US4914031A
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Subtilisin analogs
Assignee: Amgen Inc
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Filing: 1987-04-10
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Granted: 1990-04-03
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Expires: 2007-04-10
Abstract
Abstract A class of subtilisin analogs suitable for admixture to cleaning compositions and having improved stability over naturally occurring Bacillus subtilisins are prepared by expressing a modified gene encoding the subtilisin analog in Bacillus subtilis. The subtilisin analogs are characterized as having a modified calcium binding site to improve calcium binding and either an Asn or a Gly replaced in any Asn-Gly sequences present in the subtilisin.
Inventors
- 👤 Mark M. Zukowski
- 👤 Yitzhak Stabinsky
- 👤 Michael Levitt
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 23:38
CPC Classifications
C; C12; C12N; C12N15/00; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/52; C12N9/54; C12N15/09; C12N15/63; C12N15/74; C12N15/75; C12N5/00; C12P; C12P21/00
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
106 words
A subtilisin analog having an amino acid sequence of a naturally occurring Bacillus subtilisin that has been modified by having: (1) one or more of the amino acids present in the calcium binding site of the naturally occurring Bacillus subtilisin represented by Asp 41 , Leu 75 , Asn 76 , Asn 77 , Ser 78 , Ile 79 , Gly 80 , Val 81 , Thr 208 , and Tyr 214 replaced by a negatively charged amino acid; and (2) one or more of the amino acids comprising any Asn-Gly sequence of the naturally occurring Bacillus subtilisin deleted or replaced by a different amino acid.
Claim 2
Dependent Claim
106 words
A subtilisin analog having an amino acid sequence of a naturally occurring Bacillus subtilisin that has been modified by having: (1) one or more of the amino acids present in the calcium binding site of the naturally occurring Bacillus subtilisin represented by Asp 41 , Leu 75 , Asn 76 , Asn 77 , Ser 78 , Ile 79 , Gly 80 , Val 81 , Thr 208 , and Tyr 214 replaced by a negatively charged amino acid; and (2) one or more of the amino acids comprising any Asn-Gly sequence of the naturally occurring Bacillus subtilisin deleted or replaced by a different amino acid.
Claim 3
Dependent Claim
42 words
A subtilisin analog according to claim 1 wherein the analog is an analog of a naturally occurring Bacillus subtilisin selected from the group consisting of subtilisin Carlsberg, subtilisin DY, subtilisin BPN', an aprA subtilisin of Bacillus subtilis and subtilisin from Bacillus mesentericus.
Claim 4
Dependent Claim
17 words
A subtilisin analog according to claim 1 wherein the negatively charged amino acid is Asp or Glu.
Claim 5
Dependent Claim
15 words
A subtilisin analog according to claim 3 having Asn 76 replaced with Asp 76 .
Claim 6
Dependent Claim
15 words
A subtilisin analog according to claim 3 having Asn 77 replaced with Asp 77 .
Claim 7
Dependent Claim
15 words
A subtilisin analog according to claim 3 having Ile 79 replaced with Glu 79 .
Claim 8
Dependent Claim
22 words
A subtilisin analog according to claim 3 having Asn 76 replaced with Asp 76 and Asn 77 replaced with Asp 77 .
Claim 9
Dependent Claim
22 words
A subtilisin analog according to claim 3 having Asn 76 replaced with ASP 76 and Ile 79 replaced with Glu 79 .
Claim 10
Dependent Claim
25 words
A subtilisin analog according to claim 1 wherein an Asn residue in the Asn-Gly sequence is replaced by a residue of a different amino acid.
Claim 11
Dependent Claim
36 words
The analog as recited in claim 9 wherein an Asn residue in said Asn-Gly sequence is replaced by a residue of an amino acid from the group consisting of Ser, Val, Thr, Cys, Glu and Ile.
Claim 12
Dependent Claim
19 words
A subtilisin analog according to claim 10 wherein the Asn residue in the Asn-Gly sequence is replaced by Ser.
Claim 13
Dependent Claim
18 words
A subtilisin analog according to claim 11 wherein an Asn residue at position 109 is replaced by Ser.
Claim 14
Dependent Claim
18 words
A subtilisin analog according to claim 11 wherein an Asn residue at position 218 is replaced by Ser.
Claim 15
Dependent Claim
20 words
A subtilisin analog according to claim 11 wherein an Asn residue at positions 109 and 218 is replaced by Ser.
Claim 16
Dependent Claim
70 words
a subtilisin analog according to claim 14 selected from the group consisting of [Asp 76 , Ser 109 , Ser 218 ] subtilisin, [Asp 77 , Ser 109 , Ser 218 ] subtilisin, [Glu 79 , Ser 109 , Ser 218 ] subtilisin, [Asp 76 , Asp 77 , Ser 109 , Ser 218 ] subtilisin and [Asp 76 , Glu 79 , Ser 109 , Ser 218 ] subtilisin.
Claim 17
Dependent Claim
19 words
A subtilisin analog according to claim 1 wherein the Bacillus subtilisin has a naturally occurring amino acid sequence ##STR42##
Claim 18
Dependent Claim
17 words
A subtilisin analog according to claim 16, [Asp 76 , Ser 109 , Ser 218 ] subtilisin.
Claim 19
Dependent Claim
17 words
A subtilisin analog according to claim 16, [Asp 77 , Ser 109 , Ser 218 ] subtilisin.
Claim 20
Dependent Claim
17 words
A subtilisin analog according to claim 16, [Glu 79 , Ser 109 , Ser 218 ] subtilisin.
Claim 21
Dependent Claim
20 words
A subtilisin analog according to claim 16, [Asp 76 , Asp 77 , Ser 109 , Ser 218 ] subtilisin.
Claim 22
Dependent Claim
20 words
a subtilisin analog according to claim 16, [Asp 76 , Glu 79 , Ser 109 , Ser 218 ] subtilisin.
Claim 23
Dependent Claim
78 words
a subtilisin analog having an amino acid sequence of a naturally occurring Bacillus subtilisin that has been modified by having one or more of the amino acids present in the calcium binding site of the naturally occurring Bacillus subtilisin represented by Asp 41 , Leu 75 , Asn 76 , Asn 77 , Ser 78 , Ile 79 , Gly 80 , Val 81 , Thr 208 , and Tyr 214 replaced by a negatively charged amino acid.
Claim 24
Dependent Claim
78 words
a subtilisin analog having an amino acid sequence of a naturally occurring Bacillus subtilisin that has been modified by having one or more of the amino acids present in the calcium binding site of the naturally occurring Bacillus subtilisin represented by Asp 41 , Leu 75 , Asn 76 , Asn 77 , Ser 78 , Ile 79 , Gly 80 , Val 81 , Thr 208 , and Tyr 214 replaced by a negatively charged amino acid.
References Cited (Backward Citations)
US4760025A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1993-04-02 | 1993-10-02 | 1994-04-03 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-04-02 | 1997-10-02 | 1998-04-03 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-04-02 | 2001-10-02 | 2002-04-03 | $7700.00 | Window Closed / Lapsed |