WO1997032031A1 - Stereospecific bioconversion of benzyl acetoacetate to benzyl-(s)-(+)-3-hydroxybutyrate - Google Patents
Stereospecific bioconversion of benzyl acetoacetate to benzyl-(s)-(+)-3-hydroxybutyrate Download PDFInfo
- Publication number
- WO1997032031A1 WO1997032031A1 PCT/US1997/002876 US9702876W WO9732031A1 WO 1997032031 A1 WO1997032031 A1 WO 1997032031A1 US 9702876 W US9702876 W US 9702876W WO 9732031 A1 WO9732031 A1 WO 9732031A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- benzyl
- hydroxybutyrate
- acetoacetate
- bioconversion
- medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
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
Definitions
- a bioconversion process employing Candida schatavii strain MY 1831 is presented.
- benzyl cetoacetate is converted to benzyl-(S)-(+)-3-hydroxybutyrate.
- a bioconversion process employing Candida schatavii strain MY 1831 is presented.
- benzyl acetoacetate is converted to benzyl-(S)-(+)-3-hydroxybutyrate.
- a bioconversion process employing Candida schatavii strain MY 1831 is presented.
- benzyl acetoacetate is converted to benzyl-(S)-(+)-3-hydroxybutyrate.
- the present invention is directed to a fermentation process which employs a readily prepared culture medium.
- Culture medium as used herein is defined as a mixture which supports the growth of yeast cells, which mixture contains ingredients such as peptone, soy peptone, and yeast extract powder. It should be understood that the precise amounts of ingredients provided above may be optimized, or modified so long as no new components are introduced.
- the key aspect of the medium is its ability to support growth of Candida schatavii and thereby the production of benzyl-(S)-(+)-3-hydroxybutyrate by bioreduction of benzyl acetoacetate.
- the product compounds may be produced by culturing (fermenting) the above-described microorganism in the presence of an appropriate amount of benzyl acetoacetate substrate in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen, preferably under submerged aerobic conditions (e.g. shaking culture, submerged culture, etc.).
- the aqueous medium is incubated at a temperature between 26°C and 29°C, preferably 28°C.
- the aqueous medium is incubated for a period of time necessary to complete the biotransformation as monitored by HPLC, usually for a period of about 24-48 hours, on a rotary shaker operating at about 220 rpm with a throw of about 2 in.
- Submerged aerobic cultural conditions may be preferred for the production of product compounds in massive amounts.
- a shaking or surface culture in a flask or bottle is employed.
- a vegetative inoculum of the organism by inoculating a relatively small quantity of culture medium with cells produced in a "slant” and culturing said inoculated medium, also called the “seed medium”, and then to transfer the cultured vegetative inoculum aseptically to large tanks.
- the fermentation medium, in which the inoculum is produced is substantially the same as or different from the medium utilized for the production of product compounds and is generally autoclaved to sterilize the medium prior to inoculation.
- Agitation and aeration of the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentation flask, by various pumping equipment or by the passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture.
- the fermentation is usually conducted at a temperature between 20°C and 35°C, for a period of about 24 hours to 48 hours, which may be varied according to fermentation conditions and scales.
- the production cultures are incubated for about 48 hours at 28°C on a rotary shaker operating at 220 rpm.
- Preferred culturing/production media for carrying out the fermentation include the Sabouraud Dextrose medium (30 g/L; Difco).
- the biotransformation product may be recovered from the culture medium by conventional means which are commonly used for the recovery of other known biologically active substances.
- the product compounds are found filtrate, which are obtained by filtering or centrifuging the cultured broth, and accordingly can be isolated and purified by a conventional method such as concentration under reduced pressure, lyophilization, extraction with a conventional solvent, such as ethylacetate and the like, pH adjustment, treatment with a conventional resin (e.g. anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g. activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), crystallization, recrystallization, and the like.
- a preferred recovery method is solvent extraction, particularly using ethylacetate.
- the compounds of the present invention have the following structures.
- Compound (I) benzyl acetoacetate is the starting material.
- Compound (II) is the product, benzyl-(S)-(+)-3-hydroxybutyrate.
- the substrate, benzyl acetoacetate may be prepared via a synthetic process or may be purchased commercially.
- a chemical reference for desired product was prepared as follows. Methyl (S)-(+)-3-hydroxybutyrate (Aldrich) was dissolved in benzyl alcohol and treated with a catalytic amount of titanium tetraisopropoxide. The mixture was aged at 60 Q C for 24 hours. Partial concentration on a rotary evaporator and filtration afforded benzyl (S)- (+)-3-hydroxybutyrate and residual benzyl alcohol.
