic liquid detergent compos it ion formulated to give a wash liquor pH of 8.5 or more v;hen used at a rate corre¬sponding to 0.4-0.8 g/1 surfactant.
e) An enzymatic liquid detergent composition formulated to give a wash liquor ionic strength of 0.03 or less, e.g. 0.02 or less, when used at a rate corresponding to 0.4-0.8 g/1 surfactant.
f) An enzymatic liquid detergent composition formulated to give a wash liquor ionic strength of 0.01 or more, e.g. 0.02 or more, when used at a rate corresponding to 0.4-0.8 g/1 surfactant.

It was found that the subtilase variant.s of the present invention can also be usefully Incorporated in detergent composition in the form of bars, tablets, ;J1; Icks and the like for direct application to fabrics, Yiard sui:laces or any other surface. In particular, they can be Incorporated into soap or soap/synthetic compositions in bar form, wheirein they exhibit a remarkable enzyme stability. Detergent ccimposition in the form of bars, tablets, sticks and the like for direct application, are for example described in South African Patent 93/7274, incorporated herein by reference.
Accordingly, the preferred bars in accordance with this
invention comprise, in addition to the subtilase variant:
i) 25 to 80%, most preferably 25 to 70%, by weight of
detergent active which is soap or a mixture of soap and
synthetic detergent active, reckoned as anhydrous; ii) 0 to 50 % and, most preferably, 10 to 30% by weight of
water; iii) 0 to 35% and, most preferably, 0.1 to 3 0% by weight
In general, the amount of subtilase variant to be included in such compositions of the invention is such that it corresponds with a proteolytic activity of 0.1 to 100 GU/mg based on the composition, preferably 0.5 to 20GU/mg, most preferably 1.0 to 10 GU/mg, where GU/mg is glycine unit per milligram.
Many methods for introducing mutations into genes are well known in the art. After a brief discussion of cloning subtilase genes, methods for generating mutations in both random sites, and specific sites, within the subtilase gene will be discussed.

The gene encoding a subtilase may be cloned from any of the organisms indicated in Table I, especially gram-positive bacteria or fungus, by various methods, well known in the art.
5 First a genomic, and/or cDNA library ot DNA miist be C9nr.tructed using chromosomal DNA or messenger RNA from the organism that produces the subtilase to be studied. Then, if the amino-acid sequence of the subtilase is known, homologouf?, labelled oligonucleotide probes may be syntliesized .iml used to identify
3 subtilisin-encoding clones from a genomic library of bacterial DNA, or from a cDNA library. Alternatively, a labelled oligonucleotide probe containing sequences homologous to subtilase from another strain of bacteria or organism could be used as a probe to identify subtilase-encoding clones, using hybridization and washing conditions of lower stringency.
Yet another method for identifying subtilase-producing clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming protease-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for subtilase, such as skim milk. Those bacteria containing subtilase-bearing plasmid will produce colonies surrounded by a halo of clear agar, due to digestion of the skim milk by excreted subtilase.
GENERATION OF RANDOM MUTATIONS IN THE SUBTII.A.SE GENE Once the subtilase gene has been cloned into a suitable vector, such as a plasmid, several methods can be used to introduce random mutations into the gene.
One method would be to incorporate the clonecJ subtilase gene, as part of a retrievable vector, into a mutator strain of Eschericia coli.
Another method would involve generating a sin
that a portion of the subtilase gene rernainod single stranded. This discrete, single stranded region could then be exposed to any of a number of mutagenizing agents, including, but not limited to, sodium bisulfite, hydroxylamine, nitrous acid, formic acid, or hydralazine. A specific example of this method for generating random mutations is described by Shortle and
Nathans (1978, Proc. Natl. Acad. Sci. U.S.A..75 217 0-2174).
According to the shortle and Nathans method, the plasmid bearing the subtilase gene would be nicked by a restriction enzyme that cleaves within the gene. This nick would be widened into a gap using the exonuclease action of DNA polymerase I. The resulting single-stranded gap could then be mutagenized using any one of the above mentioned mutagenizing agents.
Alternatively, the subtilisin gene from a 13acillus species including the natural promoter and other control sequences could be cloned into a plasmid vector containing replicons for both E. coli and B. subtilis, a selectable phenotypic marker and the M13 origin of replication for production of single-stranded plasmid DNA upon superinfection with helper phage IRl. Single-stranded plasmid DNA containing the cloned subtilisin gene is isolated and annealed with a DNA fragment containing vector sequences but not the coding region of subtilisin, resulting in a gapped duplex molecule. Mutations are introduced into the subtilisin gene either with sodium bisulfite, nitrous acid or formic acid or by replication in a mutator strain of E_^ coli as described above.. Since sodium bisulfite reacts exclusively with cytosine in a single-stranded DNA, the mutations created with this mutagen are restricted only to the coding regions. Reaction time and bisulfite concentration are varied in different experiments such that from one to five mutations are created per subtilisin gene on average. Incubation of 10 j^g of gapped duplex DNA in 4 M Na-bisulfite, pH. 6.0, for 9 minutes at 37 °C in a reaction volume of 4 00 ^.1,
deaminates about 1% of cytosines in the single stranded region.
The coding region of mature subtilisin contains about 200 cytosines, depending on the DNA strand. Advantageously, the reaction time is varied from about 4 niiriutos (to produce a

