IE912338A1

IE912338A1 - Process for producing enantiomers of 2-aryl-alkanoic acids

Info

Publication number: IE912338A1
Application number: IE233891A
Authority: IE
Original assignee: Novo Nordisk As; Du Pont
Priority date: 1990-07-05
Filing date: 1991-07-04
Publication date: 1992-01-15
Also published as: EP0537259A1; WO1992001062A1; PT98236A; CA2086236A1; AU8204091A; DK161690D0; JPH05507625A

Abstract

One of the enantiomers of an acid is prepared by a process in which enantioselective hydrolysis of a mixture of the corresponding R and S amide is carried out in the presence of an enzymatically active biological material having enantioselective amidase activity, the biological material being derived from a strain of Rhodococcus, Serratia, Moraxella or Pseudomonas.

Description

PROCESS FOR PRODUCING ENANTIOMERS OF 2-ARYL-ALKANOIC ACIDS TECHNICAL FIELD This invention relates to a process for preparing an enantiomer of an acid by enantioselective hydrolysis of a mixture of the corresponding R and S amide in the presence of an enantioselective amidase.

It also relates to a microorganism that produces amidase and to immobilized amidase for use in said process.

BACKGROUND ART Many agrochemical intermediates and pharmaceutical products of the general formula I described below are currently marketed and used as racemic or diastereomeric mixtures. In many cases, the physiological effect 15 specifically derives from only one enantiomer/diastereomer, whereas, the other enantiomer/diastereomers are inactive or even harmful. Chemical or enzymatic techniques for enantiodifferentiation are becoming increasingly important tools for production of chemicals of high optical purity, ao Enzymatic production of optically active acids such as 2-arylalkanoic acids by enantioselective hydrolysis of the corresponding racemic amides in the presence of a microorganism or an enzyme is known from European patent application having publication No. 326,482. The microorganisms used belong to the genera Corynebacterium and Brevibacterium. The process was performed batchwise without organic solvent, and the enzymatically active material was discarded after being used once. Data in the examples of this European patent application having publication No. 326,482 indicate that the so conversion of S amide into acid ranged from about 40 to 65%, i.e. 3 5 - 60% of the S amide remained unconverted. The enantiomer excess of the S form in the acid produced was 92 SLK/ToN/NRu/KGF, 2 July 1991, IL 3450.200 97%. The microorganisms were cultivated on a fermentation substrate that included N-roethylacetamide.

European patent application having publication No. 356,912 discloses microorganisms from the genera of Pseudo5 monas, Fusarium, Rhodococcus. Brevibacterium. Micrococcus. Bacteridium and Bacillus capable of converting racemic aliphatic 2-substituted nitriles into the optically active aliphatic 2-substituted carboxylic acid. It is stated that the microorganisms are also active on aromatic 2-substituted niio triles, but no supporting data are given. The enantiomer excess of S acid resulting from the corresponding nitrile after completion of the reaction was maximum 84%. In the case of using Rhodococcus for converting a nitrile into an acid, the enantiomer excess was 35%. The fermentation substrate inis eluded nitrile.

European patent application having publication No. 348,901 relates to a process for producing an optically active a-substituted organic acid by treating a racemic a-substituted nitrile or amide with a microorganism selected among 20 the genera Alcaliaenes, Pseudomonas, Rhodopseudomonas.

Corvnebacterium. Acinetobacter. Bacillus. Mycobacterium.

Rhodococcus and Candida. This 348,901 publication does not disclose any microorganism employed in the process of this invention.

It is the object of this invention to provide a process with improved enantioselective conversion of amides, for example 2-arylalkanoic amides.

A work describing an examination of 809 strains isolated from 330 different soil samples to produce an S acid 30 from the corresponding nitrile and amide appear^ in Appl.Environ .Microbiol. 56 (1990), 3125 et seq., which was published after the priority date of this patent application.

It also is the object of this invention to provide a process by which a mixture of R and S amides being enantiomers is converted to the acid corresponding to one of the two amide enantiomers in a high yield and high purity.

SLIC/ToN/NRu/ICGF, 2 July 1991, IL 3450.200 It is also the object of this invention to provide a method of producing enantioselective amidase without the need for nitrile or amide as inducer.

STATEMENT OF THIS INVENTION This invention provides a process for producing an enantiomer of an acid by enantioselective hydrolysis of a mixture of the corresponding S and R amide in the presence of an enzymatically active biological material with enantioselective amidase activity. By this process, two sorts of w yields are important. Briefly, the two sorts of yields are, on one hand, the degree of conversion and, on the other hand, the purity of the resulting acid. The degree of conversion is the degree in which one of the two enantiomer amides is converted into the acid. The purity of the resulting acids has herein been designated enantiomer excess and is defined below. As mentioned below, this invention is superior as far as at least one of these two sorts of yields is concerned.

We have found that, surprisingly, it is possible to obtain a very high degree of conversion by the process of 20 this invention. Under certain reaction conditions, it is even possible to obtain a degree of conversion of nearly 100%.

In addition and, surprisingly, a very high enantiomer excess is obtainable by the process of this invention. Under certain reaction conditions, it is even possible to obtain an enantiomer excess of nearly 100%.

Hence, after completion of the process of this invention and removal of the amide(s) , the resulting R or S acid has a very high purity, in some cases a purity of almost 100%. Thus, from a starting mixture of an R and S amide being 30 enantiomers, optionally a racemic mixture of R and S amides, it is possible, by the process of this invention, either to produce directly a mixture of s acid and unconverted R amide essentially devoid of R acid and S amide or to produce a mixture of R acid and unconverted S amide essentially devoid SLK/ToN/NRu/KGF, 2 July 1991, IL 3450.200 of S acid and R amide. This allows for simplified separation and higher yield.

Examples of suitable acids which can be prepared by the process of this invention are compounds of the general formula I X_CR1R2-COOH (I) wherein X represents a phenyl group or a naphthyl group which groups optionally are substituted with halogen, alkyl, alkoxy or benzoyl, R1 represents hydroxy, amino or alkyl, and R2 io represents hydrogen or alkyl.

Examples of amides which can be used as starting material in the process of this invention are compounds of the general formula II X-CR1R2-CONH2 (II) is wherein R1, R2 and X each are as defined above.

Obviously, both the phenyl and naphthyl groups mentioned above may be substituted. The naphthyl group may be an a or β naphthyl group. Halogen is, preferably, chloro and fluoro. The alkyl and alkoxy groups are preferably lower 20 alkyl and lower alkoxy groups. Hereinafter the term lower indicates that the group in question contains not more than carbon atoms, preferably not more than 4 carbon atoms.

The amide used by the process of this invention may optionally have been produced in situ.

As the enzymatically active biological material used in the process of this invention has an enantioselective amidase activity, this material may herein be designated an amidase.

In one aspect of this invention, the process is characterized in that said biological material is immobilized. In a second aspect, the process is characterized in that the hydrolysis is carried out in the presence of an organic solvent.

The enantioselectivity of the amidase used in the process of this invention may be determined by hydrolysis of a racemic amide, for example, an acid of formula II, for example, racemic 2-(4-chlorophenyl)-3-methylbutyramide.

SLK/ToN/HRu/KGF, 2 July 1991, IL 3450.200 The degree of conversion obtainable by the process of this invention is preferably above about 65%, more preferred above about 90%, even more preferred above about 95%, most preferred above about 99%. The preferred high 5 degrees of conversion are obtainable by using especially preferred reaction conditions which are further illustrated in the examples below.

The enantiomer excess of the resulting enantiomer acid is preferably above about 85%, more preferred above io about 90%, even more preferred above 95%. Using especially preferred reaction conditions which are further illustrated in the examples below, the enantiomer excess of the resulting enantiomer acid may be above 99%, more preferred above about 99.5%, most preferred above about 99.9%. The enantiomer 15 excess is calculated from the concentration of R and s forms using the following equation: ((SJ - [R])/((S] + £R)) x 100% wherein [R] and [S] is the concentration of the R and S form, respectively.

