EP0944730A1

EP0944730A1 - Method for production of d-proline derivatives

Info

Publication number: EP0944730A1
Application number: EP97953805A
Authority: EP
Inventors: Martin Sauter; Daniel Venetz; Oleg Werbitzky
Original assignee: Lonza AG
Current assignee: Lonza AG
Priority date: 1996-12-16
Filing date: 1997-12-12
Publication date: 1999-09-29
Also published as: CA2274896A1; AU5757498A; CZ213199A3; WO1998027222A1; JP2001506500A

Abstract

The invention relates to new microorganisms which are capable of utilizing a proline derivative in the form of the racemate or its optically active isomers, of the general formula (I), in which R1 means alkyl, acyl or hydrogen, R2 means hydrogen or hydroxy and R3 -NH2, aryloxy or alkoxy, as the only source of nitrogen, the only source of carbon, or as the only carbon and nitrogen source. These microorganisms are used for new methods of production of D-proline derivatives.

Description

Process for the preparation of D-proline derivatives

The present invention relates to new microorganisms which are capable of a proline derivative in the form of the racemate or its optically active isomers of the general formula

wherein R ^{1 is} alkyl, acyl or hydrogen, R ^{2 is} hydrogen or hydroxy and R is -NH ₂ , aryloxy, or alkoxy to be used as the only nitrogen source, the only carbon source or the only carbon and nitrogen source. These microorganisms or their cell-free enzymes are used for a new process for the preparation of a D-proline derivative of the general formula II and a D-proline derivative of the general formula VI

1 7 wherein R, R and R have the meaning given

D-proline and its derivatives are important intermediates for the manufacture of pharmaceuticals (J Org Chetn, 1994, 59, 7496 - 7498)

Several processes are known for producing D-proline

JP-A-92183399 describes a process for the preparation of D-proline starting from (DL) -proline using microorganisms of the genus Candida or Trichospora Dies

The process has the disadvantage that the reaction time is too long and D-proline is obtained in low yield JP-A-07127354 describes 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 in poor yield is obtained

Furthermore, JP-A-07289275 describes a process for the preparation of D-proline starting from L-proline. L-proline is racemized by means of microorganisms of the genus Escherichia, which have racemase activity, to (DL) -proline, which is then raced with L -Proline-degrading microorganisms are cultivated in order to obtain D-proline. A disadvantage of this process is that the D-proline obtained in this way has to be purified further and this is associated with high losses in yield

DE-A-43 30 678 encompasses a process for the preparation of N-carbamoyl-D-proline starting from a bicyclic hydantoin. The bicyclic hydantoin is converted to N-carbamoyl-D-proline by means of the microorganisms Agrobacterium radiobacter the disadvantage that the reaction time is too long, and on the other hand that the corresponding D-proline derivative is only obtained in moderate yield

The object of the present invention is to isolate microorganisms which can be used for a simple and technically viable process for the preparation of D-proline derivatives of the general formulas II and VI. The corresponding products should be isolated in good yield with good enantiomeric purity

This object is achieved with the microorganisms and enzyme extracts therefrom, in accordance with claim 1, with the enzymes in accordance with claim 5 and with the methods in accordance with claims 6 and 10

The microorganisms according to the invention can be isolated from soil samples, sludge or waste water with the aid of customary microbiological techniques. According to the invention, these microorganisms are isolated in such a way that they are present in a medium containing a proline derivative of the general formula I in the form of the racemate or one of its optically active isomers

- as the only source of carbon and nitrogen or

- as the only nitrogen source with a suitable carbon source, or - as the only carbon source with a suitable nitrogen source, cultivated in the usual way. From the culture obtained by cultivation, those which utilize an L-prolinamide of the general formula I as the only nitrogen source are then appropriately selected The radical R ¹ in the proline derivative of the general formula I means alkyl, acyl or hydrogen. The radical R ² means hydrogen or hydroxy. The radical R means -NH ₂ , alkoxy or aryloxy

Alkyl is hereinafter referred to a C ^ alkyl group, substituted or unsubstituted, as defined Suitable substituents are for example halogen, in particular fluorine, chlorine and bromine, and examples of an unsubstituted hydroxy Cι- ₅ alkyl group are methyl, ethyl, propyl, isopropyl, butyl , Isobutyl, Tertiarbutyl, Pentyl, Isopentyl, (Isoamyl) Examples of a substituted Cι- ₅ alkyl group are chloromethyl, trifluoromethyl, 2-bromopropyl and hydroxymethyl

