EP1625104A2

EP1625104A2 - METHOD FOR PRODUCING CHIRAL alpha HYDROXYCARBOXYLIC CRYSTALLINE ACIDS

Info

Publication number: EP1625104A2
Application number: EP04706636A
Authority: EP
Inventors: Hefried Griengl; Ingrid Osprian; Hans-Egbert Schoemaker; Christoph Reisinger; Helmut Schwab
Original assignee: DSM Fine Chemicals Austria Nfg GmbH and Co KG
Current assignee: Patheon Austria GmbH and Co KG
Priority date: 2003-02-27
Filing date: 2004-01-30
Publication date: 2006-02-15
Also published as: WO2004076385A3; US20060099696A1; ATA2852003A; US7514245B2; US7390647B2; JP2006525795A; US20080206827A1; WO2004076385A2; CA2517301A1; AT412092B; US20060199256A1

Abstract

The invention relates to a method for producing chiral alpha-hydroxycarboxylic crystalline acids consisting in transforming cyanhydrins (R) or (S) into alpha-hydroxycarboxylic acids (R) or (S), respectively by enzymatic hydrolysis in the presence of Rhodococcus erythropolis NCIMB 11540.

Description

Process for the preparation of chiral α-hydroxycarboxylic acids by enzymatic hydrolysis of chiral cyanohydrins

Optically active α-hydroxycarboxylic acids are used, for example, as additives in animal feed or in the production of active pharmaceutical ingredients, vitamins and liquid crystals.

These optically active α-hydroxycarboxylic acids can also be described, for example, by Effenberger et al., Angew. Chem. 95 (1983) No. 1, page 50, advantageously convert it into N-substituted optically active α-amino acids which are otherwise very difficult to produce.

Chiral α-hydroxycarboxylic acids are now chemically, fermentatively or enzymatically accessible.

A number of different synthesis possibilities of chiral α-hydroxycarboxylic acids are therefore known from the literature.

Racemic cyanohydrins can be hydrolyzed to the desired chiral α-hydroxycarboxylic acids with the addition of suitable microorganisms.

The production of chiral α-hydroxycarboxylic acids, especially the production of optically active lactic acid or mandelic acid from racemic cyanohydrins with various microorganisms of the genera Alicaligenes, Pseudomonas, Acotobacter, Rhodococcus, Candida etc. is for example in EP 0 449 684, EP 0 527

553, EP 0 610 048, etc. described.

From this prior art it is also known that when a racemic cyanohydrin is hydrolyzed to the corresponding α-hydroxycarboxylic acid using a nitrilase, the problem arises that the enzyme is inactivated within a short time and thus the desired α-hydroxycarboxylic acid is usually obtained only in low yields and concentrations. This also applies to the use of nitrile hydratases, which combine the cyanohydrin with the corresponding converting α-hydroxyamide. The hydroxyamides can then in turn be converted to the corresponding α-hydroxycarboxylic acids.

It is also known, for example from Angew. Chem. 1994, 106, page 1615f. That optically active cyanohydrins can be hydrolyzed with concentrated hydrochloric acid without racemization to give the corresponding chiral α-hydroxycarboxylic acids. The optical purity of the chiral α-hydroxycarboxylic acids thus produced corresponds to the optical purity of the chiral cyanohydrin used, even if it is obtained in situ by enzyme-catalyzed addition of a cyanide group to a corresponding aldehyde or a ketone and is further processed without isolation or purification.

The disadvantage of this reaction is that sensitive substrates are decomposed and the occurrence of corrosion.

It was an object of the present invention to find a process in which polar nitriles such as chiral cyanohydrins can be converted into the corresponding chiral hydroxycarboxylic acids using a mild and efficient method, the hydroxycarboxylic acids having approximately the same enantiomeric purity as the cyanohydrins.

This task was unexpectedly achieved by using a special bacterium from the Rhodococcus genus.

The present invention accordingly relates to a process for the preparation of chiral α-hydroxycarboxylic acids, which is characterized in that (R) - or (S) -cyanohydrins in the presence of Rhodococcus erythropolis NCIMB 11540 by enzymatic hydrolysis into the corresponding (R) - or (S) -α- Hydroxycarboxylic acids are transferred. In the process according to the invention, (R) ~ and (SJ-cyanohydrins are converted into (R) - and SJ-α-hydroxycarboxylic acids with an optical purity of up to> 99% ee.

The starting compounds used are (R) - and (S) - cyanohydrins, which are produced by enzymatic or chemically catalyzed addition of a cyanide group to the corresponding aldehydes or ketones.

The enzymatic or chemically catalyzed addition of a cyanide group to the corresponding aldehydes or ketones can be carried out analogously to the prior art, for example analogously to EP 0 951 561, EP 0 927 766, EP 0 632 130, EP 0547 655, EP 0 326 063 etc. ,

The aldehydes and ketones cited in the prior art are suitable as starting compounds.

Examples of suitable aldehydes are aliphatic, aromatic or heteroaromatic aldehydes. Aliphatic aldehydes are to be understood as meaning saturated or unsaturated aliphatic, straight-chain, branched or cyclic aldehydes. Preferred aliphatic aldehydes are straight-chain aldehydes having in particular 2 to 18 carbon atoms, particularly preferably 2 to 12, which are saturated or mono- or polyunsaturated. The aldehyde can have both CC double bonds and CC triple bonds. The aldehyde can be unsubstituted or one or more times by groups which are inert under the reaction conditions, for example by optionally substituted aryl or heteroaryl groups, such as phenyl or indolyl groups, by C ₁ -C ₆ -alkyl, optionally substituted cycloalkyl groups which have one or more heteroatoms from the group O, S, P, or N can be substituted, halogen, ether, alcohol, acyl, carboxylic acid, carboxylic acid ester, nitro or azido groups. Examples of aromatic or heteroaromatic aldehydes are benzaldehyde or variously substituted benzaldehydes such as 2-chlorobenzaldehyde, 3,4- Difluorobenzaldehyde, 4-methylbenzaldehyde, 3-phenoxybenzaldehyde, 4-fluor-3-phenoxybenzaldehyde, further furfural, anthracene-9-carbaldehyde, furan-3-carbaldehyde, indole-3-carbaldehyde, naphthalene-1-carbaldehyde, phthalaldehyde, pyrazole -3-carbaldehyde, pyrrole-2-carbaldehyde, thiophene-2-carbaldehyde, isophthalaldehyde or pyridine aldehydes, etc.