This invention also provides a biologically pure culture of an enzymatically active microorganism with enantioselective amidase activity. In fact, this invention describes a Rhodococcus strain that produces the amidase activity constitutively, i.e. without the need for an inducer such as an amide.

Further, this invention provides immobilized, enzymatically active biological material with enantioselective amidase activity for use in the above process. Furthermore, this invention also provides biological material having enan30 tioselective amidase activity, characterized by being derived from a strain of Rhodococcus. and a method of preparing biological material having enantioselective amidase activity, characterized by comprising cultivation of an amidase producing Rhodococcus strain in a medium that does not include nitrile or amide (unsubstituted or N-substituted).

In addition, this invention also provides biologically pure cultures of enzymatically active SLK/ToN/NRu/KGF, 2 July 1991, IL 3450.200 microorganisms with enantioselective amidase activity and a method of preparing such biological material. Such microorganisms may be obtained from strains of Serratia. Moraxella or Pseudomonas.

DETAILED DESCRIPTION OF THIS INVENTION The process of this invention is performed in a manner known per se by subjecting the mixture of the R and S amide being enantiomers to the action of the biological material in question in a pertinent reaction medium. The io skilled art worker is able to determine convenient conditions for carrying out the process for this invention, due care being taken to this specification, including the examples below.

The biological material used in the process of this is invention is prepared in a manner known per se.

The biological material having amidase activity is preferably obtained in a manner known per se from a constitutive amidase producing strains of Rhodococcus. especially Rhodococcus erythropolis DP-10. This microbial strain was de20 posited under the terms of the Budapest Treaty at DSM (Deutsche Sammlung von Microorganismen und Zellkulturen GmbH, Braunschweig, Germany) on March 23, 1990 under the accession number DSM 5910. Other Rhodococcus erythropolis strains deposited on 21 February 1991 as described above include DSM 25 6374, DSM 6375 and DSM 6378 (Rhodococcus erythropolis Nos.

DP-ll, DP-26 and DP-25, respectively).

Strain dsm 5910 is capable of hydrolyzing a wide range of aliphatic and aromatic amides into their corresponding acids. The strain, however, hydrolyses both the D and L 30 form of amino acid amides such as phenylglycine amide and a number of aliphatic amino acid amides. It is highly surprising that this strain (and other strains belonging to the genus Rhodococcus) possesses the ability to perform the enantioselective hydrolysis of racemic amides according to this SLK/ToN/MRu/KGF, 2 July 1991, ll 3450.200 invention, strain DSM 5910 is advantageous in being constitutive for production of amidase, i.e. no inducers are needed for maximum expression of amidase activity.

The Rhodococcus amidase preferably has an enantio5 selectivity above 85%, more preferably above 90%, even more preferably above 95%, and most preferably above 99%.

The biological material having amidase activity is also obtainable in a mammer known per se from amidase producing strains of Serratia. Moraxella and Pseudomonas. io These strains were deposited under the terms of the Budapest treaty at NRRL (Northern Regional Research Laboratories, Peoria, II, USA) and with accession numbers noted below. Pseudomonas putida NRRL-B-18669 and Moraxella sp. NRRL-B18671 were deposited on 8 July 1990 and Pseudomonas putida 15 NRRL-B-18820. Pseudomonas sp. NRRL-B-18819 and Serratia licruefaciens NRRL-B-18821 were deposited on 9 May 1991.

According to this invention, the process is generally conducted in homogeneous or heterogeneous aqueous or aqueous-organic medium at temperature and pH conditions 20 determined as a function of the immobilized cells, whole cells or cell extract from the microorganism and the mixture of amide and the resulting acid. The enzymatic reaction may be carried out using immobilized cells under batch or continuous conditions.

The starting material for the process of this invention may be a previously prepared amide; this can be prepared, for example, by chemical or enzymatic hydrolysis of the corresponding nitrile. Alternatively, the amide may be produced in situ, for example by enzymatic hydrolysis of the corresponding nitrile.

After completion of the reaction, the enantiomer acid can be recovered from the reaction mixture and purified by conventional methods. If a mixture of R and S amide (for example a racemic mixture) is used as starting material, the 35 process results in a mixture of S acid and R amide or a mixture of R acid and S amide. After separating the amide from the acid, the amide can be racemized by known methods into SLK/ToN/NRu/KGF, 2 July 1991, IL 3450.200 racemic amide which can be recycled and hydrolyzed as already described. Racemization can be performed by refluxing the amide with an anion exchange resin that comprises quaternary ammonium functionality, for example, Amberlite IRA-400 in OH form in toluene or an other non-aqueous solvent.

The enzymatically active biological material to be used in this invention may, for example, be whole cells, cell paste, homogenized cells or a crude or purified enzyme solution. Immobilization may be carried out by known methods, io such as cross-linking, for example, with glutaraldehyde or polyazetidine according to US patent specification No. 4,892,825.

The immobilized material is preferably used in a continuous process, either in a fixed-bed column or a stirred 15 tank reactor. If organic solvent is used, it is particularly preferred to use a stirred tank reactor, where the immobilized material and the organic solvent phase are retained by a hydrophilic membrane (cf. Example 8, Fig. 1) Organic solvent to be used in the process of this 20 invention may be water-miscible (for example, dimethyl sulfoxide) or water-immiscible (for example, toluene or octane). The amount of solvent is generally 2 - 20% by weight of the reaction system.

The process of this invention may be used to pro25 duce 2-arylalkanoic acid of the general formula I.

The process of this invention is particularly suited for production of acids where the aryl group X is phenyl, p-chlorophenyl, p- isobutylpheny1, 3-benzoylphenyl, βnaphthyl or 6-methoxy-2-naphthyl, and where the group desig30 nated R is hydroxy, methyl, ethyl or isopropyl.

Some specific examples of acids that may be produced according to this invention are 2-(4-chlorophenyl)-3methylbutyric acid (hereinafter designated CPIA), (6-methoxy2-naphthyl)hydroxypropionic acid, 2-(6-methoxy-2-naphthyl)35 propionic acid, 2-(4-isobutylphenyl)propionic acid, 2-phenyl2-hydroxypropionic acid and 2-(3-benzoylphenyl)propionic acid.

SLK/ToR/NRu/KGF, 2 July 1991, IL 3450.200 ABBREVIATIONS CPIAm is 2-(4-chlorophenyl) -3-methylbutyramide, CPIA is 2-(4-chlorophenyl)-3-roethylbutyric acid, IBAm is 2(4-isobutylphenyl)propionamide, IBAC is 2-(4-isobutylphenyl) 5 propionic acid (ibuprofen), NPAm is 2-(6-methoxy-2-naphthyl)propionamide, NPAC is 2-(6-methoxy-2-naphthyl)propionic acid (naproxen), ATAm is 2-phenyl-2-hydroxypropionamide, ATAC is 2-phenyl-2-hydroxypropionic acid and HPLC is High-Performance Liquid Chromatography.

ANALYTICAL PROCEDURE In the examples that follow, the amide and acid products listed above were measured by reverse phase high performance chromatography. A Zorbax C18 column employing mobile phases of 70 - 75% methanol and 25 - 30% H20 acidified 15 with 0.1% H3PO4 or 67% acetonitrile and 33% H2o acidified with 0.1% H3PO4 was used. Chromatographic identity and quantitation of acid products were determined by comparison with authentic standards.

Chiral HPLC for the separation of CPIAm/CPIA, 20 NPAm/NPAC and LBAm/IBAC enantiomers was carried out with an Qfi-AGP column obtained from Chromtech (Sweden) . The mobile phases for separation of various enantiomers is summarized below.

