Title of Invention

PRCESS FOR PREPARING ENANTIOMERICALLY ENRICHED (S)-ETHYL 4-CHLORO-3-HYDROXY BUTYRATE"

Abstract

A process for the production of enahtiomerically enriched (5)-ethyl 4-chloro-3-hydroxybutyrate of formula 1, which is useful as intermediate for preparing medicaments and physiologically active compounds, such as hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors, commonly referred to as statins, which comprises treating p-ketoester compound, ethyl 4-chloro-3-oxobutyrate of formula 2 with a microbial strain of Penicillium funiculosum MTCC 5246 or Aspergillus ochraceous MTCC 5245, whereby P-ketoester compound is asymmetrically reduced to ethyl 4-chloro-3-hydroxybutyrate, enriched in s-enantiomer.

Full Text

Field of invention; This invention relates to a process for the production of enantiomerically enriched (S)-ethyl 4- chloro-3-hydroxybutyrate of formula 1 from corresponding p-ketoester compound, ethyl 4-chloro-3-oxobutyrate of formula 2 by a biological asymmetric reduction using a microbial strain of Penicillium funiculosum MTCC 5246 or Aspergillus ochraceous MTCC 5245. (5)-ethyl 4-chloro-3-hydroxybutyrate is useful as chiral building block and an intermediate for the production of optically active compounds having various utilities, such as medicaments, physiologically active substances, etc. More specifically, said (S)-ethyl 4-chloro-3-hydroxybutyrate is used as an intermediate in the production of hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors, commonly referred to as statin drugs. Background and prior art of the invention; Enantiomerically enriched compounds have usually been prepared by chemical synthesis in which an optically active starting compound is converted into the desired compound or by optical resolution in which a racemic compound is treated with an optically active resolving agent or by asymmetric synthesis involving optically active reagents. But, recently microorganisms have been used for the preparation of enantiomerically enriched compounds, comprising either asymmetric reduction of a prochiral compound or resolution of a racemic compound. For example, resolution of racemic ethyl 4-chloro-3-hydroxybutyrate with a microorganism, which specifically converted one of the optical isomers into 1, 2-diol compound leaving behind optically active (S)-ethyl 4-chloro-3-hydroxybutyrate has been reported (US 5776766, 1998). Ability of the microorganisms for asymmetric reduction of carbonyl compounds has been well known. There have been a number of reports on the production of optically active (5)-ethyl 4- chloro-3-hydroxybutyrate, based on such a microbial process. Various methods have been reported based on the use of a culture liquid or separated microbial cells of microorganisms, for example, belonging to genera, Candida, Debaryomyces, Saccharomyces, Pichia, Hansenula, Stemphylium, Alternaria, Corynespora, Preussia, Brevibacterium, Escherichia, Lactobacillus, Kluyveromyces, Saccharomycopsis, Stephanoascus, Phoma, Nectria, Pseudonectria, Spondylocladium, Melanospora, Metarhizium, Gliocladium, Pestalotia, Pestalotiopsis, Curvularia, Hormonema, Sydowia, Sarcinomyces, Dothiora, Xanthothecium, Dothidea, Pringsheimia, and Selenophoma (US Patents 5891685, 1999; 5700670, 1997; 5559030, 1996; 5413921, 1995; 4933282, 1990; 4710468, 1987 and Japanese Patent Publication Nos 4-7195, 6-38776, 6-209782). However, the resolution processes suffer from the drawback that theoretically a maximum of 50% yield of the desired optically active compound ethyl 4-chloro-3-hydroxybutyrate can be obtained. Moreover, separation of the desired compound from the reaction mixture is tedious. The asymmetric reduction processes with the aid of a microorganism are also disadvantageous in that the practically sufficient efficiency of the processes can not be achieved, because either the reaction rates are low or higher concentration of the product can not be accumulated or high optical purity of the product can not be obtained and that the microorganisms usable in the reactions are extremely restricted. Thus, these known processes are not necessarily satisfactory from the industrial point of