Examples of ketones are aliphatic, aromatic or heteroaromatic ketones in which the carbonyl carbon atom is unequally substituted. Aliphatic ketones are straight-chain, branched or cyclic ketones. The ketones can be saturated or mono- or polyunsaturated. You can unsubstituted or one or more times by groups inert under the reaction conditions, for example by optionally substituted aryl or heteroaryl groups such as phenyl or indolyl groups, by halogen, ether, alcohol, acyl, carboxylic acid, carboxylic acid ester, Nitro or azido groups can be substituted. Examples of aromatic or heteroaromatic ketones are acetophenone, indolylacetone, etc.

(R) - or (S) -cyanohydrins of the formula are preferred

HO CN

R1 X R2 z in the R1 and R2 are independently H, an optionally mono- or polysubstituted with inert under the reaction conditions substituent substituted C-ι-C ₆ - alkyl or alkenyl radical or an optionally mono- or polysubstituted with inert under the reaction conditions substituent substituted phenyl radical, with the proviso that R1 and R2 are not both H.

Preferred substituents which are inert under the reaction conditions are, for example, halogens, such as fluorine, bromine and chlorine, C ₁ -C ₆ -alkyl or alkoxy, ethers, esters, acetals or optionally substituted phenyl and phenyloxy. Particularly suitable for the process according to the invention are (R) - or (S) - cyanohydrins, such as (R) - or (S) -2-hydroxy-4-phenyl-butyronitrile, (R) - or (S) -2 - Chloromandelonitrile, (R) - or (S) -mandelonitrile, (R) - or (S) -4-methylmandelonitrile, (R) - or (S) -3-phenoxymandelonitrile, (R) - or (S) -2 -Hydroxy-2-methyl-heptanenitrile, (R) - or (S) -2-hydroxy-2-phenyl-propionitrile, (R) - or (S) -2-hydroxy-3-pentenenitrile, (R) - or (S) -l-hydroxy-cyclohexanenitrile, (R) - or (S) -acetophenonecynahydrin.

The corresponding (R) - or (S) - cyanohydrin is then enzymatically hydrolyzed according to the invention.

According to the invention, the enzymatic hydrolysis takes place in the presence of Rhodococcus erythropolis NCIMB 11540.

Rhodococcus erythropolis NCIMB 11540 has unexpectedly found a microorganism which is distinguished by the fact that it has a nitrile hydrate / amidase enzyme system which can hydrolyze the nitrile function of such polar nitriles as the cyanohydrins mentioned above.

Through the nitrile hydratase / amidase enzyme system from Rhodococcus erythropolis NCIMB 11540, the chiral cyanohydrins are hydrolyzed in the first step by the nitrile hydratase into the corresponding chiral hydroxyamide, which is then converted in a second hydrolysis step by the amidase into the corresponding chiral α-hydroxycarboxylic acid.

The microorganism can be used in the method according to the invention in any form, for example in the form of ground cells, crude or purified enzymes, recombinant enzymes, immobilized cells or enzymes, lyophilized cells or "resting cells".

Recombinant enzymes, resting cells or lyophilized cells are preferably used, particularly preferably recombinant enzymes or resting cells. In addition to the direct use of the nitrile hydratase / amidase-active preparations of Rhodococcus erythropolis NCIMB 11540 cells, the use of recombinant preparations expressed in a suitable microorganism, such as E. coli, Pichia pastoris, Saccharomyces, Asperagillus, K. Lactis, etc. good alternative. The corresponding genes are introduced with the aid of plasmid constructs into suitable host cells, for example in E. coli, Pichia pastoris, Saccharomyces, Asperagillus, K. Lactis host cells. By choosing an inducible promoter, both the nitrile hydratase and the amidase can be overexpressed in active form. In the case of amidase, much higher activity levels can be achieved than with corresponding fermentation of the Rhodococcus cells.

The microorganism is then suspended in the desired form in an aqueous medium, such as water or a buffer solution. Suitable buffer solutions are, for example, phosphate buffers such as K / Na phosphate buffers, PBS buffers, butyrate buffers, citrate solutions, etc.

The pH of the buffer solution used should be in a range from pH 4.5 to pH 11, preferably from 5.5 to 8.5.

The corresponding chiral cyanohydrin is then added to the suspension thus obtained. Since the chiral cyanohydrins are lipophilic compounds with limited water solubility, the use of a solubilizer as a cosolvent is necessary in order to dissolve the cyanohydrins in an aqueous medium.

Suitable solubilizers are, for example, organic solvents, surfactants, phase transfer catalysts, etc.

Organic solvents which are suitable as cosolvents for the process according to the invention are those which firstly sufficiently dissolve the substrate and secondly impair the enzyme activity as little as possible. Examples include dimethyl sulfoxide (DMSO), dimethylformamide (DMF), Ci-Cβ alcohols, such as methanol, ethanol, i-propanol, 1-butanol, 2-butanol, t-butanol or 1-pentanol, toluene or t.- Butyl methyl ether (TBME) or mixtures thereof. DMSO, DMF, ethanol, i-propanol or mixtures thereof, and particularly preferably DMSO and DMF, are preferably used as cosolvents.

The proportion of cosolvents should be between 0.5 and 20% by volume, based on the total volume of the reaction solution.

The cosolvent fraction is preferably between 1 and 15 vol% and particularly preferably between 2 and 10 vol%.

The substrate concentration in the reaction solution in the process according to the invention should be in a range from 1 g / l to 10Og / l (based on the total volume of the reaction solution), the acceptance of a sufficiently high substrate concentration being the basic prerequisite for the use of the enzymatic hydrolysis according to the invention in the preparative Scale is.

Substrate concentrations of up to 50 g / l are preferred, particularly preferably up to 25 g / l.