Acyl is defined below as acetyl, formyl, propionyl, butyryl, pentyryl, benzoyl or phenylacetyl

Alkoxy is defined below as a C] - ₅ alkoxy group, substituted or unsubstituted. Suitable substituents are, for example, the substituents mentioned above for alkyl. Examples of an unsubstituted C] - ₅ alkoxy group are methoxy, ethoxy, propoxy, isopropoxy, butoxy, pentoxy , Isopentoxy or Isobutoxy Examples of a substituted C ₅ alkoxy group are trifluoromethoxy, chloromethoxy, 2-chloropropoxy and hydroxymethoxy

Aryloxy is defined below as benzyloxy and phenyloxy

The microorganisms can use, for example, sugar, polyols, peptones, carboxylic acids or amino acids as a growth substrate as a suitable carbon source. Hexoses such as glucose or fructose, pentoses such as xylose and disaccharides such as maltose or sucrose can be used as sugars. For example, glycols, glycerol, sugar alcohols such as mannitol can be used as polyols , Sorbitol or inositol can be used. The carboxylic acids used are di- or tricarboxylic acids or their salts, such as, for example, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, azelaic acid, sebacic acid, maleic acid, fumaric acid, sorbic acid, 1,2, citric acid, agaric acid 3- propanetricarboxylic acid, citrate, fumarate, oxalate or malate As amino acids or their salts, all proteinogenic amino acids or their salts, such as aspartic acid, glutamic acid, aspartate, glutamate, glutamine, asparagine, alanine, valine, leucine, isoleucine, proline, Tryptophan, phenylalanine, methioni n, glycine, serine, tyrosine, threonine, cysteine, histidine, arginine can be used A sugar or sugar alcohol is preferably used as the carbon source

The microorganisms can use, for example, ammonium compounds or amino acids or their amides as a suitable nitrogen source. For example, ammonium chloride, ammonium sulfate, ammonium carbonate or ammonium acetate can be used as the ammonium compound. The amino acid can be alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine, glycine, serine , Tyrosine, threonine, cysteine, asparagine, glutamine, lysine, arginine or histidine. Appropriate representatives of the amino acid amides are prolinamide, alanine amide, glycinamide, serine amide and valine amide

Pipecolinic acid amide can be used as the piperidinecarboxylic acid amide, nicotinic acid amide as the pyridinecarboxylic acid amide and lactic acid amide as the hydroxycarboxylic acid amide

The selection and cultivation medium which can be used are those customary in the art, for example that described in Table 1

The active enzymes of the microorganisms are expediently induced during the cultivation and selection. L- and DL-lactic acid amide or their N-methylated derivatives such as N-methyl-L-lactic acid amide and N, N-dimethyl-L-lactic acid amide can preferably be used as the enzyme inducer the L-enantiomer used The inductor concentration is expediently between 0.05 and 10 g / 1, preferably between 0.5 and 3 g / 1

The cultivation and selection is usually carried out at a temperature of 10 to 40 ° C., preferably 25 to 35 ° C. and at a pH between pH 4 and pH 10, preferably between pH 6 and pH 8

Preferred microorganisms are L-proline-utilizing the genus Aureobacterium, Klebsiella, Aeromonas, Serratia or Pseudomonas In particular, microorganisms of the species Klebsiella pneumoniae, Aeromonas sobria, Serratia plymuthica, Pseudomonas fluorescens or Aureobacterium sp LS10 (DSM 10203), as well as their functionally equivalent variants and mutants isolated The microorganisms Aureobacterium sp LS10 were deposited on 29 August 1995 with the DSM-German Collection of Microorganisms and Cell Cultures GmbH, Mascheroderweg 1 b, D-38124 Braunschweig, according to the Budapest contract

"Functionally equivalent variants and mutants" are understood to mean microorganisms or enzymes which have essentially the same properties and functions as the original microorganisms or the enzymes obtained from them. Variants and mutants of this type can be formed accidentally, for example by UV radiation become

Taxonomic description of Aureobacterium sp LS10 (DSM 10203)

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

Agility +

Spores

Catalase +

Meso-diaminopimeline acid in the cell wall no

Peptidoglycan type B2ß, [L-Hsr] D-Glu (Hyg) - Gly - D-Orn

16S r DNA sequence similarity Sequencing of the region with the greatest variability showed 98.9% agreement with Aureobacterium luteolum Aureobacterium luteolum, however, is immobile, that is to say that the Aureobacterium sp according to the invention is a new species

The enzymes according to the invention, the L-prolinester hydrolases or L-prolinamide hydrolases, which are capable of hydrolyzing proline derivatives of the formula I can be obtained, for example, by digestion of the microorganisms which is customary in the art. For example, the ultrasonic, French press or Lysozyme method can be used In the first stage of the process according to the invention, an L-proline derivative of the general formula is expediently used

where R has the meaning already mentioned, to the corresponding proline derivative of the general formula

. 7 wherein R has the meaning given, racemized The racemization can either be carried out in a known manner, for example in accordance with EP-A-0 057 092, or the racemization takes place in a strongly alkaline environment

The second stage, the esterification, the production of amides or the alkylation or acylation on the nitrogen atom, of the racemic proline derivative to the proline derivative of the general formula I is likewise carried out in a manner known per se. The esterification can be carried out by reaction with an alcohol in accordance with the organic agent, 1976 , S 498ff, the preparation of the amides, for example by ammonolysis with ammonia according to Organikum, 1976, S 509ff, the alkylation on the nitrogen atom by reacting, for example, with an alkyl halide

Organikum, 1976, p 257 and the acylation on the nitrogen atom by reaction, for example, with a carboxylic acid halide

The conversion of the proline derivative of the general formula I to the L-proline derivative of the general formula III

is carried out according to the invention either by means of microorganisms and / or enzyme extracts therefrom or by means of enzymes with L-prolinester hydrolase or L-prolinamide hydrolysis activity. In this biotransformation, in addition to the L-proline derivative of the general formula III, the D-proline derivative of the formula II occurs isolated In principle, biotransformation is possible with all microorganisms that use a proline derivative of the general formula I in the form of the racemate or its optically active isomers as the only nitrogen source, as the only carbon source or as the only carbon and nitrogen source

The microorganisms of the genus Aureobacterium, Klebsiella, Aeromonas, Serratia or Pseudomonas described above are particularly suitable for the method. In particular, the microorganisms of the genus Aureobacterium such as Aureobacterium sp LS10 (DSM 10203) or its functionally equivalent variants and mutants are suitable for the biotransformation

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. The biotransformation is preferably carried out with resting cells

For the biotransformation, media customary in the art can be used, such as, for example, low-molar phosphate buffers, Tris buffers, or the medium described in Table 1. The biotransformation is preferably carried out in the medium in accordance with Table 1

The biotransformation is expediently carried out with a single or continuous addition of the proline derivative of the formula I in such a way that the concentration does not exceed 40% by weight, preferably 20% by weight

The pH of the medium can be in a range from 4 to 10, preferably in a range from 5 to 8. The biotransformation is expediently carried out at a temperature of 10 to 50 ° C., preferably 20 to 40 ° C.

The biotransformation is preferably carried out at a high cell density, in particular at a cell density of OD ₆₅₀ = 20 to OD ₆₅₀ = 60

The biotransformation can be carried out in the absence of the alcohols formed in the prolinester hydrolysis, preferably in the absence of ethanol, propanol, butanol, isopropanol, isobutanol, isopentyl alcohol (isoamyl alcohol) and benzyl alcohol. The corresponding alcohols can be removed, for example, by adsorption

The D-proline derivatives of the formula II or III obtained in this way can be isolated by customary workup methods

The process is preferably carried out without isolation of the racemic proline derivative of the formula V.

The proline derivatives according to formula VI are prepared by hydrolysis of the proline derivatives according to formula II. The hydrolysis is carried out in a manner customary in the art, for example according to Organikum S 476 and S 477

Examples:

Example 1:

Selection of prolinamide-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 SO ₄ 0.1 g / 1

Na ₂ HPO_ι-2 HO 2.5 g / 1

KH ₂ PO ₄ 1.0 g / 1

NaCl 3.0 g / 1

MgCl ₂ -6 H ₂ O 0.4 g / 1

CaCl -2 H ₂ O 14.5 mg / 1

FeCl ₃ -6 H ₂ O 0.8 mg / 1

Trace element solution 1.0 ml / 1

Vitamin solution 1.0 ml / 1 pH 7.0

Fructose (5 g / 1) was added as a carbon source (C source). To enrich microorganisms that are able to hydrolyze prolinamide, DL-prolinamide (1 g / 1) was the only nitrogen source (N source) to this basic medium. added Different batches were then inoculated with soil samples from different locations and incubated (30 ° C, 120 rpm) until clearly visible growth was evident. An aliquot of this culture was then inoculated into 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, more than 20 different bacterial isolates were obtained which were capable of prolinamide to be used as the only N source Example 2:

Test of the selected microorganisms for hydrolytic activity and selectivity towards prolinamide, proline esters or N-methyl proline esters

The isolates obtained with the method described in Example 1 were propagated in the medium described there, but using L-prolinamide (1 g / 1) as an N source and a small amount of yeast extract (0.2 g / 1) The cells thus produced were harvested by centrifugation and then resuspended in buffer solution (50 mM phosphate buffer, pH 7.0) and washed. After resuspending in buffer again, the ability to hydrolize L-prolinamide was tested with resting cells. A suitable amount was tested incubated on cells with the respective substrate (4 g / l) in buffer solution (50 mM phosphate buffer, pH 7.0) (30 ° C.) At different times, aliquots were taken and checked for the release of L-proline or N by thin-layer chromatography - Methyl-L-proline checked Several isolates showed the desired hydrolytic activity towards L-proline amide and various esters of L-proline and N-methyl-L-proline respectively. In the case of Proli Namid and the proline ester, the selectivity towards the respective L-enantiomers was also examined, and some strains, e.g. Klebsiella pneumoniae, Aeromonas sobria, Serratia plymuthica, Pseudomonas fluorescens and Aureobacterium sp (DSM 10203) were found with a high enantioselectivity

Example 3:

Growth and enzymatic activity of Aureobacterium sp. LS10

a) N sources

Aureobacterium sp LS 10 was grown in the minimal medium described in Table 1 with glucose (10 g / 1) as the C source and some yeast extract (0.2 g / 1). Various N sources (2 g / 1, except peptone 10 g / l) were added, such as amino acids and acid amides. Cultures which achieved a good cell density (OD ₅₀ > 0.5 in 48 h) were analyzed for their enzymatic activity (hydrolysis of L-proline butyl ester). The activity after cultivation with L-prolinamide as the N source was used as the 100% value Table 2 Enzymatic activity of Aureobacterium sp LSIO when grown with various N sources

Numerous N sources could be used for growth by Aureobacterium sp LSIO, but the enzymatic activity was strongly influenced both negatively and positively (see Table 2)

b) C sources Aureobacterium sp LSIO was grown in the minimal medium described in Table 1 with L-lactic acid amide (2 g / 1) as N source and some yeast extract (0.2 g / 1). Various C sources (10 g / 1) were added. Cultures which reached a good cell density (OD ₆₅₀ > 0.5 in 48 h) were analyzed for their enzymatic activity (hydrolysis of L-proline butyl ester). The activity of cells after growth on glucose as C - Source was used as a 100% value Table 3 Enzymatic activity of Aureobacterium sp LSIO when grown with various C sources

Aureobacterium sp LS10 was able to use a wide variety of C sources, but preferred sugar and sugar alcohols. The enzymatic activity was only influenced when amino acids were used as C sources (see Table 3)

Example 4:

Aureobacterium sp. L-prolinester hydrolase inducers. LS10

a) Inductor effect of different molecules

Aureobacterium sp LS10 was grown in the minimal medium described in Table 1 with yeast extract (2 g / 1) as the N source and glucose (10 g / 1) as the C source. In addition, the cultures were grown after reaching a sufficient cell density their enzymatic activity (hydrolysis of L-proline butyl ester) was analyzed, L-lactic acid amide or other compounds structurally related to L-lactic acid amide were added (each 1 g / 1) to test their potency as an inducer of L-proline ester hydrolase In addition to the racemate of lactic acid amide, only the N-methylated derivatives of L-lactic acid amide showed an inducing effect (see Table 4) Table 4 Enzymatic activity of Aureobacterium sp. LSIO when grown with various potential inductors

b) Optimal inductor concentration

Aureobacterium sp LSIO was grown as described under 4a). L-lactic acid amide, DL-lactic acid amide or N-methyl-L-lactic acid amide was used as an inducer in various concentrations and, after reaching a good cell density, the enzymatic activity (hydrolysis of L-proline butyl ester) was determined . The enzymatic activity at 1 g / 1 L-lactic acid amide was used as a 100% value

1 shows the enzymatic activity of Aureobacterium sp LS10 when grown with various inducers in variable concentrations

From the results it can be seen that only the L-enantiomer of lactic acid amide has an inducing effect, the optimal concentration being 0.5 to 1.5 g / l. With increasing inducer concentration, the enzymatic activity decreases again, which is probably due to the inhibitory effect of the ammonium liberated in the hydrolysis of the amide

In contrast, N-methyl-L-lactic acid amide reaches an activity plateau, since the rate of hydrolysis is likely to be significantly reduced. However, N-methyl-L-lactic acid amide is only effective at higher concentrations than L-lactic acid amide, made possible at the optimum (3.0 g / 1 ) but higher enzymatic activity

Example 5:

Influence of various parameters on the activity and stereoselectivity of the L-prolinester hydrolase from Aureobacterium sp. LSIO

Aureobacterium sp LSIO was grown in the minimal medium described in Table 1 with glucose (10 g / 1) as the C source, L-lactic acid amide (2 g / 1) as the N source and some yeast extract (0.2 g / 1) Cultured After reaching a sufficient cell density, the culture was harvested, the cells were washed and resuspended in saline solution (0.9%). Aliquots of this bacterial suspension were tested for their hydrolysis activity against L- or D-proline butyl ester by varying various parameters

a) Activity and stereoselectivity

For an optimal racemate cleavage of a proline ester, in addition to the highest possible enzymatic activity, the highest possible stereoselectivity is crucial. It was therefore investigated which parameters are important. To determine the 100% value for the enzymatic activity, the attempt was made to: where mean values were used for all parameters

Table 5 Influence of pH, temperature and cell density on activity and stereoselectivity of the L-prolinester hydrolase (E-value = quotient of the activity towards the L-prolinesters and the corresponding D-prolinesters)

The enzymatic activity of the L-prolinester hydrolase is largely temperature-dependent. An increase in temperature by 15 ° C leads to a quadrupling of the activity. Increasing pH also leads to increased activity, but only to a relatively small extent. Increasing the cell density is almost proportional Achieving increased activity The stereoselectivity, on the other hand, is largely independent of temperature. The most important parameter here is the pH value, which should be as low as possible. A high cell density also contributes slightly to improved selectivity. This experiment shows that at low pH, high cell density and high temperature the best compromise between enzymatic activity and stereoselectivity can be achieved

b) Product inhibition

Since in the hydrolysis of the L-proline butyl ester by the L-proline ester hydrolase from Aureobacterium sp. LS10 Indications of an inhibition of the enzyme activity by the butanol released during the reaction were found. Experiments were carried out to investigate the influence of various parameters on this inhibition. A test was carried out without butanol as a reference and the determined enzymatic activity as a 100% value used Table 6 Influence of temperature, cell density and butanol concentration on the activity of the L-prolinester hydrolase

The most important parameter for the inhibition of L-prolinester hydrolase by butanol turns out to be cell density. With a low cell density, 100 mM butanol leads to complete inactivation of the enzyme, while with a high cell density, higher butanol concentrations are less harmful With increasing butanol concentration the inhibitory effect increases, but only to a comparatively small extent.Also, an increase in temperature leads to increased inactivation of the enzyme.To ensure complete conversion with the best possible activity in the specific hydrolysis of L-prolinester, the pH must be low, which Cell density is high and the temperature is low

Example 6:

Influence of various alcohols on the activity of the L-prolinester hydrolase from

Aureobacterium sp. LSIO

Since butanol inhibits the enzymatic activity of the L-prolinester hydrolase (cf. Example 5), the inhibitory effect of various alcohols was investigated. Aureobacterium sp. LSIO grown in the minimal medium described in Table 1 with glucose (10 g / 1) as C source, L-lactic acid amide (2 g / 1) as N source and some yeast extract (0.2 g / 1) after reaching a sufficient cell density, the culture was harvested. After washing and resuspending the cells in saline solution (0.9%), aliquots of the

Bacteria suspension incubated at room temperature with various alcohols in various concentrations for 30 minutes. The enzymatic activity was then determined

2 shows the influence of various alcohols on the activity of the L-prolinester hydrolase

This experiment shows that, with the exception of methanol, all alcohols tested have a negative effect on the enzymatic activity. With increasing concentration, the enzyme activity continues to decrease, in extreme cases even to zero. There is also a trend that this negative effect becomes stronger with increasing hydrophobicity of the alcohols

Example 7:

Hydrolysis of various proline esters by L-prolinester hydrolase from Aureobacterium sp. LS10

The activity and stereoselectivity of the L-prolinester hydrolase towards various esters of proline was investigated depending on the pH Esters were chosen whose stability in water is sufficiently good so that the ee value is not significantly deteriorated by unspecific chemical hydrolysis, and whose hydrolysis produces alcohols which have the least possible negative influence on the enzymatic activity (cf. Example 6) For this purpose, Aureobacterium sp LSIO in the minimal medium described in Table 1 with glucose (10 g / 1) as the C source, L-lactic acid amide (2 g / 1) as the N source and some yeast extract (0.2 g / 1 ) attracted After reaching a sufficient cell density, the culture was harvested, the cells were washed and resuspended in saline solution (0.9%). The enzymatic activity against various L- or D-proline esters was then determined. The hydrolysis was determined as 100% of L-proline butyl ester used at pH 7.0 The stereoselectivity of the enzyme was determined on the basis of the ratio of the activity towards the L-proline esters and the corresponding D-proline esters n determined (E value)