view. Objects of the invention; The main objective of the present invention is to provide an improved process for the production of enantiomerically enriched ethyl 4-chloro-3-hydroxybutyrate of formula 1 by asymmetric reduction of ethyl 4-chloro-3-oxobutyrate of formula 2 with a culture broth or mycelia of a microorganism, which belong to fungal genera Penicilium or Aspergilus. Another objective is to provide a process, which leads to the production of said compound enriched in S- configuration. Yet another objective is to use a strain of Penicillium funiculosum, deposited in Microbial Type Culture Collection (MTCC, a constituent laboratory of the applicant), Institute of Microbial Technology, and Chandigarh, India, having accession number MTCC 5246. Yet another objective is to use a strain of Aspergillus ochraceous, deposited in Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India, having accession number MTCC 5245. Summary of the invention: The present invention deals with a process for the production of enantiomerically enriched (5)- ethyl 4-chloro-3-hydroxybutyrate of formula 1, which is useful as intermediate for preparing medicaments and physiologically active compounds, such as hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors, commonly referred to as statins, which comprises treating 0-ketoester compound, ethyl 4-chloro-3-oxobutyrate of formula 2 with a microbial strain of Penicillium funiculosum MTCC 5246 or Aspergillus ochraceous MTCC 5245, whereby [3-ketoester compound is asymmetrically reduced to ethyl 4-chloro-3-hydroxybutyrate, enriched in S-enantiomer. Detailed Description of the Invention Accordingly, the present invention provides an improved process for the production of enantiomerically enriched ethyl 4-chloro-3-hydroxybutyrate of formula 1 by asymmetric reduction of ethyl 4-chloro-3-oxobutyrate of formula 2 with a culture broth or mycelia of a microorganism, which belong to fungal genera Penicilium or Aspergilus. The microorganisms used in this invention include any bacterial or fungal strain of microorganisms, which can asymmetrically reduce ethyl 4-chloro-3-oxobutyrate leading to the production of enantiomerically enriched ethyl 4-chloro-3-hydroxybutyrate of S-configuration in high enantiomeric excess. Preferably, fungal strains, Penicillium funiculosum MTCC5246 and Aspergillus ochraceous MTCC 5245 were used. These microorganisms do not have ability to assimilate ethyl 4-chloro-3-oxobutyrate and therefore mycelia obtained by growing them have been used for asymmetric reduction of ethyl 4-chloro-3-oxobutyrate. The inventors have done an extensive screening of microorganisms, belonging to the class of bacterium and fungi for the purpose of preparing the said compound, enantiomerically enriched ethyl 4-chloro-3-hydroxybutyrate of S-configuration. Most of the bacteria and fungi used in the study were isolated from soil samples, but in addition some of the fungi were also isolated from minor microbial growth, which sometimes contaminated laboratory reagents. Several of the bacteria and fungi were able to asymmetrically reduce ethyl 4-chloro-3-oxobutyrate giving rise to ethyl 4-chloro-3-hydroxybutyrate having either R or S configuration in enantiomeric excess ranging from less than 10% to more than 97%. Penicillium funiculosum MTCC 5246 and Aspergillus ochraceous MTCC 5245 were preferably used because they provide said compound in desired S-configuration in high enantiomeric excess. Penicillium funiculosum and Aspergillus ochraceous were isolated from minor microbial growth, which was noticed in the waste reservoir of HPLC in the laboratory of inventors, containing acetonitrile, methanol and ammonium acetate in water as the main components. This was cultured by plating on medium containing yeast extract, KHi PO4 and agar and incubating the plates at 30 °C till extensive growth occurred. Pure cultures were obtained by repeated subculturing on the same medium. The strains designated as MTCC 5246 and MTCC 5245 were identified as Penicillium funiculosum Thorn and Aspergillus ochraceous Wilhelm, respectively based on following characteristics: Penicillium funiculosum Thorn MTCC 5246: On czapek