The possible convertible substrate concentration depends on the amount of enzyme used. For an efficient, quantitative conversion, the first hydrolysis step has to be carried out very quickly in order to avoid the disintegration of the cyanohydrin and the resulting racemization, so that relatively high cell densities are required.

It should be noted that sufficient mixing of the reaction system is guaranteed.

The amount of cells or enzyme depends on the activity of the microorganism in the form used, as well as on the substrate concentration and the cosolvent. The pH of the reaction mixture should be between 4.5 and 11, preferably between 5.5 and 8.

If necessary, a suitable acid or acidic salts, such as phosphoric acid, boric acid, citric acid, etc., can be added to the reaction mixture to adjust the pH. be added.

The enzymatic hydrolysis according to the invention is carried out at a temperature of 10 to 60 ° C., preferably at 15 to 50 ° C. and particularly preferably at 20 to 45 ° C.

After hydrolysis to the desired chiral α-hydroxycarboxylic acids, they are isolated from the reaction mixture using a known technique, such as centrifuging the cells, extracting the product after acidification with HCl (e.g. pH 2) and, if appropriate, further purifying it by activated carbon filtration and recrystallization.

The use of Rhodococcus erythropolis NCIMB 11540 according to the invention thus converts polar nitriles, such as chiral cyanohydrins, into the corresponding chiral α-hydroxycarboxylic acids in a simple and efficient manner under mild conditions, with no racemization occurring. Depending on the ee value of the cyanohydrin used, the desired α-hydroxycarboxylic acids are obtained in high optical purity of up to over 99% and in high yields of up to over 98%.

Example 1: Production of the biocatalyst

A complex standard medium (Medium A, see Table 1) was used to produce the Rhodococcus erythropolis NCIMB 11540 biomass. The stock was kept on agar plates with medium A (solidification with 15 g / l agar). The plates were sealed by wrapping parafilm on the side and stored in the refrigerator at 4 ° C.

The growth of the liquid cultures took place in 1000 ml Erlenmeyer flasks with baffles with 250 ml medium A at 30 ° C and 130rpm.

Variant I (without preculture): About half of the biomass of an agar plate was suspended in 5 ml of sterile, physiological saline. One cell suspension each was added to 250 ml of culture medium.

Variant II (with preculture): For the preculture, some biomass of an agar plate was suspended in 5 ml of sterile, physiological saline. One cell suspension each was added to 100 ml of culture medium (= preculture). After 20-24 hours of growth, 5 ml of this preculture were added to each 250 ml culture medium.

The cells were harvested by centrifugation at approx. 3000rpm for 30min at 0-4 ° C. The cells were washed once with K / Na phosphate buffer (50mM, pH 6.5). The cells were then resuspended in fresh buffer and either lyophilized after shock freezing (reactions with lyophilized cells, example 2), or this cell suspension (about 6-8% of the culture volume) was used directly for the biocatalytic reactions (reactions with resting cells, example 3). Table: Composition of Medium A

Example 2: Reactions with lyophilized cells on an analytical scale

31.6mg, 52.6mg and 105.2mg lyophilized cells were rehydrated in 10ml phosphate buffer (50mM, pH 6.5) for approx. 1 hour at 130rpm and 20-25 ° C. Each 475μl of this cell suspension was transferred to 1.5ml Eppendorf reaction vessels and 25μl of an approx. 200mM substrate solution of 2-hydroxy-4-phenyl-butyronitrile in DMSO was added (3mg, 5mg or 10mg cells / ml, S. conc. Approx 10mM, 5% DMSO). The reaction was carried out in a thermomixer at 30 ° C and 1000rpm. After 0, 2, 4, 6, 8, 10, 15, 20, 30, 60 and 120 minutes, 0.5 ml of 1N HCl was added to each Eppendorf reaction vessel. After centrifugation (5min, 13,000rpm) and appropriate dilution, the concentrations of cyanohydrin, hydroxyamide and hydroxy acid were determined by HPLC. Table 2 Hydrolysis of 2-hydroxy-4-phenylbutyronitrile by lyophilized Rhodococcus erythropolis NCIMB 11540 cells (10mg cells / ml; substrate concentration: 10mM) concentration (mM) of substrate, hydroxyamide and hydroxycarboxylic acid as a function of time (min)

Example 3: Enzymatic hydrolysis using resting cells and lyophilized cells

The biocatalyst was produced analogously to Example 1, variant I, 2 culture flasks. After 20 hours (OD ₆ = 3.5 or 1.8), cells were centrifuged from 4 x 10 ml fermentation broth and washed once with K / Na-P0 ₄ buffer (pH 6.5, 50mM). Two cell samples each were lyophilized before activity determination, the other two were used as resting cells.

The cells were resuspended in 1.8 ml K / Na-PO ₄ buffer (pH 6.5, 50 mM) (lyophilized cells were shaken for 1 hour for rehydration). The reaction was started by adding 200μl of a 200mM substrate solution in DMSO (substrate concentration approx. 20mM) and carried out at 30 ° C. and 130rpm in a shaker. After 30 min, 60 min and 17 h, 200 μl were removed and 200 μl of 1N HCl were added. After centrifugation (5 min, 13,000 rpm) and dilution, the conversions were determined by means of HPLC. Only the substrate and the two products were taken into account. (R) -2-chloromandelonitrile (ee> 99%) was used as the substrate. The results are shown in Tab. 3. Table 3: Results of the reactions of culture 1 (OD _{5 6} 3.5), substrate: (R) -2- chloromandelonitrile

1: The conversion of cyanohydrin (CH) relates to both products (hydroxyamide and hydroxy acid).

2: The conversion of the hydroxyamide (HA) relates to the amount of hydroxy acid that was formed from the hydroxyamide present.

Example 4: Enzymatic hydrolysis using different substrate concentrations

Experiment 4.1:

In experiment 4.1, the reaction was carried out on an analytical scale (reaction volume 1 ml) with 3 substrate concentrations (2.2g / l, 6.6g / l, 13.2g / l).