- Methyl (S)-(+)-3-hydroxybutyrate Aldrich
- the enantiomers were separated using a Chiralcel OD column (Chiral Technologies), employing a mobile phase comprised of a mixture of 98 % hexane and 2 % isopropanol at a flow rate of 0.6 ml/min.
- Yeast cells were preserved on slants of Sabouraud dextrose agar (Difco) kept at 4°C. Cells were obtained from the slants using a sterile loop and used to inoculate a 250-ml Erlenmeyer flask containing 50-ml of Sabouraud dextrose broth. The cultures were incubated aerobically at 28 e C for 48 hours on a shaker operated at 220 rpm. A volume of 2.5 ml of this 48 h-old culture was used to inoculate a 250-ml Erlenmeyer flask containing 50 ml of Sabouraud dextrose medium. The cultures were incubated in the same conditions as described above for 24 hours.
- the substrate for bioconversion (benzyl acetoacetate) was added to each flask in 1 ml of ethanol to give a final concentration of 1 g/1.
- the cultures were incubated for 24 hours under the same conditions.
Landscapes
- 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)
Abstract
A bioconversion process where benzyl acetoacetate is converted to benzyl-(S)-(+)-3-hydroxybutyrate.
Description
TITLE OF THE INVENTION
STEREOSPECIFIC BIOCONVERSION OF BENZYL
ACETOACETATE TO BENZYL-(S)-(+)-3-HYDROXYBUTYRATE
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
STATEMENT REGARDING FEDERALLY-SPONSORED R&D Not applicable.
REFERENCE TO MICROFICHE APPENDD
Not applicable.
FIELD OF THE INVENTION
Fermantation and biotransformation.
BACKGROUND OF THE INVENTION
A bioconversion process employing Candida schatavii strain MY 1831 is presented. In this bioconversion process benzyl cetoacetate is converted to benzyl-(S)-(+)-3-hydroxybutyrate.
SUMMARY OF THE INVENTION
A bioconversion process employing Candida schatavii strain MY 1831 is presented. In this bioconversion process benzyl acetoacetate is converted to benzyl-(S)-(+)-3-hydroxybutyrate.
BRIEF DESCRIPTION OF THE DRAWINGS Not applicable.
DETAILED DESCRIPTION OF THE INVENTION
A bioconversion process employing Candida schatavii strain MY 1831 is presented. In this bioconversion process benzyl acetoacetate is converted to benzyl-(S)-(+)-3-hydroxybutyrate.
The present invention is directed to a fermentation process which employs a readily prepared culture medium. Culture medium as used herein is defined as a mixture which supports the growth of yeast cells, which mixture contains ingredients such as peptone, soy peptone, and yeast extract powder. It should be understood that the precise amounts of ingredients provided above may be optimized, or modified so long as no new components are introduced. The key aspect of the medium is its ability to support growth of Candida schatavii and thereby the production of benzyl-(S)-(+)-3-hydroxybutyrate by bioreduction of benzyl acetoacetate.
In general, the product compounds may be produced by culturing (fermenting) the above-described microorganism in the presence of an appropriate amount of benzyl acetoacetate substrate in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen, preferably under submerged aerobic conditions (e.g. shaking culture, submerged culture, etc.). The aqueous medium is incubated at a temperature between 26°C and 29°C, preferably 28°C. The aqueous medium is incubated for a period of time necessary to complete the biotransformation as monitored by HPLC, usually for a period of about 24-48 hours, on a rotary shaker operating at about 220 rpm with a throw of about 2 in.
Submerged aerobic cultural conditions may be preferred for the production of product compounds in massive amounts. For the production in small amounts, a shaking or surface culture in a flask or bottle is employed.
Accordingly, it is desirable first to produce a vegetative inoculum of the organism by inoculating a relatively small quantity of culture medium with cells produced in a "slant" and culturing said inoculated medium, also called the "seed medium", and then to transfer the cultured vegetative inoculum aseptically to large tanks. The fermentation medium, in which the inoculum is produced, is substantially the same as or different from the medium utilized for the
production of product compounds and is generally autoclaved to sterilize the medium prior to inoculation.
Agitation and aeration of the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentation flask, by various pumping equipment or by the passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture.
The fermentation is usually conducted at a temperature between 20°C and 35°C, for a period of about 24 hours to 48 hours, which may be varied according to fermentation conditions and scales. Preferably, the production cultures are incubated for about 48 hours at 28°C on a rotary shaker operating at 220 rpm.
