Protease variants and compositions

Details Protease variants and compositions Protease variants and compositions

2000-02-24

2003-04-29

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S069100, C435S252310, C435S320100, C435S471000, C536S023200, C510S306000

Reexamination Certificate

active

06555355

ABSTRACT:

TECHNICAL FIELD
This invention relates to novel mutant protease enzymes or enzyme variants 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 describe in the art, such as in EP 130756 (GENENTECH)(corresponding to U.S. Reissue Pat. 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); U.S. Pat. No. 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); WO 94/02618 (GIST-BROCADES N.V.); and WO 96/34946 (NOVO NORDISK A/S).
However, even though a number of useful protease variants have been described, there is still a need for new improved protease variants for a number of industrial uses.
Therefore, an object of the present invention, is to provide improved protein engineered protease variants, especially for use in the detergent industry.
SUMMARY OF THE INVENTION
The present inventors have intensively studied numerous of the possible combinations of the N252, T255 and S259 residues of SAVINASE®, and identified a number of variants with increased improved wash performance.
For further details reference is made to working examples herein (vide infra).
Accordingly, the present invention relates in its first aspect to a subtilase protease variant having improved wash performance in detergents, comprising modification(s) in position(s) 252, 255 and/or 259.
Preferably a subtilase variant according to the invention comprises modifications in both positions 252 and 255, and more preferred comprises modifications in all three positions 252, 255, and 259.
In a second aspect the invention relates to a subtilase enzyme variant having improved wash performance in detergents, comprising at least one modification chosen from the group comprising:
252L+255I
252V+255A
252M+255C+259H
252S+255E+259C
252K+255S+259C; or
a variant comprising one or more conservative modification(s) in any of the above mentioned variants (e.g. a conservative modification of a 252L(hydrophobic a.a.)+255I variant include variants such as 252I(hydrophobic a.a.)+255I, and 252V (hydrophobic a.a.)+255I.
In a third aspect the invention relates to an isolated DNA sequence encoding a subtilase variant of the invention.
In a fourth aspect the invention relates to an expression vector comprising an isolated DNA sequence encoding a subtilase variant of the invention.
In a fifth 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.
Even further the invention relates to a composition comprising a subtilase variant of the invention.
Finally 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 Variants
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)
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:
Gly 195* or G195*
and insertion of an additional amino acid residue such as lysine is:
Gly 195 GlyLys or G195GK
Where a deletion in comparison with the sequence used for the numbering is indicated, an insertion in such a position is indicated as:
*36 Asp or *36D
for insertion of an aspartic acid in position 36
Multiple mutations are separated by pluses, i.e.:
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, respectively.
Proteases
Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979,
Enzymatic Reaction Mechanisms.
W. H. Freeman and Company, San Francisco, Chapter 3).
Numbering of Amino Acid Positions/residues
If no other mentioned the amino acid numbering used herein correspond to that of the subtilase BPN{grave over ( )} (BASBPN) sequence. For further description of the BPN{grave over ( )} sequence see Siezen et al.,
Protein Engng.
4 (1991) 719-737 and FIG.
1
.
Serine Proteases
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 diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977)
Bacteriological Rev.
41 711-753).
Subtilases
A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al.,
Protein Engng.
4 (1991) 719-737. They are defined by homology analysis of more than 40 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previou

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