Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-11-02
2004-11-09
Nashed, Nashaat (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S221000, C435S222000, C435S069100, C435S252300, C435S320100, C435S471000, C510S392000, C510S400000, C536S023200
Reexamination Certificate
active
06815193
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 protease variants containing a substitution of an amino acid at a residue position corresponding to position 103 of
Bacillus amyloliquefaciens
subtilisin and substituting one or more amino acids at residue positions selected from the group consisting of residue positions corresponding to positions 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177, 181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 and 275 of
Bacillus amyloliquefaciens
subtilisin; wherein when a substitution at a position corresponding to residue position 103 is combined with a substitution at a position corresponding to residue position 76, there is also a substitution at one or more residue positions other than residue positions corresponding to positions 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265, or 274 of
Bacillus amyloliquefaciens
subtilisin.
While any combination of the above listed amino acid substitutions may be employed, the preferred protease variant enzymes useful for the present invention comprise the substitution of amino acid residues in the following combinations of positions. 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 103 and at one or more of the following positions 236 and 245;
(2) a protease variant including substitutions of the amino acid residues at positions 103 and 236 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 248, 252, 257, 260, 270 and 275;
(3) a protease variant including substitutions of the amino acid residues at positions 103 and 245 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 170, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 222, 230, 232, 248, 252, 257, 260, 261, 270 and 275; or
(4) a protease variant including substitutions of the amino acid residues at positions 103, 236 and 245 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 243, 248, 252, 257, 260, 270 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
222
76
98
103
104
76
78
103
104
76
103
104
107
4
76
103
104
76
103
104
246
76
77
103
104
76
103
104
183
218
16
76
103
104
248
1
76
103
104
76
103
104
261
76
103
104
160
76
103
104
216
17
76
103
104
37
76
103
104
76
77
103
104
174
38
76
103
104
38
76
103
104
237
8
76
103
104
76
103
104
183
19
76
103
104
13
76
103
104
19
76
103
104
76
103
104
184
76
103
104
252
76
103
104
259
76
103
104
251
76
86
103
104
72
76
103
104
185
76
103
104
237
274
76
103
104
160
76
103
104
228
55
76
103
104
240
76
103
104
254
76
103
104
204
76
103
104
204
43
76
103
104
76
103
104
159
10
76
103
104
177
58
76
103
104
76
103
104
270
76
103
104
185
27
76
103
104
76
103
104
262
76
78
103
104
24
76
103
104
76
103
104
166
236
251
17
76
103
104
237
76
103
104
130
76
103
104
109
76
99
103
104
204
76
103
104
181
12
76
103
104
76
103
104
212
271
76
103
104
252
261
76
103
104
242
76
103
104
271
12
76
103
104
242
43
76
103
104
116
183
76
103
104
258
76
103
104
271
61
76
103
104
38
76
103
104
182
263
76
103
104
182
272
76
103
104
109
246
76
87
103
104
206
249
265
76
103
104
137
238
271
103
104
228
76
103
104
182
198
21
76
103
104
182
76
103
104
119
137
76
103
104
137
248
13
76
103
104
206
76
103
104
206
76
103
104
212
258
58
76
103
104
271
76
103
104
206
261
4
76
103
104
206
76
77
103
104
206
76
103
104
158
76
103
104
206
4
76
103
104
159
217
251
4
76
103
104
159
217
252
76
77
103
104
133
185
251
76
103
104
159
206
244
4
76
103
104
188
4
76
103
104
158
76
77
103
104
185
76
103
104
206
251
48
76
103
104
111
159
68
76
103
104
159
236
42
76
103
104
159
12
62
76
103
104
159
42
76
103
104
159
76
103
104
146
159
76
103
104
159
238
76
103
104
159
224
76
103
104
212
268
271
76
89
103
104
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
76
103
104
109
245
76
103
104
109
210
20
62
76
103
104
68
76
103
104
236
68
76
103
104
159
236
271
68
76
103
104
159
236
245
68
76
103
104
159
217
236
271
17
68
76
103
104
68
76
103
104
68
76
103
104
159
236
68
75
76
103
104
159
236
68
76
76
103
114
121
159
236
245
12
68
76
103
104
159
236
68
76
103
104
159
209
236
253
68
76
103
104
117
159
184
236
68
76
103
104
159
236
243
68
76
103
104
159
236
245
68
76
103
104
142
159
68
76
103
104
123
159
236
249
68
76
103
104
159
236
249
76
103
104
222
245
12
76
103
104
222
249
76
103
104
173
222
76
103
104
222
263
21
76
103
104
222
237
263
76
103
104
109
222
76
103
104
109
222
271
61
76
103
104
222
76
103
104
137
222
76
103
104
109
222
248
76
103
104
222
249
68
76
103
104
159
236
245
261
68
76
103
104
141
159
236
245
255
68
76
103
104
159
236
245
247
68
76
10
Baeck Andre C.
Caldwell Robert M.
Collier Katherine D.
Kellis, Jr. James T.
Nadherny Joanne
Genencor International Inc.
Genencor International Inc.
Moore William W.
Nashed Nashaat
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