Preferred culturing/production media for carrying out the fermentation include the Sabouraud Dextrose medium (30 g/L; Difco).
The biotransformation product may be recovered from the culture medium by conventional means which are commonly used for the recovery of other known biologically active substances. The product compounds are found filtrate, which are obtained by filtering or centrifuging the cultured broth, and accordingly can be isolated and purified by a conventional method such as concentration under reduced pressure, lyophilization, extraction with a conventional solvent, such as ethylacetate and the like, pH adjustment, treatment with a conventional resin (e.g. anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g. activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), crystallization, recrystallization, and the like. A preferred recovery method is solvent extraction, particularly using ethylacetate.
The compounds of the present invention have the following structures. Compound (I), benzyl acetoacetate is the starting material. Compound (II) is the product, benzyl-(S)-(+)-3-hydroxybutyrate.
π
A sample of Candida schatavii was deposited under the Budapest Treaty in the American Type Culture Collection, 12301
Parklawn Drive, Rockville, MD 20852, USA on and has been assigned accession number .
EXAMPLE 1 Preparation of substrate
The substrate, benzyl acetoacetate may be prepared via a synthetic process or may be purchased commercially.
EXAMPLE 2 Preparation of benzyl-(SV(+V3-hydroxybutyrate
A chemical reference for desired product was prepared as follows. Methyl (S)-(+)-3-hydroxybutyrate (Aldrich) was dissolved in benzyl alcohol and treated with a catalytic amount of titanium tetraisopropoxide. The mixture was aged at 60QC for 24 hours. Partial concentration on a rotary evaporator and filtration afforded benzyl (S)- (+)-3-hydroxybutyrate and residual benzyl alcohol.
EXAMPLE 3 Analytical procedures
An amount of 5 ul of the extract resuspended in methylene chloride was spoted on a TLC plate (Kiesel precoated silica gel 60, F254), and was developed in a chromatography chamber using a mixture of hexane/ethyl acetate (5/1 ). Starting materials (benzyl acetoacete) and product (benzyl-(S)-(+)-3-hydroxybutyrate) were observed under ultraviolet light. An authentic racemic sample of benzyl-3-hydroxybutyrate was used for reference purposes. Chirality of the produced benzyl-(S)-(+)-3-hydroxybutyrate was determined by high pressure liquid chromatography (HPLC). The enantiomers were separated using a Chiralcel OD column (Chiral Technologies), employing a mobile phase comprised of a mixture of 98 % hexane and 2 % isopropanol at a flow rate of 0.6 ml/min.
EXAMPLE 4 Shake flask fermentations
Yeast cells were preserved on slants of Sabouraud dextrose agar (Difco) kept at 4°C. Cells were obtained from the slants using a sterile loop and used to inoculate a 250-ml Erlenmeyer flask containing 50-ml of Sabouraud dextrose broth. The cultures were incubated aerobically at 28 eC for 48 hours on a shaker operated at 220 rpm. A volume of 2.5 ml of this 48 h-old culture was used to inoculate a 250-ml Erlenmeyer flask containing 50 ml of Sabouraud dextrose medium. The cultures were incubated in the same conditions as described above for 24 hours. After 24 hours of incubation, the substrate for bioconversion (benzyl acetoacetate) was added to each flask in 1 ml of ethanol to give a final concentration of 1 g/1. The cultures were incubated for 24 hours under the same conditions.
After that 24-hour incubation, the whole content of the flasks were harvested. A volume of 50 ml of ethyl acetate was added to each broth, and the mixtures were shaken for 10 minutes, followed by a 10 minute centrifugation at 2500 rpm in a bench top centrifuge. The
ethyl acetate layer was separated from the water layer and taken to dryness. After TLC and HPLC analyses, Candida schatavii strain MY 1831 was found to produce benzyl-(S)-(+)-3-hydroxybutyrate with an enantiomeric excess of 93 %.
EXAMPLE 5 NMR analyses
Identification of the benzyl-(S)-(+)-3-hydroxybutyrate was done by proton NMR. Proton NMR spectra were recorded with CD3OD (49.1 ppm) as internal standard. The resonance of the benzyl- (S)-(+)-3-hydroxybutyrate are: 1.24 ppm (3H, d), 2.52 ppm (2H,m), 4.23 ppm (lH,m), 5.18 ppm (2H,s), 7.37 ppm (5H,s).