FIG. 3 shows the enzymatic activity of the L-proline ester hydrolase with various L-proline esters as a function of the pH

Table 7 Stereoselectivity (E value) of the L-prolinester hydrolase with various prolinesters as a function of the pH

With all four proline esters tested, the enzymatic activity increases with increasing pH.The hydrolysis of L-proline butyl ester is significantly slower than the hydrolysis of the three remaining esters.For all four esters, the L-enantiomer is hydrolyzed significantly faster than the D-enantiomer, so that good until very good E values can be observed, the selectivity, depending on the ester used, changing slightly depending on the pH Example 8:

Hydrolysis of the proline isopropyl ester at different substrate concentrations

Proline isopropyl ester, which had proven to be the cheapest substrate, was to be used to investigate which product concentrations can be achieved. To this end, Aureobacterium sp LSIO was used in the minimal medium described in Table 1 with glucose (10 g / 1) as the C source, L-lactic acid amide (2 g / 1) as N source and some yeast extract (0.2 g / 1) grown. After reaching a sufficient cell density, the culture was harvested. The cells were then washed in saline solution (0.9%) and finally resuspended therein The test was carried out with DL-proline isopropyl ester as a substrate in various concentrations, while the other parameters were kept constant (25 ° C., pH 6.0, OD ₆₅₀ ca 15). At different times, aliquots were taken, which were determined by HPLC to determine their content D- and L-proline isopropyl esters were analyzed

4 shows the biotransformation with Aureobacterium sp LS10 with variation of the substrate concentration

With 60 or 120 g / 1 DL-proline isopropyl ester, a very rapid hydrolysis of the L-ester was observed, which resulted in the formation of D-ester with an ee value of over 98%. At higher substrate concentrations (240 g / 1 or 300 g / 1) the slowed

Hydrolysis rate clear With increasing concentration of released isopropanol, the reaction becomes progressively slower, so that complete hydrolysis of the L-proline isopropyl ester becomes difficult. High ee values could only be observed after a longer incubation period, although the yield of D-ester decreased slightly as an alternative the cell density was doubled in the batch (OD ^ ₀ ca 30), which resulted in a complete conversion of the L-ester after about 4 h with 240 g / 1 DL-proline isopropyl ester and an ee value of over 98% of the remaining D-proline isopropyl ester would have

Example 9:

Production of D-proline using the L-prolinester hydrolase from Aureobacterium sp.

LSIO

a) Cultivation of Aureobacterium sp LSIO on minimal medium

Aureobacterium sp LSIO was in a Chemap fermenter (working volume 5 1) in minimal medium (see Tab 1) with glucose (30 g / 1) and L-lactic acid amide (4 g / 1) as a C or N source at 30 ° C grown In order to facilitate the growth of the cells, the medium also contained a small amount of yeast extract (0.7 g / 1). During the course of the cultivation, further L-lactic acid amide was added as required by the cells until a sufficiently high cell density (OD ₆₅₀ ca 23) and a good specific enzyme activity (> 1.2 μmol L-proline butyl ester hydrolyzed / min x OD ₆₅₀ ) was achieved. The cells were separated by centrifugation, washed in saline solution (0.9%) and in it finally, finally resuspended 401.4 g of DL-proline isopropyl ester were added to the bacterial suspension and the volume was adjusted to 4.5 l by adding water. This biotransformation mixture was then incubated for 160 minutes at 25.degree. C. and pH 6.0 with stirring. The cells were then major ch centrifugation separated and the supernatant worked up. Repeated electrodialysis at pH 6.3 initially removed the D-proline isopropyl ester from L-proline. In the next step, D-proline was released from the ester by alkaline hydrolysis with sodium hydroxide solution and the sodium chloride formed