mutation frequency of about one in 200) to about 20 minutes (about 5 in 200).
After mutagenesis the gapped molecules are treated in vitro with DNA polymerase I (Klenow fragment) to make fully double-stranded molecules and fix the mutations. Competent E. coli are then transformed with the mutagenized DNA to produce an amplified library of mutant subtilisins. Amplified mutant libraries can also be made by growing the plasraid DNA in a Mut D strain of E. coli which increases the range of mutations due to its error prone DNA polymerase.
The mutagens nitrous acid and formic acid may also be used to
jroduce mutant libraries. Because these chemicals are not as
specific for single-stranded DNA as sodium bisulfite, the
lutagenesis reactions are performed ivccordiiig to the following
)rocedure. The coding portion of tiie subtil isin gene is cloned
.n M13 phage by standard methods and single stranded phage DNA
)repared. The single-stranded DNA is then reacted with 1 M
litrous acid pH. 4.3 for 15-60 minutes at 23°C or 2.4 M formic
cid for 1-5 minutes at 23°C. These ranges of reaction times
reduce a mutation frequency of from 1 in 1000 to 5 in 1000.
fter mutagenesis, a universal primer is annealed to the M13
NA and duplex DNA is synthesized using the mutagenized single-
tranded DNA as a template so that the coding portion of the
ubtilisin gene becomes fully double-strancJed. At t-his point
he coding region can be cut out of the M13 vector with
estriction enzymes and ligated into an un-mutagenized
xpression vector so that mutations occur only in the
sstriction fragment. (Myers et al ■ , Science 229 242-257
L985)) .
SNERATION OF SITE DIRECTED MUTATIONS IN THE SUBTILASE GENE ice the subtilase gene has been cloned, and desirable sites 3r mutation identified and the residue to L.ubstitute for the riginal ones have been decided, these mutations can be itroduced using synthetic oligonucleotides. These oligonucleo-.des contain nucleotide sequences flanking the desired

