Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-10-23
2002-11-19
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S220000, C435S252310, C435S320100, C435S471000, C510S392000, C536S023200
Reexamination Certificate
active
06482628
ABSTRACT:
BACKGROUND OF THE INVENTION
Serine proteases are a subgroup of carbonyl hydrolases. They comprise a diverse class of enzymes having a wide range of specificities and biological functions. Stroud, R.
Sci. Amer
., 131:74-88. Despite their functional diversity, the catalytic machinery of serine proteases has been approached by at least two genetically distinct families of enzymes: 1) the subtilisins and 2) the mammalian chymotrypsin-related and homologous bacterial serine proteases (e.g., trypsin and S. gresius trypsin). These two families of serine proteases show remarkably similar mechanisms of catalysis. Kraut, J. (1977),
Annu. Rev. Biochem
., 46:331-358. Furthermore, although the primary structure is unrelated, the tertiary structure of these two enzyme families bring together a conserved catalytic triad of amino acids consisting of serine, histidine and aspartate.
Subtilisins are serine proteases (approx. MW 27,500) which are secreted in large amounts from a wide variety of Bacillus species and other microorganisms. The protein sequence of subtilisin has been determined from at least nine different species of Bacillus. Markland, F. S., et al. (1983),
Hoppe-Seyler's Z. Physiol. Chem
., 364:1537-1540. The three-dimensional crystallographic structure of subtilisins from
Bacillus amyloliquefaciens, Bacillus licheniforimis
and several natural variants of
B. lentus
have been reported. These studies indicate that although subtilisin is genetically unrelated to the mammalian serine proteases, it has a similar active site structure. The x-ray crystal structures of subtilisin containing covalently bound peptide inhibitors (Robertus, J. D., et al. (1972),
Biochemistry
, 11:2439-2449) or product complexes (Robertus, J. D., et al. (1976),
J. Biol. Chem
., 251:1097-1103) have also provided information regarding the active site and putative substrate binding cleft of subtilisin. In addition, a large number of kinetic and chemical modification studies have been reported for subtilisin; Svendsen, B. (1976),
Carlsberg Res. Commun
., 41:237-291; Markland, F. S. Id.) as well as at least one report wherein the side chain of methionine at residue 222 of subtilisin was converted by hydrogen peroxide to methionine-sulfoxide (Stauffer, D. C., et al. (1965),
J. Biol. Chem
., 244:5333-5338) and extensive site-specific mutagenesis has been carried out (Wells and Estell (1988)
TIBS
13:291-297)
SUMMARY OF THE INVENTION
It is an object herein to provide a protease variant containing a substitution of an amino acid at one or more residue positions corresponding to residue positions selected from the group consisting of 62, 212, 230, 232, 252 and 257 of
Bacillus amyloliquefaciens
subtilisin.
While any combination of the above listed amino acid substitutions may be employed, the preferred protease variant enzymes of the present invention comprise the substitution of amino acid residues in the following combinations. All of the residue positions correspond to positions of
Bacillus amyloliquefaciens
subtilisin:
(1) a protease variant including substitutions of the amino acid residues at position 62 and at one or more of the following positions 103, 104, 109, 159, 213, 232, 236, 245, 248 and 252;
(2) a protease variant including substitutions of the amino acid residues at position 212 and at one or more of the following positions 12, 98, 102, 103, 104, 159, 232, 236, 245, 248 and 252;
(3) a protease variant including substitutions of the amino acid residues at position 230 and at one or more of the following positions 68, 103, 104, 159, 232, 236 and 245;
(4) a protease variant including substitutions of the amino acid residues at position 232 and at one or more of the following positions: 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 212, 213, 217, 230, 236, 245, 248, 252, 257, 260, 270 and 275;
(5) a protease variant including substitutions of the amino acid residues at position 232 and at one or more of the following positions 103, 104, 236 and 245;
(6) a protease variant including substitutions of the amino acid residues at position 232 and 103 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 212, 213, 217, 230, 236, 245, 248, 252, 257, 260, 270 and 275;
(7) a protease variant including substitutions of the amino acid residues at position 232 and 104 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 212, 213, 217, 230, 236, 245, 248, 252, 257, 260, 270 and 275;
(8) a protease variant including substitutions of the amino acid residues at position 232 and 236 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 212, 213, 217, 230, 236, 245, 248, 252, 257, 260, 270 and 275;
(9) a protease variant including substitutions of the amino acid residues at position 232 and 245 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 212, 213, 217, 230, 236, 245, 248, 252, 257, 260, 270 and 275;
(10) a protease variant including substitutions of the amino acid residues at position 232, 103, 104, 236 and 245 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 212, 213, 217, 230, 236, 245, 248, 252, 257, 260, 270 and 275;
(11) a protease variant including substitutions of the amino acid residues at position 252 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248 and 270;
(12) a protease variant including substitutions of the amino acid residues at position 252 and at one or more of the following positions 103, 104, 236 and 245;
(13) a protease variant including substitutions of the amino acid residues at positions 252 and 103 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248 and 270;
(14) a protease variant including substitutions of the amino acid residues at positions 252 and 104 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248 and 270;
(15) a protease variant including substitutions of the amino acid residues at positions 252 and 236 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248 and 270;
(16) a protease variant including substitutions of the amino acid residues at positions 252 and 245 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248 and 270;
(17) a protease variant including substitutions of the amino acid residues at positions 252, 103, 104, 236 and 245 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248 and 270; and
(18) a protease variant including substitutions of the amino acid residues at position 257 and at one or more of the following positions 68, 103, 104, 205, 209, 210, 232, 236, 245 and 275. More preferred protease variants are substitution sets selected from the group consisting of residue positions corresponding to positions in Table 1 of Bacillus amyloliquefaciens subtilisin:
TABLE 1
76
103
104
212
271
76
103
104
252
261
76
103
104
212
258
4
76
103
104
159
217
252
12
62
76
103
104
159
76
103
104
212
268
271
76
87
103
104
212
271
76
103
104
212
245
271
76
103
104
134
141
212
271
76
103
104
212
236
243
271
2
Baeck Andre C.
Caldwell Robert M.
Collier Katherine D.
Kellis, Jr. James T.
Nadherny Joanne
Achutamurthy Ponnathapu
Genencor International Inc.
Ito Richard T.
Moore William W.
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