Electrodialysis (pH 6.3) separated. The D-proline solution thus obtained was evaporated to dryness and contamination by a yellow dye was removed by washing the D-proline crystals with ethanol _bs (30 minutes at 60 ° C.) In this way, 64.0 g of D-proline (53.7% of theory) were ultimately isolated, which had both a very good content (> 99% titrimetric) and an excellent optical purity (ee> 99% according to HPLC, [α] _D - 83.7 for c = 4 in acetic acid)

b) Cultivation of Aureobacterium sp LS 10 on complete medium

Aureobacterium sp LS10 was in a Chemap fermenter (working volume 2 1) in mineral medium (see Tab 1) with glucose (20 g / 1) as a C source and yeast extract (15 g / 1) as a N source at 30 ° C dressed In addition, the medium contained N-methyl-L-lactic acid amide (3 g / 1) as an inducer. After reaching a sufficient cell density (OD ₆₅₀ ca 30), the cells were harvested by centrifugation After washing and resuspending in saline solution, the cells had good activity (approx. 1.1 μmol L-proline butyl ester hydrolyzed / min × OD ₆₅₀ ) and part of it (OD ₆₅ o = 21 for V = 1.15 1) was used for the racemate cleavage of 228.2 g of DL-proline isopropyl ester. After 5.5 hours, the hydrolysis of the L-proline ester was complete and the cells were separated by centrifugation. 90% of the D-proline ester used was still detectable in solution, one of which was very high good optical purity (ee value = 99% by HPLC) was determined

Example 10:

Chemical syntheses

10.1 Racemization of L-Proline

To obtain racemic proline, 160 g (1.39 mol) of L-proline was dissolved in 600 ml (10.49 mol) of acetic acid and 20 g (277 mmol) of butanal were added. This solution was dissolved in a

Autoclave heated to 100 ° C and held at this temperature for two hours. After cooling, the mixture was concentrated as far as possible and racemic proline was crystallized by adding 700 ml of acetone. 144.4 g of proline were obtained after drying the crystals (90.3% of theory) Th), whose identity was confirmed by NMR spectroscopy and which was almost racemic ([α] D24 = - 0.5 ° for c = 5 in 1 N HC1)

Alternatively, proline could also be racemized in an alkaline aqueous solution (see part of the one-pot reaction in 10 3)

10.2 Esterification of DL-Proline 300 g (2.61 mol) of DL-proline and 1000 ml (13.06 mol) of isopropanol were placed in a three-necked flask for the preparation of racemic proline isopropyl ester. This mixture was cooled to 10 ° C. and then slowly While maintaining the temperature, 380 ml (5.22 mol) of thionyl chloride were added dropwise. After the addition had ended, the mixture was first stirred at room temperature for 90 min and then the solution was heated to reflux for a few hours. After cooling and distilling off the remaining solvent, 521.9 g were obtained of a brown oil, the HPLC analysis of which showed that a conversion of 95% was achieved Using the same procedure, other proline esters such as the proline methyl, proline ethyl and proline butyl esters and the corresponding esters of N-methylproline could also be produced

10.3 One-pot reaction for the production of DL-prolinester

Using the racemization of L-proline in aqueous alkaline solution, DL-proline isopropyl ester from L-proline can also be prepared in a one-pot process. 80 g (695 mmol) of L-proline in 26 ml of 4 N NaOH (104 mmol ) and 148.5 g of water was dissolved. This solution was then heated in an autoclave to 160 ° C., a pressure of about 5 bar being reached, and kept at this temperature for 14 hours. The water was then passed through as well as possible Distillation removed, using the effect of azeotropic distillation after the addition of 250 ml of isopropanol to remove residual amounts of water. After isopropanol was removed as well as possible by distillation, 78.5 g (2.43 mol) Isopropanol was added and the mixture was cooled to 10 ° C. While maintaining a temperature of 5-10 ° C., 103.5 g (0.87 mol) of thionyl chloride were then added dropwise. The mixture was then slowly added to 84 over a period of 2.5 hours ° C heated After filtering off a white precipitate (NaCl) and distilling off the solvent, 138.4 g of a yellowish oil were obtained. According to HPLC analysis, about 70% of the L-proline used was now in the form of DL-proline isopropyl ester

10.4 Production of N-methyl-proline

To prepare N-methyl-proline, 115.1 g (1.0 mol) of L-proline in 375.8 g of 98%

Formic acid (8.0 mol) dissolved and then 329.1 g of 36.5% formaldehyde (4.0 mol) were slowly added dropwise

The reaction then took place for four hours at 60 ° C. After the solvents had been separated off by distillation, 184.5 g of an orange-colored oil remained. This was dissolved in 100 ml of water and the pH was adjusted to 6.3 (pl) with NaOH so that N-methyl-L-proline was present as the hermaphrodite ion. The water was removed by distillation and the residue was taken up in 180 ml of ethanol. Residues of water were removed by azeotropic distillation