mutation sites; mutant nucleotides are inserted during oligo¬nucleotide synthesis. In a preferred method, a single stranded gap of DNA, bridging the subtilase gene, is created in a vector bearing the subtilase gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in by DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase, A specific example of this method is described in Morinaqa eL..al..,., (1984, Biotech-nology 2 646-639). According to Moiinaga .et: .a.L^, a fragment within the gene is removed using restriction ondonuclease. The vector/gene, now containing a gaj:), is then denatured and hybridized to a vector/gene which, instead of: c'ontaininc) a gap, has been cleaved with another restriction endonuclease at a site outside the area involved in tlie gap. A single-stranded region of the gene is then available for hybridization with mutated oligonucleotides, the remaining gap is filled in by the Klenow fragment of DNA polymerase I, the insertions are ligated with T4 DNA ligase, and, after one cycle of replication, a double-stranded plasmid bearing the desired mutation is produced. The Morinaga method obviates the additional manipulation of constructing new restriction sites, and therefore facilitates the generation of mutations at multiple sites. U.S. Reissue Patent number Ji4,606 by Estell et al. , issued May 10, 1994, is able to int,reduce oligonucleotides bearing multiple mutations by performing minor alterations of the cassette, however, an even greater variety of mutations can be introduced at any one time by the Morinaga method, because a multitude of oligonucleotides, of various lengths, can be introduced.
According to the invention, a mutated subtilase gene prodviced by methods described above, or any alternativG methods knoun in the art, can be expressed, in enzyme form, using an expr-esiision vector. An expression vector genei.-ally falJ.s under the Jefinition of a cloning vector, since an expression vector usually includes the components of a typical cloning vector,

namely, an element that permits autonomous replication of the vector in a microorganism independent of the genome of the microorganism, and one or more phenotypic markers for selection purposes. An expression vector includes control sequences 5 encoding a promoter, operator, ribosome binding site, trans¬lation initiation signal, and, optionally, a repressor gene or various activator genes. To permit the secretion of the ex¬pressed protein, nucleotides encoding a "signal sequence" may be inserted prior to the coding sequence ol; the gene. For ex-
10 pression under the direction of ccmtrol. secjuences, a target gene to be treated according to the i.nvi.Mitian i^ operably linked to the control sequences in the proper reading frame. Promoter sequences that can be incorporated into plasmid vec¬tors, and which can support the transcription of the mutant
15 subtilase gene, include but are not limited to the prokaryotlc B-lactamase promoter (Villa-Kamarof i;, e,t:_iy,. (1978) Proc. Natl. Acad. Sci. U.S.A. 75 3727-3731) and the tac promoter (DeBoer, et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80 21-25). Further references can also be found in "Useful proteins from recombi-
20 nant bacteria" in Scientific American (1980) 242 74-94.
According to one embodiment B. subtil is is transformed by an expression vector carrying the mutated DNA. If expression is to take place in a secreting microorganism such as B. subtj.lis a signal sequence may follow the tranfilat Lou initiation signal 25 and precede the DNA sequence of interest. The signal sequence acts to transport the expression product to the cell wall where it is cleaved from the product upon secretion. The term "control sequences" as defined above is intended to include a signal sequence, when it is present.
30 Other host systems known to the skilled person are also contemplated for the expression and production of the protease variants of the invention. Such host system'-; comprise fungi, including filamentous fungi, plant, avian and mammalian i;:ells, as well as others.

B. subtilis 309 and 147 are variants of Bacillus lentus^
deposited with the NCIB and accorded the accession numbers NCIB 5 10309 and 10147, and described in US Patent No. 3,723,250 incorporated by reference herein.
E. coli MC 1000 (M.J. Casadaban and S.N. Cohen (1980); J. Mol. Biol. 138 179-207), was made r',iii* by conventional methods and is also described in US Patent Application Serial No. 039,298.
10 Proteolytic Activity
In the context of this invention proteolytic activity is expressed in Kilo NOVO Protease Units (KNPii) . The activity is determined relatively to an enzyme standard (SAVINASE'") , and the determination is based on the digestion of a dimethyl
15 casein (DMC) solution by the proteolytic enzyme at standard conditions, i.e. 50 °C, pH 8.3, 9 min. reaction time, 3 min. measuring time. A folder AF 220/1 is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference.
20 A GU is a Glycine Unit, defined as the proteolytic enzyme activity which, under standard conditions, during a 15-minutes' incubation at 40 deg C, with N-acetyl casein as substrate, produces an amount of NH2-group equivalent to 1 /umole of glycine.
25 Enzyme activity can also be measured using the PNA assay, according to reaction with the soluble substrate succinyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol, which is described in the Journal of American Oil Chemists Society, Rothgeb, T.M., Goodlander, B.D., Garrison, P.H., and Smith,
30 L.A. , (1988) .