Claims
1. A method of converting benzyl acetoacetate is converted to benzyl-(S)-(+)-3-hydroxybutyrate comprising:
(a) cultivating a microorganism in a medium containing benzyl acetoacetate; and
(b) recovering the benzyl-(S)-(+)-3-hydroxybutyrate.
2. The method of claim 1 wherein the microorganism is Candida schatavii strain MY 1831 ATCC ).
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US1269296P | 1996-03-01 | 1996-03-01 | |
| US60/012,692 | 1996-03-01 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1997032031A1 true WO1997032031A1 (en) | 1997-09-04 |
Family
ID=21756238
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1997/002876 Ceased WO1997032031A1 (en) | 1996-03-01 | 1997-02-25 | Stereospecific bioconversion of benzyl acetoacetate to benzyl-(s)-(+)-3-hydroxybutyrate |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO1997032031A1 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4933282A (en) * | 1985-11-28 | 1990-06-12 | Nippon Gohsei Kagaku Kogyo Kabushiki Kaisha | Process for preparing an optically active γ-halo-β-hydroxybutyric acid ester |
-
1997
- 1997-02-25 WO PCT/US1997/002876 patent/WO1997032031A1/en not_active Ceased
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4933282A (en) * | 1985-11-28 | 1990-06-12 | Nippon Gohsei Kagaku Kogyo Kabushiki Kaisha | Process for preparing an optically active γ-halo-β-hydroxybutyric acid ester |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5407826A (en) | Isolated cultures of microorganisms of Clonostachys Cylindrospora, Gliocladium and Nectria Gliocladioides | |
| US5766894A (en) | Production of vitamin B6 by fermentation | |
| CA2116003C (en) | Arylalkanoic acid resolution | |
| US5958743A (en) | Stereospecific bioconversion of benzyl acetoacetate to benzyl-(s)-(+)-hydroxybutyrate | |
| US7141412B2 (en) | Process for preparing optically active 3-hydroxypyrrolidine derivatives by enzymatic hydroxylation | |
| CA1109406A (en) | Production of monorden | |
| WO1997032031A1 (en) | Stereospecific bioconversion of benzyl acetoacetate to benzyl-(s)-(+)-3-hydroxybutyrate | |
| EP0434393A2 (en) | Process for preparation of R-(-)-3-halogeno-1,2-propanediol by treatment with microorganism | |
| US6451587B1 (en) | Microbial asymmetric reduction of 2-chloro-1-[-6-(2,5-dimethyl-pyrrol-1-yl)-pyridin-3-yl]-ethanone | |
| JP3023179B2 (en) | Stereoselective microbial reduction process | |
| US5232853A (en) | Method for producing (2r,3s)-3-hydroxy-2-methylbutyrate by microbial reduction | |
| Chartrain et al. | Asymmetric bioreduction of benzyl acetoacetate to its corresponding alcohol, benzyl (S)-(+)-3-hydroxybutyrate by the yeast Candida schatavii MY 1831 | |
| EP0481712B1 (en) | Process for production of optically active methylsuccinic acid | |
| JP2693536B2 (en) | Process for producing (7R) -cyclopenta [d] pyrimidine derivative | |
| EP1325146B1 (en) | Process for preparing optically active 4-hydroxy-2-pyrrolidinone and n-substituted 4-hydroxy-2-pyrrolidinones by enzymatic hydroxylation | |
| AU620595B2 (en) | Process for the preparation of macrolide compounds | |
| HK1038768A1 (en) | Microbial process for preparation of optically active 3-hydroxypyrrolidine derivatives | |
| JPH0592967A (en) | Novel natural substance with 10-membered ring lactone structure | |
| Matzinger et al. | Asymmetric microbial reduction of ethyl and isoprophyl α, 1, 3-trioxo-2-isoindolinebutyrate | |
| JPH04166093A (en) | Production of optically active benzhydrol derivative using microorganism | |
| JPH0539B2 (en) | ||
| JPH01199582A (en) | Novel antibiotic fo-125a4, a5 or b and production thereof | |
| JP2003093046A (en) | Useful microorganism for transformation | |
| JP2003125793A (en) | Method for producing optically active 1,2-trans-cyclohexanediol | |
| HK1010740B (en) | Microbial process for the production of trans-4-hydroxyl-l-proline |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AK | Designated states |
Kind code of ref document: A1 Designated state(s): CA JP US |
|
| AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
| DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
| NENP | Non-entry into the national phase |
Ref country code: JP Ref document number: 97531054 Format of ref document f/p: F |
|
| 122 | Ep: pct application non-entry in european phase | ||
| NENP | Non-entry into the national phase |
Ref country code: CA |