The biocatalyst was prepared according to Example 1, Variant II, 21 fermentation medium, harvest after 20 hours (ODs ₄₆ 6.1). The cells from 8 x 10 ml fermentation solution were centrifuged off in culture tubes. The cell mass obtained was washed once with 2 ml of K Na phosphate buffer (pH 6.5, 50 mM). The contents of 2 tubes were lyophilized to determine the dry weight. Weight: 1. 37 mg lyophilized cells / 10 ml fermentation solution

2. 30mg (This amount corresponded approximately to the use of cells / ml in the following reactions.)

The contents of the remaining 6 tubes were resuspended in 950 μl buffer (OD approx. 40) and transferred to Eppendorfers. 50 μl of differently concentrated substrate solutions (3 concentrations, parallel batches, 5% DMSO as cosolvent) were added to each of these cell suspensions. The Eppendorfers were shaken on a thermomixer at 30 ° C and 100 orpm. To check the turnover, 200 μl were taken in each case and 200 μl of 1 N HCl were added. After centrifugation (5 min, 13,000 rpm) and dilution, the conversions were determined using HPLC. The following concentrations (R) -2-chloromandelonitrile were used: a. Substrate solution: 11mg (R) -2-chloromandelonitrile in 250μl DMSO (approx. 260mM) substrate concentration in the batch: 2.2g / l (13.1mM) b. Substrate solution: 33mg (R) -2-chloromandelonitrile in 250μl DMSO (approx. 290mM) Substrate concentration in the batch: Substrate concentration: 6.6g / l (39.4mM) c. Substrate solution: 66mg (R) -2-chloromandelonitrile in 250μl DMSO (approx. 1580mM) Substrate concentration in the batch: 13.2g / l (78.8mM)

A comparison of the approaches ac is shown in Table 4.1 based on the formation of 2-chloromandelic acid (in%). The hydroxy acid was formed quantitatively in all batches. It was shown that higher substrate concentrations are also accepted without problems.

Table 4.1: Comparison of approaches a-c based on the formation of (R) -2- chloromandelic acid in%

Experiment 4.2:

In experiment 4.2, the reaction was carried out with 2 different substrate concentrations (10 g / l, 20 g / l) on a 5 ml scale.

The biocatalyst was prepared according to Example 1, Variant II, 21 fermentation medium, harvest after 19 hours (OD ₅₄₆ 8.4). The cells were resuspended in approx. 140 ml buffer (Resting cells, OD _{5 6} 52). 4.75 ml each of this cell suspension was used for the enzymatic reactions. Two different concentrations of (R) -2-chloromandelonitrile were investigated in parallel. The reaction was started by adding 250 μl of substrate solution and carried out in culture tubes in a shaking cabinet at 30 ° C. and 130 rpm. To check the turnover, 200 μl were taken in each case and 200 μl of 1 N HCl were added. After centrifugation (5 min, 13,000 rpm) and dilution, the conversions were determined using HPLC.

a. 50mg (R) -2-chloromandelonitrile, dissolved in 250μL DMSO ([S] = 60mM, 10g / L,

Cosolvens: 5% DMSO) All of the cyanohydrin was converted to the hydroxyamide after only 30 minutes, and about 40% of the hydroxy acid was formed after 2 hours. After 20 hours the implementation was quantitative. b. 100mg (R) -2-chloromandelonitrile, dissolved in 250μL DMSO ([S] = 120mM, 20g / l,

Cosolvens: 5% DMSO) All of the cyanohydrin was converted to the amide after 30 minutes, and 36% of hydroxy acid was formed after 18 hours. After 43 hours, 41% of hydroxy acid had formed, after which no further reaction took place.

Experiment 4.3:

In experiment 4.3, reactions with 10 g / l and 15 g / l (r?) - 2-chloromandelonitrile were carried out. Furthermore, the ee of the hydroxy acid formed was determined after complete conversion.

The biocatalyst was prepared according to Example 1, Variant II, 2.75I fermentation medium, harvested after 20 hours. The cells were resuspended in approx. 200 ml buffer (Resting cells, OD _{5 6} 44). 4.85 ml each of this cell suspension were used for the enzymatic reactions.

Two different concentrations of (R) -2-chloromandelonitrile were investigated in parallel. The reaction was started by adding 150 μl of substrate solution and carried out in culture tubes in a shaker at 40 ° C. and 150 rpm. The sales were checked by means of HPLC. When the conversion was complete, the hydroxy acid was extracted after acidification and the ee was determined.

a. 50mg (R) -2-chloromandelonitrile, dissolved in 150μl DMSO ([S] = 60mM, 10g / l, cosolvent: 3% DMSO)

Already after 30 minutes all of the cyanohydrin had been converted to the amide, after 2 hours 80% hydroxy acid had been formed, after 19 hours the hydrolysis to the hydroxy acid was complete (product ee> 99%). b. 75mg (R) -2-chloromandelonitrile, dissolved in 150μl DMSO ([S] = 90mM, 15g / l, cosolvent: 3% DMSO).

Already after 30 minutes all of the cyanohydrin had been converted to the amide, after 2 hours approx. 60% hydroxy acid had been formed, after 19 hours the hydrolysis to the hydroxy acid was complete (product ee = 99%).

Example 5: Enzymatic hydrolysis using different co-solvents

Experiment 5.1:

In reactions on a 50 ml scale, DMSO was compared with EtOH as cosolvent. 5% cosolvent was used, but the substrate concentration was only 4 g / l (R) -2-chloromandelonitrile.