For the generation of enzyme variants c^ccording to the invention the same materials and methods as described in i.a. WO 89/06279 (Novo Nordisk A/S) , EP 130,756 (Genentech), EP 5 479,870 (Novo Nordisk A/S), EP 214,435 (Henkel), WO 87/04461 (Amgen), WO 87/05050 (Genex), EP application no. 87303761 (Genentech), EP 260,105 (Genencor), WO 88/06624 (Gist-Brocades NV), WO 88/07578 (Genentech), WO 88/08028 (Genex), WO 88/08033 (Amgen), WO 88/08164 (Genex), Thomas et al. (1985) Nature, 318
10 375-3 7 6; Thomas et al. (198 7) J^„_Mo_l_. Biol. , 19 3, 803-813;
Russel and Fersht (1987) Nature 328 496-500. Other methods well established in the art may also be used,
Construction and Expression of
15 A vector suited to a synthetic gene coding i.ar subtilase 309 and its mutants was constructed. It is essentially a pUC19 plasmid [Yanish-Perron and Messing (1985) Gene; 33 103-119], in which the multiple cloning site has been replaced by a linker containing the restriction sites used to seprirate five sub--
20 fragments constituting the.gene. The new linker was inserted into EcoRI - Hindlll cut pUC19 thereby destroying these sites. The details of this construction are described in WO 92/19729 on pages 25-26 and in figure 1 (sheets 1/7-7/7) thereof, the content of which is hereby included by reference.
25 Each subfragment was made from 6 to 12 oligonucleotides. The oligonucleotides were synthesiaed on an automatic DNA synthesizer using phosphoramidite chemistry on a controlled glass support [Beaucage and Carruthers (1901); Tetrahedron Letters 22 1859-1869].
30 The five subfragments were isolated on a 2% agarose gel and inserted into pSX191. The seguence was verified by dideoxynu-cleotide sequencing. Fragments A-E were isolated and ligated

together with Kpnl-BamHI cut pSX191. The ligation mixtures were used to transform competent E coli MCIOOCJ r",m* selecting for ampicillin resistance. The 850 bp Kpnl-Eiamlfl fragment that constitutes the part of the subtilisin 309 gene coding for the 1 mature part of the enzyme was then used to replace the wild type gene on pSX212 giving rise to pSX222, which was then transformed into a competent B. subtil is strain. After fermentation of the transformed strain and purification of the enzyme it was shown that the product v/as indistinguishable from the wild type product.
Protease variants derived from the synthetic gene are made by using oligonucleotides with altered sequenct? at the place (s) where mutation is wanted (e.g. with sequences as given below) and mixing them with the rest of the oligonuclo-otides appropri¬ate to the synthetic gene. Assembly of the variant gene is carried out with the variant materials in a manner otherwise analogous to that described above. Further information on syn¬thetic genes generally is available in Agarval et al (1970); Nature; 227 27-34.
A Kpnl site was introduced into the beginning of the subtilase 309 synthetic gene encoding the mature part of the enzyme. The method used is called oligonucleotide directed double-strand break repair mutagenesis and is describcMi by Mandecki (1986) Proc. Nat. Acad. Sci. USA 83 7177-7101. pSXl72 is opened with Ncol at the beginning of the mature part of the subtilase 3 09 gene and is mixed with the oligonucleotide NOR 789 (see WO 92/19729) , heated to 100°C, cooled to 0"C, and transfor-med into E. coli. After retransformation, the recombinants can be screened by colony hybridisation using 32-P-labelled NOR 789. The recombinants that turned out to be positive during the screening had the Kpnl site introduced right in front of Ncol oy changing two bases without changing the amino acid sequence. ;)SX172 is described in EP Patent Publication Ho. 405 901. The
The synthetic gene is inserted between Kpnl and BamHI on pSX212, giving rise to pSX222.
Examples of mutations and corresponding sequences of oligonu¬cleotides are as follows:

