WO1997035025A1 - Alcaligenes eutrophus strains which synthesize polymers of hydroxy alkanoic acids - Google Patents
Alcaligenes eutrophus strains which synthesize polymers of hydroxy alkanoic acids Download PDFInfo
- Publication number
- WO1997035025A1 WO1997035025A1 PCT/US1997/004441 US9704441W WO9735025A1 WO 1997035025 A1 WO1997035025 A1 WO 1997035025A1 US 9704441 W US9704441 W US 9704441W WO 9735025 A1 WO9735025 A1 WO 9735025A1
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- WO
- WIPO (PCT)
- Prior art keywords
- alcaligenes eutrophus
- ncimb
- strain
- mls
- alkanoic acids
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
- C12P7/625—Polyesters of hydroxy carboxylic acids
Definitions
- This invention relates to microorganisms and processes using them.
- PHA hydroxyalkanoic acids
- PVB/HV hydroxybutyric acid and hydroxy val eric acid
- suitable nutrients for example glucose and propionic acid (which acts as a source of HV units) as carbon sources.
- HB/HV copolymer (or in the substantial absence of a carbon nutrient metabolizable to HV monomer units by the organism, HB polymer) more rapidly than typical members of the species.
- the invention comprises strains of Alcaligenes eutrophus having the characteristics of that deposited with the National Collection of Industrial and Marine Bacteria Limited, 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland, UK on 27 July 1993 under Deposit No. NCIMB 40585 and variants, derivatives and mutants thereof.
- the characteristics of the organism are in accordance with the description in Bergy's Manual of Systematic
- the invention also comprises a process for producing a polymer of hydroxybutyric acid (for example a copolymer of hydroxybutyric acid and hydroxyvaleric acid) by cultivating a strain of Alcaligenes eutrophus according to the invention in a suitable medium. Cultivation of strain may be carried out by known means, for example as disclosed in European Patent 69,497.
- the polymer may be recovered by known methods, for example those disclosed in European Patent 145,233.
- NCIMB 40585 and an organism closely related to it, D9-10 were investigated as above with the following results.
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Novel Alcaligenes eutrophus strains are provided which synthesize PHA polymer more rapidly than typical members of the species. Further provided are methods of producing PHA polymer by cultivating said Alcaligenes eutrophus strains in a suitable medium.
Description
ALCALIGENES EUTROPHUS STRAINS WHICH SYNTHESIZE
POLYMERS OF HYDROXYALKANOIC ACIDS
This invention relates to microorganisms and processes using them.
It is known for example from our European Patent 69,497 to produce polymers of hydroxyalkanoic acids (PHA) for example polymers and copolymers of hydroxybutyric acid and hydroxy val eric acid (PHB/HV) by cultivating Alcaligenes eutrophus on a substrate containing suitable nutrients, for example glucose and propionic acid (which acts as a source of HV units) as carbon sources. It is preferred to conduct at least part of the process under limitation of at least one nutrient other than a carbon source, for example a nitrogen or phosphorus source, in order to maximize the ratio of polymer to non-polymer cellular material produced.
We have now identified a variant of Alcaligenes eutrophus which we have deposited with the National Collection of Industrial and Marine Bacteria Limited which synthesizes
HB/HV copolymer (or in the substantial absence of a carbon nutrient metabolizable to HV monomer units by the organism, HB polymer) more rapidly than typical members of the species.
The invention comprises strains of Alcaligenes eutrophus having the characteristics of that deposited with the National Collection of Industrial and Marine Bacteria Limited, 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland, UK on 27 July 1993 under Deposit No. NCIMB 40585 and variants, derivatives and mutants thereof. The characteristics of the organism are in accordance with the description in Bergy's Manual of Systematic
Bacteriology Eighth Edition, pages 273-275.
The invention also comprises a process for producing a polymer of hydroxybutyric acid (for example a copolymer of hydroxybutyric acid and hydroxyvaleric acid) by cultivating a strain of Alcaligenes eutrophus according to the invention in a suitable medium. Cultivation of strain may be carried out by known means, for example as disclosed in European Patent 69,497.
The polymer may be recovered by known methods, for example those disclosed in European Patent 145,233.
