US4493893A
Expired / Lapsed
US
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Process for cloning the gene coding for a thermostable alpha-amylase into Escherichia coli and Bacillus subtilis
Assignee: Genencor International Wisconsin Inc
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Filing: 1983-03-11
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Granted: 1985-01-15
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Expires: 2003-03-11
Abstract
Abstract An improved process for producing a thermostable alpha-amylase enzyme is described. The gene coding for the alpha-amylase is incorporated into a chimeric plasmid which is produced in multiple copies by a host microorganism.
Inventors
- 👤 Jonathan R. Mielenz
- 👤 Susan Mickel
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-25 18:16
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/24; C12N9/2402; C12N9/2405; C12N9/2408; C12N9/2411; C12N9/2414; C12N9/2417; C12N15/00
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
72 words
A process for constructing a chimeric plasmid having a gene coding for a thermostable alpha-amylase which comprises: (a) cutting naturally-occurring plasmid DNA present in a strain of B. stearothermophilus having a gene coding for a thermostable alpha-amylase to obtain a linear DNA sequence containing the alpha-amylase coding gene; (b) cutting a suitable vector to obtain a second linear DNA sequence; and (c) joining the linear DNA sequences of Steps (a) and (b).
Claim 2
Dependent Claim
72 words
A process for constructing a chimeric plasmid having a gene coding for a thermostable alpha-amylase which comprises: (a) cutting naturally-occurring plasmid DNA present in a strain of B. stearothermophilus having a gene coding for a thermostable alpha-amylase to obtain a linear DNA sequence containing the alpha-amylase coding gene; (b) cutting a suitable vector to obtain a second linear DNA sequence; and (c) joining the linear DNA sequences of Steps (a) and (b).
Claim 3
Dependent Claim
29 words
The process of claim 1 wherein the vector is a plasmid selected from the group consisting of E. coli plasmids pBR322, pBR325 and pWL625; and B. subtilis plasmid pC194.
Claim 4
Dependent Claim
29 words
The process of claim 1 wherein the strain of B. stearothermophilus is selected from the group consisting of B. stearothermophilus, ATCC Nos. 31,195; 31,196; 31,197; 31,198; 31,199; and 31,783.
Claim 5
Dependent Claim
16 words
The process of claim 1 wherein the cutting is accomplished by means of a restriction endonuclease.
Claim 6
Dependent Claim
13 words
The process of claim 4 wherein the endonuclease is the enzyme Hind III.
Claim 7
Dependent Claim
17 words
The process of claim 1 wherein the linear DNA sequences are joined by means of a ligase.
Claim 8
Dependent Claim
13 words
The process of claim 6 wherein the ligase is T 4 DNA ligase.
Claim 9
Dependent Claim
64 words
A process for constructing a chimeric plasmid having a gene coding for a thermostable alpha-amylase which comprises: (a) cutting a chimeric plasmid, prepared by the method of claim 1, to obtain a linear DNA sequence containing the alpha-amylase coding gene; (b) cutting a suitable vector to obtain a second linear DNA sequence; and (c) joining the linear DNA sequences of Steps (a) and (b).
Claim 10
Dependent Claim
29 words
The process of claim 8 wherein the vector is a plasmid selected from the group consisting of E. coli plasmids pBR322, pBR325 and pWL625; and B. subtilis plasmid pC194.
Claim 11
Dependent Claim
29 words
The process of claim 8 wherein the strain of B. stearothermophilus is selected from the group consisting of B. stearothermophilus, ATCC Nos. 31,195; 31,196; 31,197; 31,198; 31,199; and 31,783.
Claim 12
Dependent Claim
16 words
The process of claim 8 wherein the cutting is accomplished by means of a restriction endonuclease.
Claim 13
Dependent Claim
13 words
The process of claim 11 wherein the endonuclease is the enzyme Hind III.
Claim 14
Dependent Claim
17 words
The process of claim 8 wherein the linear DNA sequences are joined by means of a ligase.
Claim 15
Dependent Claim
13 words
The process of claim 13 wherein the ligase is T 4 DNA ligase.
Claim 16
Dependent Claim
56 words
A chimeric plasmid having a gene coding for a thermostable alpha-amylase having the same thermostable characteristics as those of the alpha-amylase produced by B. stearothermophilus ATCC #31,783, which plasmid comprises a DNA sequence containing the alpha-amylase gene cut from naturally-occurring plasmid DNA present in a strain of B. stearothermophilus and the DNA of a suitable vector.
Claim 17
Dependent Claim
56 words
A chimeric plasmid having a gene coding for a thermostable alpha-amylase having the same thermostable characteristics as those of the alpha-amylase produced by B. stearothermophilus ATCC #31,783, which plasmid comprises a DNA sequence containing the alpha-amylase gene cut from naturally-occurring plasmid DNA present in a strain of B. stearothermophilus and the DNA of a suitable vector.
Claim 18
Dependent Claim
30 words
The chimeric plasmid of claim 15 wherein the strain of B. stearothermophilus is selected from the group consisting of B. stearothermophilus, ATCC Nos. 31,195; 31,196; 31,197; 31,198; 31,199; and 31,783.
Claim 19
Dependent Claim
30 words
The chimeric plasmid of claim 15 wherein the vector is a plasmid selected from the group consisting of E. coli plasmids pBR322, pBR325 and pWL625; and B. subtilis plasmid pC194.
Claim 20
Dependent Claim
11 words
A microorganism comprising at least one chimeric plasmid of claim 15.
Claim 21
Dependent Claim
11 words
The microorganism of claim 18 wherein the microorganism is a bacterium.
Claim 22
Dependent Claim
11 words
The microorganism of claim 19 wherein the bacterium is E. coli.
Claim 23
Dependent Claim
11 words
The microorganism of claim 19 wherein the bacterium is B. subtilis.
Claim 24
Dependent Claim
48 words
A process for preparing a thermostable alpha-amylase which comprises the steps of: (a) introducing at least one chimeric plasmid of claim 15 into a host microorganism; (b) culturing the microorganism containing the chimeric plasmid in a suitable medium; and (c) isolating the alpha-amylase produced by the cultured microorganism.
References Cited (Backward Citations)
US4237224A
Cited
EP0034470A2
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1988-01-15 | 1988-07-16 | 1989-01-15 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1992-01-15 | 1992-07-16 | 1993-01-15 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1996-01-15 | 1996-07-16 | 1997-01-15 | $7700.00 | Window Closed / Lapsed |