US5041377A
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Subtilisin crystallization process
Assignee: Danisco US Inc
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Filing: 1990-11-13
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Granted: 1991-08-20
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Expires: 2010-11-13
Abstract
Abstract Crystalline subtilisin is produced by adding a halide salt, such as sodium chloride or calcium chloride, to a concentrated subtilisin solution (at least about 40 g/1). This process does not produce amorphous subtilisin even at high salt concentrations in the solution. Optionally, subtilisin seed crystals also may be added to the concentrate to speed up the crystallization process.
Inventors
- 👤 Todd Becker
- 👤 Virgil B. Lawlis, Jr.
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 15:23
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/52; C12N9/54; Y; Y10; Y10S; Y10S435/00; Y10S435/803; Y10S435/814; Y10S435/816; Y10S435/8215; Y10S435/822; Y10S435/832; Y10S435/839
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
72 words
A process for the preparation of subtilisin crystals comprising: (i) preparing a subtilisin solution by removing cells and suspended solids from a fermentation mixture produced by fermentation of a subtilisin-producing bacterium; (ii) forming a concentrated solution by concentrating the subtilisin solution such that the subtilisin is present in a concentration of at least about 40 g/l; and (iii) adding a halide salt in an amount effective to cause formation of subtilisin crystals.
Claim 2
Dependent Claim
72 words
A process for the preparation of subtilisin crystals comprising: (i) preparing a subtilisin solution by removing cells and suspended solids from a fermentation mixture produced by fermentation of a subtilisin-producing bacterium; (ii) forming a concentrated solution by concentrating the subtilisin solution such that the subtilisin is present in a concentration of at least about 40 g/l; and (iii) adding a halide salt in an amount effective to cause formation of subtilisin crystals.
Claim 3
Dependent Claim
14 words
The process of claim 1 wherein halide salt is added to the fermentation mixture.
Claim 4
Dependent Claim
14 words
The process of claim 1 wherein halide salt is added to the subtilisin solution.
Claim 5
Dependent Claim
14 words
The process of claim 1 wherein halide salt is added to the concentrated solution.
Claim 6
Dependent Claim
25 words
The process of claim 1 wherein the concentrating step (ii) is carried out such that the concentration of subtilisin is at least about 80 g/l.
Claim 7
Dependent Claim
14 words
The process of claim 1 wherein the salt is sodium chloride or calcium chloride.
Claim 8
Dependent Claim
29 words
The process of claim 4 wherein the salt is sodium chloride and is added to form a solution having from about 400 to about 500 mM of sodium chloride.
Claim 9
Dependent Claim
29 words
The process of claim 4 wherein the salt is calcium chloride and is added to form a solution having from about 50 to about 200 mM of calcium chloride.
Claim 10
Dependent Claim
16 words
The process of claim 1 further comprising adding seed crystals of subtilisin to the concentrated solution.
Claim 11
Dependent Claim
24 words
The process of claim 1 wherein said step (iii) of forming subtilisin crystals is performed at a pH between about 5.2 and about 5.8.
Claim 12
Dependent Claim
11 words
The process of claim 1 wherein the bacterium is a bacillus.
Claim 13
Dependent Claim
11 words
The process of claim 10 wherein the bacillus is B. subtilis.
Claim 14
Dependent Claim
18 words
The process of claim 5 wherein the concentration of subtilisin is between about 80 and about 100 g/l.
Claim 15
Dependent Claim
11 words
The process of claim 1 wherein the salt is sodium iodide.
Claim 16
Dependent Claim
11 words
The process of claim 1 wherein the salt is potassium chloride.
Claim 17
Dependent Claim
11 words
The process of claim 1 wherein the salt is potassium iodide.
Claim 18
Dependent Claim
50 words
A process for the purification of subtilisin comprising forming a concentrated subtilisin solution having a subtilisin concentration of at leas about 40 g/l and adding a halide salt to a subtilisin solution wherein the amount of halide salt is an amount effective to cause crystallization of subtilisin from the solution.
Claim 19
Dependent Claim
50 words
A process for the purification of subtilisin comprising forming a concentrated subtilisin solution having a subtilisin concentration of at leas about 40 g/l and adding a halide salt to a subtilisin solution wherein the amount of halide salt is an amount effective to cause crystallization of subtilisin from the solution.
Claim 20
Dependent Claim
34 words
The process of claim 17 wherein the halide salt concentration in the solution is from about 0.01 to about 2 M, and the subtilisin concentration in the solution is at least about 40 g/l.
Claim 21
Dependent Claim
36 words
A composition comprising a subtilisin solution having a subtilisin concentration in the solution of at least about 40 g/l, containing a halide salt present in an amount effective to cause crystallization of subtilisin from the solution.
Claim 22
Dependent Claim
36 words
A composition comprising a subtilisin solution having a subtilisin concentration in the solution of at least about 40 g/l, containing a halide salt present in an amount effective to cause crystallization of subtilisin from the solution.
Claim 23
Dependent Claim
34 words
A composition according to claim 19 wherein the salt concentration in the solution is from about 0.01 to about 2 M and the subtilisin concentration in the solution is at least about 60 g/l.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1994-08-19 | 1995-02-18 | 1995-08-20 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1998-08-19 | 1999-02-18 | 1999-08-20 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2002-08-19 | 2003-02-18 | 2003-08-20 | $7700.00 | Window Closed / Lapsed |