EXAMPLE
A 250 ml baffled flask containing 50 ml of a solution of composition:
0.5 M Phosphate buffer 20.0 mls/L
36% Ammonium sulphate 5.0 mls/L
40% Magnesium sulphate (.7 H2O) 0.5 mls/L
0.97% Ferric chloride 0.1 mls/L
* Trace elements (Fisons) 1.0 mls/L
and 1 ml of a 50% by weight glucose solution was sterilized by autoclaving at a temperature of 121°C for 20 minutes at approximately 2 bars absolute pressure. A freeze dried sample of the organism to be tested was suspended in sterile water and stood in an ampoule containing beads at -70°C. A bean was introduced aseptically to the above medium at room temperature and allowed to incubate for 8 hours at 30°C whilst shaking the flask.
Sufficient culture from the incubation was then added to a similarly sterilized baffled flask containing 50 ml of a solution of composition:
0.5 M Phosphate buffer 40.0 mls/L
36% Ammonium sulphate 10.0 mls/L
40% Magnesium sulphate (.7 H2O) 1.0 mls/L
0.97% Ferric chloride 0.2 mls/L
Trace elements (Fisons) 2.0 mls/L
and 0.4 ml of a 50% w/w solution of glucose to give an optional density at 600 μm between 0.10 and 0.2. The flasks were incubated for 5 days at 30°C with shaking thus reaching glucose starvation. This ensures that they are free from accumulated hydroxybutyrate polymer.
The cells were then recovered by centrifuging and resuspended in 1 ml of a medium of composition:
0.5 M Phosphate buffer 20.0 mls/L
40% Magnesium sulphate (.7 H2O) 0.5 mls/L
0.97% Ferric chloride 0.1 mls/L
Trace elements (Fisons) 1.0 mls/L
* Fisons Trace elements
Ca 720 ppm
Zn 22 ppm
Mn 25 ppm
Cu 5 ppm
Sufficient of the resuspension was added to another similarly sterilized 250 ml non baffled flask containing 50 ml of medium C containing glucose (1 ml 50% w/w solution) or sodium gluconate (1.5 ml of 33% w/w solution) to give an initial optical density at 436 nm of 0.5. The flask was then held at 30°C with shaking for at least 4 hours and the initial rate of polymer accumulation was estimated by the average rate of increase in optical density over the first 3 to 4 hours. NCIMB 40529, a strain of Alcaligenes eutrophus of high efficiency in the process,
NCIMB 40585 and an organism closely related to it, D9-10 were investigated as above with the following results.
The following data was obtained.
Substrate (1%)
Glucose Gluconate
Alcaligenes eutrophus NCIMB 40529 0.85 1.50
(average of 80 results)
Alcaligenes eutrophus D9-10 1.41 1.40
(average of 30 results)
Alcaligenes eutrophus NCIMB 40585 1.40 1.37
(average of 10 results)
The results show that Alcaligenes eutrophus NCIMB 40585 was very efficient in the conversion of glucose to polymer. The results with the related organism D9-10 were similar but NCIMB 40585 proved more stable in practical use and is considered more robust.
Claims
1. A strain of Alcaligenes eutrophus having the rapid PHA production characteristic of Alcaligenes eutrophus NCIMB 40585.
2. The Alcaligenes eutrophus strain of claim 1 , wherein said strain comprises NCIMB 40585.
3. The Alcaligenes eutrophus strain of ciaim 2, wherein said strain comprises a variant of NCIMB 40585.
4. The Alcaligenes eutrophus strain of claim 2, wherein said strain comprises a derivative of NCIMB 40585.
5. The Alcaligenes eutrophus strain of claim 2, wherein said strain comprises a mutant of NCIMB 40585.
6. A method of producing PHA polymer by cultivating an Alcaligenes eutrophus strain having the rapid PHA production characteristic of Alcaligenes eutrophus NCIMB 40585.