The filtrates were concentrated to approximately 400 ml using an Amicon CH2A UF unit equipped with an Amicon SIYIO UF cartridge. The UF concentrate was centrifuged and filtered prior to absorption at room temperature on a Bacitracin af-5 finity column at pH 7. The protease was eluted from the Bacitracin column at room temperature using 2 5% 2-propanol and 1 M sodium chloride in a buffer solution with 0.01 dimethyl-glutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to pH 7.
10 The fractions with protease activity from the Bacitracin purification step were combined and applied to a 7 50 ml Sephadex G25 column (5 cm dia.) equilibrated with a buffer containing 0.01 dimethylglutaric acid, 0.2 M boric acid and 0.002 m calcium chloride adjusted to pH 6.5.
15 Fractions with proteolytic activity from the Sephadex G2 5 column were combined and applied to a 150 ml CM Sepharose CL 6B cation exchange column (5 cm dia.) equilibrated v/ith a buffer containing 0.01 M dimethylglutaric acid, 0.? M boric acid, and 0,002 M calcium chloride adjusted to pH 6.5.
20 The protease was eluted using a linear gradient of 0-0.1 M sodium chloride in 2 litres of the same buffer (0-0.2 M sodium chloride in case of Subtilisin 147).
In a final purification step protease containing fractions from the CM Sepharose column were combined and concentrated in an 25 Amicon ultrafiltration cell equipped with a GR81PP membrane (from the Danish Sugar Factories Inc.).
By using the techniques of Example 1 for the construction and the above isolation procedure the following subtilisin 309 variants were produced and isolated;

Stability in Detergent CompositionB Comprising Enzyme Variants
Example Dl:
An (isotropic) aqueous detergent liquid according to an embodi-5 ment of the invention is formulated to contain:

Table III.
Residual enzyme activity (in percentage ol original activity) after storage at 37'C for Example DI comprising the BLS309 variant S57P+R170L+N218S.

From Table III it is evident that the variant S57P+R170LH-N218S exhibits a remarkably improved stability in this type; of detergent. Moreover, the variant S57P-i-R170Lt-N218S possesses excellent compatibility towards lipase.
15 Table IV.
Residual lipase activity (in percentage of original activity) after storage at 37'C for Example Dl comprising the BLS309 variant S57P+R170L+N218S and LIPOLASE*.

From the above Table IV it is apparent that, in addition to the stability of the protease, the compatibility of the protease is also improved.
Example D2; 30 A non-aqueous detergent liquid according to an embodiment of the invention is formulated using 38.5% C13-C15 linear primary

mol/mol propylene oxide, 5% triacetin, 3 0% socMura tripliosphate, 4% soda ash, 15.5% sodium perborate monohydrate containing a minor proportion of oxoborate, 4% TAED, 0.25% EDTA of which 5 0.1% as phosphonic acid, Aerosil 0.6%, SCMC 1%, and 0.6% protease. The pH is adjusted to a value between 9 and 10, e.g. about 9.8.
Example D3:
Structured liquid detergents can for example contain, in
10 addition to a protease as described herein, 2-15% nonionic surfactant, 5-40% total surfactant, comprising nonionic and optionally anionic surfactant, 5-3 5% phospliate-containing or non-phosphate containing builder, 0.2-0.0% polymeric thickener, e.g. cross-linked acrylic polymer witli m.w. over 10*, at least
15 10% sodium silicate, e.g. as neutral waterglass, alk=ali (e.g. potassium-containing alkali) to adjust to desired pH, preferably in the range 9-10 or upwards, e.g. above pH 11, with a ratio sodium cation: silicate anion (at; free silica) (by weight) less than 0.7:1, and viscosity oE 0.3-30 Pas (at 20°C
20 and 20*-') .
Suitable examples contain about 5% nonionic surfactant C13--15 alcohol alkoxylated with about 5 EO groups per mole and with about 2.7 PO groups per mole, 15-2 3% neutral waterglass with 3.5 weight ratio between silica and sodium oxide, 13-19% KOH, 25 8-23% STPP, 0-11% sodium carbonate, 0.5% Carbopol 941 (TM).
Protease may be incorporated at for example 0.5%.

