WO1999007873A1 - METHOD FOR PRODUCING CYCLIC α-AMINO ACIDS FREE FROM ENANTIOMERS OR THEIR N-PROTECTED DERIVATIVES BY MEANS OF A D-SPECIFIC AMINOACYLASE - Google Patents

Abstract

The invention relates to new microorganisms capable of utilising a N-protected cyclic amino acid derivative, selected from the compounds of the general formula (I), in the form of the racemate or one of its optical isomers, where A together with -N- and -CH is a possibly substituted 4-, 5-, 6-, or 7-membered heterocyclic ring and R<1> is a possibly substituted alkyl, alkoxy, aryl or aryloxy, and/or are capable of hydrolizing a N-protected cyclic amino acid derivative, selected from among the compounds of the general formula (I), as well as to enzyme extracts thereof. The invention also relates to a new method for producing N-protected cyclic L-amino acid derivatives and cyclic D-amino acids by using said microorganisms.

Description

 Process for the preparation of enantiomerically pure cyclic α-amino acids or their N-protected derivatives using a D-specific aminoacylase.

The present invention relates to microorganisms which are capable of one or more N-protected cyclic amino acid derivatives selected from the compounds of the general formula

wherein A together with -N- and -CH represent an optionally substituted 4-, 5-, 6- or 7- membered saturated heterocyclic ring and R ¹ each represents optionally substituted alkyl, alkoxy, aryl or aryloxy, in the form of the racemate or one of the optically active isomers as the only C source, as the only N source or as the only C and N source, and / or are able to hydrolyze them. These microorganisms or the enzyme extracts therefrom are used for a new process for the production of N-protected cyclic L-amino acid derivatives and / or cyclic D-amino acids are used

D-amino acids such as D-Prohn are important intermediates for the manufacture of pharmaceuticals (J Org Chem 1994, 59, 7496-7498)

Several processes are known for the preparation of D-amino acids. JP-A 01 005 488 encompasses a process for the preparation of D-amino acid acylases which, for example, hydrolyze N-benzyloxycarbonylmethionine in D-methionine, N-benzyloxycarbonyl-L-methionine being obtained. This D On the one hand, amino acid aciases do not hydrolyze cych N-protected amino acids, on the other hand, they badly hydrolyze the N-benzyloxycarbonyl group (Z group) JP-A 07 289 275 describes a process for the preparation of D-proline by reacting L-Prohn using microorganisms of the genus Escherichia with racemase activity to (DL) -proline, which is then used by microorganisms that utilize the unprotected L-proline, is metabolized to obtain D-proline D-proiin is obtained in poor yield

JP-A 07 127 354 comprises a process for the preparation of D-proline starting from ornithine by means of microorganisms of the species Proteus mitajiri. A disadvantage of this process is that on the one hand the educt ornithine is too expensive, and on the other hand that D-proline is inferior Yield is obtained

JP-A 92 183 399 discloses a process for the preparation of D-proline starting from (DL) -proline using microorganisms of the genus Candida or trichospora. This process also has the disadvantage that D-proline is obtained in low yield

The object of the present invention is to provide a simple and technically viable method for producing both N-protected cych L-amino acid derivatives and cych D-amino acids. The corresponding products are to be isolated in good enantiomeric purity

This object is achieved with the microorganisms according to claim 1 and the method according to claim 5

The microorganisms according to the invention can be isolated, for example, from soil samples, sludge or wastewater, in particular contaminated soil or clear sludge, with the aid of customary microbiological techniques cyclic amino acid derivatives of the general formula I, in the form of the racemate or one of the optically active isomers, __

wherein A together with -N- and -CH and R ^{1 have} the meaning given, namely

• as the only C and N source or

• as the only N source with a suitable C source or

• as the only C source with a suitable N source

The microorganisms are expediently first enriched in a corresponding liquid culture and the anaerobic or aerobic microorganisms are isolated from the culture obtained, the desired N-protected cychino amino acid derivatives as the only N source, as the only C source or as the only C and Can utilize the N source and / or are able to hydrolyze it

Examples of optionally substituted saturated 4-membered heterocyclic rings are azetidine. Methyl azetidine, diazetidine