agar, colorless colonies reaching 25-35 mm in dimeter in a week, moderately dense, conspiously funiculose, rarely almost velutinous, margins low to moderately deep, irregular, mycelium, white to peach or brownish orange; condiogenesis moderate to heavy, greyish to dull green, reverse pale, brown or more commonly deeply pigmented, brownish red to red. On malt agar, colonies conspiously funiculose, occasionally floccose; margins entire subsurface to deep, mycelium usually white, less commonly brownish red, cnidiogenesis moderate to heavy. Conidiophores from well defined funicles, stripes short with walls smooth to finely roghned, bearing terminal biverticillate penicillin, metulae inverticils of 5-7, closely appressed, almost parallel, phialides closely apppressed acerrose, with gradually tapering collula; conidia cylindrical to ellipsoidal, typically small, smooth-walled, borne in short columns. Rapid growth in 37 °C compared to 25 °C, floccose, conidia enmass pale grayish green, exceptionally shoet- striped, closely appressed penillis and often with very small cylindroidal conidia are distinguishing features of Penicillium funiculosum. Aspergillus ochraceous Wilhelm MTCC 5245: Colonies on czapek agar having restricted growth of 3-4 cm in about 10 days, plane to slightly furrowed basal mycelium which is tough and usually submerged but raised in central areas, colorless to dull yellow-orange and producing abundant light ochraceus conidial heads; reverse yellow to greenish brown or reddish purple shades; exudates usually limited, ranging colorless to amber with slight mushroom odor; sclerotia abundant. Conidial heads globose and turning into two or three divergent and compact columns in age; conidiophores conspicuously pigmented in dull yellow to light brown shades, vesicles globose, thin-walled; sterigmata covering the entire vesicle, in two series, conidia globose to subglobose, smooth to roughened. Sclerotia white to pale pink when young but becoming lavender to vinaceous purple at maturity, globose to cylindrical, borne singly, in definite clusters or more rarely as confluent masses. Conidial heads in dull yellowish cream, or ochraceous shades and scattered and globose to cylindrical and sclerotia in pink to vinaceous purple when mature and globose to subglobose conidia distinguishes the fungus from the rest of the species belonging to Aspergillus. Accordingly, the present invention provides a process for the production of enantiomerically enriched (5)-ethyl 4-chloro-3-hydroxybutyrate, which comprises: contacting ethyl 4-chloro-3-oxobutyrate, under agitation at pH 6 to 8.0, but preferably at pH 6.5 at temperature 10-45 °C, but preferably at 30 °C with Penicilliwn funiculosum MTCC 5246, grown in a known manner in a conventional nutrient medium at pH between 5.0 to 7.5, at temperature between 25 to 45 °C for at least a period of 12-120 h., separating the biomass by conventional methods and recovering (S)-ethyl 4-chloro-3-hydroxybutyrate by conventional solvent extraction methods. In another embodiment of the present invention, the microorganism used is Aspergillus ochraceous MTCC 5245, grown in a known manner in a conventional nutrient medium at pH between 5.0 to 7.5, at temperature between 25 to 45°C for at least a period of 12-120 h., separating the biomass by conventional methods and recovering (S)-ethyl 4-chloro-3-hydroxybutyrate by conventional solvent extraction methods. In an embodiment of the present invention, the conventional nutrient medium used may be any medium in which these microorganisms can grow. The medium contains a carbon source such as glucose, starch, sucrose, a nitrogen source, such as urea, peptone, casein, yeast extract, meat extract, corn steep liquor, ammonium sulfate, ammonium acetate, ammonium nitrate, ammonium phosphate and may further contain an inorganic salt, such as phosphate, magnesium salt, potassium salt, manganese salt, iron salt, zinc salt, copper salt and may further contain vitamins. In addition to above, the medium may also contain ethyl 4-chloro-3-oxobutyrate. In another embodiment of the present invention, fermentation broth or mycelia of Penicillium funiculosum MTCC 