From Table V it is evident that the R170L variant exhibits a remarkably improved stability in this type of: detergent.

From Table VI it is evident that the variant S57P+R170L+N218S 30 exhibits a remarkably improved stability in this type of detergent. Moreover the variant S57P+R170l,vN218S possesses excellent compatibility towards limate.

Example D6:
Soap bars were produced containing 49.7 wt.% 80/20 tallow /coconut soap, 49.0% water, 20% sodium citrate, 1.0% citric acid and 0.031% protease. After preparation of the £:oap bars 5 they were stored at ambient temperature and after specific time intervals samples were taken and measured for prcit:ease activity. The stability data are given below;

Wash Performance of Detergent Compositions Comprising Enzyme Variants
The following examples provide results from a number of washing tests that were conducted under the conditions indicated

Experimental conditions
Table IX: Experimental conditions for evaluation of Subtilisin 309 variants.

Further the pH is adjusted to 9.5, which is low for a powder detergent.

a is the initial slope for the reference enzyme.
c is the enzyme concentration in mg/1
ARMAX IS THE Theoretical maximum wash effect of the enzyme in remmision units (c-»oo) ,

As it can be seen from Table XI all the Subtilisin 309 variants of the invention exhibits an improvement in wash performance.

1. A process for preparing a subtilase variant comprising one or more of the subtitutions in subtilase subgroup
a) I-Sl:
pos. 129: P129V, P129I, P129L, P129M, P129F, P129W
pos. 131: G131V, G131I, G131L, G131M, G131F, G131P, G131W
pos. 136: K136V, K136I, K136L, K136M, K136F, K136P, K136W,
K136G, K136C, K136S, K136A, K136T, K136Y, K136Q,
K136H, K136N,
pos. 159: S159V, S159I, S159L, S159M, S159F, S159P,'S159W,
pos. 164: T164V, T164I, T164L, T164M, T164F, T164P, T164W,
pos. 167: Y167I, yi67L, Y167M, Y167F, Y167P, Y167W,
pos. 170: K170V, K170I, K170L, K170M, K170F, K170P, K170W,
K170G, K170C, K170S, K170A, K170T, K170Y, K170Q,
K170H, K170N,
pos. 171: Y171V, Y171I, Y171L, Y171M, Y171F, Y171P, Y171W,
pos. 194: (in BASBPN) P194V, P194r, P194L. P194M, P194F,
(in BSS168) S194V, S194I. S194L, S194M, S194F,
S194P, S194W,
(in all other I-Sl svibtilases) A194V, A194I,
A194L, A194M. A194F, A194P, A194W
pos. 195: E195V, E195I, E195L, E195M. E195F, E195P. E195W,
E195G, E195C, E195S, E195A, E195T, E195Y. E195Q,
E195H, E19SN.
b) I-S2:
pos. 129: (in BLS309 and BAPB92) P129V, P129I, P129L,
P129M, P129F, P129W
(in BLS147) T129V, T129I, T129L, T129M, T129F,
T129P, T129W,
(in BYSYAB) S129V, S129I, S129L, S129M, S129F,
S129P, S129W, pos. 131: (in BLS147 and BYSYAB) G131V, G131I, G131L,
G131M, G131F, G131P, G131W,
(in BLS309 and BAPB92) P131V, P131I, P131L,
P131M, P131F, P131W,