Examples of optionally substituted saturated 5-membered heterocyclic rings are proline, hydroxyproline, pyrazolidine, methylpyrazolidine, imidazolidine, pyroglutamate, oxazolidine, isoxazolane, thiazolidine and triazolidine

Examples of optionally substituted saturated 6-membered heterocyclic rings are piperidine. 3-methylpiperidine, piperazine, pipecolin, 3-methylpiperazine, morpholine

Examples of optionally substituted 7-membered heterocyclic rings are ε-capiolactam and azetine The radical R ¹ in the N-protected cychino amino acid derivative of the general formula 1 means alkyl, alkoxy, aryl or aryloxy

Alkyl here means a substituted or unsubstituted C 1 -C alkyl group, preferably a substituted or unsubstituted C 1 -C alkyl group. Suitable substituents are, for. B hydroxy or halogens such as F, Cl. Br or J Examples of a Ci-is-alkyl group are methyl, chloromethyl, trifluoromethyl, co-hydroxyalkyl, ethyl, propyl, butyl, iso-butyl, iso-propyl and stearyl ω-hydroxyalkyl group in the following means a hydroxymethyl, hydroxyethyl, Hydroxypropyl or Hydroxybutyl Group Preferably alkyl means methyl

Alkoxy means a substituted or unsubstituted C 1 ₈ alkoxy group, preferably a substituted or unsubstituted C 4 alkoxy group. Suitable substituents are, for example, the substituents mentioned for alkyl. Examples of an alkoxy group are methoxy, chloromethoxy, trifluoromethoxy, ethoxy, propoxy, butoxy, tert -butoxy, iso-butoxy and stearoxy

A phenyl or benzyl group, substituted or unsubstituted, for example 4-methoxybenzyl or 4-methoxyphenyl, is used as the aryl. A phenyloxy or benzyloxy group, substituted or unsubstituted, is used as aryloxy. Examples of an aryloxy group are benzyloxy, 4-methoxybenzyloxy or 4-nitrobenzyloxy. Aryloxy preferably means benzyloxy

The compounds of the general formula I are preferably N-protected cyclic D-amino acid derivatives

Particularly preferred N-protected cyclic D-amino acid derivatives are N-protected D-proline derivatives, for example N-acetyl and / or N-benzyloxycarbonyl-D-proline (N-Z-D-proline), and N-protected D-pipecolinic acid derivatives

The microorganisms can use, for example, sugar, sugar alcohols, or carboxylic acids as a growth substrate as a suitable C source. Hexose, for example glucose and fructose, pentoses, for example ribose, or disaccharides, for example sucrose, can be used as sugars. Amino carboxylic acids, mono-, di - or tricarboxylic acids or their salts can be used. For example, mannitol or glycerin can be used as sugar alcohols. For example, proline, glutamate, lysine, glycine or their salts can be used as amino carboxylic acids. Acetic acid, acetate, lactic acid can be used as mono-, di- or tricarboxylic acids. Lactate, succinic acid, succinate, malic acid, malate, citric acid, citrate or their salts are used

The microorganisms can, for example, ammonium, nitrate, urea as a suitable N source. Use glycine or lysine

The media customary in the art, for example the medium described in Table 1, can preferably be used as the selection and growth medium. The medium described in Table 1 is preferably used

The active enzymes of the microorganisms are expediently induced during the cultivation and selection. An N-protected cyclic amino acid derivative according to formula I, preferably its D-isomer, is expediently used as the enzyme inducer

The cultivation and selection is usually carried out at a temperature of 5 to 100 ° C., expediently from 10 to 75 ° C., preferably from 15 to 40 ° C., in particular from 20 to 35 ° C. and at a pH between pH 0 and pH 12, expediently from pH 4 to 10, preferably between pH 5 and pH 9