5246 or Aspergillus ochraceous MTCC 5245 isolated by centrifilgation or filtration and suspended in a buffer at pH 6 to 8.0, but preferably at pH 6.5 may be used. In another embodiment of present invention, purified or partially purified components of Penicillium funiculosum MTCC 5246 or Aspergillus ochraceous MTCC 5245 may be used and optionally glucose and cofactor NADPH may be added. In yet another embodiment of the present invention, an enzyme such as glucose dehydrogenase or glucose-6-phosphate dehydrogenase or similar such enzyme systems may be added for cofactor recycling. In yet another embodiment of the present invention, the solvents used for the extraction of (S)- ethyl 4-chloro-3-hydroxybutyrate may be such as diethyl ether, ethyl acetate, chloroform, dichloromethane or similar water immiscible solvents. The microorganisms were isolated in the following manner. Purified cultures of several bacterial and fungal strains were screened for their ability to asymmetrically reduce ethyl 4-chloro-3- oxobutyrate. The bacterial strains were grown in liquid medium at 37 °C for 12-24 h, while the fungal strains were grown at 30 °C for 2-5 days (as required). Mycelia were isolated by centrifugation and washed with phosphate buffer at pH 6.5. The washed mycelia were suspended in 2 ml of same buffer at a concentration of 0.5 g/ml. The cell suspension was incubated with 2 mg of ethyl 4-chloro-3-oxobutyrate at 30 °C (fungal) or 37 °C (bacterial) at 200 rpm for 24 h, after which the samples were extracted with ethyl acetate and organic layer analyzed by thin layer chromatography for the formation of ethyl 4-chloro-3-hydroxybutyrate. The configuration of the product and enantiomeric excess was determined by resolving R and S-enantiomers on HPLC using a chiral column, as described in Example 1 below. The microorganisms, which produced ethyl 4-chloro-3-hydroxybutyrate of -configuration in enantiomeric excess of more than 95%, were selected. The selected microorganisms were identified as Penicillium funiculosum and Aspergillus ochraceous based on their morphological and physiological characteristics described above. These have been deposited with Microbial Type Culture Collection, Institute of Microbial Technology, Sector 39,Chandigarh, India and have been assigned accession number MTCC 5246 and MTCC 5245. respectively. The present invention is illustrated in more detail by the following examples, but should not be construed to be limited thereto. Example 1 Penicillium funiculosum MTCC 5246 was cultivated for 4 days at 30 °C in 100 ml sterile medium, consisting of 0.2% peptone, 0.2% yeast extract, 0.2% KH2?O4, 2% glucose, pH 6.0-6.5). The fungal mycelia were isolated by filtration or centrifugation and washed with 200 mM phosphate buffer at pH 6.5. These were re-suspended in 100 ml of the same buffer containing 2 gm glucose. Ethyl 4-chloro-3-oxobutyrate, 2.5 gm was added to the suspension and contents stirred for 5 hr while maintaining the temperature of reaction mixture at 30 °C. The pH of this reaction mixture was maintained at 6.5 by addition of sodium carbonate. The progress of the reaction was monitored by thin layer chromatography. At the end of the reaction, ethyl acetate (25 ml) was added and the contents stirred for another 45 min. The organic layer was separated by centrifugation and dried over anhydrous sodium sulfate. Evaporation of solvents by use of rotary evaporator produced (5)-ethyl 4-chloro-3-hydroxybutyrate in 96% yield. Characterization data for (5)-ethyl 4-chloro-3-hydroxybutyrate is given below: 'H NMR of: 1.26 (3H, i,J = 7.3 Hz, OCH2CH3); 2.56-2.70 (2H, m, H-2); 3.62 (2H, d, J = 4.9 Hz, H-4); 4.08-4.29 (3H, m, H-3 and OCH2CHs). Determination of S-configuration and enantiomeric excess of ethyl 4-chloro-3-oxobutyrate: Enantiomeric excess was determined by chiral HPLC using 250 x 4.6 mm chiralcel OB-H (Diacel, Japan) column. Elution was done with isopropanol/ hexane (4:96) at flow rate of 0.5 ml/min and detection was done at 217- Enantiomeric excess (e.e.) was determined to be about 98%. The absolute configuration was assigned as S based on negative sign of rotation and comparison of HPLC data with