Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-10-23
2001-11-06
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S220000, C435S221000, C435S222000, C435S091500, C435S320100, C536S023200, C424S094640
Reexamination Certificate
active
06312936
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:
76
103
104
212
245
271
68
76
103
104
159
236
76
103
104
212
236
243
271
76
103
104
109
245
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
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
123
159
236
249
68
76
103
104
159
236
249
76
103
104
222
245
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
103
104
159
174
204
236
245
68
76
103
104
159
204
236
245
68
76
103
104
133
159
218
236
245
68
76
103
104
159
232
236
245
68
76
103
104
159
194
203
236
245
12
76
103
104
222
245
76
103
104
232
245
24
68
76
103
104
159
232
236
245
68
103
104
159
232
236
245
252
68
76
103
104
159
213
232
236
245
260
12
76
103
104
222
244
245
12
76
103
222
210
245
12
76
103
104
130
222
245
68
103
104
159
232
236
245
248
252
68
103
104
159
232
236
245
68
103
104
140
159
232
236
245
252
43
68
103
104
159
232
236
245
252
43
68
103
104
159
232
236
245
43
68
103
104
159
232
236
245
252
68
87
103
104
159
232
236
245
252
275
12
76
103
104
130
222
245
248
262
12
76
103
104
130
215
222
245
12
76
103
104
130
222
227
245
262
12
76
103
104
130
222
245
261
76
103
104
130
222
245
12
76
103
104
130
218
222
245
262
269
12
57
76
103
104
130
222
245
251
12
76
103
104
130
170
185
222
243
245
12
76
103
104
130
222
245
268
12
76
103
104
130
222
210
245
68
103
104
159
232
236
245
257
68
103
104
116
159
232
236
245
68
103
104
159
232
236
245
248
68
68
103
104
159
232
236
245
68
103
104
159
203
232
236
245
68
103
104
159
232
236
237
245
68
76
79
103
104
159
232
236
245
68
103
104
159
183
232
236
245
68
103
104
159
174
206
232
236
245
68
103
104
159
188
232
236
245
68
103
104
159
230
232
236
245
68
98
103
104
159
232
236
245
68
103
104
159
215
232
236
245
68
103
104
159
232
236
245
248
68
76
103
104
159
232
236
245
68
76
103
104
159
210
232
236
245
68
76
103
104
159
232
236
245
257
76
103
104
232
236
245
257
68
103
104
159
232
236
245
257
275
68
103
104
159
224
232
236
245
257
76
103
104
159
232
236
245
257
68
76
103
104
159
209
232
236
245
68
76
103
104
159
211
232
236
245
12
68
76
103
104
159
214
232
236
245
68
76
103
104
159
215
232
236
245
12
68
76
103
104
159
232
236
245
20
68
76
103
104
159
232
236
245
259
68
87
76
103
104
159
232
236
245
260
68
76
103
104
159
232
236
245
261
76
103
104
232
236
242
245
68
76
103
104
159
210
232
236
245
12
48
68
76
103
104
159
232
236
245
76
103
104
232
236
245
76
103
104
159
192
232
236
2
Baeck Andre C.
Caldwell Robert M.
Collier Katherine D.
Kellis, Jr. James T.
Nadherny Joanne
Achutamurthy Ponnathapu
Genencor International Inc.
Genencor Intl. Inc.
Moore William W.
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