Biomass from 8 times 250 ml (minus 80 ml, see experiment 4.1) fermentation medium (OD ~ 6.1) was harvested after 20 hours. The cells were suspended in 100 ml K / Na phosphate buffer (pH 6.5, 50mM). This cell suspension (OD 60) was used for the enzymatic reactions. The reactions of (R) -2-chloromandelonitrile, dissolved in DMSO or EtOH, were carried out in 100 ml Schlifferienmeyer flasks at 150rpm and 30 ° C. To check the turnover by means of HPLC, 200 μl of sample were mixed with 200 μl of 1N HCl, centrifuged (5 min, 13,000 rpm) and diluted before the measurement. After complete conversion, the ee of the product was determined.

a. 50 ml cell suspension was mixed with 200 mg (f?) - 2-chloromandelonitrile (> 99%) dissolved in 2300 μl DMSO and 200 μl 0.1% H ₃ P0 ₄ . Substrate concentration: 4g / L (24mM), 5% DMSO as cosolvent product-ee of (R) -2-chloromandelic acid: 97% b. 50 ml of cell suspension are mixed with 200 mg (R) -2-chloromandelonitrile (> 99%) dissolved in 2300 μl EtOH and 200 μl 0.1% H ₃ P0 ₄ . Substrate concentration: 4g / l (24mM), 5% EtOH as cosolvent product ee of (R) -2-chloromandelic acid:> 99%

Experiment 5.2:

Here the solvents DMSO, EtOH and 'PrOH were used again in a proportion of 5%, the substrate concentration was 10 g / l (R) -2-chloromandelonitrile. The reaction was carried out on a 5 ml scale.

The preparation of the biocatalyst example 4, experiment 4.2 (ODs ₆ 8.4). 4.75 ml of the cell suspension (resting cells, OD ₅₄₆ 52) were used for the enzymatic reactions. Reactions of (R) -2-chloromandelonitrile (> 99%) dissolved in DMSO, EtOH and i-PrOH were carried out in culture tubes at 150rpm and 30 ° C (parallel batches).

To check the turnover by means of HPLC, 200 μl of sample were mixed with 200 μl of 1N HCl, centrifuged (5 min, 13,000 rpm) and diluted before the measurement. After complete conversion, the ee of the product was determined.

a. 50mg (R) -2-chloromandelonitrile, dissolved in 250μl DMSO ([S] = 60mM, 10g / l, cosolvent: 5% DMSO)

All of the cyanohydrin was converted to the hydroxyamide after only 30 minutes, and about 40% of the hydroxy acid was formed after 2 hours. After 20 hours the conversion was quantitative (product ee = 95%).

b. 50mg (R) -2-chloromandelonitrile, dissolved in 250μl EtOH, ([S] = 60mM, 10g / l, cosolvent: 5% EtOH)

Already after 30 minutes all of the cyanohydrin has been converted to the amide, after 3 hours 35% hydroxy acid has been formed, after 19 hours the hydrolysis to the hydroxy acid is closed 94% completed, after 28 hours the implementation is practically complete (product ee =

97%).

c. 50mg (R) -2-chloromandelonitrile, dissolved in 250μl / -PrOH, ([S] = 60mM, 10g / l, cosolvent: 5% / -PrOH)

The entire cyanohydrin was converted to the amide after only 30 minutes, 8% hydroxy acid was formed after 3 hours, 64% hydroxy acid was formed after 44 hours (product ee = 92.3)

Example 6: Enzymatic hydrolysis at different temperatures

Experiment 6.1:

In experiment 6.1, the course of the reaction was compared at reaction temperatures of 30 ° C., 35 ° C. and 40 ° C. The batches were carried out on a 5 ml scale with a substrate concentration of 10 g / l (R) -2-chloromandelonitrile. After complete conversion, the ee of the product was determined.

The biocatalyst was produced in accordance with Example 4, Experiment 4.2, (OD ₅₄₆ 8.4). 4.75 ml of the cell suspension (resting cells, OD ₅₄₆ 52) were used for the enzymatic reactions. Reactions of 50mg (R) -2- chloromandelonitrile (> 99%) ([S] = 60mM, 10g / l), dissolved in 250μl DMSO (5%), were carried out at 3 different temperatures (30 ° C, 35 ° C, 40 ° C) carried out in culture tubes at 150rpm (parallel batches).

To check the turnover by means of HPLC, 200 μl of sample were mixed with 200 μl of 1N HCl, centrifuged (5 min, 13,000 rpm) and diluted before the measurement. After complete conversion, the ee of the product was determined. a. T = 30 ° C

After only 30 minutes, all of the cyanohydrin had become the amide _. implemented, after 2 hours 42% hydroxy acid was formed, after about 20 hours the hydrolysis to (R) -2-chloromandelic acid was complete (product ee = 95%).

b. T = 35 ° C

After only 30 minutes, all of the cyanohydrin had been converted to the amide, 65% of hydroxy acid had been formed after 2 hours, and hydrolysis to (R) -2-chloromandelic acid was complete after 19 hours (product ee = 96.5%).

c. T = 40 ° C

Already after 30 minutes all of the cyanohydrin had been converted to the amide, after 2 hours 86% hydroxy acid had been formed, after 19 hours the hydrolysis to (R) -2-chloromandelic acid was complete (product ee = 97.9%).

Experiment 6.2:

In experiment 6.2, the temperature was increased to 50 ° C.

The biocatalyst was prepared in accordance with Example 4, Experiment 4.3 (OD _{5 6} 44). 4.85 ml of the cell suspension (resting cells, OD ₅₄₆ 44) were used for the enzymatic reactions. Reactions of 50mg (R) -2- chloromandelonitrile (> 99%) ([S] = 60mM, 10g / l), dissolved in 150μl DMSO (3%), were carried out at 3 different temperatures (30 ° C, 40 ° C, 50 ° C) carried out in culture tubes at 150rpm (parallel batches).

Already after 30 minutes all of the cyanohydrin had been converted to the amide, after 2 hours 42% hydroxy acid had been formed, after 19 hours the hydrolysis to the hydroxy acid was complete (product ee> 99%).

b. T = 40 ° C

Already after 30 minutes all of the cyanohydrin had been converted to the amide, after 2 hours 80% hydroxy acid had been formed, after 19 hours the hydrolysis to the hydroxy acid was complete (product ee> 99%).

c. T = 50 ° C

After only 30 minutes, all of the cyanohydrin had been converted to the amide, 92% of the hydroxy acid had been formed after 2 hours, and hydrolysis to the hydroxy acid was complete after 19 hours (product ee> 99%).

Example 7: Reactions of cyanohydrins from aldehydes with Rhodococcus erythropolis NCIMB 11540 on a semi-preparative scale

K / Na phosphate buffer (50mM, pH 6.5) was used for all reactions. The reaction was monitored using HPLC. After sampling, 1 N HCI was added to stop the biocatalytic reaction (parallel samples). After centrifugation (5 min, 13,000 rpm), the mixture was diluted with HPLC mobile phase.

For working up, the biomass was centrifuged off at 4 ° C. and 3000 rpm for 30 min and once with H ₂ 0 dist. washed. After acidifying the supernatant with 1 N HCl to pH 2, extraction was carried out 3-4 times with TBME. Experiment 7.1:

The biocatalyst was produced in accordance with Example 1, variant II. 31 fermentation medium, harvest after 20 hours (ODs ₄₆ 5.9). The cells were resuspended to about 180 ml in buffer (Resting cells, OD54 ₆ 80).

0.6g (R) -2-chloromandelonitrile (ee> 99%), dissolved in 1.5ml DMSO, 60ml of this cell suspension was added. The hydrolysis was carried out at 30 ° C. and 150 rpm in the shaker cabinet. After 30 minutes the cyanohydrin was completely hydrolyzed, after 17 hours the conversion to (R) -2-chloromandelic acid was complete. Crude yield: 0.73g (109%) Product ee:> 99%

Experiment 7.2:

In experiment 7.2 some reaction parameters were varied. The standard conditions were 10 g / l substrate and DMSO as cosolvent (here 2.5%). A second batch was carried out with 15 g / l substrate, another with 10 g / l substrate and DMF as cosolvent.

The biocatalyst was produced in accordance with Example 1, variant II. 31 fermentation medium, harvest after 20 hours (ODs ₄ 6 6.8). The cells were resuspended to about 190 ml buffer (Resting cells, OD54 ₆ 69). Three implementations were carried out.

Batch A: 0.3 g (R) -2-chloromandelonitrile (ee> 99%), dissolved in 750 μl DMSO, 30 ml of this cell suspension were added. The hydrolysis was carried out at 40 ° C. and 150 rpm in the shaker. After 30 minutes the cyanohydrin was completely hydrolyzed, after 5 hours the conversion to (R) -2-chloromandelic acid was complete. Crude yield: 0.31g (93%) Product ee:> 99% Batch B: 0.3 g (R) -2-chloromandelonitrile (ee> 99%), dissolved in 750 μl DMF, 30 ml of this cell suspension were added. The hydrolysis was carried out at 40 ° C. and 150 rpm in the shaker. After 30 minutes the cyanohydrin was completely hydrolyzed, after 5 hours the conversion to (R) -2-chloromandelic acid was complete. Crude yield: 0.30g (90%) Product ee: 98.5%

Batch C: 0.45g (R) -2-chloromandelonitrile (ee> 99%), dissolved in 750μl DMSO, 30ml of this cell suspension was added. The hydrolysis was carried out at 40 ° C. and 150 rpm in the shaker. After 30 minutes the cyanohydrin was completely hydrolyzed, after 5 hours the conversion to (R) -2-chloromandelic acid was practically complete. ^'

Crude yield: 0.45g (90%) product ee:> 99%

Experiment 7.3:

Here 2 batches with different substrate concentrations (batch A 10g / l, batch B 15g / l) were carried out. Both implementations were almost equally quick and were complete after 2 hours.

The biocatalyst was produced in accordance with Example 1, variant II. 31 fermentation medium, harvest after 20 hours (OD ₅₄₆ 1.2). The cells were resuspended in approximately 180 ml of buffer (Resting cells, OD ₅₄₆ 70). Two implementations were carried out.

Batch A: 0.8 g (R) -2-chloromandelonitrile (ee> 99%), dissolved in 1.6 ml DMSO, was added to the cell suspension (80 ml). The hydrolysis was carried out at 50 ° C. and 150 rpm in the shaker. After 15 minutes the cyanohydrin was completely hydrolyzed, after 2 hours the conversion to (R) -2-chloromandelic acid was complete. Crude yield: 0.85g (95%) Product ee:> 99%

Batch: 1.2g (R) -2-chloromandelonitrile (ee> 99%), dissolved in 1.6ml DMSO, were added to the cell suspension (80ml). The hydrolysis was carried out at 50 ° C. and 150 rpm in the shaker. After 30 minutes the cyanohydrin was completely hydrolyzed, after 2 hours the conversion to (R) -2-chloromandelic acid was complete. Crude yield: 1.26g (94%) Product ee: 98.9%

Experiment 7.4:

The biocatalyst was produced according to Example 1, variant II, 2.5I fermentation medium, harvest after 20 hours (OD ₅₄₆ 6.9). The cells were resuspended in approx. 160 ml buffer (Resting cells, OD ₅₄₆ 63).

1.3 g of (R) -2-chloromandelonitrile (ee> 99%), dissolved in 2.5 ml of DMSO, were added to 140 ml of this cell suspension. The hydrolysis was carried out at 40 ° C. and 150 rpm in the shaker. After 15 minutes the cyanohydrin was completely hydrolyzed, after 3 hours the conversion to (R) -2-chloromandelic acid was complete. Crude yield: 1.43g (98%) product ee:> 99%

Experiment 7.5:

In this reaction, 1 g of mandelonitrile was hydrolyzed to the corresponding hydroxy acid at a substrate concentration of 8 g / l.

The biocatalyst was produced according to Example 1, Variant II, 21 fermentation medium, harvest after 20 hours (OD ₅₄₆ 8.4). The cells were resuspended in approx. 120 ml buffer (Resting cells, OD ₅₄₆ 74). The reaction was carried out after freezing the biocatalyst overnight. 1.0 g (R) - (+) - mandelonitrile, dissolved in 2.4 ml DMSO, was added to the cell suspension (120 ml). The hydrolysis was carried out at 40 ° C. and 150 rpm in the shaker. After 15 minutes the cyanohydrin was completely hydrolyzed, after 5 hours the conversion to (R) -mandelic acid was complete. Crude yield: 1.16g (100%) Product ee: 93%

Example 8: Reactions of cyanohydrins from ketones with Rhodococcus erythropolis NCIMB 11540 on a semi-preparative scale

K / Na phosphate buffer (50mM, pH 6.5) was used. The reaction was monitored using DC.