Chiral HPLC Separation of Amide and Acid Enantiomers Enantiomers CPIAm, CPIA NPAm, NPAC IBAm, IBAC Mobile Phase 95% 0.01 M Phosphate Buffer (pH 6.0):5% Ethanol 95% 0.04 M Phosphate Buffer (pH 5.6):5% Etanol 96% 0.02 M Phosphate Buffer (pH 5.2):4% Ethanol SLK/ToN/NRu/KGF, 2 July 1991, Il 3450.200 Chiral HPLC for the separation of ATAm and ATAC enantiomers was carried out with a Resolvosil BSA-7 column (Alltech, Inc.). The mobile phase employed for ATAm/ATAC enantiomer composition was 0.01 M phosphate buffer (pH 6.0). 5 Enantiomeric composition, purity and chromatographic identity of the above amide and acids were determined by comparison with authentic standard enantiomers or racemic mixtures.

PRODUCTION EXAMPLE Chemical hydrolysis of R,8-CP1N to R,S-CPIAm io A suspension containing 9.69 g of racemic 2-(4-chlorophenyl)-3-methylbutyronitrile (50 mmoles), 75 ml of dioxane, 75 ml of water and 25.2 g of Amberlite IRA-400 (OH) resin was stirred and heated to reflux for 64 hours. After filtration of the resin, the filtrate was evaporated to dryness and dried in a vacuum oven at 50 - 55*C to give 9.8 g of crude racemic CPlAm. The composition of the recovered material was determined by HPLC.

The recovered material contained 3.6 mmoles of racemic 2-(4-chlorophenyl)-3-methylbutyronitrile and 46.4 mmoles of racemic CPI Am.

EXAMPLE 1 Preparation of biological material with enantioselective amidase activity Microorganisms used are Rhodococcus ervthropolis DSM 5910 (DP-10), Rhodococcus ervthropolis DSM 6374 (DP-11), Rhodococcus ervthropolis DSM 6378 (DP-25), Rhodococcus SLK/ToN/NRu/KGF, 2 July 1991, IL 3450.200 ervthropolis DSM 6375 (DP-26), Pseudomonas putida NRRL-B18669 (13-5S-ACN-2a), Moraxella sp. NRRL-B-18671 (3L-A-1-5la-1), Pseudomonas putida NRRL-B-18820 (2D-ll-5-lb), Pseudomonas sp. NRLL-B-18819 (2D-11-5-1C) and Serratia lique5 faciens NRRL-B-18821 (MOB IM/N3).

The growth medium used for the cultivation of Pseudomonas, Serratia and Moraxella strains was made up of the following constituents. 3Z1 10 kh2po4 8.85 Sodium citrate 2.25 MgSO4.7H2O 0.5 FeSO4.7H2O 0.05 Glucosea 10.0 15 Trace element solution0 1.0 ml Vitamin solutiond 1.0 ml Nitrile 2.7 g a Added after autoclaving. • b Trace element solution: 10 ml of 20 FeCl2.4H2O, 0.019 g of CoCl2.6H2O, % HCl, 1.5 g of 0.07 g of ZnCl2, 0.062 g of H3BO3/ 0.036 g of NaMoO4.2H2O, 0.024 g of NiCl2.6H2O and 0.017 g of CuC12.2H2O. The FeCl2.4H2O is dissolved in HCl and distilled water is added to 1 liter.

Vitamin solution: 0.01 g of biotin, 0.01 g of folic acid, 0.05 g of pyridoxine.HCl, 0.025 g of riboflavin, 0.025 g of thiamine HCl, 0.025 g of nicotinic acid, 0.025 g of pantothenic acid, 0.0065 g of vitamin B12, 0.025 g of p-aminobenzoic acid and 0.025 g of thioacetic acid. 1,4-dicyanobutane (Pseudomonas putida 13-5S-ACN-2a), 2-methylglutaronitrile (Pseudomonas sp. 2D-ll-5-ic, Pseudomonas putida 2D-ll-5-lb, Serratia liguefaciens MOB IM/N3, Moraxella sp. 3L-A-l-5-la-l).

SLK/lWNRu/KGF, 2 July 1991, IL 3450.200 A 10 ml volume of the above medium (PR/glucose) was inoculated with 0.1 ml of frozen stock culture. Following overnight growth at room temperature (22 - 25 *C) on a shaker at 250 RPM, the 10 ml inoculum was added to 990 ml of fresh s medium in a 2 liter flask. The cells were grown for 18 - 24 hours at room temperature with magnetic stirring at a rate high enough to cause bubble formation in the medium. Cells were harvested by centrifugation, washed once with 0.85% saline and the concentrated paste immediately placed in a io -70*C freezer for storage.

EXAMPLE 2 Preparation of biological material with enantioselective amidase activity The growth medium used for cultivation of Rhodococcus 15 ervthropolis DP-10 was made up of the following constituents: 3/1 KH2PO4 9.00 Na2HPO4.2H2O 21.00 NaCl 0.50 CaCl2«2H2O 0.02 MgSO4·7H20 0.30 (nh4)2so4 3.50 Yeast extract (Difco) 0.50 Glucose* 3.75 Trace element solution** 10.00 pH value 7.5 * added after autoclaving ** trace element solution SL-7 (cf. DSM, Catalogue of strains 1983, p. 296) These constituents were dissolved in 900 ml of water, the pH value was adjusted to 7.5 by addition of phosphoric SlK/ToN/NRu/KGf, 2 July 1991, IL 3450.200 acid/sodium hydroxide, and the solution was made up to 1 liter by addition of water. 100 ml of the above growth medium was added to a 500 ml Erlenmeyer flasks and autoclaved. Glucose was added to the 5 cooled medium from a sterile stock solution (20%, weight/ volume). Erlenmeyer flasks inoculated with Rhodococcus erythropolis DSM 5910 were incubated at 30’C on a shaker for 8 hours.

Cells were harvested by centrifugation (at 20,000 rpm io for 15 minutes on a Sorvall® centrifuge), washed twice with 0.1 M phosphate buffer of pH value 7. The washed cell pellet (= cell paste) was stored at 4*C or frozen (-25*C).

EXAMPLE 3 Immobilization of biological material with enantioselective is amidase activity Fresh cell paste obtained as described in Example 2 was immobilized as previously described (US patent specification No. 4,892,825, Example 11).

EXAMPLE 4 Hydrolysis of S-CPIAm to S-CPIA by Rhodococcus erythropolis DP-10 A 75 mg sample of dried, immobilized Rhodococcus erythropolis DP-10 was added to 3 ml of phosphate buffer (100 mM, pH value: 7.0) and incubated for 1 hour at 4*C. The 25 immobilized cell suspension was warmed to room temperature and 6.3 mg (29.8 /moles) of S-CPIAm in 120 μΐ of dimethyl sulfoxide (hereinafter designated DMSO) was added. After incubation with agitation at 50’C for 48 hours, the reaction was acidified with 3M H2SO4 to a pH value of 3.0. Four SLt/ToM/MRu/KGF, 2 July 1991, IL 3450.200 volumes of methylene chloride were added and the suspension was agitated for 15 - 30 minutes. The methylene chloride layer was removed and evaporated to dryness under a stream of nitrogen gas and the residue was suspended in 3 ml of metha5 nol. The composition of the methanol solution was determined by HPLC.

The extracted supernatant contained 0.5 μ-mole of CPIAm and 28.3 mmoles of CPIA.

Measurement of the enantiomeric composition by chiral io HPLC showed that the enantiomer excess of S-CPIA was 99%.

COMPARATIVE EXAMPLE Hydrolysis of R-CPIAm by Rhodococcus ervthropolis DP-10 A 75 mg sample of dried immobilized Rhodococcus ervthropolis DP-10 was added to 3 ml of phosphate buffer (100 15 mM, pH value: 7.0) and incubated for 1 hour at 4*C. The immobilized cell suspension was wanned v to room temperature and 6.3 mg (29.8 /imoles) of R-CPIAm in 120 μΐ of DMSO was added. After incubation with agitation at 50*C for 48 hours, the reaction was acidified with 3M H2SO4 to a pH value of 20 3.0. Four volumes of methylene chloride were added and the suspension was agitated for 15 - 30 minutes. The methylene chloride layer was removed and evaporated to dryness under a stream of nitrogen gas. The composition of the methanol solution was determined by HPLC.

The extracted supernatant contained 22.7 /xmoles of CPIAm and less than 0.5 /xmole of CPIA. , The amount of acid corresponded to the amount present as an impurity in the starting material.

Incomplete recovery of R-CPIAm was most likely due to so experimental errors and/or adsorption of substrate to cells and/or the support material.

SLK/ToM/NRu/KGF, 2 July 1991, IL 3450.200 EXAMPLE 5 Hydrolysis of R,S-CPIAm to S-CPIA by Rhodococcus erythropolis DP-10 A 75 mg sample of dried immobilized Rhodococcus 5 erythropolis DP-10 was added to 3 ml of phosphate buffer (100 mM, pH value: 7.0) and incubated for 1 hour at 4*C. The immobilized cell suspension was warmed to room temperature and 6.3 mg (29.8 /moles) of R,S-CPIAm in 120 /tl of DMSO was added. After incubation with agitation at 50’C for 48 hours, io the reaction was acidified with 3M H2SO4 to a pH value of 3.0. Four volumes of methylene chloride were added and the suspension was agitated for 15 - 30 minutes. The methylene chloride layer was removed and evaporated to dryness under a stream of nitrogen gas. The composition of the methanol is solution was determined by HPLC.

The extracted supernatant contained 12.5 /moles of CPIAm and 11.8 /moles of CPIA.

Measurement of the enantiomeric composition by chiral HPLC showed that the enantiomer excess of R-CPIAm was 100% and that the enantiomer excess of S-CPIA was 100%.

EXAMPLE 6 Hydrolysis of R,S-CPIAm to S-CPIA by Rhodococcus erythropolis DP-10 The procedure of Example 5 was repeated with 59.8 25 /moles of R,S-CPIAm and a 72 hour incubation. The composition of the methanol solution was determined by HPLC.

The extracted supernatant contained 31.9 /moles of CPIAm and 21.8 /moles of CPIA.

Measurement of the enantiomeric composition of the extracted supernatant by chiral HPLC showed 4.8 Mmoles of SCPIAm, 27.1 Mmoles of R-CPIAm and 21.8 Mmoles of S-CPIA.

SLK/ToN/NRu/KGF, 2 July 1991, IL 3450.200 The enantiomer excess of s-CPIA was 100%.

EXAMPLE 7 Hydrolysis of S-CPIAm to CPIA in toluene by Rhodococcus ervthropolis DP-10 A 300 mg sample of dried immobilized Rhodococcus ervthropolis DP-10 was added to 12 ml of phosphate buffer (100 mM, pH value: 7.0) and the mixture was incubated for 1 hour at 4*C. The immobilized cell suspension was warmed to room temperature and the phosphate buffer was removed by io decantation. 12 ml of toluene saturated with 100 mM phosphate buffer (pH value: 7.0) was added. Toluene saturated buffer was prepared by mixing equal volumes of toluene and 100 mM phosphate buffer (pH value: 7.0), shaking, allowing the phase to separate and removing the aqueous layer. After the addition of 118 ^tmoles of S-CPIAm (19.7 ^moles/2 ml), the toluene suspension was incubated with agitation at 30*C. A 2 ml sample was removed at 48 hours and 120 hours and each sample was evaporated to dryness under a stream of nitrogen gas. Each sample was resuspended in 2 ml of methanol and the composition of the methanol solution was determined by HPLC.

After 48 hours, the toluene supernatant samples contained 11.4 /xmoles of CPIAm and 5.7 /xmoles of CPIA and after 120 hours it contained 1.9 /xmoles of CPIAm and 9.4 /xmoles of CPIA.

EXAMPLE 8 s-CPIAm hydrolysis to S-CPIA in buffer/octane by Rhodococcus erythropolis DP-10 A 500 mg sample of dried immobilized Rhodococcus ervthropolis DP-10 was added to a biphasic solution SLK/ToN/NRu/KGF, 2 July 1991, IL 3450.200 consisting of 16 ml of phosphate buffer (100 mM, pH value: 7.0) and 4 ml of n-octane. The immobilized cell suspension was incubated for 3 hours at 50 *C. After the addition of 10 mg (47.4 mmoles) of S-CPIAm and incubation for 6 hours at 50 *C and 600 rpm, 0.5 ml aliquots of each phase was removed. Each aliquot was adjusted to a pH value of 2.0 with 10N HCl. Four volumes of methylene chloride were added to each aliquot and the suspensions were agitated for 15 minutes. The methylene chloride layer was removed and evaporated to io dryness under a stream of air and each residue was resuspended in 0.5 ml of methanol. The composition of the methanol solution was determined by HPLC.

The extracted supernatants contained: Compound CPIAm CPIA Buffer phase 0.5 /xmoles 19.8 ^moles Octane phase o 1.9 /xmoles EXAMPLE 9 Continuous S-CPIAm hydrolysis to S-CPIA in buffer/octane by 20 Rhodococcus ervthropolis DP-10 A 3.74 g sample of dried immobilized Rhodococcus erythropolis DP-10 was added to a reactor consisting of a Millipore ultrafiltration cell with a hydrophilic membrane (PTGC 043 10) installed at the bottom. The immobilized cells 25 were incubated in 50 ml of phosphate buffer ' (100 mM, pH value: 7.0) plus 20 ml of n-octane at 50*C for 2 hours. After the addition of 64 mg of S-CPIAro (303.3 ^moles), the reactor was closed and the peristaltic pump which delivered phosphate buffer (100 mM, pH value: 7.0) pre-saturated with S-CPIAm was 30 started. The liquid feed rate was adjusted to 20 ml/hour to give a 3.5 hour hydraulic retention time for the reacting system. The hydrophilic membrane selectively passes the SLK/ToN/MRu/KGF, 2 July 1991, IL 3450.200 buffer phase and retained the n-octane and immobilized Rhodococcus erythropolis DP-10. The reactor configuration is shown schematically in Figure 1.

The reactor effluent was collected at time intervals, 5 was extracted as described in Example 7 and was analyzed by HPLC for CPIAm and CPIA.

The sample analysis translated into the following rates at the exit: Elapsed time, Minutes CPIAm, umoles/hour CPIA, umoles/hour 12.5 14.5 115 9.1 24.1 180 4.4 32.7 EXAMPLE 10 is Resin racemization of S-CPIAm A suspension containing 1.0 g of S-CPIAm (4.7 mmoles), 1.0 g of Amberlite IRA-400 (OH) resin and 25 ml of toluene was stirred and heated to reflux for 40 hours. After removal of the resin by filtration, the filtrate was evaporated to 20 give 0.94 g of crystalline solid. The enantiomeric composition of the recovered material was measured by chiral HPLC and showed 54% S-CPIAm and 46% R-CPIAm.

EXAMPLE 11 Hydrolysis of S-CPIAm, R-CPIAm and R,S-CPIAm by Rhodococcus 25 erythropolis DP-10 A 50 mg sample of frozen cell paste of Rhodococcus ervthropolis DP-10 was added to 1 ml of phosphate buffer (lOOmM, pH value: 7.0) at room temperature. Then 9.4 μπιοίβ of S-CPIAm or R, S-CPIAm in 40 μΐ of dimethylsulf oxide was added.

After incubation at 50*C for 48 hours, the reactions were SLK/ToW/NRu/KGF, 2 July 1991, IL 3450.200 acidified with 3M H2SO4 to a pH value of 3. Four volumes of methylene chloride were added and the suspension was agitated for 15-30 min. The methylene chloride layer was removed and evaporated to dryness under a stream of nitrogen gas and the residue was suspended in 1 ml of methanol. The composition of the methanol solution was determined by reverse-phase HPLC and chiral HPLC and is shown in Table l.

TABLE 1 S-CPIAm, R-CPIAm and R,S-CPIAm Hydrolysis by Rhodococcus io erythropolis dp-10 Substrate (gmole added) HPLC Analysis fumole recovered) Reverse-Phase Chiral CPIAm CPIA S-CPIAm R-CPIAm S-CPIA R-CPIA S-CPIAm (9.4) NDa 9.1 NDa NDa 9.1 NDa R-CPIAm (9.4) 8.2NDa,b nt NT NT NT R,S-CPIAm (9.4) 4.7 4.6 TRd 4.7 4.6 NDa a ND = None detected. b Data corrected for R-CPIA impurity in R-CPIAm starting material. c NT = Not tested. d TR = Trace amount detected.

Enantiomer excess of S-CPIA recovered was 100%.

SLX/ToH/KRu/KGF, 2 July 1991. Il 3450.200 EXAMPLE 12 Hydrolysis of R,S-ATAm to R-ATAC by Rhodococcus erythropolis DP-10 A 50 mg sample of frozen cell paste of Rhodococcus 5 erythropolis DSM 5910 (DP-10) was added to l ml of phosphate buffer (lOOmM, pH value: 7.0) at room temperature. Then 12.1 Mmole of R,S-ATAm in 40 μΐ of dimethyl sulfoxide was added. After incubation at 28*C with agitation for 48 hours, the reaction suspension was centrifuged to remove cell debris and io the supernatant was passed through a 0.2 μ membrane filter. The composition of the clarified supernatant was determined by reverse-phase HPLC and chiral HPLC and is shown in Table 2.

TABLE 2 R,S-ATAm Hydrolysis by Rhodococcus erythropolis DP-10 HPLC Analysis (nmole recovered) Substrate Reverse-Phase__Chiral_ (gmole added) ATAm ATAC S-ATAm R-ATAro S-ATAC R-ATAC 20 _ R,S-ATAm 3.3 4.2 3.3 TRa 0.3 3.9 (12.1) a TR = Trace amount detected.

Incomplete recovery of R,S-ATAm was most likely due to experimental errors and/or adsorption of substrate to cells.

Enantiomer excess of R-atac recovered was 86%.

SLK/ToM/NRu/KGF, 2 July 1991, IL 3450.200 EXAMPLE 13 Hydrolysis of S-CPIAm, R-CPIAm and R,S-CPIAm by Rhodococcus ervthropolis DP-11 A 50 mg sample of frozen cell paste of Rhodococcus s ervthropolis DP-11 was added to l ml of phosphate buffer (lOOmM, pH value: 7.0) at room temperature. In the same manner as Example 11, 9.4 gmole of S-CPIAm or R-CPIAm or R,SCPIAm was added. Following the same incubation and extraction protocols as in Example 11, the composition of the extracted io supernatant was determined by reverse-phase and chiral HPLC.

The results are shown in Table 3.

TABLE 3 s,CPIAm, R-CPIAm and R,S-CPIAm Hydrolysis by Rhodococcus ervthropolis DP-11 _ HPLC Analysis (nmole recovered) Substrate Reverse-Phase _Chiral_ (gmole added) CPIAm CPIA S-CPIAm R-CPIAm S-CPIA R-CPIA S-CPIAm (9.4) 4.7 4.7 TRa NDb 4.7 NDb R-CPIAm (9.4) 8.3 NDb'c NTd NTd NTd NTd R,S-CPIAm 6.3 2.8 1.8 4.5 2.8 NDb (9.4) a TR = Trace amount detected.

ND = None detected. c Data corrected for R-CPIA impurity in R-CPIAm starting SLK/ToN/NRu/KGF, 2 July 1991, IL 3450.200 material. d NT = Not tested.

Enantiomer excess of S-CPIA recovered was 100%.

EXAMPLE 14 Hydrolysis of R,S-ATAm to R-ATAC by Rhodococcus erythropolis DP-11 A 50 mg sample of frozen cell paste of Rhodococcus erythropolis DP-11 was added to 1 ml of phosphate buffer (100 mM, pH value: 7.0) at room temperature. In the same manner as 10 in Example 12, 12.1 gmole of R, S-ATAm was added. Following the same incubation, centrifugation and filtration protocols as in Example 12, the composition of the extracted supernatant was determined by reverse-phase HPLC and chiral HPLC.

The results are shown in Table 4.

TABLE 4 R,S-ATAm Hydrolysis by Rhodococcus erythropolis DP-11 HPLC Analysis fttmole recovered) Substrate Reverse-Phase Chiral (gmole added) ATAm ATAC S-ATAm R-ATAm S· -ATAC R-ATAC R,S-ATAm 4.8 1.2 3.4 1.4 NDa 1.2 (12.1) as a ND = Trace amount detected.

SLK/ToN/MRu/KGF, 2 July 1991, IL 3450.200 Incomplete recovery of R,S-ATAm was most likely due to experimental errors and/or adsorption of substrate to cells. Enantiomer excess of R-ATAC recovered was 100%.

EXAMPLE 15 Hydrolysis of S-CPIAm, R-CPIAm and R,S-CPIAm by Rhodococcus erythropolis DP-25 A 50 mg sample of frozen cell paste of Rhodococcus ervthropolis DP-25 was added to 1 ml of phosphate buffer (lOOroM, pH value: 7.0) at room temperature. In the same io manner as Example 11, 9.4 gmole of S-CPIAm or R-CPIAm or R,SCPIAm was added. Following the same incubation and extraction protocols as in Example 11, the composition of the extracted supernatant was determined by reverse-phase and chiral HPLC.

The results are shown in Table 5.

TABLE 5 S-CPIAm, R-CPIAm and R,S-CPIAm Hydrolysis by Rhodococcus erythropolis DP-25 Substrate (gmole added) HPLC Analysis (umole recovered) Reverse-Phase Chiral CPIAm CPIA S-CPIAm R-CPIAm S-CPIA R-CPIA S-CPIAm (9.4) 6.0 3.2 1.7 4.3 3.2 NDa R-CPIAm (9.4) 8.4 NDa'b NTC NTC NTC NTC R,S-CPIAm (9.4) 6.2 3.1 1.8 4.4 3.1 NDa SLK/ToN/NRU/KGF, 2 July 1991, IL 3450.200 a ND = None detected. b Corrected for R-CPIA impurity in R-CPIAm starting material . c NT = Not tested.

Enantiomer excess of S-CPIA recovered was 100%.

EXAMPLE 16 Hydrolysis of R,S-ATAa to R-ATAC by Rhodococcus ervthropolis DP-25 A 50 mg sample of frozen cell paste of 24 Rhodococcus io erythropolis was added to 1 ml of phosphate buffer (lOOmM, pH value: 7.0) at room temperature. In the same manner as Example 5, 12.1 gmole of R,s-ATAm was added. Following the same incubation, centrifugation and filtration protocols as in Example 12, the composition of the extractant supernatant 15 was determined by reverse-phase HPLC and chiral HPLC. The results are shown in Table 6.

TABLE 6 R,s-ATAm Hydrolysis by Rhodococcus ervthropolis dp-25 HPLC Analysis fumole recovered) Substrate Reverse-Phase Chiral (Mmole added) ATAm ATAC S-ATAm R-ATAm S-ATAC R-ATAC R,S-ATAm 9,7 1.8 6.2 3.5 NDa 1.8 (12.1) SlK/ToN/NRu/KGF, 2 July 1991, IL 3450.200 a ND = None detected.

Enantiomer excess of R-ATAC recovered was 100%.

EXAMPLE 17 Hydrolysis of S-CPIAm, R-CPIAm and R,s-CPIAm by Rhodococcus 5 erythropolis DP-26 A 50 mg sample of frozen cell paste of Rhodococcus erythropolis DP-26 was added to 1 ml of phosphate buffer (lOOmM, pH value: 7.0) at room temperature. In the same manner as Example 11, 9.4 gmole of S-CPIAm or R-CPIAm or R,Sio CPIAm was added. Following the same incubation and extraction protocols as in Example 11, the composition of the extracted supernatant was determined by reverse-phase and chiral HPLC.

These results are shown in Table 7.

TABLE 7 is Hydrolysis of S-CPIAm, R-CPIAm and R,S-CPIAm by Rhodococcus erythropolis DP-26 HPLC Analysis fumole recovered) Substrate Reverse-Phase _Chiral_ (umole added) CPIAm CPIA S-CPIAm R-CPIAm S-CPIA Λ R-CPIA S-CPIAm (9.4) 4.6 4.7 TRa 4.6 4.7 ND^ R-CPIAm (9.4) 8.4 NDb,C NTd NTd NTd NTd R,S-CPIAm 5.7 3.2 1.5 4.2 3.2 NDb (9.4) SLK/ToN/NRu/KGF, 2 July 1991, ll 3450.200 a TR = Trace detected. b ND = None detected. c Data corrected for R-CPIA impurity in R-CPIAm starting material.

Enantiomer excess of S-CPIA recovered was 100%.

EXAMPLE 18 Hydrolysis of R,S-ATAm to R-ATAC by Rhodococous ervthropolis DP-2 6 A 50 mg sample of frozen cell paste of Rhodococcus io ervthropolis DP-26 was added to 1 ml of phosphate buffer (lOOmM, pH value: 7.0) at room temperature. In the same manner as in Example 12, 12.1 μιηοΐβ of R,S-ATAm was added.

Following the same incubation, centrifugation and filtration protocols as in Example 12, the composition of the extracted is supernatant was determined by reverse-phase and chiral HPLC.

The results are shown in Table 8.

TABLE 8 R,S-ATAm Hydrolysis by Rhodococcus ervthropolis DP-26 HPLC Analysis iumole recovered) Substrate Reverse-Phase Chiralt (Mmole added) ATAm ATAC S-ATAm R-ATAro S-ATAC R-ATAC R,S-ATAm 8.5 3.0 6.2 2.3 NDa 3.0 (12.1) SLK/ToN/MRu/ttf, 2 July 1991, IL 3450.200 a ND = None detected.

Enantiomer excess of R-ATAC recovered was 100%.

EXAMPLE 19 Hydrolysis of R,S-NPAm to s-npac by Pseudomonas putida 13-585 ACN-2a A 25 mg sample of frozen cell paste of Pseudomonas putida 13-5S-ACN-2a was added to l ml of phosphate buffer (100 mM) , pH value: 7.2) at room temperature. Then 1 gmole of R,S-NPAm in 40 μΐ of dimethylsulfoxide was added. After inio cubation at 28’C with agitation for 48 hours, the reaction was acidified to a pH value of 3 with 3M H2so4. Four volumes of methylene chloride were added and the suspension was agitated for 30 min. The methylene chloride layer was removed and evaporated to dryness under a stream of nitrogen. The is residue was redissolved in 1 ml of methanol. The composition of the extracted supernatant was determined by reverse-phase HPLC and chiral HPLC and is shown in Table 9.

TABLE 9 R,S-NPAm Hydrolysis by Pseudomonas putida 13-5S-ACN-2a H PLC Analysis (umole recovered) Substrate Reverse-Phase Chiral (μιιιοίβ added) NPAro NPAC S-NPAm R-NPAm S-NPAC R-NPAC R,S-NPAm (1.0) 0.42 0.44 NDa 0.42 0.44 NDa SLK/ToM/NRu/KGF, 2 July 1991, IL 3450.200 a ND = None detected.

Enantiomer excess of S-NPAC recovered was 100%.

EXAMPLE 20 Hydrolysis of R,S-IBAm to S-IBAC by Pseudomonas putida 13-5S5 ACN-2a A 50 mg sample of frozen cell paste of Pseudomonas putida 13-5S-ACN-2a was added to 1 ml of phosphate buffer (100 mM) , pH value: 7.0) at room temperature. Then 9.8 pinole R,S-IBAm in 40 pi of dimethylsulfoxide was added. After inlo cubation at 28 *C with agitation for 48 hours, the reaction was acidified with 3M H2SO4 to a pH value of 3. Four volumes of methylene chloride were added and the suspension was agitated for 15 - 30 minutes. The methylene chloride layer was removed and evaporated to dryness under a stream of nitrogen is gas and the residue was suspended in 1 ml of acetonitrile. The composition of the acetonitrile solution was determined by reverse phase HPLC and chiral HPLC and is shown in Table .

TABLE 10 R,s-IBAm Hydrolysis by Pseudomonas putida i3-5S-ACN-2a HPLC Analysis (umole recovered) Substrate Reverse-Phase Chiral (pmole added) IBAm IBAC S-IBAm R-IBAm S-IBAC R-IBAC R,S-IBAm 4.8 1.3 1.4 3.4 1.3 NDa (9.8) SLK/ToN/HRu/KGF, 2 July 1991, IL 3450.200 a ND = None detected.

Incomplete recovery of R,S-IBAm was most likely due to experimental errors and/or adsorption of substrate to cells. Enantiomer excess of S-IBAC recovered was 100%.

EXAMPLE 21 Hydrolysis of R,S-NPAm to S-NPAC by Pseudomonas putida 2D-115-lb A 20 mg sample of frozen cell paste of Pseudomonas putida 2D-ll-5-lb was added to 1 ml of phosphate buffer (100 io mM) , pH value: 7.2) at room temperature. In the same manner as Example 19, 1 Mmole of R,S-NPAm in 40 μΐ of dimethylsulfoxide was added. Following incubation for 24 hours and the same extraction protocol as in Example 19, the composition of the extracted supernatant was determined by reverse-phase HPLC and chiral HPLC and is shown in Table 11.

TABLE 11 R,S-NPAm Hydrolysis by Pseudomonas putida. 2D-ii-5-ib HPLC Analysis fumole recovered) Substrate Reverse-Phase Chiral (Mmole added) NPAm NPAC S-NPAm R-NPAm S -NPAC R-NPAC R,S-NPAm 0.60 0.40 0.05 0.55 0.39 0.01 (1-0) SLK/ToM/NRu/KGF, 2 July 1991, IL 3450.200 Enantiomer excess of s-NPAC recovered was 95%.

EXAMPLE 22 Hydrolysis of R,S-IBAu to S-IBAC by Pseudomonas putida 2D-115-lb A 50 mg sample of frozen cell paste of Pseudomonas putida 2D-ll-5-lb was added to 1 ml of phosphate buffer (100 mM, pH value: 7.0) at room temperature. In the same manner as Example 20, 9.8 Mmole of R,S-IBAm in 40 μΐ of dimethylsulfoxide was added. Following the same incubation io and extraction protocols as in Example 20, the composition of the extracted supernatant was determined by reverse-phase HPLC and chiral HPLC and is shown in Table 12.

TABLE 12 R/S-NPAa Hydrolysis by Pseudomonas putida 2D-11-5-lb HPLC Analysis (Mmole recovered) Substrate Reverse-Phase Chiral (Mmole added) « IBAm IBAC S-IBAm R-IBAm S-IBAC R-IBAC R f S IB Am 3.8 2.1 0.6 3.2 2.0 0.1 (9.8) Incomplete recovery of R,S-IBAm was most likely due to experimental errors and/or adsorption of substrate to cells.

Enantiomer excess of S-IBAC recovered was 90%.