Preferred microorganisms are microorganisms corresponding to the microorganisms with the designation MIS1 12, MIS125, MIS132, MIS211, MIS213, MIS221, MIS222. MIS231, MIS242, MIS253 Particularly preferred are microorganisms of the genus Arthrobacter, such as the species Arthrobacter oxydans GS121, Arthrobacter sp GS132, Arthrobacter pascens or ramosus GS131 (DSM 1 1637), Arthrobacter pascens or ramosus GS134, and their variant organisms and mutant mutants GS131 were deposited on 30.06.1997 with the German Collection of Microorganisms and Zellkultur GmbH, Mascheroderweg lb, D-38124 Braunschweig, according to the Budapest Treaty under the deposit number DSM 1 1637 "Flinktionally equivalent λ ^ arianten and mutants" are understood to mean microorganisms which are derived from the described original organisms and whose properties and functions essential to the invention essentially match them. Variants and mutants of this kind can be formed accidentally, for example by UV radiation become

Properties of the microorganisms Arthrobacter oxydans GS121

Characterization Gram-positive, coryne-shaped rods, strictly aerobic, no acid or gas formation from glucose mobility spores

Catalase + meso-diaminopimelic acid in the cell wall no peptidoglycan type A3α, L-Lys-L-Ser-L-Thr-L-Ala

16S rDNA sequence similarity Sequencing of the area with the greatest variability gave 100.0% with Arthrobacter oxydans

Properties of the microorganisms Arthrobacter pascens or ramosus GS131 (DSM 11637)

Characterization Gram-positive, coryne-shaped rods, strictly aerobic, none

Acid or gas formation from glucose mobility spores

Catalase +

Strong hydrolysis meso-diaminopimelic acid in the cell wall no Peptidoglycan type A3α, L-Lys-L-Ala ₂ . ₃

16S rDNA sequence similarity Sequencing of the area with the greatest variability resulted in 98.3% with Arthrobacter pascens / A ramosus (highest value)

A reasonably reliable species assignment is not possible in many cases with the genus Arthrobacter. The descriptions are usually based only on the type strain. The trunk belongs with certainty in the closer Arthrobacter globiformis / Arthrobacter ramosus group The 16S rDNA sequences are identical to those of the GS134 strain

Properties of the microorganisms Arthrobacter oxydans GS132

Characterization Gram-positive, coryne-shaped rods, strictly aerobic, none

Acid or gas formation from glucose mobility spores

Catalase + meso-diaminopimeline acid in the cell wall no Peptidoglycan type A3. L-Lys-L-Ser-L-Thr-L-Ala

16S rDNA sequence similarity Sequencing of the area with the greatest variability resulted in 97.2% with Arthrobacter nicotinovorans (highest value)

The type of peptidoglycan found has hitherto been described only for Arthrobacter oxydans cell wall and 16S rDNA sequence data indicate that the strain represents a species of the genus Arthrobacter which has not previously been described

Properties of the microorganisms Arthrobacter pascens or ramosus GS134

Characterization Gram-positive, coryne-shaped rods, strictly aerobic, none

Acid or gas formation from glucose

agility

Spores

Catalase + meso-diaminopimeline acid in the cell wall no

Peptidoglycan type A3, L-Lys-L-Ala _2-3

16S rDNA sequence similarity Sequencing of the area with the greatest variability resulted in 98.3% with Arthrobacter pascens / Arthrobacter ramosus (highest value)

A reasonably reliable species assignment is not possible in many cases with the genus Arthrobacter. The descriptions are usually based only on the type strain The strain belongs with certainty in the closer Arthrobacter globiformis / Arthrobacter ramosus group The 16S rDNA sequences are identical to those of strain GS131

The process according to the invention for the preparation of N-protected cych L-amino acid derivatives of the general formula II and / or of a cych D-amino acid of the general formula III

wherein A together with -N- and -CH and R ^{1 have} the meaning given, comprises the reaction of a racemic N-protected cych aminosaur derivative of the general formula

wherein A together with -N- and -CH and R ^{1 have} the meaning given, by means of the microorganisms already described and / or an enzyme preparation thereof, into a cyclic D-amino acid (formula III) and / or the N- protected cychschen L-amino acid derivatives (formula II), and optionally isolation of these compounds

The racemic compounds described above are used as N-protected cyclic amino acid derivatives of the formula I. Particularly preferred N-protected cyclic amino acid derivatives are NZ-proline (R = benzyloxy), N-acetyl-proline (R ¹ = methyl) and NZ-pipecolinic acid. The educts, the racemic N-protected cyclic amino acid derivatives of the general formula I such as, for example, NZ- (DL) -proline, are commercial compounds

