Protease variants and compositions

Details Protease variants and compositions Protease variants and compositions

1998-11-19

2003-08-12

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S069100, C435S221000, C435S222000, C435S252300, C435S320100, C435S471000, C510S350000, C536S023200

Reexamination Certificate

active

06605458

ABSTRACT:

CROSS-REFERENCE TO RELATED APPLICATIONS
This application claim priority under 35 U.S.C. 119 of Danish application 1332/97 filed Nov. 21, 1997, the contents of which are fully incorporated herein by reference.
TECHNICAL FIELD
This invention relates to novel mutant protease enzymes or enzyme variants, comprising insertions in one or more active site loops, useful in formulating detergent compositions and exhibiting improved wash performance in detergents; 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.
BACKGROUND OF THE INVENTION
In the detergent industry enzymes have for more than 30 years been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially most important enzymes are proteases.
An increasing number of commercially used proteases are protein engineered variants of naturally occurring wild type proteases, e.g. DURAZYM® (Novo Nordisk A/S), RELASE® (Novo Nordisk A/S), MAXAPEM® (Gist-Brocades N.V.), PURAFECT® (Genencor International, Inc.).
Further a number of protease variants are described in the art, such as in EP 130756 (GENENTECH)(corresponding to US Reissue Patent No. 34,606 (GENENCOR)); EP 214435 (HENKEL); WO 87/04461 (AMGEN); WO 87/05050 (GENEX); EP 260105 (GENENCOR); Thomas, Russell, and Fersht (1985)
Nature
318 375-376; Thomas, Russell, and Fersht (1987)
J. Mol. Biol.
193 803-813; Russel and Fersht
Nature
328 496-500 (1987); WO 88/08028 (Genex); WO 88/08033 (Amgen); WO 95/27049.(SOLVAY S.A.); WO 95/30011 (PROCTER & GAMBLE COMPANY); WO 95/30010 (PROCTER & GAMBLE COMPANY); WO 95/29979 (PROCTER & GAMBLE COMPANY); US 5.543.302 (SOLVAY S.A.); EP 251 446 (GENENCOR); WO 89/06279 (NOVO NORDISK A/S); WO 91/00345 (NOVO NORDISK A/S); EP 525 610 Al (SOLVAY); and WO 94/02618 (GIST-BROCADES N.V.).
However, even though a number of useful protease variants have been described, there is still a need for new improved proteases or protease variants for a number of industrial uses.
Therefore, an object of the present invention, is to provide improved proteases or protein engineered protease variants, especially for use in the detergent industry.
SUMMARY OF THE INVENTION
The present inventors have identified that it is possible to construct variants of BLSAVI (Savinase®), having improved wash performance in detergent, as compared to the parent wildtype BLSAVI, by introducing at least one insertion in at least one of the active site loops in said BLSAVI.
It is predicted that it will be possible to make similar variants in other subtilases, which are similar to BLSAVI.
Further it is predicted that it is possible to isolate from nature and identify naturally occurring parent or wildtype subtilases, having improved wash performance in a detergent, as compared to BLSAVI, by specifically screening for such parent wildtype subtilases comprising at least one active site loop, which is longer than the corresponding active site loop in BLSAVI.
Accordingly, in a first aspect the invention relates to an isolated subtilase enzyme,having improved wash performance in a detergent, as compared to BLSAVI, having an amino acid sequence which is at least 40% identical to the amino acid sequence of the mature BLSAVI, and characterized by that at least one of the active site loops, in said isolated subtilase, is longer than the corresponding active site loop in BLSAVI, whereby such active site loops regions, in said isolated subtilase, is having the minimum amino acid length as specified from the group below comprising:
(a) the region (both of the end amino acids included) between amino acid residue from 33 to 43 is at least 11 amino acid long (i.e. at least one amino acid insertion, as compared to BLSAVI);
(b) the region (both of the end amino acids included) between amino acid residue 95 to 103 is at least 9 amino acids long (i.e. at least one amino acid insertion, as compated to BLSAVI);
(c) the region (both of the end amino acids included) between amino acid residue 125 to 132 is at least 8 amino acids long (i.e. at least one amino acid insertion, as compared to BLSAVI);
(d) the region (both of the end amino acids included) between amino acid residue 153 to 173 is at least 21 amino acids long (i.e. at least one amino acid insertion, as compared to BLSAVI);
(e) the region (both of the end amino acids included) between amino acid residue 181 to 195 is at least 15 amino acids long (i.e. at least one amino acid insertion, as compared to BLSAVI);
(f) the region (both of the end amino acids included) between amino acid residue 202 to 204 is at least 3 amino acids long (i.e. at least one amino acid insertion, as compared to BLSAVI); and
(g) the region (both of the end amino acids included) between amino acid residue 218 to 219 is at least 3 amino acids long (i.e. at least one amino acid insertion, as compared to BLSAVI).
In a second aspect the invention relates to an isolated DNA sequence encoding a subtilase variant of the invention.
In a third aspect the invention relates to an expression vector comprising an isolated DNA sequence encoding a subtilase variant of the invention.
In a fourth aspect the invention relates to a microbial host cell transformed with an expression vector according to the fourth aspect.
In a further aspect the invention relates to the production of the subtilisin enzymes of the invention by inserting an expression vector according to the fourth aspect into a suitable microbial host, cultivating the host to express the desired subtilase enzyme, and recovering the enzyme product.
Further the invention relates to a composition comprising a subtilase variant of the invention.
Even further the invention relates to the use of the mutant enzymes for a number of industrial relevant uses, in particular for use in cleaning compositions and cleaning compositions comprising the mutant enzymes, especially detergent compositions comprising the mutant subtilisin enzymes.
Definitions
Prior to discussing this invention in further detail, the following term will first be defined.
Nomenclature of Amino Acids
A=Ala=Alanine
V=Val=Valine
L=Leu=Leucine
I=Ile=Isoleucine
P=Pro=Proline
F=Phe=Phenylalanine
W=Trp=Tryptophan
M=Met=Methionine
G=Gly=Glycine
S=Ser=Serine
T=Thr=Threonine
C=Cys=Cysteine
Y=Tyr=Tyrosine
N=Asn=Asparagine
Q=Gln=Glutamine
D=Asp=Aspartic Acid
E=Glu=Glutamic Acid
K=Lys=Lysine
R=Arg=Arginine
H=His=Histidine
X=Xaa=Any amino acid
Nomenclature of Nucleic Acids
A=Adenine
G=Guanine
C=Cytosine
T=Thymine (only in DNA)
U=Uracil (only in RNA)
Nomenclature of Varianrs
In describing the various enzyme variants produced or contemplated according to the invention, the following nomenclatures have been adapted for ease of reference:
Original amino acid(s) position(s) substituted amino acid(s)
In the case when the original amino acid residue may be any amino acid residue, a short hand notation may at times be used indicating only the position and substituted amino acid,
Position Substituted Amino Acid
Such a notation is particular relevant in connection with modifications) in homologous subtilases (vide intra).
Similarly when the identity of the substituting amino acid residue(s) is immaterial,
Original Amino Acid Position
When both the original amino acid(s) and substituted amino acid(s) may comprise any amino acid, then only the position(s) is indicated, e.g.: 170.
When the original amino acid(s) and/or substituted amino acid(s) may comprise more than one, but not all amino acid(s), then the selected amino acids are indicated inside brackets &

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