The residue was then slurried in 500 ml of ethanol and kept at 8 ° C. for a few hours. Insoluble constituents were then filtered off and the filtrate was evaporated to dryness After redissolving in 120 ml of ethanol, N-methyl-L-proline was crystallized out by adding 95 ml of ether. After the crystals had been separated off and dried, 74.6 g of N-methyl-L-proline remained, the identity of which was confirmed by GC analysis

10.5 Synthesis of N-methyl-prolinamide

In order to produce the corresponding amide from N-methyl-proline, 12.9 g (100 mmol) of N-methyl-L-proline and 100 ml (2.47 mol) of methanol were cooled to 0 ° C. and slowly 14 while maintaining the temperature , 3 g (120 mmol) of thionyl chloride was added dropwise. After stirring for about 90 min at room temperature, the mixture was heated to reflux for 2.5 hours. After filtering off an insoluble impurity and concentrating to dryness, 15.1 g of an oily residue remained Half of it was added to 20 ml of 50% K ₂ CO ₃ (0 ° C.) and the N-methyl-L-proline methyl ester, which was uncharged under these conditions, was extracted with a total of 60 ml of ether. The 4.1 g of residue which remained after drying to dryness were dissolved in a total of 60 ml of ammoniacal methanol and heated in an autoclave for seven days (40-80 ° C.). The residue obtained after concentration to dryness was then dissolved in 20 ml of methanol. An insoluble impurity was determined by F iltration separated and the filtrate was finally evaporated to dryness. 3.0 g of N-methyl-L-prolinamide were obtained, the identity of which was confirmed by GC analysis

Claims

Claims:

1. Microorganisms, characterized in that they are capable of proline derivatives, in the form of the racemate or optically active isomers, selected from the compounds of the general formula

1 7 "in which R is alkyl, acyl or hydrogen, R is hydrogen or hydroxy and R is -NH ₂ , aryloxy or alkoxy, as the only nitrogen source, as the only carbon source or as the only carbon and nitrogen source, and enzyme extracts therefrom

2. Microorganisms according to claim 1 or 2 of the genus Aureobacterium, Klebsiella, Aeromonas, Serratia or Pseudomonas

3. Microorganisms according to claim 3 of the genus Aureobacterium

4. Microorganisms according to at least one of claims 1 to 3 of the species Aureobacterium sp LSIO (DSM 10203) and its functionally equivalent variants and mutants

5. enzyme with L-prolinester hydrolase and / or L-prolinamide hydrolase activity, obtainable from microorganisms according to one of claims 1 to 4 and capable of proline derivatives, in the form of the racemate or optically active isomers, selected from the compounds of the general formula

1 7 T • in which R, R and R have the meaning given, to hydrolysers, and also functionally equivalent variants and mutants thereof

6. Process for the preparation of D-proline derivatives of the general formulas

and or

wherein R, R and R ^'have the meaning given in claim 1, comprising the

Implementation of a proline derivative of the general formula

wherein R, R and R ^'have the meaning given, by means of a microorganism and / or enzyme preparation according to claim 1 or an enzyme according to claim 5 to an L-proline derivative of the general formula III, and optionally isolation of this compound and / or of this reaction resulting D-proline derivative of the formula II

7. The method according to claim 6, characterized in that the proline derivative of the general formula

wherein R, R and R have the meaning given, is prepared in that in a first step an L-proline derivative of the general formula

wherein R has the meaning given, to a proline derivative of the general formula

wherein R has the meaning given, racemizes and this in the second stage

Proline derivative of the general formula

where R, R and R have the meaning given, transferred

8. The method according to claim 6, characterized in that the reaction of the proline derivative of the general formula I by means of microorganisms of the species

Aureobacterium sp LS IO (DSM 10203) or with its functionally equivalent variants and mutants

9. The method according to claim 6, characterized in that one carries out the biotransformation at a pH of 4 to 10 and at a temperature of 10 to 50 ° C.

10. Process for the preparation of a D-proline derivative of the general formula

I 7 wherein R and R have the meaning given in claim 1, characterized in that in a first step an L-proline derivative of the general formula

wherein R ^{2 has} the meaning given, to the corresponding proline derivative of the general formula

7 wherein R has the meaning given, racemizes it in a second stage into a proline derivative of the general formula

wherein R ¹ , R ² and R ^{3 have} the meaning given, and this in a third stage by means of a microorganism and / or enzyme preparation according to claim 1 or an enzyme according to claim 5 in an L-proline derivative of the general formula

transferred, with the biotransformation in addition to the L-proline derivative of the general

Formula III the D-proline derivative of the general formula

is obtained, which is hydrolyzed in a fourth stage to the product of formula VI.