an authentic sample of 98.4% e.e. purchased from Aldrich, Fine Chemicals, USA. Example 2 Aspergillus ochraceous MTCC 5245 was cultivated for 4 days at 30 °C in 100 ml medium, consisting of 0.2% peptone, 0.2% yeast extract, 0.2% KH2PO4, 2% glucose, pH 6.0-6.5). The fungal mycelia were isolated by filtration or centrifugation and washed with 200 mM phosphate buffer at pH 6.5. These were re-suspended in 100 ml of the same buffer containing 2 gm glucose. Ethyl 4-chloro-3-oxobutyrate, 2.5 gm was added to the suspension and contents stirred for 5 hr while maintaining the temperature of reaction mixture at 30 °C. The pH of this reaction mixture was maintained at 6.5 by addition of sodium carbonate. The progress of the reaction was monitored by thin layer chromatography. At the end of the reaction, ethyl acetate (25 ml) was added and the contents stirred for another 45 min. The organic layer was separated by centrifugation and dried over anhydrous sodium sulfate. Evaporation of solvents by use of rotary evaporator produced (5)-ethyl 4-chloro-3-hydroxybutyrate in 97% yield. Characterization and determination of enantiomeric excess of the product (5)-ethyl 4-chloro-3-hydroxybutyrate was done as shown in Example 1. Enantiomeric excess (e.e.) was determined to be about 96%. The absolute configuration was assigned as S. Example 3 This example pertains to the use of components of Penicillium funiculosum for the production of ethyl 4-chloro-3-hydroxybutyrate. Cultures of Penicillium funiculosum were maintained on potato dextrose agar medium. Cultures were grown at 30 °C in 100 ml medium containing 2% peptone, 2% yeast extract, 2% KF^PC^ and 2% glucose at pH 6-6.5 for 4 days. Mycelia were isolated by centrifugation at 4 °C and washed with 200 mM phosphate buffer at pH 6.5. These were ruptured by any of the following methods: 1. A suspension of mycelia in 50% w/v in phosphate buffer pH 6.5 at 4 °C was subjected to a pressure of 1200 psi for 5 min in minicell (French press, SLM Aminco, SLM Instruments Incorporation, New Jersey) pre-cooled to 4 °C . 2. A suspension of mycelia in 50% w/v in phosphate buffer pH 6.5 cooled to 4 °C was disrupted by glass beads (0.45 urn) in 1:1 v/v ratio at 4 °C for 5 min in Dyano Mill (KDL, Switzerland). 3. Buffer was removed from the cell suspension by filtration under reduced pressure. The cell mass thus obtained was frozen (-20 °C, CCU/liquid nitrogen bath) and mixed with silica-gel (200-400 mesh; cell mass/silica gel in the ratio of 2:1) and ground in pestle mortar. These were then suspended in chilled phosphate buffer pH 6.5 at final concentration of 50% w/v. The extract was obtained by centrifugation at 15,000 rpm for 45 min at 4 °C. NADPH, 784 nmol; Glucose dehydrogenase, 14 units and Glucose 2 g were added followed by addition of ethyl 4-chloro-3-oxobutyrate (2 gm). The contents were gently stirred at 30 °C and the progress of the reaction monitored by TLC. Extraction, purification, characterization and determination of configuration and enantiomeric excess of the product (5)-ethyl 4-chloro-3-hydroxybutyrate was done as shown in Example 1. Example 4 This example pertains to the use of partially purified components of Penicillium funiculosum for the production of ethyl 4-chloro-3-hydroxybutyrate. Cell free extract was prepared as shown in example 3. Solid ammonium sulfate was added in small portions to the cell free extract at 4 °C to 20% saturation. Stirring was continued for another 1 h and then the precipitate removed by centrifugation. The supernatant was then brought to 75% saturation with solid ammonium sulfate. The pH of the lysate was adjusted with 0.1 N NaOH during fractionation. The precipitate so obtained was dissolved in 2.5 ml phosphate buffer 50 mM pH 6.5. Desalting was done using PD-10 columns (PD-10, Sephadex, Sigma Aldrich, USA; Product No. 5-4805) using manufacturer's protocol. The desalted lysate volume 3.5 ml was diluted to 10 ml with same buffer and again subjected to centrifugation at 15,000 rpm for 15 min before loading on the Q-Sepharose Fast Flow column (1.5 x 35 cm) equilibrated with 50 mM phosphate buffer pH 6.5, followed by washing with 100 ml of same buffer. The