pos. 136: E136V, E136I, E136L, E136M, E136F, E136P, E136W, E136G, E136C, E136S, E136A, E136T, E136Y, E136Q, E136H, E136N,
pos. 159: (in BLS147) Q159V, Q159I, Q159L, Q159M, Q159F, Q159P, Q159W,
(in all other I-S2 subtilases) G159V, G159I, G159L, G159M, G159F, G159P, G159W,
pos. 164: (in BLS147) G164V, G164I, G164L, G164M, G164F, G164P, G164W,
(in BYSYAB) S164V, S164I, S164L, S164M, S164F, S164P, S164W,
(in BLS309 and BAPB92) S164V, S164I, S164L, S164M, S164F, S164P, S164W,
pos. 167: Y167A, Y167H, Y167N, Y167P, Y167C, Y167W, Y167Q, Y167S, Y167T, Y167G, Y167I, Y167L, Y167M, Y167F,
pos. 170: (in BLS309 and BLS147) R170W, R170A, R170H, R170N, R170P, R170Q, R170S, R170T, R170V, R170I, R170L, R170M, R170F, R170G, R170C,
(in BAPB92) R170W, R170A, R170H, R170N, R170P, R170Q, R170S, R170T, R170L, R170F, R170G, R170C, (in all other I-S2 subtilases) R170W, R170A, R170H, R170N, R170P, R170Q, R170S, R170T, R170Y, R170V, R170I, R170L, R170M, R170F, R170G, R170C,
pos. 171: Y171A, Y171H, Y171N, Y171P, Y171C, Y171W, Y171Q, Y171S, Y171T, Y171G, Y171V, Y171I, Y171L, Y171M, Y171F,
pos. 194: (in BLS147) P194V, P194I, P194L, P194M, P194F, P194W,
(in all other I-S2 subtilases) A194V, A194I, A194L, A194M, A194F, A194P, A194W,
pos. 195: (in BLS147) E195V, E195I, E195L, E195M, E195F, E195P, E195W, E195G, E195C, E195S, E195A, E195T, E195Y, E195Q, E195H, E195N,
(in all other I-S2 subtilases) G195V, G195I, G195L, G195M, G195F, G195P, G195W,
c) Thermitase:
pos. 129: T129V, T129I, T129L, T129M, T129F, T129P, TI29W

pos. 131: G131V, G131I, G131L, G131M, G131F, G131P, G131W,
pos. 136: Q136V, Q136I, Q136L, Q136M, Q136F, Q136P, Q136W,
pos. 159: T159V, T159I, T159L, T159M, T159F, T159P, T159W,
pos. 164: A164V, A164I, A164L, A164M, A164F, A164P, A164W,
pos. 167: Y167I, Y167L, Y167M, Y167F, Y167P, Y167W,
pos. 170: Y170V, Y170I, Y170L, Y170M, Y170F, Y170P, Y170W
pos. 171: Y171V, Y171I, Y171L, Y171M, Y171F, Y171P, Y171W,
pos. 194: S194V, S194I, S194L, S194M, S194F, S194P, S194W.
said process comprising culturing a microbial host, which is transformed with vector comprising a DNA sequence encoding the subtilase variant unde conditions conductive to the expression and secretion of said subtilase varian and recovering the subtilase variant.
The process for preparing a detergent composition for removing proteinaceou stains comprising adding to a mixture of surfactants, builders and othe processing ingredients of the kind as herein before described, a protease whic is a subtilase variant of the kind as herein before described in an amount c 0.01% to 5% by weight of the composition.
A process for preparing a subtilase variant substantially as herein describe with reference to the accompanying drawings.


744-mas-1996 abstract.pdf

744-mas-1996 assignment.pdf

744-mas-1996 claims.pdf

744-mas-1996 correspondence -others.pdf

744-mas-1996 correspondence -po.pdf

744-mas-1996 description (complete).pdf

744-mas-1996 drawings.pdf

744-mas-1996 form-2.pdf

744-mas-1996 form-26.pdf

744-mas-1996 form-4.pdf

744-mas-1996 form-6.pdf

744-mas-1996 petition.pdf

Patent Number 194793
Indian Patent Application Number 744/MAS/1996
PG Journal Number 30/2009
Publication Date 24-Jul-2009
Grant Date 02-Jan-2006
Date of Filing 06-May-1996
Name of Patentee M/S. NOVOZYMES
Applicant Address KROGSHOJVEJ 36, DK-2800 BAGSVAERD,
# Inventor's Name Inventor's Address
PCT International Classification Number C11D10/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA