MXPA01003893A - Method for producing chiral carboxylic acids from nitriles with the assistance of a nitrilase or microorganisms which contain a gene for the nitrilase - Google Patents

Abstract

The invention relates to nucleic acid sequences which code for a polypeptide with nitrilase activity, to nucleic acid constructs containing the nucleic acid sequences, and to vectors containing the nucleic acid sequences or the nucleic acid constructs. The invention also relates to amino acid sequences which are coded by the nucleic acid sequences, and to microorganisms containing the nucleic acid sequences, the nucleic acid constructs or vectors containing the nucleic acid sequences or the nucleic acid constructs. In addition, the invention relates to a method for producing chiral carboxylic acids from the racemic nitriles.

Description

METHOD FOR PRODUCING OUIRAL CARBOXYLIC ACIDS FROM NITRILE WITH THE ASSISTANCE OF A NITRILASE OR MICROORGANISMS THAT CONTAIN A GENE FOR NITRILASE BRIEF DESCRIPTION OF THE INVENTION The invention relates to nucleic acid sequences encoding a polypeptide having nitrilase activity, for nucleic acid constructs comprising the nucleic acid sequences, and for vectors comprising nucleic acid sequences or nucleic acid sequences. nucleic acid constructs. The invention further relates to amino acid sequences that are encoded by the nucleic acid sequences, and for microorganisms comprising the nucleic acid sequences, the nucleic acid constructs or vectors comprising the nucleic acid sequences or the nucleic acid constructs . The invention further relates to a process for preparing chiral carboxylic acids from racemic nitriles. The chiral carboxylic acids are compounds in demand for organic chemical synthesis. They are initial materials for a large number of active pharmaceutical ingredients or active ingredients for crop protection. The chiral carboxylic acids can be used for the classical resolution of racemate by diastereomeric salts. R - (-) - or S - (-) - mandelic acid [sic] are used, for example, for resolution of racemates or racemic amines. R- (-) - mandelic acid is additionally used as an intermediate to synthesize semisynthetic antibiotics and a large number of agricultural products. Various different synthetic routes for chiral carboxylic acids are described in the literature. Thus, for example, optically active amino acids are obtained industrially by fermentation processes. This causes the disadvantage that a specific process must be developed for each amino acid. This is because chemical or enzymatic processes are used to be able to prepare a maximum range of different compounds. A disadvantage of the chemical processes is that the stereocenter normally must be constructed in complicated, multi-stage syntheses, not widely applicable [sic]. The enzymatic synthesis of chiral carboxylic acids is [sic] found in a number of patents or patent applications. W092 / 05275 describes the synthesis of enantiomeric a-hydroxy-a-alkyl or a-alkylcarboxylic acids in the presence of biological materials. EP-B-0 348 901 claims a process for preparing a-substituted optically active organic acids using microorganisms of the genus Alcaligenes, Pseudomonas, Rhodopseudomonas, Corynebacterium sp. strain KO-2-4, Acinetobacter, Bacillus, Mycobacterium, Rhodococcus and Candida. The preparation of La-amino acids using microorganisms is claimed in EP-B-0 332 379. The preparation of α-hydroxycarboxylic acids, specifically the preparation of optically active lactic acid or mandelic acid, using various microorganisms, such as microorganisms of the genera Alcaligenes , Aureobacterium, Pseudomonas, Rhodopseudomonas, Corynebacterium, Acinetobacter, Caseobacter, Bacillus, Mycobacterium, Rhodococcus, Brevibacterium, Nocardia, Variovorax, Arthrobacter and Candida or using enzymes are described in EP-A-0 348 901 or the US equivalent US 5,283,193, EP-A-0 449 648, EP-B-0 473 328, EP-B-0 527 553 or the US equivalent US 5,296,373, EP-A-0 610 048, EP-A-0 610 049, EP-A 0 666 320 or W097 / 32030. The disadvantages of these processes are that they often lead to products with only low optical purity and / or that proceed with only low space-time yields. This leads to economically unattractive processes. Still attempts to increase productivity by adding substances such as sulfite, disulfite, dithionite, hypophosphite or phosphite (see EP-A 0 486 289) or by the use of microorganisms having an increased resistance to α-hydroxy nitriles (see W097 / 32030) leads to a negligible increase in productivity. It is an object of the present invention to develop an easy, cost-effective, broadly applicable process for preparing optically active chiral carboxylic acids that does not have the above disadvantages. We have found that this object is achieved by the process according to the invention for preparing chiral carboxylic acids of the general formula I which comprises converting racemic nitriles of the general formula II in the presence of an amino acid sequence that is encoded by a nucleic acid sequence selected from the group of a) a nucleic acid sequence having the sequence depicted in SEQ. FROM IDENT. NO .: 1, b) nucleic acid sequences that are derived from the nucleic acid sequences depicted in SEQ. FROM IDENT. NO .: 1 as a result of the degeneracy of the genetic code, c) derived from the nucleic acid sequence represented in SEC. FROM IDENT. DO NOT . : 1, which encodes polypeptides having the amino acid sequences represented in SEC. FROM IDENT. DO NOT . : 2 and have at least 80% homology at the amino acid level, with negligible reduction in the enzymatic action of the polypeptides. or a disorganized latent growing microorganism which comprises a nucleic acid sequence of the aforementioned group or a nucleic acid construct that binds a nucleic acid of the group to one or more regulatory signals, and wherein at least 25 mmoles of nitrile are converted per h and per mg of protein or 25 mmoles of nitrile are converted by h and g of dry weight into the chiral carboxylic acids, wherein the substituents and variables in formulas I and II have the following meanings: * an optically active center R1, R2 R3 independently of each other hydrogen, substituted or unsubstituted, branched or unbranched C1-C10 alkyl, C2-C10 alkenyl, substituted or unsubstituted aryl, hetaryl, OR4 or NR4R5 and wherein the radicals R1, R2 and R 3 are always different R 4 hydrogen, unsubstituted or unsubstituted, branched or unbranched C 1 -C 4 alkyl, C 2 -C 10 alkenyl, C 1 -C 4 alkylcarbonyl, C 2 -C 10 alkenylcarbonyl , aryl, arylcarbonyl, hetaryl or hetarylcarbonyl, R5 hydrogen, substituted or unsubstituted Cx-C10 alkyl, branched or unbranched, C2-C10 alkenyl, aryl or hetaryl. R1, R2 R3 in the compounds of the formulas I and II are, independently of one another, hydrogen, unsubstituted or branched or unbranched C? -C10 alkyl, branched or unbranched, unsubstituted or substituted aryl C2-C10 alkenyl, hetaryl , OR4 or NR4R5 and wherein the radicals R1, R2 and R3 are always different. Alkyl radicals which may be mentioned are substituted or unsubstituted, branched or unbranched C1-C10 alkyl chains such as, for example, methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl, -methylpropyl, 1,1-dimethylethyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, 1,1-dimethylpropyl, 1,2 -dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3 -dimethylbutyl, 3, 3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1, 1, 2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl , n-heptyl, n-octyl, n-nonyl or n-decyl. Methyl, ethyl, n-propyl, n-butyl, i-propyl or i-butyl are preferred. Alkenyl radicals which may be mentioned are branched or unbranched C2-C10 alkenyl chains such as, for example, ethenyl, propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methylpropenyl, 1-pentenyl, 2- pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, l-methyl-3-butenyl, 2-met l-3-butenyl, 3-methyl-3-butenyl, 1, l-dimethyl-2-propenyl, 1,2-dimethyl- 1-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl, 1-ethyl-2-propenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5- hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 3-methyl-1-pentenyl, 1 4-methyl-1-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl , 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, l-methyl-3-pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, l-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1, l-dimethyl-2-butenyl, 1, l-dimethyl-3 -butenyl, 1,2-dimethyl-1-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-d-methyl-1-butenyl, 1,3-dimethyl -2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-1-butenyl, 2,3-dimethyl-2-butenyl, 2, 3 -dimethyl-3-butenyl, 3, 3-dimethyl-l-butenyl, 3, 3-dimethyl-2-butenyl, 1-ethyl-l-butenyl, l-ethyl-2-butenyl, l-ethyl-3-butenyl , 2-ethyl-l-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1, 1, 2-trimethyl-2-propenyl, 1-ethyl-l-methyl-2-propenyl, l -ethyl-2-methyl-1-propenyl, 1-ethyl-2-methyl-2-propenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl , 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, nonenyl or decenyl. Ethenyl, propenyl, butenyl or pentenyl are preferred. The aryl radicals which may be mentioned are substituted and unsubstituted aryl radicals containing from 6 to 20 carbon atoms in the ring or ring system. The latter may comprise aromatic rings that are fused together or aromatic rings linked by alkyl, alkylcarbonyl, alkenyl or alkenylcarbonyl chains, carbonyl, oxygen or nitrogen. The aryl radicals may, where appropriate, also be linked via the C 1 -C 4 alkyl, C 3 -C 8 alkenyl, C 3 -C 6 alkynyl or C 3 -C 8 cycloalkyl chain to the basic structure. Phenyl or naphthyl is preferred. Hetaryl [lacuna] which may be mentioned are single or fused aromatic ring systems substituted or unsubstituted with one or more 3- to 7-membered heteroaromatic rings which may contain one or more heteroatoms such as N, O or S and may, wherein it is appropriate to link via the C 1 -C 10 alkyl, C 3 -C 8 alkenyl or C 3 -C 8 cycloalkyl chain to the basic structure. Examples of hetaryl radicals of this type are pyrazole, imidazole, oxazole, isooxazole [sic], thiazole, triazole, pyridine, quinoline, isoquinoline, acridine, pyrimidine, pyridazine, pyrazine, phenazim, purine or pteridine. The hetaryl radicals can be linked to the basic structure by means of the heteroatoms or by means of various carbon atoms in the ring or ring system or by means of the substituents. Pyridine, imidazole, pyrimidine, purine, pyrazine or quinoline are preferred. Suitable substituents for radicals R1, R2 or R3, for example, one or more substituents such as halogen, such as fluorine, chlorine or bromine, thio [sic], nitro, amino, hydroxyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkyl or other aromatics or other rings are not saturated or unsaturated aromatics or ring systems. Preference is given to alkyl radicals such as C 1 -C 6 alkyl such as methyl, ethyl, propyl or butyl, aryl such as phenyl, halogen such as chlorine, fluorine or bromine, hydroxyl or ammonium. R4 in the radicals OR4 or NRR5 is hydrogen, substituted or unsubstituted, branched or unbranched C1-C10 alkyl, C2-C10 alkenyl, C ^ C ^ alkylcarbonyl, C2-C10 alkenylcarbonyl, aryl, arylcarbonyl, hetaryl or hetarylcarbonyl. Alkyl radicals which may be mentioned are substituted or unsubstituted, branched or unbranched C 1 -C 4 alkyl chains such as, for example, methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl , 2- methylpropyl, 1,1-dimethylethyl, n-pentyl, 1-methylbutyl, 2- methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, 1,1-dimethylpropyl, 1,2 - 10-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3, 3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1, 1, 2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-l-methylpropyl, l-ethyl-2-methylpropyl, n-heptyl , n-octyl, n-nonyl or n-decyl. Methyl, ethyl, n-propyl, n-butyl, i-propyl or i-butyl are preferred. Alkenyl radicals which may be mentioned are branched or unbranched C2-C10 alkenyl chains Such as, for example, ethenyl, propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methylpropenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl , 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2 - 25 methyl-3-butenyl, 3-methyl-3-butenyl, 1, l-dimethyl-2- ? fym ^ - \ riti ím (t propenyl, 1, 2-dimethyl-1-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl, 1-ethyl-2-propenyl, 1-hexenyl , 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 3-methyl-1-pentenyl, 4-methyl-1-pentenyl, -methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, l-methyl-3-pentenyl, 2-methyl-3-pentenyl, 3-methyl -3-pentynyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1, 1-dimethyl -2-butenyl, 1, l-dimethyl-3-butenyl, 1,2-dimethyl-1-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl -1-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-l-butenyl, 2,3-dimethyl -2-butenyl, 2,3-dimethyl-3-butenyl, 3, 3-dimethyl-l-butenyl, 3,3-dimethyl-2-butenyl, 1-ethyl-l-butenyl, l-ethyl-2-butenyl , l-ethyl-3-butenyl, 2-ethyl-1-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1, 1, 2-trimethyl-2-propenyl, l-ethyl-l-methyl-2-propenyl, l-ethyl-2-methyl-l-propenyl, l-ethyl-2-methyl-2-propenyl, 1- heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, nonnyl or decenyl. Ethenyl, propenyl, butenyl or pentenyl are preferred. The alkylcarbonyl radicals which may be mentioned are substituted or unsubstituted, branched or unbranched Ci-Cio alkylcarbonyl chains such as, for example, methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, 1-methylethylcarbonyl, n-butylcarbonyl, 1-methylpropylcarbonyl, -methylpropylcarbonyl, 1,1-dimethylethylcarbonyl, n-pentylcarbonyl, 1-methylbutylcarbonyl, 2-methylbutylcarbonyl, 3-methylbutylcarbonyl, 2,2-dimethylpropylcarbonyl, 1-ethylpropylcarbonyl, n-hexylcarbonyl, 1,1-dimethylpropylcarbonyl , 1,2-dimethylpropycarbonyl, 1-methypentylcarbonyl, 2-methylpentylcarbonyl, 3-methylenylcarbonyl, 4-methylpentylcarbonyl, 1,1-dimethylbutylcarbonyl, 1 ^ 2-dimethylbutylcarbonyl, 1,3-dimethylbutylcarbonyl, 2,2-dimethylbutylcarbonyl, 2,3-dimethylbutylcarbonyl, 3,3-dimethylbutylcarbonyl, 1-ethylbutylcarbonyl , 2-ethylbutylcarbonyl, 1, 1, 2-trimethylpropylcarbonyl, 1,2,2-trimethylpropylcarbonyl, 1-ethyl-1-methylpropylcarbonyl, 1-ethyl-2-methylpropylcarbonyl, n-heptylcarbonyl, n-octylcarbonyl, n-nonylcarbonyl or n-decylcarbonyl. Methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, n-butylcarbonyl, i-propylcarbonyl or i-butylcarbonyl are preferred. Alkenylcarbonyl radicals which may be mentioned are branched or unbranched C2-C10 alkenylcarbonyl chains such as, for example, ethenylcarbonyl, propenylcarbonyl, 1-butenylcarbonyl, 2-butenylcarbonyl, 3-butenylcarbonyl, 2-methylpro-penylcarbonyl, 1-pentenylcarbonyl, 2-pentenylcarbonyl, 3-pentenylcarbonyl, 4-pentenylcarbonyl, 1-methyl-1-butenylcarbonyl, 2-methyl-1-butenylcarbonyl, 3-methyl-1-butenylcarbonyl, 1-methyl-2-butenylcarbonyl, 2-methyl-2- butenylcarbonyl, 3-methyl-2-butenylcarbonyl, l-methyl-3-butenylcarbonyl, 2-methyl-3-butenylcarbonyl, 3-methyl-3-butenylcarbonyl, 1, l-dimethyl-2-propenylcarbonyl, 1,2-dimethyl- 1-propenylcarbonyl, 1,2-dimethyl-2-propenylcarbonyl, 1-ethyl-1-propenylcarbonyl, 1-ethyl-2-propenylcarbonyl, 1-hexenylcarbonyl, 2-hexenylcarbonyl, 3-hexenylcarbonyl, 4-hexenylcarbonyl, 5-hexenylcarbonyl, 1-methyl-1-pentenylcarbonyl, 2-methyl-1-pentenylcarbonyl, 3-methyl-1-pentenylcarbonyl, 4-methyl-1-pentenylcarbonyl, 1-methyl-2-pe ntenylcarbonyl, 2-methyl-2-pentenylcarbonyl, 3-methyl-2-pentenylcarbonyl, 4-methyl-2-pentenylcarbonyl, l-methyl-3-pentenylcarbonyl, 2-methyl-3-pentenylcarbonyl, 3-methyl-3-pentenylcarbonyl, 4-methyl-3-pentenylcarbonyl, l-methyl-4-pentenylcarbonyl, 2-methyl-4-pentenylcarbonyl, 3-methyl-4-pentenylcarbonyl, 4-methyl-4-pentenylcarbonyl, 1,1-dimethyl-2-butenylcarbonyl, 1, 1-dimethyl-3-butenylcarbonyl, 1,2-dimethyl-1-butenylcarbonyl, 1,2-dimethyl-2-butenylcarbonyl, 1,2-dimethyl-3-butenylcarbonyl, 1,3-dimethyl-1-butenylcarbonyl, 1,3-dimethyl-2-butenylcarbonyl, 1,3-dimethyl-3-butenylcarbonyl, 2,2-dimethyl-3-butenylcarbonyl, 2,3-dimethyl-1-butenylcarbonyl, 2,3-dimethyl-2-butenylcarbonyl, 2,3-dimethyl-3-butenylcarbonyl, 3,3-di-ethyl-1-butenylcarbonyl, 3,3-dimethyl-2-butenylcarbonyl, 1-ethyl-1-butenylcarbonyl, 1-ethyl-2-butenylcarbonyl, 1-ethyl 3-butenylcarbonyl, 2-ethyl-l-butenylcarbonyl, 2-ethyl-2-butenylcarbonyl, 2-ethyl-3-butenylcarbonyl, 1, 1, 2-trimethyl-2-p carbolyl, l-ethyl-l-methyl-2-propenylcarbonyl, 1-ethyl-2-methyl-l-propenylcarbonyl, l-ethyl-2-methyl-2-propenylcarbonyl, 1-heptenylcarbonyl, 2-heptenylcarbonyl, 3- heptenylcarbonyl, 4-heptenylcarbonyl, 5-heptenylcarbonyl, 6-heptenylcarbonyl, 1-octenylcarbonyl, 2-octenylcarbonyl, 3-octenylcarbonyl, 4-octenylcarbonyl, 5-octenylcarbonyl, 6-octenylcarbonyl, 7-octenylcarbonyl, nonenylcarbonyl or decenylcarbonyl. Ethenylcarbonyl, propenylcarbonyl, butenylcarbonyl or pentemylcarbonyl are preferred. The aryl radicals which may be mentioned are substituted and unsubstituted aryl radicals containing from 6 to 20 carbon atoms in the ring or ring system. The latter may comprise aromatic rings that are fused together or aromatic rings linked by alkyl, alkylcarbonyl, alkenyl or alkenylcarbonyl chains, carbonyl, oxygen or nitrogen. The aryl radicals can, where appropriate, also be linked via the C1-C10 alkyl, C3-C8 alkenyl, C3-C6 alkynyl or C3-C8 cycloalkyl chain to the basic structure. Phenyl or naphthyl is preferred.

The arylcarbonyl radicals which may be mentioned are substituted and unsubstituted arylcarbonyl radicals containing from 6 to 20 carbon atoms in the ring or ring system. The latter may comprise aromatic rings which are fused together or aromatic rings which are linked by means of alkyl, alkylcarbonyl, alkenyl or alkenylcarbonyl chains, carbonyl, oxygen or nitrogen. Phenylcarbonyl or naphthylcarbonyl is preferred. Hetaryl [lacuna] which may be mentioned are single or fused aromatic ring systems substituted or unsubstituted with one or more 3- to 7-membered heteroaromatic rings which may contain one or more heteroatoms such as N, O or S and may, wherein it is appropriate to link via the C ± -C 10 alkyl, C3-C8 alkenyl or C3-C8 cycloalkyl chain to the basic structure. Examples of heteplo radicals of this type are pyrazole, imidazole, oxazole, isooxazole [sic], thiazole, triazole, pyridine, quinoline, isoquinoline, acridine, pyrimidine, pyridazine, pyrazine, phenazine, purine or fteridine. The heteryl radicals can be linked to the basic structure by means of the hetero atoms or by means of various carbon atoms in the ring or ring system or by means of the substituents. Pyridine, imidazole, pyrimidine, punna, pyrazine or quinoline are preferred.

Suitable substituents for the radicals R4 are, for example, one or more substituents such as halogen, such as fluorine, chlorine or bromine, thio [sic], nitro, amino, hydroxyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl or other aromatic or other non-aromatic saturated or unsaturated rings or ring systems. Preference is given to alkyl radicals such as alkyl such as methyl, ethyl, propyl or butyl, halogen such as chlorine, fluorine or bromine, hydroxyl or amino. The radical R4 is preferably hydrogen. R5 in the radical NR4R5 is hydrogen, substituted or unsubstituted, branched or unbranched C1-C10 alkyl, C2-C10 alkenyl, aryl or heteryl, wherein the alkyl, alkenyl, aryl and heteryl radicals have the meanings mentioned above. Preference is given to hydrogen or C1-C10 alkyl such as methyl, ethyl or propyl. Suitable substituents for the radicals R5 are, for example, one or more substituents such as halogen, such as fluorine, chlorine or bromine, thio [sic], nitro, amino, hydroxyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl or other aromatic or other non-aromatic saturated or unsaturated ring system. Preference is given to alkyl radicals such as C 1 -C 6 alkyl such as methyl, ethyl, propyl or butyl, aryl such as phenyl, halogen such as chlorine, fluorine or bromine, hydroxyl or amino.

It is further possible for two adjacent R4 or R5 substituents to form together another substituted or unsubstituted, saturated or partially saturated aromatic ring with 5 to 6 ring atoms which may contain one or more heteroatoms such as O, N or S. It is advantageous for one of the substituents R1, R2 or R3 in formulas I and II be aplo, such as phenyl. It is further preferred for one of the substituents R1, R2 or R3 in the formulas I and II to be hydroxyl and to be one hydrogen or methyl. The process according to the invention is advantageously carried out at a pH of 4 to 11, preferably 4 to It is further advantageous to use 0.01 to 10% by weight of nitrile or 0.01 to 10% by weight of a corresponding aldehyde or ketone and 0.01 to 10% by weight of hydrocyanic acid in the process. The process is advantageously carried out with an excess of hydrocyanic acid. In some circumstances, this leads to hydrocyanic acid contents that are higher than those established. Various amounts of nitrile can be used in the reaction, depending on the nitrile. The smallest amounts (= amounts between 0.01 to [sic] 5% by weight) of nitrile are advantageously used for nitriles (cyanohydrins) which are in equilibrium with the corresponding aldehydes and hydrocyanic acid. Since the aldehyde is usually toxic to microorganisms or enzymes. Also volatile nitriles are advantageously employed in amounts between 0.01 to [sic] 5% by weight. The reaction is delayed with larger amounts of cyanohydrin or nitrile. In the case of nitriles that only have low or virtually no solvent property, or nitriles that dissolve in only very small amounts in aqueous medium, it is possible and advantageous to use larger amounts than those previously established. To increase the conversion and production, the reaction is advantageously carried out with the controlled addition of the racemic nitrile. The product can be isolated after the end of the reaction or continuously removed in a bypass. The above-mentioned appropriate aldehydes or ketones mean compounds that form the nitrile after the reaction between the aldehyde or ketone and the hydrocyanic acid, where it has been appropriately acid catalyzed. The reaction between the aldehyde and the hydroxyanic acid results in cyanohydrin having the advantage that it is in equilibrium with the aldehyde and the hydroxyanic acid. The established equilibrium with cyanohydrin means that it is possible with an enzyme that converts only one enantiomer of the nitrile however to obtain a 100% theory yield because the racemic nitrile is continuously refilled. With all the other nitriles, the nitrile not converted by the enzyme (= "wrong" or another enantiomer) is advantageously racemized by a chemical reaction and is returned to the process to be able to reach a theoretical yield of 100%, or is discarded or purifies and hydrolyzes 5 chemically with retention of the stereocenter. The process according to the invention is advantageously carried out at a temperature between 0 ° C to [sic] 80 ° C, preferably between 10 ° C to [sic] 60 ° C, particularly preferably between 15 ° C to [sic] 50 ° C. The racemic nitriles in the process according to the invention mean nitriles consisting of a 50:50 mixture of the two enantiomers or any other mixture with enrichment of one of the two enantiomers in the mixture. The chiral carboxylic acids in the process of According to the invention means those which show an enantiomeric enrichment. The process preferably results in enantiomeric purities of at least 90% ee, preferably min. 95% ee, particularly preferably min. 98% ee, preferably very particularly of min. 99% us The process according to the invention makes it possible to convert a large number of racemic nitriles into the chiral carboxylic acids. It is possible in the process to convert at least 25 mmoles nitplo / h x mg protein or at least 25 mmoles of nitrile / h x g in dry weight of the microorganisms, preferably at least 30 mmoles of nitrile / hx mg of protein or at least 30 mmoles of nitrile / hxg by dry weight, particularly preferably at least 40 mmoles of nitrile / hx mg of protein or at least 40 mmoles of nitrile / hxg in dry weight, very particularly preferably at least 50 mmoles of nitrile / hx mg of protein or at least 50 mmoles of nitrile / hxg by dry weight. It is possible to use growing cells comprising the nucleic acids, constructs or nucleic acid vectors according to the invention for the process according to the invention. Although latent or disorganized cells can be used. Disorganized cells mean, for example, cells that have been made permeable by a treatment with, for example, solvents, or cells that have been disintegrated by an enzymatic treatment, by a mechanical treatment (for example French press or ultrasound) or by any another method The unrefined extracts obtained in this form are suitable and advantageous for the process according to the invention. Purified or partially purified enzymes can also be used for the process. Immobilized microorganisms or enzymes are equally suitable and can be used advantageously in the reaction. The chiral carboxylic acids prepared in the process according to the invention can be isolated Advantageously, the aqueous reaction solution is extracted or crystallized or extracted and crystallized. For this purpose, the aqueous reaction solution is acidified with an acid such as a mineral acid, (for example, HCl or H2SO4) or an organic acid, advantageously at pH values below 2, and then extracted with an organic solvent. The extraction can be repeated several times to increase the yield. The organic solvents that can be used are in principle all solvents that show a boundary phase with water, where appropriate after the addition of salts. Advantageous solvents are solvents such as toluene, benzene, hexane, methyl tert-butyl ether or ethyl acetate. After concentration of the organic phase, the products can normally be isolated in good chemical purities, meaning a chemical purity of more than 90%. After extraction, the organic phase with the product can, however, also be only partially concentrated, and the product can be crystallized. For this purpose, the solution is advantageously cooled to a temperature of 0 ° C to 10 ° C. The crystallization can also take place directly from the organic solution. The crystallized product can be recovered again in the same or a different solvent for renewed crystallization and can be crystallized once again. The crystallization 2. 2 Subsequent at least one time can, depending on the position of the eutectic composition, further increase the enantiomeric purity of the product. The chiral carboxylic acids can, however, also be crystallized from the aqueous reaction solution immediately after acidification with an acid at a pH advantageously below 2. This advantageously causes the aqueous solution to be concentrated by heating to reduce its volume in 10 to 90%, preferably 80 to 80%, particularly preferably 30 to 70%. The crystallization is preferably carried out with cooling. Temperatures between 0 ° C and [sic] 10 ° C are preferred for crystallization. Direct crystallization of the aqueous solution is preferred for cost reasons. Is It is also preferred to prepare the chiral carboxylic acids by means of extraction and, where appropriate, subsequent crystallization. With these preferred types of processing, the product of the process according to the invention can be isolated in yields of 60 to 100%, preferably 80 to 100%, particularly preferably 90 to 100%, based on the nitrile used for the reaction. The isolated product has a high chemical purity of > 90%, preferably > 95%, particularly preferably > 98%. In addition, the product [sic] has a high enantiomeric purity, which can be further increased by crystallization. The products obtained in this form are suitable as initial material for organic synthesis to prepare drugs or agrochemicals or for racemate resolution. The invention further relates to an isolated nucleic acid sequence encoding a polypeptide having nitrilase activity, selected from the group of: a) a nucleic acid sequence having the sequence depicted in SEQ. FROM IDENT. DO NOT . : 1, b) nucleic acid sequences that are derived from the nucleic acid sequence depicted in SEQ. FROM IDENT. DO NOT. : 1 as a result of the degeneracy of the genetic code, c) derived from the nucleic acid sequence represented in SEC. FROM IDENT. DO NOT . : 1, which encodes polypeptides having the amino acid sequences represented in SEC. FROM IDENT. DO NOT . : 2 and have at least 95% homology at the amino acid level, with negligible reduction in the enzymatic action of the polypeptides. Homologs of the nucleic acid sequence according to the invention with the sequence SEQ. FROM IDENT. DO NOT. : 1 means, for example, allelic variants that have at least 95% homology at the amino acid level derived, Preferably at least "97% homology, preferably very particularly at least 98% homology, over the entire sequence range. It is possible and advantageous for the homologies to be larger over the regions that are part of the sequences. The amino acid sequence derived from SEC. FROM IDENT. NO .: 1 is seen in SEC. FROM IDENT. NO .: 2. The allelic variants comprise, in particular, functional variants that are obtainable by deletion, insertion or substitution of nucleotides of the sequence represented in SEC. FROM IDENT. DO NOT . : 1, and should make a negligible reduction in the enzymatic activity of the synthesized proteins derived for the introduction of one or more genes within organisms nevertheless obtained [sic]. The invention thus also refers to sequences of amino acids that are encoded by the group of nucleic acid sequences described above. The invention advantageously relates to amino acid sequences encoded by the sequence SEQ. FROM IDENT. NO .: 1. SEC counterparts. FROM IDENT. DO NOT . : 1 too means, for example, fungal or bacterial homologs, single-stranded DNA or RNA truncated sequences of the DNA sequence encoding or not encoding. SEC counterparts. FROM IDENT. DO NOT . : 1 have at the DNA level a homology of at least 60%, preferably at least 70%, particularly Preferably at least 80%, preferably very particularly of at least 90%, over the entire DNA region indicated in SEQ. FROM IDENT. NO .: 1. SEC counterparts. FROM IDENT. DO NOT . : 1 additionally means derivatives such as, for example, promoter variants. The promoters that precede the established nucleotide sequences can be modified by one or more nucleotide exchanges per insertion (s) and / or deletion (s), however adversely affecting the functionality or effectiveness of the promoters. The promoters can also have their effectiveness increased by modifying their sequence or be completely replaced by more effective promoters even from organisms of different species. Derivatives also means variants whose nucleotide sequences in the region of -1 to -200 opposite the start codon or 0 to 1000 base pairs after the high codon have been modified in such a way that the expression of the gene y / or protein expression is altered, preferably increased. The SEC. FROM IDENT. DO NOT . : 1 or its homologs can be advantageously isolated by methods known to the worker skilled in the art from bacteria, preferably from Gram-negative bacteria, preferably particularly from bacteria of the genus Alcaligenes, preferably very particularly from bacteria of the genus and species Alcaligenes faecalis.

The SEC. FROM IDENT. DO NOT . : 1 or its homologs or parts of these sequences can be isolated from other fungi or bacteria for example using conventional hybridization processes or the PCR technique. These DNA sequences hybridize under standard conditions with the sequences according to the invention. Hybridization is preferably carried out with short oligonucleotides from the conserved regions, for example from the active center, and these can be identified in a manner known to the skilled worker by comparisons with other nitrile or nitrile hydratase. However, it is also possible to use longer fragments of the nucleic acids according to the invention or the complete sequences for hybridization. These standard conditions vary depending on the nucleic acid oligonucleotide [sic] used, the longer fragment or complete sequence, or depending on what type of nucleic acid, DNA or RNA, is used [sic] for hybridization. Thus, for example, the fusion temperatures of the DNA: DNA hybrids are about 10 ° C lower than those of the DNA: RNA hybrids of the same length. Standard conditions mean, for example depending on the nucleic acid temperatures between 42 and 58 CC in an aqueous buffer with a concentration between 0.1 to [sic] 5 x SSC (1 X SSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.2 ) or additionally in the presence of 50% formamide, such as, for example, 42 ° C in 5 x SSC, 50% formamide. Hybridization conditions for DNA: DN hybrids advantageously comprise 0.1 x SSC and temperatures between about 20 ° C to [sic] 45 ° C, preferably 5 between about 30 ° C to [sic] 45 ° C. Hybridization conditions for the DNA: RNA hybrids preferably comprise 0.1 x SSC and temperatures between about 30 ° C to [sic] 55 ° C, preferably between about 45 ° C to [sic] 55 ° C. These temperatures The established for the hybridization are melting temperatures calculated by way of example for a nucleic acid with a length of approximately 100 nucleotides and a G + C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization are described in relevant textbooks of genetics, such as, for example, Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be calculated by formulas known to the skilled worker, for example depending on the length of the acids nucleic acids, the nature of hybrids or content content of G + C. The skilled worker can find additional information on hybridization in the following textbooks: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Hames and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press k. ^^. . ^ -JS ^ s.j & faith. »« & Ñ ^ r * and £ £ & ^ at Oxford University Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford. The nucleic acid construct according to the invention means the sequence nitrilase gene. SEC. FROM IDENT. NO. 1 and its homologs, which have been advantageously functionally linked to one or more regulatory signals to increase gene expression. These regulatory sequences are, for example, sequences for which the inductors or repressors bind and thus regulate the expression of the nucleic acid. In addition to these new regulatory sequences, it is also possible for the natural regulation of these sequences to be present in front of the current structural genes and, where appropriate, have been genetically modified so that the natural regulation is switched off and the expression of the genes has been increased. The nucleic acid construct can, however, also have a simpler structure, ie no additional regulatory signals have been inserted in front of the sequence SEC. FROM IDENT. NO .. 1 or its counterparts, and the natural promoter with its regulation has not been erased. In contrast, the natural regulatory sequence is mimicked in such a way that regulation no longer takes place, and the expression of the gene is increased. The nucleic acid construct can additionally advantageously comprise one or more sequences * «Improver JEfea, which makes possible the increased expression of the nucleic acid sequence, functionally linked to the promoter. It is also possible to insert advantageous original sequences at the 3 'end of the DNA sequences, such as other regulatory or terminating elements. The nucleic acids according to the invention can be present in one or more copies in the construction. The construct may also further comprise markers such as antibiotic resistance or genes that complement auxotrophy where appropriate for the selection of the construct. Advantageous regulatory sequences for the process according to the invention are, for example, present in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3, gal , trc, ara, SP6,? -PR or the? -PL promoter, which are advantageously used in Gram-negative bacteria. Further advantageous regulatory sequences are in, for example, the Gram positive promoters amy and SP02, in the promoters of fungus or yeast ADCl, MFa, AC, P-60, CYCl, GAPDH, TEF, rp28, ADH. Also advantageous in this connection are the carboxylase pyruvate and methanol oxidase promoters of, for example, Hansenula. It is also possible to use artificial promoters for regulation.

The nucleic acid construct is advantageously inserted into a vector such as, for example, a plasmid, phage or other DNA for expression in a host organism. Which makes optimal expression of the 5 genes in the possible host. These vectors represent a further development of the invention. Examples of suitable plasmids in E. Coli are pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-Bl,? Gtll or pBdCI, in Streptomyces are pIJIOl, pIJ364, pIJ702 or pIJ361, in Bacillus are pUBUO, pC194 or pBD214, in Corynebacterium are pSA77 or pAJ667, in fungi are pALSl, pIL2 or pBBllβ, in yeasts are 2uM, pAG-1, YEp6, YEpl3 or pEMBLYe23 or in Vegetables are pLGV23, pGHlac +, pBIN19, pAK2004 or pDH51. These plasmids represent a small selection of possible plasmids. Additional plasmids are well known to the skilled worker and can be found, for example, in the book Cloning Vectors (eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). The nucleic acid construct also advantageously further contains, for the expression of the other genes present, 3 'and / or 5' terminal regulatory sequences, to increase expression, which are selected for optimal expression depending on the host organism and gene. or selected genes.

These regulatory sequences are tried to make possible the specific expression of the genes and of [sic] the expression of the protein. This may mean, for example depending on the host organism, that the gene is expressed or overexpressed only after induction, or that it is expressed and / or overexpressed immediately. Regulatory sequences or factors may also positively influence, and thus increase, the expression of the introduced genes. So, the improvement of the regulatory elements can advantageously take place at the level of transcription, using strong transcription signals such as promoters and / or enhancers. However, it is also possible to improve translation, for example, by improving the stability of mRNA mixtures. In another embodiment of the vector, the vector comprising the nucleic acid construct according to the invention or the nucleic acid according to the invention can also be advantageously introduced into the form of a linear DNA within the microorganisms and integrated by heterologous or homologous recombination within the genome of the host organism. This linear DNA may consist of a linearized vector such as a plasmid or solely of the nucleic acid or nucleic acid construct. For optimal expression of heterologous genes in the Organism, it is advantageous to modify the acid sequences nucleic to agree with the use of the codon specifically used in organisms. The use of the codon can be easily established on the basis of computer analysis or other known genes in the relevant organism. Suitable host organisms for the nucleic acid according to the invention or the construction of nucleic acid are in principle all prokaryotic or eukaryotic organisms. Advantageously used host organisms are microorganisms such as bacteria, fungi or yeasts. It is advantageous to use Gram positive or Gram negative bacteria, preferably bacteria of the family Enterobacteriaceae or Nocardiaceae, preferably particularly bacteria of the genera Escherichia, Pseudomonas or Rhodococcus. Particular preference is given to the genus and species Escherichia coli. The host organism according to the invention further comprises preferably at least one proteinaceous agent to bend the polypeptides it has synthesized and, in particular, the nucleic acid sequences having nitrilase activity described in this invention and / or the genes encoding this agent , the amount of this agent present being greater than that corresponding to the basic amount in the microorganism considered. The genes that code for this agent are present on the chromosome or in JíUi sákZ extrachromosomal elements such as, for example, plasmids.

EXAMPLES Example 1: Purification of the nitrilase of Alcaligenes faecalis 1650 1. Production of the cells Alcaligenes faecalis 1650 was cultured with agitation in culture medium A at 30 ° C for a period of 8 hours.

Culture medium A: Yeast extract 5 g / 1 Peptone 3.5 g / 1 KH2PO4 5 g / 1 MgSO4 0.2 g / 1 FeSO4 0.03 g / 1 NaCl 1 g / 1 Butyronitrile 1 g / 1 200 ml of this preculture was used to inoculate a 10 1 burner containing 8 1 of fresh A medium.

The pH, the temperature, the air flow rate and the agitation speed were 7.2, 30 ° C, 300 1 / h and 300 rpm. After 22 h, 81 g of wet biomass were obtained. This corresponds to a dry cell weight of 3.8 g / 1 and an optical density at 600 nm of 8. 2. Determination of the enzymatic activity for mandelonitrile The cells were obtained as described in Example 1 and washed twice in 10 mM Na / K phosphate buffer, pH 7.2. 40 mg dry weight of cells were resuspended in 20 ml of 10 M Na / K phosphate buffer, and pH 6.8, and the reaction was started by adding 8.3 mM mandelonitrile. The reaction was carried out at 40 ° C with stirring. The kinetics of racemate resolution were followed by sampling and subsequently removing cells with the aid of high performance liquid chromatography (ODS Hypersil). In this case they determined mandelonitrilo, benzaldehyde, mandela ida and mandelic acid. The results are shown in Figure 1 [conversion of mandelonitrile to mandelic acid, batch]. The rate of mandelic acid formation is 41.3 U / g dry weight of cells with 30% conversion, wherein 1 U is defined as the formation of 1 μmol of mandelic acid per minute at 40 ° C. 3. Determination of the enzymatic selectivity for mandelonitrile The cells were obtained as described in Example 1 and rinsed twice in 10 mM Na / R phosphate buffer, pH 7.2. 40 mg dry weight of cells were resuspended in 20 ml of 10 mM Na / K phosphate buffer, pH 6.8, and the reaction was started by adding 8.3 mM mandelonitrile. The reaction was carried out with stirring at 30 ° C. The kinetics were followed by taking samples and subsequently removing cells with the help of high performance liquid chromatography (Nucleodex ß-PM). S - (+) - and R - (-) mandelic acid was determined in this case. The optical purity of the formed R - (-) - mandelic acid (eeR-MA) was 98% at 50% conversion. The selectivity of the enzyme (= E) was 499 at 50% conversion. 4. Purification Unless otherwise indicated, 10 mM DTT was present in all regulators during purification.

Step 1: Cell disruption The cells were obtained as described in Example 1 of two fermentations of 10 1 in each case, and the rotation was stopped and washed twice with 1 1 of regulator 0.1 M Tris / HCl, pH 7.2. The yield was approximately 162 g in wet weight of cells. In each case 81 g of cells were resuspended by wet weight in 160 ml of 0.1 M Tris / HCl buffer, pH 7.2, and disorganized four times in a Menton-Gaulin [sic] under 750 bar. The homogenate was then centrifuged at 30,000 g for 39 min, and the pellet was discarded. The supernatant (140 ml) had a remaining activity of 73%, as shown in Table 1.

Step 2: Ion exchange chromatography The supernatant was diluted to 400 ml with buffer A (20 mM Tris / HCl, pH 8.5) and centrifuged once more at 23,000 g for 20 min. 350 ml were then loaded onto a column (diameter 5 cm, height 22 cm, volume 432 ml, Q-Sepharose Fast Flow from Pharmacia) in regulator A. Initially 10% regulator B (as regulator A with 1 M NaCl) was used to wash at a flow rate of 20 ml / min (Total load and wash volume corresponded to 1.5 1).

The relationship was increased to 60% B linearly during the course of 90 min. 100% regulator B was then used to wash from 91 to 120 min. 100 fractions of 40 ml were collected. Nitrilase eluted between fractions 50 and 60. The fractions were combined and concentrated to a volume of 10 ml by ultrafiltration through a 10 kDa membrane (Amicon).

Step 3: Molecular bed chromatography The ion exchange chromatography concentrate (step 2) was further purified in two portions each of 5 ml by molecular-bed chromatography.

(Superdex 200 prep grade, Pharmacia, separation degree from 10 to 600 kDa, diameter 2.6 cm, height 60 cm, volume 325 ml).

The detection took place at 280 nm. The column is equilibrated in 20 mM phosphate buffer, pH 7.4, 5 mM DTT and 150 mM NaCl and was operated with a flow rate of 1.5 ml / min. 40 fractions were collected. The activity that hydrolyzes nitrile was found in fractions 3 to 5.

Step 4: Ion exchange chromatography The combined fractions of the molecular-bed chromatography (step 3) were further purified by ion exchange chromatography on a Mono Q column. (column volume 1 ml, Mono Q HR515, Pharmacia). The regulator A used was 20 mM Tris / HCl, pH 8.5, 5 mM DTT, and regulator B was the same regulator as in A with 1 M of NaCl The flow rate was 1 ml / min. The active fraction of the molecular-bed chromatography (approximately 100 ml) was diluted to a conductivity of about 6 mS / cm and loaded directly onto the Mono Q column, and the protein was thus absorbed. The column was washed with 5% regulator B after loading. The column was eluted with a gradient of 5% to 40% B in 30 min, followed by 100% B for 10 minutes. Nitrilase eluted in fractions 17 and 18 of the gradient. Steps 1-4 of the purification are shown in Table I.

Table I: Purification scheme ^ s ^ J ^ '-.

The active fractions (= AF, Table I) of the molecular-bed chromatography (step 3) and the ion exchange chromatography on Mono Q (step 4) were fractionated by SDS-PAGE as shown in Figure 2.

Stage 5: High reverse phase (lacuna) liquid chromatography (RP) The active fraction (fractions 17 and 18) of the Mono Q chromatography (step 4) was checked by homogeneity by RP chromatography and further purified to prepare for tripsiona cleavage . The separation was carried out with an Abi ed column (3 cm) on a Hewlett-Packard apparatus (HP 1090). The mobile phase used was regulator A: water with 0.1% TFA and regulator B: acetonitrile with 0.1% TFA. Volume injected 0.1 ml, flow rate 0.5 ml / min. The elution gradient had the following profile: Nitrilase eluted between 12 and 13 minutes, this corresponds to a band of 37 kDa in the SDS-PAGE. This band was partially sequenced using the Applied Biosystems 494 Procise protein sequencer. The N-ter sequence of 39 amino acids obtained in this form is referred to as SEC. FROM IDENT. DO NOT. : 3 later. The sequence is included in the attached list of sequences and is: Met Gln Thr Arg Lys lie Val Arg Ala Ala Ala Gl Gln Ala Ala Ser Pro Asn Tyr Asp Leu Wing Thr Gly Val Asp Lys Thr lie Glu Leu Wing Arg Gln Wing Arg Asp Glu Gly.

Preparation of tryptic peptides The Mono Q chromatography sample (step 4) was pretreated as follows: the protein (approximately 0.6 mg) was • precipitated with 12.5% TCA and the pellet was washed three times with 1 ml of ether / ethanol (1: 1). The pellet was dissolved in 0.2 ml of 1 ml of ether / ethanol (1: 1). The pellet was dissolved in 0.2 ml of 6M guanidine HCl, 25 mM tris / HCl, pH 8.5. 2.6 μl of a 1 M solution of DTT was added to this solution to reduce the disulfite bridges [sic]. The sample was shaken in the dark for 1 hour. The protein was then reacted with 1.5 μl of a solution of 4-vinylpyridine (35%) in the dark for 2 hours. The reaction was stopped by incubating with 2.6 μl of a 1 M solution of DTT for 1 hour. The vinyl pyrillete enzyme [sic] was purified by RP-HPLC as described above. The retention time was now between 10 and 12 minutes. The active fraction, identified by its molecular weight, was collected and concentrated to 0.02 ml. This was adjusted to 0.2 ml by adding 0.01 ml of acetonitrile and 0.1 M of Tris / HCl, pH 8.5. The pH was corrected by also adding approximately 0.05 ml of 0.1 M NaOH. The sample (estimated amount of protein 0.3 mg) was mixed with 0.032 ml of a solution of 1 mg / ml trypsin in 0.1 M Tris / HCl, pH 8.5, 5% acetonitrile, and incubated at 37 ° C overnight. The digestion was stopped with 0.01 ml of acetic acid, followed by centrifugation. The supernatant was separated by RP-HPLC over C18 (eluent system: regulator A: water, 0.1% TFA, regulator B: acetonitrile, 0.1% TFA). The peptides (detection at 205 nm and 280 nm) were collected and sequenced. The Applied Biosystems 494 Procise protein sequencer was used. The inteRNA peptide sequence of 21 amino acids is ^ j ^ jl ^ i ^^ s ^ &HÉ referred to below as SEC. FROM IDENT. DO NOT . : 4 and the inteRNA peptide sequence of 11 amino acids is referred to as SEC. FROM IDENT. DO NOT. : 5. The SEC. FROM IDENT. DO NOT. : 4 and 5 are included in the attached list of sequences and are: SEC. FROM IDENT. DO NOT. : 4 Glu Glu Pro Wing Glu Gln Gly Val Gln Ser Lys He Wing Ser Val Ala He Ser His Pro Gln SEC. FROM IDENT. DO NOT . : 5 Glu Glu Pro Wing Glu Gln Gly Val Gln Ser Lys 6. Activity of the purified nitrilase for mandelonitrile The activity of the purified nitrilase for mandelonitrile was investigated as described in Example 2. The specific activity of the purified protein for mandelonitplo was 12,380 U / g of protein.

Example 2: Cloning of the nitrilase from Alcaligenes faecalis 1650 Nucleotide probes were derived from the peptide sequences SEC. FROM IDENT. DO NOT. : 3 and 4 described in Example 1 and were synthesized. The nucleotide probe derived from SEC. FROM IDENT. DO NOT. : 3, the N-terminal peptide sequence was a 23 mer 64-fold degenerate (in the sequence of the nucleotide probe, A, C, G or T are replaced by N; A or G by R; C or G by S ). The tall ^^^ percentage of GC in the Alcalifenes strains described in the literature (Wade et al., 1992, Nucí Acids Res., 20, 2111-2118) meant that in the case of glutamine and isoleucine the selection of the Third codon position is predetermined. The nucleotide probe, which is referred to below as SEC. FROM IDENT. DO NOT. : 6, is the 5 'primer, for the subsequent PCR, where S = C or G and N = A, C, G or T, and is: SEC. FROM IDENT. DO NOT . : 6 5 '-ATGCAGACNAGNAARATCGTSCG-3' A 20 mer 256-fold degenerate was derived as a nucleotide probe from the SEC. FROM IDENT. NO .: 4, the peptide sequence inteRNA (in the sequence of the nucleotide bases, A, C, G or T is replaced by N; A or G by R; C or G by S). The high percentage of GC in the Alcaligenes strains means that in the case of lysine the selection of the third position of the codon was predetermined. This nucleotide probe is the 3 'primer for the subsequent PCR and is referred to below as SEC. FROM IDENT. NO .: 7. It is included in the attached list of sequences and is: SEC. FROM IDENT. DO NOT . : 7 5 '-TNGCSACNGANGCRATCTTG-3' This pair of primers, SEC. FROM IDENT. DO NOT . : 6 and 7, was used to carry out the PCR on chromosomal DNA of Alcaligenes faecalis 1650. The isolation of chromosomal DNA took place after oß ??; cell lysis treatment with lysosim and proteinase K by the classical method known to the skilled worker (Ausubel, F. M. et al. (1994) Current protocols in molecular biology, John Wiley and Sons). 5 PCR using Pwo polymeric comprised denaturation at 95 ° C for 3 min .; 35 cycles with denaturation at 95 ° C for 1 min, quenching of the primer at 58 ° C for 1 min 30 sec and polymerization at 72 ° C for 1 min 30 sec; and a polymerization concluding at 72 ° C during 5 min Under these conditions, a fragment of approximately 1 kb in size was amplified from the chromosomal DNA from Alcaligenes faecalis 1650. To clone the PCR product, a restriction cleavage site Xbal and two additional nucleotides (5'-AATCTAGA and 5'-ATTCTAGA) were attached to each of the primers mentioned above, and the PCR reaction was repeated under the above-mentioned conditions. Once again there was amplification of a fragment of approximately 1 kb of size, which after purification and Xbal digestion, was ligated into pUC18 analogously digested. After transformation of E. coli JM109 and isolation of the resulting plasmid, the DNA was purified by sequencing and subsequent genomic Southern staining. The methods of molecular and microbiological biology for the isolation of complete nitrilasa gene (nit) took place by classical methods [sic] known to the skilled worker. The complete nitrilase sequence is represented in the SEC. FROM IDENT. DO NOT.: 1.

Example 3: Homology with other proteins, identification of the homologous sequence Comparison with the sequences of the protein database SWISSPROT showed that the nitrilase gene in this invention has 11 to 96% homology with the known nitrilalas at the amino acid level. The largest sequence of homologies was found with the aryl acetonitrile-specific nitrilase of Alcalignes [sic] faecalis JM3 (Nagasawa et al., Eur. J. Biochem, 1990, 194, 765-772). The two nitrilase genes have a 93.2% identity at the nucleotide level over a region of 1071 bp. The derived amino acid sequence has a 96.1% identity over a region of 356 amino acids. The smallest homology of 11.4% over a region of 534 amino acids was found with Rhodococcus erythropolis SK92 nitrilase (EP-A-0 719 862). Example 4: Heterologous expression of nitrilase in E. coli The nit gene was amplified for cloning into the expression vector pJOE2702. The 5 'primer selected in this case for PCR was the aforementioned SEC. FROM IDENT. DO NOT. : 3, with a Ndel splitting site with overlaps with the translation start being joined at the 5 'end. This primer is referred to below as SEC. FROM IDENT. DO NOT. : 8 and is included in the attached list of sequences. The selected 3 'primer was a 24 mer of the 3' region of the nit gene, being linked with cleavage sites BamHI [sic] adjacent to the stop codon. It is referred to later as SEC. FROM IDENT. DO NOT. : 9 and is included in the subsequent list of sequences. 5 '-TTAATCATATGCAGACAAGAAAAATCGTCCG-3, (= SECTION DE IDENT.NO .: 8) 5' -AAGGATCCTCAAGACGGCTCTTGCACTAGCAG-39 (= SEC. ID. NO .: 9) PCR using Pwo polymerase comprised a denaturation at 94 ° C for 3 min .; 25 cycles with a denaturation at 93 ° C for 1 min, a primer quenching at 55 ° C for 1 min 30 sec and a polymerization at 72 ° C for 1 min 30 sec, and a final polymerization at 72 ° C for 5 min . The resulting PCR fragment was purified, digested with Ndel / BamHI and integrated into the analogously digested vector pJOE2702 (Volff et al., 1996, Mol.Microbiol., 21 (5), 1037-1047). The resulting plasmid was called pDHE 19.2 and is represented in Figure 3. The integration by means of the Ndel / BamHI cleavage sites means that in the plasmid pDHE19.2 the nit gene is under transcriptional control of the rhap promoter which is present in pJOE2702 and originates from the positively regulated rhaBAD L-rhamnose in E. coli (Egan &Schleif, 1994, J. Mol. Biol., 243, 821-829). The termination of the transcription of the nit gene and the initiation of translation also takes place by means of vector sequences. In addition, the plasmid contains a gene that confers resistance to Ampicillin ApR. The heterologous expression of the nitrilase is shown with the E. coli strain JM109 which contains a plasmid pDHE19.2. For this purpose, strain JM109 (pDHE19.2) was cultured in TB culture medium with 100 μg / ml ampicillin (Tartof, Hobbs 1987 [sic] with shaking at 37 ° C. At an OD600 of 1.7, the culture was transferred 1: 200 into the fresh TB medium which contains 0.2% (w / v) L-rhamnose to induce the nitrilase, and was cultivated with shaking at 30 ° C. After 8 hours, the cells were harvested, washed with 10 mM Na / K phosphate buffer, pH 7.2, resuspended in the same regulator at an OD600 of 10, and then disrupted [sic] of the treatment with ultrasound. Example 5: Determination of the nitrilase activity of the recombinant strain [sic] E. coli JM109 (pDHE19.2) 1. Production of the cells E. coli JM109 (pDHE19.2) was cultured in TB medium + 100 μg / ml of ampicillin with agitation at 37 ° C for 6 hours. At an OD600 of 4, 100 ml of this preculture was used to inoculate a 10 1 burner containing 8 1 of fresh TB medium + 100 μg / ml of ampicillin + 2 g / l of L-rhamnose. He JAÉ ^ ..-. ^ A ^ ¿. ^ ^ 1 pH, temperature, l., Air flow rate and stirring speed were 7.2, 30 ° C, 300 1 / h and 400-650 rpm. The cells were harvested after 16 hours. The optical density at 600 nm at this time was 18, which corresponds to a dry cell weight of 7.8 g / l. 2. Determination of the specific activity for mandelonitrile The cells were obtained as described in Example 1 and washed in 10 mM Na / K phosphate buffer, pH 7.2. 2 mg dry weight of cells were resuspended in 1 ml of 10 mM Na / K phosphate buffer, pH 7.2, and the reaction was started by adding 8.3 mM mandelonitrile. The reaction was carried out with stirring at 40 ° C. The kinetics were followed by taking samples and subsequent high performance liquid chromatography (ODS Hypersil). Mandelonitrile, benzaldehyde, mandelamide and mandelic acid were determined. The rate of formation of mandelic acid is 403 U / g dry weight of cells with a conversion of 30%, 1 U being defined as the formulation of 1 μmol of mandelic acid per minute at 40 ° C.

Example 6: Synthesis of R-mandelic acid by hydrolysis of mandelonitrile using E. coli JM109 (pDHE19.2) in suspension Mandelonitrile in a concentration of 1.3 g / l was measured over the course of 10 hours within a volume of 1 1 10 M Na / K phosphate buffer, pH 7.2, which contained the strain E. coli JM109 (pDHE19.2) in a concentration of 2 g / 1 while stirring with a propeller stirrer at 40 ° C. The measurement was controlled by the nitrile consumption. The R-mandelic acid consumption rate was followed as described in Example 5. The results are shown in Figure 4. Example 7: Isolation of R-mandelic acid by extraction of the reaction mixture from the hydrolysis of mandelonitrile by E. Coli [sic] JM109 (pDHE19.2) in suspension. The aqueous mandelic acid reaction mixture obtained in Example 6 was centrifuged to remove the cells, the pH was adjusted to 2 as an acid and extracted three times with methyl tert-butyl ether (MTBE). After removal of the organic solvent from the mandelic acid extract by evaporation, the resulting white crystals of mandelic acid were redissolved and investigated for chemical and optical purity by high performance liquid chromatography. The chemical purity was 99%, and the optical purity of R-mandelic acid was 97.4% ee.

I -L Example 8: Isolation of R-mandelic acid by crystallization with cooling from the reaction mixture of mandelonitrile hydrolysis by E. Coli [sic] JM109 (pDHE19.2) in suspension. The aqueous mandelic acid reaction mixture obtained in Example 6 was centrifuged to remove the cells, concentrated to 40% of the initial volume with heating and stirring and adjusted to pH 2 with an acid. The mandelic acid was crystallized upon cooling in an ice bath, and the resulting white crystals of mandelic acid were filtered off with suction and dried. The crystals were redissolved and investigated for chemical and optical purity by high performance liquid chromatography. The chemical purity was 99.1%, and the optical purity of R-mandelic acid was 99.8% ee.

Example 9: Conversion of various nitriles The E. coli strain (see Example 6) or the initial strain Alcaligenes was used to convert various nitriles. Alcaligenes cells were cultured in 400 ml of Alcaligenes medium (see media A above) at 30 ° C and 160 rpm for 16 hours (= h) [sic]. The cells were harvested by centrifugation (4 ° C and 5000 rpm, 30 min). Portions of 150 μl of a cell suspension were pipetted into each Sis- '- y-frt ... ytí .- &&«- ^ fc.ji = faith of the wells of the microtitre plate. The plate was then centrifuged. The supernatant was aspirated and the cell pellets were washed twice with Na2HP04 (1.42 g / l in Finnaqua, pH 7.2). The substrate solution (150 μl) was then pipetted, and the cells resuspended. One substrate was added to each row 12 holes on the microtiter plate. A row with the substrate solution but no cells was used as control (= white). The microtiter plates were left in an incubator with shaking at 200 rpm and 30 ° C for 2 hours. The centrifugation of cells was then stopped and the amount of NH 4 ions produced in the supernatant was determined using a Biomek apparatus. The measurement took place at 620 nm using a calibration chart constructed with various NH40H solutions (see Figure 5). The substrates used were mandelonitrile (= 1), 2-phenylpropionitrile (= 2), 2-phenylbutyronitrile (= 3), benzyl cyanide (= 4), 4-chlorobenzyl cyanide (= 5), 4-bromobenzyl cyanide (= 6), propionitrile (= 7) , 2-methylbutyronitrile (= 8,2-cyanobutane), geranonitrile (= 9), valeronitrile (= 10), 3-cyanopyridine (= 11), 3-biphenyl-2-hydroxybutyronitrile [sic] (= 12), 4-flourobenzyl cyclic [sic] (= 13, 4-fluorophenylacetro-nitrile [sic]) and a- (3-heptyl) -nitro-tpacetonitrile (= 14). A 0.2 molar standard solution in methanol was made for each of the substrates, and this was diluted to 10 mM with Na2HP04 (1.42 g / 1 in Fmnequa, pH 7.2). Cell suspensions were standardized at 2 g / 1 dry biomass Table II shows the means for a microtitre plate row in the conversion Table II: Conversion of various nitriles with nitrilase 1650 Figure 6 shows the results of the conversion as activities.

G ^ | igM ^^ LIST (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME-: BASF A ktie ng that 11 s ch aft (B) STREET: C ar 1 - B os ch - S trasse 38 (C) CITY: Ludwigshafen (D) ) STATE: Rheinland Palatinate (E) COUNTRY: Federal Republic of Germany (ii) TITLE OF THE APPLICATION: METHOD TO PRODUCE CARBOXYLIC ACIDS QUIRALES FROM NITRILE WITH THE ASSISTANCE OF A NITRILASE OR MICROORGANISMS CONTAINING A GENE FOR NITRILASE (iü) NUMBER OF SEQUENCES: 9 (iv) READING FORM ON THE COMPUTER: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: compatible with an IBM PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.25 (EPO) (2) INFORMATION FOR SEC. FROM IDENT. NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1071 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear n) TYPE OF MOLECULE: DNA (genomic) ni) HYPOTHETICAL: NO m ) [sic] ANTICIPATION: NO) ORIGINAL SOURCE: (A) ORGANIZATION: Alcaligenes faecalis (B) CEPA: 1650 ix) FEATURES: (A) NAME / KEY: CDS (B) LOCATION: 1. .1071 xi DESCRIPTION OF THE SEQUENCE SEC OF IDENT. NO: 1: ATG CAG ACÁ AGA AAA ATC GTC CGG GCA GCC GCC GTA CAG GCC GCC TCT 48 Met Gln Thr Arg Lys He Val Arg Ala Wing Wing Val Gln Wing Wing Ser 1 5 10 15 ccc AAC TAC GAT CTG GCA ACG GGT GTT GAT AAA ACC ATT GAG CTG GCT 96 Pro Asn Tyr Asp Leu Wing Thr Gly Val Asp Lys Thr He Glu Leu Wing 20 25 30 CGT CAG GCC CGC GAT GAG GGC TGT GAC CTG ATC GTG TTT GGT GAA ACC 144 Arg Gln Wing Arg Asp Glu Gly Cys Asp Leu He Val Phe Gly Glu Thr 35 40 45 TGG CTG CCC GGA TAT CCC TTC CAC GTC TGG CTG GGC GCA CCG GCC TGG 192 Trp Leu Pro Gly Tyr Pro Phe His Val Trp Leu Gly Wing Pro Wing Trp 50 55 60 TCG CTG AAA TAC AGT GCC CGC TAC TAT GCC AAC TCG CTC TCG CTG GAC 240 Ser Leu Lys Tyr Ser Wing Arg Tyr Tyr Wing Asn Ser Leu Ser Leu Asp 65 70 75 80 AGT GCA GAG TTT CAA CGC ATT GCC CAG GCC GCA CGG ACC TTG GGT ATT 288 Be Wing Glu Phe Gln Arg He Wing Gln Wing Wing Arg Thr Leu Gly He 85 90 95 TTC ATC GCA CTG GGT TAT AGC GAG CGC AGC GGC GGC AGC CTT TAC CTG 336 Phe He Wing Leu Gly Tyr Ser Glu Arg Ser Gly Gly Ser Leu Tyr Leu 100 105 110 GGC CAA TGC CTG ATC GAC GAC AAG GGC GAG ATG CTG TGG TCG CGT CGC 384 Gly Gln Cys Leu He Asp Asp Lys Gly Glu Met Leu Trp Ser Arg Arg 115 120 125 AAA CTC AAA CCC ACG CAT GTA GAG CGC ACC GTA TTT GGT GAA GGT TAT 432 Lys Leu Lys Pro Thr HlS Val Glu Arg Thr Val Phe Gly Glu Gly Tyr 130 135 140 GCC CGT GAT CTG ATT GTG TCC GAC ACA GAA CTG GGA CGC GTC GGT GCT 480 Wing Arg Asp Leu He Val Being Asp Thr Glu Leu Gly Arg Val Gly Wing 145 150 155 160 CTA TGC TGC TGG GAG CAT TTG TCG CCC TTG AGC AAG TAC GCG CTG TAC 528 Leu Cys Cys Trp Glu His Leu Ser Pro Leu Ser Lys Tyr Wing Leu Tyr 165 170 175 TCC CAG CAT GAA GCC ATT CAC ATT GCT GCC TGG CCG TCG TTT TCG CTA 576 Ser Gln His Glu Wing He HlS Wing Wing Trp Pro Ser Phe Ser Leu 180 185 190 TAC AGC GAA CAG GCC CAC GCC CTC AGT GCC AAG GTG AAC ATG GCT GCC 624 Tyr Ser Glu Gln Wing His Wing Leu Ser Wing Lys Val Asn Met Wing Wing W TCG CAA ATC TAT TCG GTT GAA GGC CAG TGC TTT ACC ATC GCC GCC AGC 672 Ser Gln He Tyr Ser Val Glu Gly Gln Cys Phe Thr He Wing Wing Ser 210 215 220 AGT GTG GTC ACC CAG GAG ACG CTA GAC ATG CTG GAA GTG GGT GAA CAC 720 Val Val Thr Gln Glu Thr Leu Asp Met Leu Glu Val Gly Glu His 225 230 235 240 AAC GCC CCC TTG CTG AAA GTG GGC GGC GGC AGT TCC ATG ATT TTT GCG 768 Asn Wing Pro Leu Leu Lys Val Gly Gly Gly Ser Ser Met He Phe Wing 245 250 255 CCG GAC GGA CGC AC CTG GCT CCC CT CTG CCT CAC GAT GCC GG GGC 816 Pro Asp Gly Arg Thr Leu Wing Pro Tyr Leu Pro His Asp Wing Glu Gly 260 265 270 TTG ATC ATT GCC GAT CTG AAT ATG GAG GAG ATT GCC TTC GCC AAA GCG 864 Leu He He Wing Asp Leu Asn Met Glu Glu He Wing Phe Wing Lys Wing 275 280 285 ATC AAT GAC CCC GTA GGC CAC TAT TCC AAA CCC GAG GCC ACC CGT CTG 912 He Asn Asp Pro Val Gly His Tyr Ser Lys Pro Glu Wing Thr Arg Leu 290 295 300 GTG CTG GAC TTG GGG CAC CGA GAC CCC ATG ACT CGG GTG CAC TCC AAA 960 Val Leu Asp Leu Gly His Arg Asp Pro Met Thr Arg Val His Ser Lys 305 310 315 320 AGC GTG ACC AGG GAA GAG GCT CCC GAG CAG GGT GTG CAA AGC AAG ATT 1008 Ser Val Thr Arg Glu Glu Ala Pro Glu Gln Gly Val Gln Ser Lys He 325 330 335 GCC TCA GTC GCT ATC AGC CAT CCA CAG GAC TCG GAC ACA CTG CTA GTG 1056 Wing Ser Val Wing He Ser His Pro Gln Asp Ser Asp Thr Leu Leu Val 340 345 350 CAA GAG CCG TCT TGA 1071 Gln Glu Pro Ser 355 2) INFORMATION FOR SEC. FROM IDENT. NO: 2 (i) CHARACTERISTICS OF THE SEQUENCE: A LENGTH: 356 amino acids TYPE: amino acid D) TOPOLOGY: linear ii) TYPE OF MOLECULE: Protein x i DESCRIPTION OF SEQUENCE SEC IDENT. NO: 2: Met Gln Thr Arg Lys He Val Arg Ala Wing Wing Val Gln Wing Wing 1 5 10 15 Pro Asn Tyr Asp Leu Wing Thr Gly Val Asp Lys Thr He Glu Leu Wing 20 25 30 Arg Gln Wing Arg Asp Glu Gly Cys Asp Leu He Val Phe Gly Glu Thr 35 40 45 Trp Leu Pro Gly Tyr Pro Phe His Val Trp Leu Gly Wing Pro Wing Trp 50 55 60 Ser Leu Lys Tyr Ser Wing Arg Tyr Tyr Wing Asn Ser Leu Ser Leu Asp 65 70 75 80 Ser Wing Glu Phe Gln Arg He Wing Gln Wing Wing Arg Thr Leu Gly He 85 90 95 Phe He Wing Leu Gly Tyr Ser Glu Arg Ser Gly Gly Ser Leu Tyr Leu 100 105 110 Gly Gln Cys Leu He Asp Asp Lys Gly Glu Met Leu Trp Ser Arg Arg 115 120 125 Lys Leu Lys Pro Thr His Val Glu Arg Thr Val Phe Gly Glu Gly Tyr 130 135 140 Wing Arg Asp Leu He Val Ser Asp Thr Glu Leu Gly Arg Val Gly Wing 145 150 155 160 Leu Cys Cys Trp Glu His Leu Ser Pro Leu Ser Lys Tyr Wing Leu Tyr 165 170 175 Ser Gln His Glu Wing He His Wing Wing Wing Trp Pro Ser Phe Ser Leu 180 185 190 Tyr Ser Glu Gln Wing His Wing Leu Ser Wing Lys Val Asn Met Wing Wing 195 200 205 Ser Gl n He Tyr Ser Val Glu Gly Gln Cys Phe Thr He Ala Wing Ser 210 215 220 Ser Val Val Thr Gln Glu Thr Leu Asp Met Leu Glu Val Gly Glu His 225 230 235 240 Asn Ala Pro Leu Leu Lys Val Gly Gly Gly Ser Met He Phe Wing 245 250 255 Pro Asp Gly Arg Thr Leu Wing Pro Tyr Leu Pro His Asp Wing Glu Gly 260 265 270 Leu He He Wing Asp Leu Asn Met Glu Glu He Wing Phe Wing Lys Wing 275 280 285 He Asn Asp Pro Val Gly His Tyr Ser Lys Pro Glu Wing Thr Arg Leu 290 295 300 Val Leu Asp Leu Gly His Arg Asp Pro Met Thr Arg Val His Ser Lys 305 310 315 320 Ser Val Thr Arg Glu Glu Pro Wing Glu Gln Gly Val Gln Ser Lys He 325 330 335 Ala Ser Val Ala He Ser His Pro Gln Asp Ser Asp Thr Leu Leu Val 340 345 350 Gln Glu Pro Ser 355 ) INFORMATION FOR SEC. DE IDENT NO: 3 i) CHARACTERISTICS OF THE LONGITUDE SEQUENCE 39 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Peptide (iii) HYPOTHETIC: NO (iii) [sic] ANTISENTIDO: NO (v) TYPE OF FRAGMENT: N Terminus (vi) ORIGINAL SOURCE: (A) ORGANISM: Alcaligenes faecalis (B) CEPA: 1650 (vii) IMMEDIATE SOURCE: (B) CLON: Nitrilasa xi) DESCRIPTION OF SEQUENCE ID SEC. NO: 3: Met Gln Thr Arg Lys He Val Arg Ala Wing Wing Val Gln Wing Wing Ser 1 5 10 15 Pro Asn Tyr Asp Leu Wing Thr Gly Val Asp Lys Thr He Glu Leu Wing 20 25 30 Arg Gln Wing Arg Asp Glu Gly 35 INFORMATION FOR SEC. FROM IDENT. NO: 4 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (n) TYPE OF MOLECULE: Peptide (m) HYPOTHETIC: NO (m) [sc] ANTICIPATION: NO (v) TYPE OF FRAGMENT: Internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Alcaligenes faecalis (B) CEPA: 1650 (vl) IMMEDIATE SOURCE: (B) CLON: Nitrilasa XI DESCRIPTION OF SEQUENCE SEC IDENT. NO: 4 Glu Glu Wing Pro Glu Gln Gly Val Gln Ser Lys He Wing Ser Val Wing 1 5 10 15 He Ser His Pro Gln 20 INFORMATION FOR SEC. FROM IDENT. NO: 5 CHARACTERISTICS OF THE SEQUENCE A LENGTH 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Peptide (iii) HYPOTHETIC: NO (iii) [sic] ANTICIPATION: NO (v) TYPE OF FRAGMENT: Internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Alcaligenes faecalis (B) CEPA: 1650 (vii) IMMEDIATE SOURCE: (B) CLON: Nitrilasa (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 5: Glu Glu Pro Wing Glu Gln Gly Val Gln Ser Lys 1 5 10 INFORMATION FOR SEC. FROM IDENT. NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear & m ..? .g ^. í 'S' - ~ iS-5 --- S ^ ¡B lt-? í? lSSi (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO (iii) [sic] ANTI-SENSE: NO ( vi) ORIGINAL SOURCE: (A) ORGANISM: Alcaligenes faecalis (B) CEPA: 1650 (v l) IMMEDIATE SOURCE: (B) CLON: Nitrilasa (xi) DESCRIPTION OF THE SEQUENCE: SEC D E IDENT. NO: 6: ATGCAGACNA GNAARATCGT SCG 2 3 (2) INFORMATION FOR SEC. FROM IDENT. NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) [sic] ANTICIPATION: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Alcal genes faecalis (B) CEPA: 1650 (vile) IMMEDIATE SOURCE: ¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡! NO: 7: TNGCSACNGA NGCRATCTTG 20) INFORMATION FOR SEC. IDENT. NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) CHAIN FORM: Single (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO (iii) [sic] ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Alcaligenes faecalis (B) CEPA: 1650 (vn) IMMEDIATE SOURCE: (B) CLON: Nitrilasa xi DESCRIPTION OF SEQUENCE SEC DE IDENT. NO: 8: TTAATCATAT GCAGACAAGA AAAATCGTCC G 3 1) INFORMATION FOR SEC. IDENT. NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: HF. * 3 »& ij & ? t l l? £ ^ í ^ Á? ^^ (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear ll) TYPE OF MOLECULE: DNA (genomic) m) HYPOTHETICAL: NO m) [sic] ANTISENTIDO: NO vi) SOURCE ORIGINAL: (A) ORGANISM: Alcal genes faecalis (B) CEPA: 1650 vil) IMMEDIATE SOURCE: (B) CLON: Nitrilasa xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 9: AAGGATCCTC AAGACGGCTC TTGCACTAGC AG 32 ? &l l lllllllllllllllllllllllllllllllllllllllllllllllllllllllll