elution was done with a linear gradient of 0-1 M NaCl in 5 column volumes of the buffer at 2 ml/min. The fractions containing the activity were combined (30 ml). NADPH, 784 nmol; Glucose dehydrogenase, 14 units and Glucose 2 g were added followed by addition of ethyl 4-chloro-3-oxobutyrate (Ig). The contents were gently stirred at 30 °C and the progress of the reaction monitored by TLC. Extraction, purification, characterization and determination of configuration and enantiomeric excess of the product (S)-ethyl 4-chloro-3-hydroxybutyrate was done as shown in Example 1. We Claim: 1. A process for preparation of enantiomerically enriched ethyl (S)-4-chloro-3-hydroxybutyrate of formula 1 by asymmetric reduction of ethyl 4-chloro-3-oxobutyrate of formula 2 with mycelia of fungal strain Penicillium, which comprises the following steps; (Formula Removed) a) growing Penicillium in a medium containing peptone, yeast extract, KH2PO4, and glucose or any other medium containing a carbon source such as glucose, starch, sucrose, a nitrogen source, such as urea, peptone, casein, yeast extract, meat extract, corn steep liquor, ammonium sulfate, ammonium acetate, ammonium nitrate, ammonium phosphate and may further contain an inorganic salt, such as phosphate, magnesium salt, potassium salt, manganese salt, iron salt, zinc salt, copper salt and may further contain vitamins; b) isolating the fungal mycelia obtained from step (a) by centrifugation or filtration followed by washing with suitable buffer, such as phosphate buffer, pH 6.0-8.0; c) suspending the mycelia obtained from step (b) in a buffer such as phosphate buffer, pH 6.0-8.0 containing glucose; d) adding ethyl 4-chloro-3-oxobutyrate with constant stirring to the suspension obtained from step (c) while maintaining the temperature of reaction mixture at 10 to 45 °C; e) maintaining the pH of the reaction mixture of step (d) between 6.0 to 8.0; f) adding any water immiscible solvent, such as ethyl acetate, diethyl ether, dichloromethane or chloroform with stirring to the mixture obtained from step (d); g) centrifuging the mixture obtained from step (f) to separate the organic layer and the cell components; h) drying of organic layer obtained in step (g) over sodium sulfate or magnesium sulfate; i) evaporating the solvents obtained from step (h) to obtain the desired ethyl (S)-4-chloro-3-hydroxybutyrate; 2. The process as claimed in claim 1 wherein cell-free extract obtained from mycelia of fungal strain Penicillium is used for the production of ethyl (S)-4-chloro-3-hydroxybutyrate, which comprises following steps: a. maintaining cultures of Penicillium on potato dextrose agar medium and b. growing at 30 °C in a medium containing peptone, yeast extract, KH2PO4 and glucose or any other medium containing a carbon source such as glucose, starch, sucrose, a nitrogen source, such as urea, peptone, casein, yeast extract, meat extract, corn steep liquor, ammonium sulfate, ammonium acetate, ammonium nitrate, ammonium phosphate and may further contain an inorganic salt, such as phosphate, magnesium salt, potassium salt, manganese salt, iron salt, zinc salt, copper salt and may further contain vitamins; c. isolating mycelia obtained from step (d) by centrifugation or Alteration and washing with appropriate buffer, such as phosphate buffer, pH 6.0-8.0; d. rupturing mycelia obtained from step (e) by a known method, such as by ultra-sonication or with glass beads in Dynomill; e. obtaining the cell free extract by removing the solid mass by centrifugation; f. adding ethyl 4-chloro-3-oxobutyrate with constant stirring while maintaining the temperature of reaction mixture at 10 to 45 °C; g. maintaining the pH of the reaction mixture of step (f) between 6.0 to 8.0; h. adding any water immiscible solvent, such as ethyl acetate, diethyl ether, dichloromethane or chloroform with stirring to the mixture obtained from step (f); i. centrifuging the mixture obtained from step (h) to separate the organic layer and the cell components; j. drying of organic layer obtained in step (i) over sodium sulfate or magnesium sulfate; k. evaporating the solvents obtained from step (j) to obtain the desired