Modified subtilisins and detergent compositions containing same

Details Modified subtilisins and detergent compositions containing same Modified subtilisins and detergent compositions containing same

2000-10-02

2003-10-14

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S221000, C435S222000, C435S069100, C435S252310, C435S320100, C435S471000, C536S023200, C510S300000, C510S320000

Reexamination Certificate

active

06632646

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to novel mutant enzymes or enzyme variants useful in formulating detergent compositions in exhibiting improved wash performance, cleaning and detergent compositions containing said enzymes, mutated genes coding for the expression of said enzymes when inserted in a suitable host cell or organism and methods of selecting the amino acid residues to be changed in a parent enzyme in order to perform better in a given wash liquor under specified conditions.
BACKGROUND OF THE INVENTION
In the detergent industry, enzymes have been implemented in washing formulations for more than 20 years. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially, proteases are most important.
Although proteases have been used in the detergent industry for more than 20 years, it is still not exactly known which physical or chemical characteristics are responsible for a good washing performance or ability of a protease.
The currently used proteases have been found by isolating proteases from nature and testing them in detergent formulations.
Bacillus 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). Bacteria of the Bacillus species secrete two extracellular species of protease, a neutral, or metalloprotease, and an alkaline protease which is functionally a serine endopeptidase, 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. Joliffe et al. (1980, J. Bacterial 141:1199-1208) has suggested that Bacillus proteases function in cell wall turnover.
Subtilisin
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, N.Y., pp. 271-272).
The bacterial serine proteases have molecular weights in the range of 20,000 to 45,000. They are inhibited by diisopropylfluorophosphate, but in contrast to metalloproteases, are resistant to ethylene diamino tetraacetic acid (EDTA) (although they are stabilized at high temperatures by calcium ions). 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).
In relation to the present invention a subtilisin is a serine protease produced by Gram-positive bacteria or fungi. A wide variety of subtilisins have been identified and the amino acid sequence of a number of subtilisins have been determined. These include at least six subtilisins from Bacillus strains, namely, subtilisin 168, subtilisin BPN′, subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus and mesentericopeptidase (Kurihara et al., 1972, J. Biol. Chem. 247:5629-5631; Wells et al., 1983, Nucleic Acids Res. 11:7911-7925; Stahl and Ferrari, 1984, J. Bacteriol. 159:811-819, Jacobs et al., 1985, Nucl. Acids Res. 13:8913-8926; Nedkov et al., 1985, Biol. Chem. Hoppe-Seyler 366:421-430, Svendsen et al., 1986, FEBS Lett 196:228-232), one subtilisin from an actinomycetales, thermitase from Thermoactinomyces vulgaris (Meloun et al., 1985, FEBS Lett. 1983: 195-200) and one fungal subtilisin, proteinase K from Tritirachium album (Jany and Mayer, 1985, Biol. Chem. Hoppe-Seyler 366:584-492).
Subtilisins are well-characterized physically and chemically. In addition to knowledge of the primary structure (amino acid sequence) of these enzymes, over 50 high resolution X-ray structures of subtilisin have been determined which delineate the binding of substrate, transition state, products, at least three different protease inhibitors and define the-structural consequences for natural variation (Kraut, 1977, Ann. Rev. Biochem. 46:331-358).
In the context of this invention, a subtilisin variant or mutated subtilisin protease means a subtilisin 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 from which said mutated subtilisin protease is produced when expressed in a suitable host.
Random and site-directed mutations of the subtilisin gene have both arisen from knowledge of the physical and chemical properties of the enzyme and contributed information relating to subtilisin's catalytic activity, substrate specificity, tertiary structure, etc. (Wells et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84; 1219-1223; Wells et al., 1986, Phil. Trans. R. Soc. Lond. A. 317:415-423: Hwang and Warshel, 1987, Biochem. 26:2669-2673; Rao et al., 1987, Nature 328:551-554).
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 U.S. Pat. No. 4,760,025 (GENENCOR)) relating to site specific or randomly generated mutations in “carbonyl hydrolases” and subsequent screening of the mutated enzymes for various properties, such as k
cat
/k
m
ratio, pH-activity profile and oxidation stability. Apart from revealing that site-specific mutation is feasible and that mutation of subtilisin BPN′ in certain specified positions, i.e.
−1
Tyr,
32
Asp,
155
Asn,
104
Tyr,
222
Met,
166
Gly,
64
His,
169
Gly,
189
Phe,
33
Ser,
221
Ser,
217
Tyr,
156
Glu or
152
Ala, provide for enzymes exhibiting altered properties, this application does not contribute 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 of subtilisin Carlsberg and two mutants thereof. In this application no reason for mutation of
158
Asp to
158
Ser and
161
Ser to
161
Asp is provided.
In International Patent Publication No. WO 87/04461 (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
109
Asn and the
218
Asn residues in subtilisin BPN′.
International patent publication No. WO 87/05050 (GENEX) discloses random mutation and subsequent screening of a large number of mutants of subtilisin BPN′ for improved properties. In the application, mutations are described in positions
218
Asn,
131
Gly,
254
Thr,
166
Gly,
116
Ala,
188
Ser,
126
Leu and
53
Ser.
In EP Application No. 87303761 (GENENTECH) it is described how homology 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′ led the inventors to select a number of positions susceptible to mutation with an expectation of obtaining mutants with altered properties. The positions so identified are:
124
Met,
222
Met,
104
Tyr,
152
Ala,
156
Glu,
166
Gly,
169
Gly,
189
Phe,
217
Tyr. Also
155
Asn,
21
Tyr,
22
Thr,
24
Ser,
32
Asp,
33
Ser,
36
Asp,
46
Gly,
48
Ala,
49
Ser,
50
Met,
77
Asn,
87
Ser,
94
Lys,
95
Val,
96
Leu,
107
Ile,
110
Gly,
170
Lys,
171
Tyr,
172
Pro,
197
Asp,
199
Met,
204
Ser,
213
Lys and
221
Ser. The positions are identified as being expected to influence various properties of the enzyme. In addition, a number of mutations are exemplified to support th

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