SLK/TON/NRU/ICGF, 2 July 1991, IL 3450.200 EXAMPLE 23 Hydrolysis of R,S-NPAm to S-NPAC by Pseudomonas sp. 2D-11-5lo A 20 mg sample of frozen cell paste of Pseudomonas sp. 5 strain 2D-ll-5-lc, was added to 1 ml of phosphate buffer (100 mM, pH value: 7.2) at room temperature. In the same manner as Example 19, 1 /mole of R,S-NPAm in 40 /:1 of dimethyl sul fox ide was added. Following the same incubation and extraction protocols as in Example 19, the composition of the extracted io supernatant was determined by reverse-phase HPLC and chiral HPLC and is shown in Table 13.

TABLE 13 R/S-NPAm Hydrolysis by Pseudomonas sp. 2D-li-5-ic H PLC Analysis fumole recovered) Substrate Reverse-Phase Chiral (gmole added) NPAm NPAC S-NPAm R-NPAm S-NPAC R-NPAC R,S-NPAm (1.0) 0.54 0.48 NDa 0.54 0.47 0.01 aND = Not detected.

Enantiomer excess of S-NPAC recovered was 96%.

SLK/ToN/NRu/KGF, 2 July 1991, IL 3450.200 EXAMPLE 24 Hydrolysis of R,S-NPAm to NPAC by Serratia liquefaciens mob IM/N3 A 20 mg sample of frozen cell paste of Serratia lique5 faciens MOB IM/N3 was added to 1 ml of phosphate buffer (lOOmM, pH value: 7.2) at room temperature. In the same manner as Example 19, 1 gmole of R,S-NPAro in 40 μΐ of dimethyl sulfoxide was added. Following the same incubation and extraction protocols as in Example 19, the composition of the io extracted supernatant was determined by reverse-phase HPLC and chiral HPLC and is shown in Table 14.

TABLE 14 R,s-NPAm Hydrolysis by Serratia liquefaciens MOB IM/N3 HPLC Analysis iumole recovered) Substrate Reverse-Phase Chiral (gmole added) NPAro NPAC S-NPAm R-NPAm S-NPAC R-NPAC R,S-NPAm (1.0) 20 0.64 0.26 0.20 0.44 0.25 0.01 Enantiomer excess of S-NPAC recovered was 92%.

SLK/ToN/NRu/KGF, 2 July 1991, IL 3450.200 EXAMPLE 25 Hydrolysis of R,S-IBAm to s-iBAC by serratia liquefacieps MOB IM/N3 A 50 mg sample of frozen cell paste of Serratia liaue5 faciens MOB IM/N3 was added to 1 ml of phosphate buffer (lOOmM), pH value: 7.0) at room temperature. In the same manner as Example 20, 9.8 Mmole of R,s-IBAm in 4 0 μΐ of dimethylsulf oxide was added. Following the same incubation and extraction protocols as in Example 20, the composition of the io extracted supernatant was determined by reverse-phase HPLC and chiral HPLC and is shown in Table 15.

TABLE 15 R,S-IBAm Hydrolysis by Serratia liquefaclens, MOB IM/N3 _HPLC Analysis' (umole recovered) Substrate Reverse-Phase__Chiral_ (Umole added) IBAm IBAC S-IBAm R-IBAm S-IBAC R-IBAC R,S-IBAm (9.8) 4.9 0.7 2.0 2.9 0.7 NDa ________ a ND = None detected.

SLK/ToK/NRuZKGF, 2 July 1991, IL 3450.200 Incomplete recovery of R,S-IBAm was most likely due to experimental errors and/or adsorption of substrate to cells.

Entantiomer excess of S-IBAC recovered was 100%.

EXAMPLE 26 Hydrolysis of R,s-ATAm to R-ATAC by Moraxella sp. 3L-A-1-5la-l A 50 mg sample of frozen cell paste of Moraxella sp. 5 3L-A-l-5-la-l was added to 1 ml of phosphate buffer (100 mM, pH value: 7.0) at room temperature. Then 12.1 gmole of R,SATAm in 40 μΐ of dimethylsulfoxide was added. After incubation at 28 *C with agitation for 48 hours, the reaction suspension was centrifuged to remove cell debris and the io supernatant was passed through a 0.2 μ membrane filter. The composition of the supernatant was determined by reversephase HPLC and chiral HPLC and is shown in Table 16.

TABLE 16 R/S-ATAm Hydrolysis by Moraxella sp. 3L-A-l-5-la-l Substrate HPLC Analysis (ixmole recovered) Reverse-Phase Chiral (gmole added) ATAm ATAC S-ATAro R-ATAm S -ATAC R-ATAC R,S-ATAm 7.6 1.5 4.9 2.7 NDa 1.5 (12.1)a ND = None detected. A Incomplete recovery of R,S-ATAm was roost likely due to experimental errors and/or adsorption of substrate to cells.

Entantiomer excess of S-ATAC recovered was 100%.

SLK/ToN/NRu/KCF, 2 July 1991, IL 3450.200 EXAMPLE 27 Comparison of CPIAm Bioconversion by Rhodococcus erythropolis DP-10 and Brevibacterium sp. R312 Fifty mg samples of frozen cell paste of Rhodococcus 5 erythropolis DP-10 and Brevibacterium sp. R312 were added to 1 ml volumes of phosphate buffer (lOOmM, pH value: 7.0) at room temperature. Then 9.4 pmole of S-CPIAm of R,S-CPIAm in 40 pi of dimethylsulfoxide was added. After incubation at 50 ’C for 2, 5, 8, 16 and 24 hours, the reactions were io acidified with 3M h2so4 to a pH value of 3.0. Four volumes of methylene chloride were added and the residues were suspended in 1 ml of methanol. The composition of the methanol solutions were determined by reverse-phase HPLC. The results expressed as specific activity are shown in Table 17.

TABLE 17 S-CPIAm and R,S-CPAm Hydrolysis by Rhodococcus erythropolis DP-io and Brevibacterium sp. R312 Bioconversion Specific Activity (pmoles Strain Time CPIA/mo Cellsa/hr x 102) hours S-CPIAm R,S-CPIAm R. erythropolis DP-10 2 5 8 16 24 5.2 5.6 5.2 4.1 2.9 6.5 6.5 4.4 2.1 1.6 Brevibacterium sp. 2 2.4 3.3 R312 5 3.0 3.6 8 3.0 3.1 16 2.8 2.2 24 2.8 1.7 SLK/ToH/NRu/KGF, 2 July 1991, IL 3450.200 a Dry cell weight.

Higher specific activities were obtained for both SCPIAm and R,S-CPIAm hydrolysis to S-CPIA with Rhodococcus ervthropolis DP-10.

SLK/ToK/NRu/KGF, 2 July 1991, IL 3450.200

Claims

1. CLAIMS i

1. A process for preparing an enantiomer of an acid by enantioselective hydrolysis of a mixture of the corresponding R and S amide in the presence of an enzymatically active 5 biological material with enantioselective amidase activity, characterized in that the bioloigical material is derived from . 1' a strain of Rhodococcus. Serratia. Moraxella or Pseudomonas, I with the proviso that the biological material is not derived from Rhodococcus sp. AK 32 (FERM BP-1046) or from Pseudomonas 10 fluorescens NRRL B 981 or IFO 3081.

2. A process, according to Claim 1, characterized in that said acid has the general formula I X-CR 1 R 2 -COOH (I) wherein X represents a phenyl group or a naphthyl group which 15 groups optionally are substituted with halogen, alkyl, alkoxy or benzoyl, R 1 represents hydroxy, amino or alkyl, and R 2 represents hydrogen or alkyl.

3. A process, according to Claim 2, wherein R 1 is hydroxy or alkyl. 20

4. A process, according to claim 2 or 3, wherein R 2 is hydrogen.

5. A process, according to any one of the Claims 2 - 4, wherein said alkyl groups and alkoxy groups contain not more than 10 carbon atoms, preferably not more than 4 carbon 25 atoms.

6. A process, according to any one of the preceding claims, wherein said biological material is derived from a strain of Rhodococcus.