ethyl (S)-4-chloro-3-hydroxybutyrate; 3. The process as claimed in claim 1 wherein the partially purified components obtained from fungal strain Penicillium is used for the production of ethyl (S)-4-chloro-3-hydroxybutyrate comprising: a. maintaining cultures of Penicillium on potato dextrose agar medium and b. growing at 30 °C in a medium containing peptone, yeast extract, KH2PO4 and glucose or any other medium containing a carbon source such as glucose, starch, sucrose, a nitrogen source, such as urea, peptone, casein, yeast extract, meat extract, corn steep liquor, ammonium sulfate, ammonium acetate, ammonium nitrate, ammonium phosphate and may further contain an inorganic salt, such as phosphate, magnesium salt, potassium salt, manganese salt, iron salt, zinc salt, copper salt and may further contain vitamins; c. rupturing mycelia obtained from step (b) by known method, such as by ultra-sonication or with glass beads in Dynomill; d. obtaining the cell free extract from step (c); e. adding solid ammonium sulfate in small portions to the cell free extract obtained from step (d) at 4°C to 20% saturation; stir and then remove precipitate by centrifugation; f. adding solid ammonium sulfate to the supernatant obtained in step (e) at 4°C to 75% saturation; g. adjusting the pH of the lysate obtained in step (f) to 6.0 to 8.0 during fractionation; h. dissolving the precipitate obtained from step (f) in phosphate buffer; i. desalting of the dissolved precipitate obtained from step (h) by known method; j. diluting the desalted lysate volume obtained from step (i) with phosphate buffer; k. centrifuging the product obtained from step (j) and then loading on the Q- Sepharose column equilibrated with appropriate buffer; 1. washing the column in step (k) with the buffer; m. eluting the column in step (1) with a linear gradient of buffer containing 0-1 M NaCl; n. combining the fractions obtained from step (m) containing the activity; o. adding ethyl 4-chloro-3-oxobutyrate with constant stirring while maintaining the temperature of reaction mixture at 10 to 45 °C; p. maintaining the pH of the reaction mixture of step (o) between 6.0 to 8.0; q. adding any water immiscible solvent, such as ethyl acetate, diethyl ether, dichloromethane or chloroform with stirring to the mixture obtained from step (o); r. centrifuging the mixture obtained from step (q) to separate the organic layer and the cell components; s. drying of organic layer obtained in step (r) over sodium sulfate or magnesium sulfate; t. evaporating the solvents obtained from step (s) to obtain the desired ethyl (S)-4-chloro-3-hydroxybutyrate; 4. The process as claimed in claim 1-3, wherein the strain of Penicillium used is Penicillium funiculosum MTCC 5246; 5. The process as claimed in claim 1-3, wherein fungal strain Penicillium is substituted with another fungal strain Aspergillus; 6. The process as claimed in claim 5, wherein the strain of Aspergillus used is Aspergillus ochraceous MTCC 5245; 7. The microorganisms deposited at MTCC, Chandigarh having accession numbers Penicillium funiculosum MTCC 5246 and Aspergillus ochraceous MTCC 5245 are isolated from soil samples or contaminated laboratory reagents in chandigarh, India; 8. The process as claimed in claim 1-6, wherein the temperature of the reaction mixture is kept preferably at 30 degree C; 9. The process as claimed in claim 1-6, wherein the pH of the reaction mixture is kept preferably at 6.5; 10. The process as claimed in claim 1-6, wherein enantiomerically enriched ethyl (S)-4-chloro-3-hydroxybutyrate is recovered after the reaction by two phase extraction using solvents, such as ethyl acetate, diethyl ether, chloroform, dichloromethane or similar water immiscible solvents; 11. The process as claimed in 1-6 wherein the said process leads to the production of said compound enriched in S-configuration in high enantiomeric excess of about 96-98%; 12. The process for the preparation of enantiomerically enriched ethyl (S)-4-chloro-3-hydroxybutyrate substantially as herein described with reference to the examples and drawings accompanying this specification.

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