Claims (15)

1. CLAIMS 1. An isolated nucleic acid sequence encoding a polypeptide having nitrilase activity, selected from the group of: a) a nucleic acid sequence having the sequence presented in SEQ. FROM IDENT. NO .: 1, b) nucleic acid sequences that are derived from the nucleic acid sequence depicted in SEQ. FROM IDENT. NO .: 1 as a result of the degeneracy of the genetic code, c) derived from the nucleic acid sequence represented in the SEC. FROM IDENT. NO .: 1, which encodes polypeptides having the amino acid sequence represented in SEQ. FROM IDENT. NO: 2 and have at least 95% homology at the amino acid level, with negligible reduction in the enzymatic action of the polypeptides.
2. 2. The amino acid sequence encoded by the sequence depicted in claim 1.
3. 3. The amino acid sequence according to claim 2, encoded by the sequence depicted in SEQ. FROM IDENT. NO .: 1. The nucleic acid construct comprising a nucleic acid sequence according to claim 1, characterized in that the nucleic acid sequence is linked to one or more regulatory signals.
4. í-ítóÜ &Kiri-, ü < , S & L í El
5. 5. The vector comprising a nucleic acid sequence according to claim 1, or a nucleic acid construct according to claim 4.
6. 6. The microorganism comprising at least one acid sequence nucleic acid according to claim 1 or at least one nucleic acid construct according to claim 4.
7. The microorganism according to claim 6, characterized in that the microorganism is a bacterium of the genera Escherichia, Pseudomonas or Alcaligenes.
8. 8. A process for preparing chiral carboxylic acids of the general formula I

   which comprises converting racemic nitriles of the general formula II

   in the presence of the amino acid sequence according to claim 2 or 3 or a growing, latent or destabilized microorganism according to claims 6 or 7, and wherein the

   "^ X inri ifjfffffmi -? Tifrr ^^ * jñit? I? L l least 25 mmoles of nitrile are converted by h and per mg of protein or 25 mmoles of nitrile are converted by h and g of dry weight into carboxylic acids chirals, wherein the substituents and the variables in formulas I and II have the following meanings: * an optically active center R1, R2, R3 independently of one another hydrogen, Ci-Cio substituted or unsubstituted, branched or unbranched alkyl , C2-C? alkenyl, substituted or unsubstituted aryl, hetaryl, OR4 or NR4R5 and wherein the radicals R1, R2 and R3 are always different R4 hydrogen, C?-C? alkyl or substituted or unsubstituted, branched or unbranched, C2-C? alkenyl, Ci-Cio alqu alkylcarbonyl, C2-C? al alkenylcarbonyl, aryl, arylcarbonyl, hetaryl or hetarylcarbonyl, R5 hidrógeno hydrogen, substituted or unsubstituted Ci-C alquilo alquilo alkyl, branched or non-branched branched, C2-C? alkenyl, aryl or hetaryl
9. 9. The conformation process ad with claim 8, characterized in that one of the substituents R 1, R 2 or R 3

   is OR4.
10. 10. The process according to claim 8 or 9, characterized in that one of the substituents R, R or R3 is aryl.
11. 11. The process according to any of claims 8 to 10, characterized in that the process is carried out in an aqueous reaction solution at a pH between 4 to [sic] 11.
12. The process according to any of the claims 8 to 11, characterized in that 0.01 to 10% by weight of nitrile or 0.01 to 10% by weight of a corresponding aldehyde or ketone and 0.01 to 10% by weight of hydrocyanic acid are reacted in the process.
13. 13. The process according to any of claims 8 to 12 characterized in that the process is carried out at a temperature between 0 ° C to [sic] 80 ° C.
14. The process according to any of claims 8 to 13, characterized in that the chiral carboxylic acid is isolated from the reaction solution in yields of 60 to 100% by extraction or crystallization or extraction and crystallization.
15. 15. The process according to any of claims 8 to 14, characterized in that the chiral carboxylic acid has an optical purity of at least 90% ee.