Patent US5334520A successfully refreshed from Google Patents.
US5334520A
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Production of poly-beta-hydroxybutyrate in transformed escherichia coli
Assignee: James Madison University
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Filing: 1990-05-25
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Granted: 1994-08-02
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Expires: 2010-05-25
Abstract
Abstract Methods are provided for enhancing the production of PHB from a transformed E. coli host which includes the genes coding for the PHB biosynthetic pathway. By inserting the genes coding for PHB into a host which includes a lactose utilization system, a low cost minimal medium including whey can be used as the fuel and carbon source for PHB production. A plasmid which codes for the PHB biosynthetic pathway plus four hundred extra bases on either side of the first and last genes in the pathway has been inserted into the host and has been shown to produce a larger amount of PHB accumulation in a shorter period of time than other plasmid constructs. CaCl2 has been shown to be an effective agglomerating agent for agglomerating PHB which has been produced in a transformed E. coli host.
Inventors
- 👤 Douglas E. Dennis
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 09:23
CPC Classifications
C; C12; C12P; C12P7/00; C12P7/62; C12P7/625; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/52
Claims Analysis
Total Claims: 21
Claim 1
Independent Claim
51 words
An Escherichia coli bacterial host transformed by a vector consisting essentially of a deoxyribonucleic acid sequence encoding the poly-beta-hydroxybutyrate biosynthetic pathway of Alcaligenes eutrophus wherein the bacterial host has a lactose utilization system and grows in minimal medium containing whey and wherein the bacterial host is Escherichia coli strain ATCC 68329.
Claim 2
Dependent Claim
51 words
An Escherichia coli bacterial host transformed by a vector consisting essentially of a deoxyribonucleic acid sequence encoding the poly-beta-hydroxybutyrate biosynthetic pathway of Alcaligenes eutrophus wherein the bacterial host has a lactose utilization system and grows in minimal medium containing whey and wherein the bacterial host is Escherichia coli strain ATCC 68329.
Claim 3
Dependent Claim
60 words
A method for producing poly-beta-hydroxybutrate, comprising the steps of: providing a culture of the bacterial host of claim 1; growing said culture in a growth medium comprising minimal medium and whey for a period greater than twenty four hours, said bacterial host producing intra-cellular poly-beta-hydroxybutyrate; lysing said bacterial host in said culture to release said poly-betahydroxybutyrate; and collecting said poly-beta-hydroxybutyrate.
Claim 4
Dependent Claim
21 words
The method as recited in claim 2 in which said Escherichia coli bacterial host is grown in a growth medium comprising:
Claim 5
Dependent Claim
32 words
6% Na 2 HPO 4 ; 0.3% KH 2 PO 4 ; 0.1% ammonium chloride; 0.05% sodium chloride; 58.84% water; 0.012% magnesium sulfate; 0.0005% thiamine; 0.01% casamino acids; and, 40% whey solution.
Claim 6
Dependent Claim
32 words
6% Na 2 HPO 4 ; 0.3% KH 2 PO 4 ; 0.1% ammonium chloride; 0.05% sodium chloride; 58.84% water; 0.012% magnesium sulfate; 0.0005% thiamine; 0.01% casamino acids; and, 40% whey solution.
Claim 7
Dependent Claim
39 words
The method as recited in claim 2 wherein said minimal medium is approximately twenty percent of said growth medium, said whey is approximately forty percent of said growth medium, and water is approximately forty percent of said growth medium.
Claim 8
Dependent Claim
50 words
A method as recited in claim 2 wherein said step of collecting includes the step of exposing said released poly-beta-hydroxybutyrate to an ionic reagent selected from the group consisting of magnesium sulfate, magnesium chloride, magnesium acetate, and calcium chloride, said ionic reagent being of sufficient concentration to agglomerate said poly-beta-hydroxybutyrate.
Claim 9
Dependent Claim
24 words
A method as recited in claim 5 wherein said ionic reagent is calcium chloride at a concentration ranging between one molar and one millimolar.
Claim 10
Dependent Claim
18 words
A method as recited in claim 6 wherein said calcium chloride has a concentration of approximately ten millimolar.
Claim 11
Independent Claim
96 words
A method for agglomerating poly-beta-hydroxybutyrate which has been intracellularly produced in a culture of Escherichia coli (strain ATCC 68329) bacterial host which has been transformed by a vector the vector containing a deoxyribonucleic acid sequence coding for the poly-beta-hydroxybutyrate biosynthetic pathway from Alcaligenes eutrophus, comprising the steps of: lysing said bacterial hosts to release said poly-beta-hydroxybutyrate; and adding to said released poly-beta-hydroxybutyrate a sufficient quantity of an ionic reagent selected from the group consisting of magnesium sulfate, magnesium chloride, magnesium acetate and calcium chloride, said sufficient quantity of said ionic reagent agglomerating granules of said poly-beta-hydroxybutyrate.
Claim 12
Independent Claim
96 words
A method for agglomerating poly-beta-hydroxybutyrate which has been intracellularly produced in a culture of Escherichia coli (strain ATCC 68329) bacterial host which has been transformed by a vector the vector containing a deoxyribonucleic acid sequence coding for the poly-beta-hydroxybutyrate biosynthetic pathway from Alcaligenes eutrophus, comprising the steps of: lysing said bacterial hosts to release said poly-beta-hydroxybutyrate; and adding to said released poly-beta-hydroxybutyrate a sufficient quantity of an ionic reagent selected from the group consisting of magnesium sulfate, magnesium chloride, magnesium acetate and calcium chloride, said sufficient quantity of said ionic reagent agglomerating granules of said poly-beta-hydroxybutyrate.
Claim 13
Dependent Claim
14 words
A method as recited in claim 8 wherein said ionic reagent is calcium chloride.
Claim 14
Dependent Claim
23 words
A method as recited in claim 8 wherein said calcium chloride is present at a concentration ranging between one molar and one millimolar.
Claim 15
Dependent Claim
20 words
A method as recited in claim 10 wherein said calcium chloride is present at a concentration of approximately 10 mM.
Claim 16
Dependent Claim
23 words
A plasmid designated as p4A and deposited with the American Type Culture Collection in Escherichia coli strain HMS 174 under accession number 68329.
Claim 17
Dependent Claim
23 words
A plasmid designated as p4A and deposited with the American Type Culture Collection in Escherichia coli strain HMS 174 under accession number 68329.
Claim 18
Dependent Claim
22 words
The method recited in claim 8 further comprising the step of adding a nucleating agent to said quantity of said ionic reagent.
Claim 19
Dependent Claim
25 words
A method as recited in claim 10 wherein said calcium chloride is present at a concentration ranging from about 5 mM to about 30 mM.
Claim 20
Dependent Claim
23 words
A plasmid designated as pTZ-18U PHB and deposited with the American Type Culture Collection in Escherichia coli strain HMS174 under accession number 69064.
Claim 21
Dependent Claim
23 words
A plasmid designated as pTZ-18U PHB and deposited with the American Type Culture Collection in Escherichia coli strain HMS174 under accession number 69064.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1997-08-01 | 1998-01-31 | 1998-08-02 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2001-08-01 | 2002-01-31 | 2002-08-02 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2005-08-01 | 2006-01-31 | 2006-08-02 | $7700.00 | Window Closed / Lapsed |