7. The method of claim 6, wherein said Alcaligenes eutrophus strain comprises NCIMB 40585.
8. The method of claim 6, wherein said Alcaligenes eutrophus strain comprises a variant of NCIMB 40585.
9. The method of claim 6, wherein said Alcaligenes eutrophus strain comprises a derivative of NCIMB 40585.
10. The method of claim 6, wherein said Alcaligenes eutrophus strain comprises a mutant of NCIMB 40585.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU23360/97A AU2336097A (en) | 1996-03-19 | 1997-03-19 | Alcaligenes eutrophus strains which synthesize polymers of hydroxy alkanoic acids |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9605704.7 | 1996-03-19 | ||
| GBGB9605704.7A GB9605704D0 (en) | 1996-03-19 | 1996-03-19 | Microorganisms and processes using them |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1997035025A1 true WO1997035025A1 (en) | 1997-09-25 |
Family
ID=10790615
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1997/004441 WO1997035025A1 (en) | 1996-03-19 | 1997-03-19 | Alcaligenes eutrophus strains which synthesize polymers of hydroxy alkanoic acids |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU2336097A (en) |
| GB (1) | GB9605704D0 (en) |
| WO (1) | WO1997035025A1 (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0052459A1 (en) * | 1980-11-18 | 1982-05-26 | Imperial Chemical Industries Plc | Beta-hydroxybutyrate polymers |
| EP0069497A2 (en) * | 1981-07-07 | 1983-01-12 | Imperial Chemical Industries Plc | Copolyesters and process for their production |
| WO1991000917A1 (en) * | 1989-07-10 | 1991-01-24 | Massachusetts Institute Of Technology | Method for producing novel polyester biopolymers |
| DE3937649A1 (en) * | 1989-11-11 | 1991-05-16 | Boehringer Ingelheim Kg | POLYESTER BASED ON 4-HYDROXYALKANSAURES AND METHOD FOR THE PRODUCTION THEREOF |
| WO1991018995A1 (en) * | 1990-05-25 | 1991-12-12 | Imperial Chemical Industries Plc | Hv/hb copolymer production |
-
1996
- 1996-03-19 GB GBGB9605704.7A patent/GB9605704D0/en active Pending
-
1997
- 1997-03-19 AU AU23360/97A patent/AU2336097A/en not_active Abandoned
- 1997-03-19 WO PCT/US1997/004441 patent/WO1997035025A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0052459A1 (en) * | 1980-11-18 | 1982-05-26 | Imperial Chemical Industries Plc | Beta-hydroxybutyrate polymers |
| EP0069497A2 (en) * | 1981-07-07 | 1983-01-12 | Imperial Chemical Industries Plc | Copolyesters and process for their production |
| WO1991000917A1 (en) * | 1989-07-10 | 1991-01-24 | Massachusetts Institute Of Technology | Method for producing novel polyester biopolymers |
| DE3937649A1 (en) * | 1989-11-11 | 1991-05-16 | Boehringer Ingelheim Kg | POLYESTER BASED ON 4-HYDROXYALKANSAURES AND METHOD FOR THE PRODUCTION THEREOF |
| WO1991018995A1 (en) * | 1990-05-25 | 1991-12-12 | Imperial Chemical Industries Plc | Hv/hb copolymer production |
Non-Patent Citations (3)
| Title |
|---|
| DOI, YOSHIHARU ET AL: "Production and characterization of unusual copolyesters by Alcaligenes eutrophus", POLYM. PREPR. (AM. CHEM. SOC., DIV. POLYM. CHEM.) (1988), 29(1), 588-9 CODEN: ACPPAY;ISSN: 0032-3934, 1988, XP000675013 * |
| EGGINK, GERRIT ET AL: "Oleic acid as a substrate for poly-3- hydroxyalkanoate formation in Alcaligenes eutrophus and Pseudomonas putida", IND. CROPS PROD. (1992), 1(2-4), 157-63 CODEN: ICRDEW;ISSN: 0926-6690, 1992, XP000674879 * |
| GOSTOMSKI P A ET AL: "Effect of glucose and NH-4+ levels on poly(beta-hydroxybutyrate) Production and growth in a continuous culture of Alcaligenes eutrophus.", BIOTECHNOLOGY PROGRESS 12 (2). 1996. 234-239. ISSN: 8756-7938, XP000675067 * |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2336097A (en) | 1997-10-10 |
| GB9605704D0 (en) | 1996-05-22 |
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