7. A process, according to the preceding claim, wherein 30 said strain belongs to Rhodococcus ervthropolis and, SLK/ToN/NRu/KGF, 2 July 1991, IL 3450.200 preferably, is strain DSM 5910, 6374, 6375 or 6378, or a variant or mutant thereof.

8. A process, according to any one of the Claims l - 5, wherein said biological material is derived from a strain of 5 Serratia.

9. A process, according to the preceding claim, wherein said strain belongs to Serratia liquefaciens and, preferably, is strain MOB IM/N3, or a variant or mutant thereof.

10. A process, according to any one of the Claims 1-5, io wherein said biological material is derived from a strain of Moraxella.

11. A process, according to the preceding claim, wherein said strain belongs to Moraxella sp. 3L-A-l-5-la-l, or a variant or mutant thereof. 15

12. A process, according to any one of the preceding claims 1-5, wherein said biological material is derived from a strain of Pseudomonas putida, preferably

13. -5S-ACN-2a or 2D-ll-5-5-lb, or from Pseudomonas sp. 2D-ll-5-lc or a variant or mutant thereof. 20 13. A process, according to any one of the Claims 1-12, wherein said resulting enantiomer acid is in S form.

14. A process, according to any one of the Claims 1-12, wherein said resulting enantiomer acid is in R form.

15. A process, according to any one of the Claims 1 2S through 7, wherein said starting amide is 2-(4-chlorophenyl)3-methylbutyramide (CPIAm) and the biological material is derived from a strain of Rhodococcus♦ SLK/ToN/NRu/KGF, 2 July 1991, ll 3450.200

16. A process, according to the preceding claim, wherein said resulting enantiomer acid is in S form.

17. A process, according to any one of the Claims l through 7, 10 and 11, wherein said starting amide is 25 phenyl-2-hydroxypropionamide (ATAm) and the biological material is derived from a strain of Rhodococcus or Moraxella.

18. A process, according to the preceding claim, wherein said resulting enantiomer acid is in R form.

19. A process, according to any one of the Claims 1 io through 5, 8, 9 and 12, wherein said starting amide is 2-(6methoxy-2-naphthyl)propionamide (NPAm) and the biological material is derived from a strain of Serratia or Pseudomonas.

20. A process, according to the preceding claim, wherein said resulting enantiomer acid is in S form. 15

21. A process, according to any one of the Claims 1 through 5, 8, 9 and 12, wherein said starting amide is 2-(4isobutylphenyljpropionamide (IBAm) and the biological material is derived from a strain of Serratia or Pseudomonas.

22. A process, according to the preceding claim, wherein 20 said resulting enantiomer acid is in S form.

23. A process, according to any one of the preceding claims, characterized in that the enantiomer excess is at least 85%, preferably at least 90%, more preferred at least about 95%, even more preferred at least about 99%, most 25 preferably at least 99.5%.

24. A process, according to any one of the preceding claims, characterized in that the degree of conversion is above about 65%, more preferred above about 90%, even more preferred above about 95%, most preferred above about 99%. SIK/ToH/NRu/KGF, 2 July 1991, U 3450.200

25. A process, according to any one of the preceding claims, characterized in that said biological material is immobilized.

26. A process, according to the preceding claim, wherein 5 said material is immobilized by cross-linking with glutaraldehyde .

27. A continuous process, according to any one of the preceding claims, wherein a solution or suspension containing the reactant amide mixture passes through a reactor in which io the immobilized biological material is retained.

28. A process, according to any one of the preceding claims, characterized in that the hydrolysis is carried out in the presence of an organic solvent.

29. A process, according to the preceding claim, wherein is the amount of organic solvent makes up 2 through 20% by weight of the total reaction system.

30. A process, according to any one of the 2 preceding claims, wherein said solvent is a water-miscible organic solvent, preferably dimethyl sulphoxide. 20

31. A process, according to Claim 28 or 29, wherein said solvent is a water-immiscible organic solvent, preferably an aromatic or aliphatic hydrocarbon with 6 through 9 carbon atoms, most preferably toluene or octane.

32. A process, according to any one of the Claims 28 - 31, 25 wherein said biological material is immobilized.

33. A process, according to the preceding claim, wherein the reaction system contains an aqueous phase and a solvent SLK/ToN/NRu/KGF, 2 July 1991, IL 3450.200 phase, and whereby the solvent phase and the immobilized material are retained by a hydrophilic membrane.

34. A process, according to any one of the Claims 2-33, wherein X is phenyl, p-chlorophenyl, p-isobutylphenyl, 35 benzoylphenyl, β-naphthyl or 6-methoxy-2-naphthyl.

35. A process, according to Claims 2 - 34, wherein R 1 is hydroxy, methyl, ethyl or isopropyl.

36. A process, according to Claim 35, wherein the acid of formula I is 2-(4-chiorophenyl)butyric acid, 2-(4-chloroio phenyl)-3-methylbutyric acid, (6-methoxy-2-naphthyl)hydroxypropionic acid, 2-(4-isobutylphenyl)propionic acid, 2-phenyl2-hydroxypropionic acid or 2-(3-benzoylphenyl)propionic acid.

37. A process, according to any one of the preceding claims comprising enantioselective hydrolysis of racemic is amide.

38. A process, according to the preceding claim, wherein said hydrolysis is followed by racemization of the unconverted amide and recycling of the racemized amide.

39. A process, according to any one of the preceding 20 claims, wherein said biological material comprises enzymatically active microbial cells in whole or disrupted form.

40. A method for racemizing a R or S amide comprising contacting the amide with a strongly basic ion exchange resin substantially in the absence of water. 25

41. A method, according to the preceding claim, wherein said resin is a strongly basic gel-type resin.

42. A method, according to the preceding claim, wherein said resin comprises quaternary ammonium functionality. SLK/ToN/NRu/KGF, 2 July 1991, IL 3450.200

43. A biologically pure culture of a material derived from a strain of Rhodococcus, Serratia, Moraxella or Pseudomonas. with the proviso that the biological material is not derived from Rhodococcus sp. AK 32 (Ferm BP-1046) or from Pseudomonas 5 fluorescens NRRL B 981 or IFO 3081.

44. A biologically pure culture of a Rhodococcus strain according to Claim 43, characterized by constitutive production of enantioselective amidase.

45. A culture, according to the preceding claim, of a w strain of Rhodococcus ervthropolis, preferably strain DSM 5910, 6374, 6375 or 6378 or a variant or mutant thereof.

46. Immobilized, enzymatically active biological material with enantioselective amidase activity for use in the process according to any one of the preceding process claims. 15

47. A preparation, according to the preceding claim, immobilized by cross-linking with glutaraldehyde.

48. Biological material derived from a strain of Rhodococcus. Serratia, Moraxella or Pseudomonas, with the proviso that the biological material is not derived from 20 Rhodococcus sp. AK 32 (FERM BP-1046) or from Pseudomonas fluorescens NRRL B 981 or IFO 3081, characterized by having enantioselective amidase activity.

49. Biological material, according to the preceding claim, derived from Rhodococcus ervthropolis, and most preferably 25 strain DSM 5910, 6374, 6375 or 6378 or a variant or mutant thereof.

50. A method of preparing biological material having enantioselective amidase activity, characterized by comprising SLK/ToN/KRu/KGF, 2 July 1991, IL 3450.200 cultivation of an amidase producing strain of Rhodococcus in a medium not containing nitrile or amide.

51. A.process according to any of claims 1-39, substantially as described herein by way of Example.

52. An enantiomer of an acid when prepared by a process according to any of claims 1-39 or 51.

53. A method according to any of claims 40-42, substantially as described herein by way of Example.

54. An R or S amide racemized by a method according to any of claims 40-42 or 53.

55. A method according to claim 50, substantially as described herein by way of Example.

56. Biological material prepared by a method according to claim 50 or 55.

57. Any novel feature or combination of features described herein.