In principle, biotransformation is possible with all microorganisms that utilize and / or hydrolyze an N-protected cyclic amino acid derivative in the form of the racemate or its optically active isomers as the only N source, as the only C source or as the only C and N source Enzyme extracts from these microorganisms are also suitable. Biotransformation is preferably carried out with the microorganisms described above, in particular with the microorganisms described above of the species Arthrobacter pascens or ramosus GS131 (DSM 1 1637) or with their functions! equivalent variants and mutants

After the microorganisms have been grown conventionally, the biotransformation can be carried out with resting cells (non-growing cells which no longer require a carbon and energy source) or with growing cells under aerobic or anaerobic conditions. The biotransformation is preferably carried out with resting cells under aerobic conditions

Media which are customary in the art can be used for the biotransformation, for example low-molar buffers such as low-molar phosphate buffer, Tris buffer or the medium described in Table 1. The biotransformation is preferably carried out in the medium according to Table 1

The biotransformation is expediently carried out by adding an N-protected amino acid derivative so that the concentration does not exceed 50% by weight, preferably 20% by weight. The N-protected amino acid derivative is expediently added once or continuously

The pH of the medium can be in a range from 3 to 12, preferably from 5 to 9. The biotransformation is expediently carried out at a temperature of 10 to 70 ° C., preferably 20 to 50 ° C. According to the method of the invention, an N-protected cyclic amino acid derivative is converted into a cyclic D-amino acid. An N-protected L-amino acid derivative is obtained. The cyclic D-amino acid and the N-protected L-amino acid derivative can be obtained in good yield and Enantiomeric purity (ee greater than 98%) can be isolated

The N-protected L-amino acid derivative obtained in this way and / or the D-amino acid obtained in this way can be isolated by customary workup methods, such as, for example, by extraction

Examples

Example 1: Selection of N-Z-D-Proline-utilizing microorganisms

First, a minimal medium (see Table 1) was produced that met the growth requirements of many microorganisms

Table 1:

Minimal medium

Na ₂ S0 ₄ 0.1 g / 1

Na HP0 -2 H ₂ 0 2.5 g / 1

KH P0 ₄ 1.0 g / 1

NaCl 3.0 g / 1

MgClrό H ₂ 0 0.4 g / 1

CaCl ₂ -2 H ₂ 0 14.5 mg / 1

FeCl ₃ -6 H ₂ 0 0.8 mg / 1

Trace element solution 1.0 ml / 1

Vitamin solution 1.0 ml / 1 pH 7.0 To enrich microorganisms that are able to hydrolyze N-protected forms of cyclic D-amino acids, the following C and N sources were added to this basic medium

(A) N-acetyl-D-Prohn (5 g / 1), ammonium sulfate (2 g / 1) (B) fructose (10 g / 1), N-Z-D-proline (2 g / 1)

Different batches were then inoculated with soil samples from different locations and incubated (30 ° C, 120 rpm) until clearly visible growth could be seen. An aliquot of these cultures was then incubated in an equal volume of fresh medium and incubated again until it became cloudy This process was repeated three times. The enriched microorganisms were then separated and purified on a solid medium (same composition as liquid medium, only addition of 20 g / 1 agar agar). In this way, about 20 different bacterial isolates were obtained which were able to

(A) N-acetyl-D-proline as the only C source or

(B) N-Z-D-proline as the only N source

to recycle

Example 2:

Test of the selected microorganisms for hydrolytic activity against N-protected cych amino acids

(A) Test with microorganisms that use N-acetyl-D-Pro5in as the only C source

The isolates obtained with the method described in Example 1 (A) were multiplied in the medium described there. The cells produced in this way were harvested by centrifugation and then resuspended in saline solution (8.5 g / l) and washed after resuspending in saline solution the ability to hydrolysis of N-protected cych amino acids was tested with resting cells For this purpose, a suitable amount of cells with 25 mM N-acetyl- (DL) -proline, NZ- (DL) -proline and NZ- (DL) -pipecolinic acid or 12.5 mM N-acetyl- (DL) -pipecolinic acid incubated in buffer solution (50 mM phosphate buffer, pH 7.0) under rubble (30 ° C). At various times, aliquots were removed and checked for the release of proline or pipecolinic acid by thin layer chromatography. In the presence of air-oxygen the When the proline released by the hydrolysis was metabolized, the majority of the experiments were carried out in the absence of oxygen (reaction in gas-tight tubes under a nitrogen atmosphere)

Ten isolates (the bacterial strains MIS112, MIS125, MIS132, MIS211, MIS213, MIS221, MIS222, M1S231, MIS242, MIS253) basically showed the ability to hydrolyze various of these substrates. The results obtained are summarized in Table 2

Table 2:

Enzymatic activity towards N-protected derivatives of cyclic

amino acids

Activity +++ very good, ++ good, + massive, (+) hardly, - none, ng not tested The different strains showed quite different substrate patterns. While most strains preferred NZ derivatives, MIS221 reacted only with N-acetyl derivatives. Also on the basis of macroscopic (colony morphology and staining), microscopic (cell morphology, motility) and physiological (API - Test by the company BioMerieux) Analyzes all these strains differed from each other. All strains showed fundamentally higher activity in the presence of oxygen and preferred proline versus pipecolic acid derivatives

The enantiomeric purity of both the N-protected and the free amino acids was then determined by HPLC (NZ-proline, NZ-pipecolic acid) or GC (proline, pipecolinic acid, N-acetyl-proiin). The conversion of the individual reactions, based on the Racemic starting material was estimated on the basis of the data from the enantiomer determination, whereby exact quantification is not possible with these methods. The ee values that could be achieved with some strains are shown in Tables 3 and 4

Table 3:

Enantiose activity towards N-acetyl-proline

- no product available In all cases, a clear selectivity of the hydrolysis towards N-acetyl-D-proline can be seen. Under aerobic conditions, the reaction proceeds better and leads to the formation of enantiomerically pure N-acetyl-L-proline in some batches. The utilization of the released proline is likely to be favored Implementation by the bacterial cells In the absence of oxygen, D-proline also accumulates. In one case (MIS231) enantiomerically pure D-proline could be observed, in the other approaches there was at least an enrichment of the desired enantiomer rather a depletion of D-proline caused by the racemization of D-proline, since the hydrolysis of the acetyl derivative appears to be very selective

Table 4:

Enantioselectivity towards N-Z proline

Praktisch alle Ansätze zeigten sehr selektive Hydrolyse von N-Z-D-Prolin In Anwesenheit von Sauerstoff lauft die Reaktion absolut selektiv, so dass am Ende das gesamte, in die Reaktion eingesetzte N-Z-L-Prolin in enantiomerenreiner Form vorlag

Practically all of the batches showed very selective hydrolysis of NZD-proline. In the presence of oxygen, the reaction proceeds absolutely selectively, so that in the end the entire NZL-proline used in the reaction was in enantiomerically pure form

The specificity of the enzymatic reaction can also be observed with derivatives of amino acids other than proline. As shown in Figure 1, the reaction with N-Z- (DL) -pipecolinic acid is relatively slow, but here too the D-enantiomer is selective from the strains MIS132 and MIS222 hydrolyzed

Figure 1 shows the reaction of MIS132 and MIS222 with N-Z- (DL) -pipecolinic acid

This is also confirmed by tests with the strains MIS21 1 and MIS242, where D-pipecolinic acid with high optical purity (ee> 95%) is formed. However, the sales in these tests were relatively low (<10%)

(B) Test with microorganisms that use N-Z-D-proline as the only N source

The isolates obtained with the method described in Example 1 (B) were multiplied in the medium described there. The cells produced in this way were harvested by centrifugation and then resuspended in saline solution (8.5 g / l) and washed. After resuspending in saline solution the ability to hydrolysis of N-protected cycho amino acids was tested with resting cells. A suitable amount of cells with 100 mM NZ-DL-proline or 50 mM NZ-DL-pipecolic acid in buffer solution (50 mM phosphate buffer, pH 7.0) below

Shake incubated (30 ° C). At different times, aliquots were taken and checked for the release of proline or pipecolinic acid by thin layer chromatography. Four isolates (the bacterial strains GS121, GS131, GS132 and GS134) showed hydrolytic activity against both NZ-proline and NZ-pipecolinic acid. Since GS131 showed the highest activity, this isolate was examined in more detail. Example 3:

Growth of bacterial strain GS131 (DSM 11637)

In order to characterize bacterial strain GS131 in more detail, the growth of this strain was investigated with different C and N sources. For this purpose, the basic medium described in Table 1 was used and the C sources were added to 10 g / 1 each and the N sources to a final concentration of 40 mM. The cultures were then incubated at 30 ° C. GS131 was able to utilize a wide variety of C and N sources and even showed good growth in most of the media examined.

Table 5:

Growth of bacterial strain GS131 with different C and N sources.

Beispiel 4:

Example 4:

Induction of D-aminoacylase from bacterial strain GS131 (DSM 11637)

Bacterial strain GS131 was grown under variation of different media components or their concentration at 30 ° C. The cells produced in this way were harvested by centrifugation and then resuspended in saline solution (8.5 g / l) and washed. After resuspending in saline solution, the ability to hydrolyze N-Z-D-proline was examined with resting cells under aerobic conditions.

Table 6:

Induction of D-aminoacylase from bacterial strain GS131

Growth +++ good, ++ massive, + poor activity + present, - none, ng not tested

It was shown that the presence of N-protected cyclic amino acids slowed growth almost continuously, with NZD-proline still being best tolerated in low concentrations. Similarly, D-aminoacylase could only be induced by administration of NZD-proline in low concentrations, whereby both glucose and L-proline were accepted as the C source Example 5:

Enantioselectivity of D-aminoacylase from bacterial strain GS131 (DSM 11637)

Bacterial strain GS131 was propagated in the medium described in Example 1 (B). The cells produced in this way were harvested by centrifugation and then resuspended in saline solution (8.5 g / l) and washed. After resuspending in saline solution, the ability to Hydrolysis of N-protected cych amino acids was tested. A suitable amount of cells with the respective substrate (10 mM NZL-proline or NZD-proline or 5 mM NZL-pipecolic acid or N-ZD-pipecolic acid) in buffer solution (50 mM phosphate buffer , pH 7.0) (30 ° C.) Aliquots were removed at various times and the wetting of the substrates was monitored by HPLC

Figure 2 shows the enantioselectivity of D-aminoacylase from bacterial strain GS 131

For both N-protected amino acids tested, the D-enantiomer was hydrolyzed, while the L-enantiomer remained untouched. The enzyme therefore shows very good enantioselectivity. However, N-Z-D-proline was hydrolyzed considerably faster than N-Z-D-pipecolinic acid

Example 6:

Production of NZL-Proline by Racemate Cleavage Using D-Aminoacylase from Bacterial Strain GS131 Bacterial Strain GS131 was propagated in the medium described in Example 1 (B). The cells thus produced were harvested by centrifugation and then in saline solution (8.5 g / 1) Resuspended and washed After resuspending in saline solution, the ability to hydrolize N-protected cych amino acids was tested with resting cells. For this purpose, a suitable amount of cells with 10 mM NZ- (DL) -proline in buffer solution (50 mM phosphate buffer , pH 7.0) incubated (30 ° C.) Aliquots were removed at various times and the conversion of the substrate was monitored by HPLC Figure 3 shows the production of NZL-proline from NZ- (DL) -proline using the D-aminoacylase from bacterial strain GS 131

After only three hours, the entire amount of N-Z-D-proline was completely hydrolyzed, while N-Z-L-proline remained untouched for 26 hours

Example 7: Synthesis of N-benzyloxycarbonyl derivatives of cyclic amino acids

(A) Synthesis of N-Z- (DL) -proline

100.0 g (DL) -proline were dissolved in 217 ml of 4 N NaOH. This solution was treated with thermostatic treatment (5 - 10 ° C) and pH-Stase (pH = 10.0 - 1 1.0) benzyl chloroformate ( Z-Cl) was added dropwise A total of 158.1 g Z-Cl and 251.2 g 4 N

NaOH added In addition, 150 ml of distilled water were added in order to maintain the agitation of the reaction mixture. The mixture was then acidified to pH = 2.4 with concentrated hydrochloric acid (76 ml) and then NZ- (DL) -proline was added in several steps a total of 584 ml of butyl acetate extracted. The combined organic phases were concentrated in vacuo and the NZ- (DL) -proline obtained was dissolved in

Ethyl acetate dissolved and crystallized by adding hexane. In this way, 187.4 g of N-Z- (DL) -proline (86.5% of theory) were obtained

The same procedure was followed for the synthesis of N-Z-D-proline or N-Z-L-proline

(B) Synthesis of N-Z-L-pipecolic acid

10.0 g of L-pipecolic acid was dissolved in 13 ml of 4 N NaOH. 14.5 g of benzyl chloroformate (Z-Cl) were added dropwise to this solution while being thermostatted (5-10 ° C.). The pH was kept at 11.5 by means of a corresponding addition of 4 N NaOH via a pH stat 10 ml of water were added to the reaction to make it easier to work up, and the pH was adjusted to 7.0 by adding concentrated hydrochloric acid (about 1 g). Impurities were removed by extraction twice with 40 ml each of butyl acetate the pH of the aqueous phase was brought to pH 2.01 by adding a further 6.67 g of concentrated hydrochloric acid. NZL-pipecolic acid was then obtained by extraction three times (40 ml each) with butyl acetate. The organic phases were combined, dried over sodium sulfate and finally until evaporated to dryness After dissolving the solid in 10 ml of ethyl acetate and adding 9 ml of hexane, NZL-pipecolic acid was crystallized by cooling to a temperature <10 ° C. After filtration and drying, 13.22 g of NZL-pipecolinic acid (64.9% of theory Th) received

An analogous procedure was followed for the synthesis of N-Z- (DL) -pipecolinic acid or N-Z-D-pipecolinic acid

Claims

Claims: Microorganisms, characterized in that they are capable of one or more N-protected cyclic amino acid derivatives selected from compounds of general formula I.

wherein A together with -N- and -CH represent an optionally substituted 4-, 5-, 6- or 7-membered saturated heterocyclic ring and R ¹ each represents optionally substituted alkyl, alkoxy, aryl or aryloxy, in the form of the racemate or one utilize and / or hydrolyze the optical isomers as the only C source, as the only N source or as the only C and N source, and enzyme extracts from it Microorganisms according to claim 1, wherein the compound of formula I is an N-protected cyclic D-amino acid derivative Microorganisms according to one of claims 1 or 2, wherein the compound of formula I is an N-protected D-proline derivative, preferably N-acetyl or N-benzyloxycarbonyl-D-proline, or an N-protected D-pipecolinic acid derivative Microorganisms according to one of claims 1 to 3, which are capable of an N-protected D-proline derivative, preferably N-acetyl or N-benzyloxycarbonyl-D-Pro n, as the only C source, as the only N source or as the only one To utilize the C and N sources, and / or are capable of hydrolyzing an N-protected D-proline derivative, preferably N- or N-benzyloxycarbonyl-D-proline and / or an N-protected D-pipe colic acid derivative Microorganisms according to one of claims 1 to 4 of the genus Arthrobacter Microorganisms according to claim 5 of the species Arthrobacter ramosus GS131 (DSM 11637), as well as its functionally equivalent variants and mutants Process for the preparation of N-protected cych L-amino acid derivatives of the general formula II and / or of cych D-amino acids of the general formula III

wherein A together with -N- and -CH and R ^{1 have} the meaning given in claim 1, comprising the reaction of a racemic N-protected cychino amino derivative of the general formula

wherein A together with -N- and -CH and R ^{1 have} the meaning given, by means of microorganisms and / or enzyme preparations according to claim 1 to give the compounds of the formula III and / or the N-protected cych L-amino acid derivatives of the formula which result from this reaction II, and optionally isolation of these compounds . A method according to claim 7, characterized in that the biotransformation is carried out by means of microorganisms of the species Arthrobacter pascens or ramosus GS131 (DSM 1 1637) or with their functionally equivalent variants and mutants ) Method according to one of claims 7 or 8, characterized in that the biotransformation is carried out with the addition of the N-protected amino acid derivatives so that the concentration does not exceed 50% by weight 0 Method according to one of claims 7 to 9, characterized in that the biotransformation is carried out at a temperature of 10 to 70 ° C and at a pH of 3 to 12