For working up, the biomass was centrifuged off at 4 ° C. and 6000 rpm for 20 min and once with H ₂ 0 dist. washed. After acidifying the supernatant with 1 N HCl to pH 2, extraction was carried out 3-4 times with TBME.

The biocatalyst is produced according to Example 1, variant II, 2L fermentation medium, harvest after 20 hours. The cells were resuspended in approx. 60 ml buffer (Resting cells, OD ₅₄₆ 60).

300 mg (S) -acetophenone cyanohydrin (25% acetophenone, ee 94%), dissolved in 1 ml DMSO, were added to the cell suspension. After 20 hours the reaction was complete. DC and the product (containing 1-phenylethanol and traces of other impurities) was extracted. The reaction proceeded without loss of enantiomeric purity. Crude yield: 357mg Example 9: Generation of enzyme preparations of nitrile hydratase and amidase for the hydrolysis of substituted cyanohydrins by means of recombinant expression in E. coli

The pMS470 plasmid system was used to express the nitrile hydratase and to express the amidase. In addition to the replicative elements, a selectable ampicillin resistance and the lac repressor gene lad, this plasmid has an inducible tac promoter which allows controlled overexpression of the cloned open reading frames.

Expression plasmid for Rhodococcus erythropolis NCIMB 11540 nitrile hydratase:

The plasmid map can be seen in Figure 1. The plasmid is called pMS470Nhse7.3. In addition to the two gene segments of nitrile hydratase (α and β subunit), it also contains a third open reading frame which codes for an activator protein.

Expression plasmid for the Rhodococcus erythropolis NCIMB 11540 amidase:

In contrast to the nitrile hydratase, only a single reading frame is necessary for the expression of the Rhidococcus erythropolis NCIMB 11540 amidase and this was cloned into the plasmid pMS470-33 / 3/1/11 behind the tac promoter. Figure 2 shows the plasmid map of this construct.

Fermentation of the recombinant nitrile hydratase and amidase

The fermentation of the two enzymes was basically carried out according to the general protocol, worked out for the overexpression of enzymes in the pMS470 system.

It was:

- Inoculated from an overnight culture (ONC) into a main culture with LB medium and antibiotic in the shake flask. - allowed to grow into the exponential phase

- at OD ₆ oo (optical density at 600nm) 0.8 to 1.5 induced with IPTG (isopropylthiogalactopyranoside)

- further induced for 18h (protein expression)

- harvested (centrifugation) and digested (ultrasound)

Expression of nitrile hydratase

The E. coli B BL21 cells transformed with pMS470Nhase7.3 (or pMSNhasetactac7.3) were separated on LB ampicillin plates and an ONC of 100 ml LB ampicillin medium was inoculated with a single colony. The next morning, a main culture consisting of 250 ml LB-ampicillin medium in a 1000 ml baffle flask was inoculated to an OD ₆ oo of 0.01 to 0.03 (Beckmann photometer). The growth temperature was controlled at 25 ° C., since at higher temperatures only insoluble inclusion bodies are formed. After reaching the induction density Beckmann photometer) the cultures were induced by adding IPTG to a concentration of 0.1 mM. In addition, the media were supplemented with 0.1 mM ammonium iron (III) citrate. After reaching an OD ₆ oo> 4, the cultures were harvested (centrifugation at approx. 3000 * g for 15 min) and washed once with approx. 100 ml PBS buffer. The cell pellet was then resuspended in PBS buffer (approx. 5ml total volume) and disrupted with an ultrasound probe (BRANSON Sonifier 250, 60% power setting, constant sonication; 5 times 30s with 1 minute break for cooling) (visual check of completeness under the Microscope). The crude lysates thus obtained had a typical activity of approx. 100-250 U / ml (approx. 350-500U / ml for pMSNhasetactac7.3), analyzed with methacrylonitrile as the substrate under the conditions listed below. For preservation, the lysates were stored at -20 ° C. Storage at room temperature is associated with a rapid loss of activity.

Activity determination: Raw lysates were diluted 1:10 with PBS buffer immediately before the activity determination. 1.4 ml of a 40 mM methacrylonitrile solution in PBS buffer were mixed with 20 μl of the diluted lysate and incubated at 28 ° C. (Ependorf Thermomixer 5436). At the time of 0, 1, 2, 5, 10 and 15 minutes, 200 μl samples were taken and the enzyme reaction in these samples was stopped immediately with 800 μl 0.17% phosphoric acid. After centrifugation (16000 * g, 10 minutes) the samples were measured spectrophotometrically (Perkin Elmer UVΛ / IS spectrometer Lambda Bio) at 224nm. The increase in absorbance was correlated with the increase in the concentration of methacrylamide, using an ε value of 0.57 l * mmo ¹ * crτϊ ¹ .

Expression of the amidase

The E. coli B BL21 cells transformed with pMS470-33 / 3/1/11 were isolated on LB-ampicillin plates and an ONC of 100 ml LB-ampicillin medium was inoculated with a single colony. The next morning, a main culture consisting of 250 ml SOC ampicillin medium in a 1000 ml baffle flask was inoculated to an OD ₆ oo of 0.01 to 0.03 (Beckmann photometer). The growth temperature was adjusted to 30 ° C because the fermentation at 37 ° C only leads to the formation of insoluble and inactive protein. After reaching the induction density (OD ₆ oo = 1 Beckmann photometer), the cultures were induced by adding IPTG to a concentration of 0.3mM. After an induction time of 16 h, the cells were harvested (centrifugation 3000 ^* g, 10 min) and washed with sodium phosphate buffer (0.1 M, pH = 7). The pellet obtained was resuspended to a total volume of approx. 5 ml in the wash buffer and disrupted to completion with an ultrasound probe (BRANSON Sonifier 250, 60% power setting, constant sonication; 5 times 30 s with 1 min break for cooling) with constant cooling (visual check of completeness Under the microscope). The crude lysates obtained in this way were frozen at -20 ° C for preservation. The lysates had an activity of approx. 75 U / ml, determined with acetamide (40mM) as substrate in PBS buffer at 37 ° C

(Determination of released ammonium using the indophenol blue method).

Determination of amidase activity:

The following solutions were used:

Substrate solution: 40 mM acetamide in PBS

Solution A: 10% (w / v) phenol in ethanol (95%)

Solution B: 0.5% (w / v) nitroprusside sodium in ddH ₂ 0

Solution C: 100g tri-sodium citrate and 5g of sodium hydroxide in 550 ml water

Solution D: 600ml commercial sodium hypochlorite solution diluted to 1000ml

Ammonium standards: 0, 80, 120, 200, 280, 400μg / l ammonium sulfate in water

1.4ml of substrate solution were incubated with 10μl enzyme dilution (1:10 in PBS) at 30 ° C (Eppendorf Thermomixer 5436). In 100 µl samples, the enzyme reaction was stopped after 0, 1, 2, 5, 10 and 15 minutes with 20 µl solution A. After taking the last sample, the solutions thus obtained were diluted with 400 μl of water. For calibration purposes, ammonium standard solutions (500 ml each) were mixed with 20 μl solution A. 20 μl of solution B and 50 μl of a mixture of 4 parts of solution C with 1 part of solution D were then pipetted into samples and standards. Good mixing was ensured by vortexing. The samples and standards treated in this way were left at 37 ° C. for 15 minutes. The resulting blue color was quantified 1:10 with water in a spectrophotometer (Perkin Elmer UV / VIS spectrometer Lambda Bio) after dilution of all samples and standards at 640nm. By correlating the increase in blue color over time with the absorbance values of the standards, the activity (μmol of released ammonium per minute) could be calculated. Example 10: Hydrolysis using the recombinant enzyme

In these reactions, cloned Rhodococcus erythropolis NCIMB 11540 nitrile hydratase was used as the crude lysate of E. coli clone 7.3 (prepared according to Example 9).

Execution:

50μL crude lysate were diluted with 425μL buffer (K / Na-P0 ₄ buffer, pH 7, 50mM) and 25μL of an approx. 200mM substrate solution in DMSO (5mg protein / mL S. conc. Approx. 10mM, 5% DMSO ). The reaction was carried out in a thermomixer at 30 ° C and 1000rpm. After 0, 2, 4, 6, 8, 10, 15, 20, 30, 60 and 120 minutes an Eppendorfer was mixed with 0.5 ml 1N HCl. After centrifugation (5min, 13,000rpm) and appropriate dilution, the concentrations of cyanohydrin and hydroxyamide were determined by HPLC. The activity of the nitrile hydratase was determined on the basis of the rate of formation of the hydroxyamide (slope in the initial region). 2-Hydroxy-4-phenyl-butyronitrile was used as the standard substrate (100% activity). The activity in the hydrolysis of the other substrates was compared with the activity on 2-hydroxy-4-phenyl-butyronitrile (Table 5).

Results:

The activity of the nitrile hydratase in the crude lysate of the E. coli clone 7.3 during the hydrolysis of 2-hydroxy-4-phenylbutyronitrile was approximately 0.3 μmol * mg ^"1 * min ^{" 1} . Table 5 shows an activity comparison on the different substrates. Table 5: Comparison of the activity of nitrile hydratase from E. coli clone 7.3 on the various substrates.

Implementation of (/?) - 2-chloromandelonitrile on a semi-preparative scale

50 ml of a crude lysate of the cloned nitrile hydratase from Rhodococcus erythropolis NCIMB 11540 (E. coli clone 7.3, produced according to Example 9) were diluted with 100 ml buffer (K / Na-P0 ₄ buffer, 50 mm, pH 6.5). After adding 1.0 g of (R) -2-chloromandelonitrile (ee> 99%) in 1.5 ml DMSO, the suspension was shaken at 150 rpm and 30 ° C. After complete conversion, the cell debris was centrifuged off and the product was extracted continuously with CH ₂ CI _{2 for} 4 days, ee of the crude product:> 99% yield after purification: 0.91 g (82%)

Claims

claims:

1. A process for the preparation of chiral α-hydroxycarboxylic acids, characterized in that (R) - or (S) -cyanohydrins in the presence of Rhodococcus erythropolis NCIMB 11540 by enzymatic hydrolysis in the corresponding (R) - or (S) - α-hydroxycarboxylic acids be transferred.

2. The method according to claim 1, characterized in that (R) - or (S) - cyanohydrins are used as starting materials which are obtained by enzymatic or chemically catalyzed addition of a cyanide group and the corresponding aliphatic, aromatic or heteroaromatic aldehydes or ketones.

3. The method according to claim 1, characterized in that (R) - or (S) - cyanohydrins of the formula

HO CN

R1 X R2 /,) in which R1 and R2 independently of one another are H, a C ₁ -C ₆ -alkyl or alkenyl radical which is optionally substituted one or more times with substituents which are inert under the reaction conditions or an optionally one or more times with substituents which are inert under the reaction conditions substituted phenyl radical, with the proviso that R1 and R2 are not both H, are used.

4. The method according to claim 1, characterized in that the microorganism Rhodococcus erythropolis NCIMB 11540 is used in the form of ground cells, crude or purified enzymes, recombinant enzymes, immobilized cells or enzymes, lyophilized cells or "resting cells".

5. The method according to claim 1, characterized in that the microorganism Rhodococcus erythropolis NCIMB 11540 is suspended in an aqueous medium and the suspension thus obtained is mixed with the corresponding chiral cyanohydrin in the presence of a solubilizer as a cosolvent.

6. The method according to claim 5, characterized in that organic solvents, surfactants or phase transfer catalysts are used as solubilizers.

7. The method according to claim 6, characterized in that DMSO, DMF, -CC ₆ alcohols, TMBE or mixtures thereof are used as the organic solvent.

8. The method according to claim 5, characterized in that the cosolvent fraction is between 0.5 and 20 vol% based on the total volume of the reaction solution.

9. The method according to claim 1, characterized in that the pH of the reaction mixture is between 4.5 and 11.

10. The method according to claim 1, characterized in that the hydrolysis is carried out at a temperature between 10 and 60 ° C.

11. The method according to claim 4, characterized in that an enzyme obtained by expression of the pMS470 plasmid system in a suitable host cell is used as the recombinant enzyme.