NL8802014A - Method for the enzymatic preparation of optically active amino acids - Google Patents

Abstract

Method for the racemization of an amino acid amide having the general formula: <IMAGE> where R represents an alkyl or aryl group which may or may not be substituted, and where the amino acid amide is brought into contact with a biocatalyst having amino acid amide racemase activity. The invention further relates to the enzymatic preparation of optically active amino acids from amino acid amides by bringing the amino acid amide into contact with a bicatalyst which also has L- or D- amino peptidase activity.

Description

METHOD FOR THE ENZYMATIC PREPARATION OF OPTICALLY ACTIVE AMINO ACIDS

The invention relates to a method for the enzymatic racemization of an amino acid anti-the general formula

wherein R represents an unsubstituted or substituted alkyl or aryl group.

The invention also relates to a method for the enzymatic preparation of optically active amino acids from amino acid amides.

With the method according to the invention, both optically active L-amino acids and optically active D-amino acids can be obtained.

There is a strong need for efficient processes for the industrial preparation of optically active compounds. The importance of the industrial preparation of optically active compounds is determined by the growing demand for selective products in the pharmaceutical, food and agrochemical industry. The reason for this lies in the fact that only one of the two optical isomers has a clear effect in the desired applications.

However, the other optical isomer can exhibit no, hardly any activity, or even undesirable side activity. There is therefore a need for stereoselective preparation methods for these compounds.

Known methods of preparation for optically active compounds include fermentation, asymmetric synthesis and chemical or enzymatic resolution processes. This has been described in reviews, such as in Biotechnology, Ed. H.-J. Rehm and G. Reed, Verlag Chemie, Florida-Basel, vol 6a, by G. Schmidt-Kastner and P. Egerer, p.

387-421 (1984) and in the Symposium Proceedings: 'Biocatalysts in Organizational Syntheses', Noordwijkerhout, April 14-17, 1985; ELsevier SciencePublishers by E, M. Meijer et al.

With regard to one class of optically active compounds, namely the optically active amino acids, it can be said that as far as the naturally occurring amino acids are concerned, the so-called 'natural amino acids' (almost all with exclusively the L-configuration) - microbial fermentation in particular is a suitable preparation method in addition to a number of enzymatic resolution methods and enzymatic asymmetric syntheses.

The natural amino acids are mainly used in the food industry and in the pharmaceutical industry, for example, as a component in infusion liquids.

It is known that certain microorganisms, such as Aspergillus oryzae, Aspergillus parasiticus, Mycobacterium phlei, Aeromonas proteolytica. Bacillus subtilis. Bacillus stearothermophilus, Enterobacter cloaca and Pseudomonas putida can form enzymes capable of hydrolyzing amino acid amides in an aqueous medium to form an amino acid.

An excellent example of an enzymatic resolution method using an enzyme activity from one of the above mentioned species is described in US-A-3,971,70Q, US-A-4,172,846 and in US-A-4,080,259. Thereby, shown in Scheme I, the racemic D, L-amino acid amide is contacted with an L-stereoselective aminopeptidase-containing preparation. Only the L-amino acid amide is hydrolysed and a mixture of L-amino acid and D-amino acid amide is formed. The L-amino acid can be extracted from this by a number of methods, such as, for example, crystallization, extraction and using an ion exchanger. The D-amino acid amide can also be precipitated by reaction with, for example, benzaldehyde as Schiff base. After recovery of the Schiff base, the D-amino acid amide can be recovered and the D-amino acid can be recovered therefrom both by chemical (acid) hydrolysis and by enzymatic hydrolysis (see, e.g., EP-A-179,523).

In the preparation of a desired enantiomer, an equal amount of undesired enantiomer is also obtained. Moreover, when the D-amino acid is the desired enantiomer, the preparation is cumbersome.

Scheme I: D, L-amino acid amide L-aminopeptidase

Β-amino acid amino

L-amino acid

D-Schiff base D-amino acid

In addition to the natural amino acids mentioned above, the non-natural amino acids are also of industrial importance. Non-natural amino acids include both the optical antipodes (D-amino acids) of the natural amino acids and the amino acids, which, because of their chemical composition (depending on the R ”substituent), do not occur in nature. The named D-amino acids in particular are often interesting due to the fact that they can interfere with normal cell metabolism. Because of this, these natural amino acids can often be used as building blocks for pharmaceutical and agrochemical products. For example, D-phenylglycine and D-p-hydroxy-phenylglycine are used as building blocks for broad-spectrum antibiotics as resp. Ampicillin and Amoxycillin.

No methods of preparation are yet known for directly preparing the D-amino2H using a stereoselective D-aminopeptidase analogous to the above-described preparation method for L-amino acids.

NL-A-88.01864 of the Applicant describes a process for the preparation of biocatalysts with stereoselective enzyme activity. In addition to the constitutively present L-aminopeptidase activity in Pseudomonas putida ATCC 12633, an inducible stereoselective D-aminopeptidase activity can be obtained (the respective mutant has been deposited with NCIB under no .......).

By classical genetic techniques such as UV irradiation or alkylating reagents (e.g. nitrosoguanidine) or recombinant DNA techniques, a mutant can again be obtained from the mutant described above with only the inducible D-aminopeptidase activity (L-aminopeptidase negative mutant).

The use of such a L-aminopeptidase negative mutant with only D-aminopeptidase activity or purified enzyme fractions with only D-aminopeptidase activity, originating from the mutant originally obtained, leads - directly to the method described for the L-aminopeptidase - to direct formation of D-amino acids from racemic D, L-amino acid amides.

In either case, the drawback remains that up to 50% of the racemic starting material can be utilized for the recovery of the corresponding L or D amino acid.

Enzymatic preparation methods are preferred in view of the - in contrast to chemical methods - milder reaction conditions of these enzymatic processes, the high stereo selectivity and the usually broad substrate specificity. The latter means that the same enzyme preparation can be used for the preparation of several different amino acids.

Surprisingly, it has now been found that, inter alia, via the method as described in NL-A-88.01864 from Applicant, a biocatalyst having an amino acid amide racemase activity can be obtained.

No such activity was known. Based on the racemase activity, optically active amino acids can be produced much more efficiently than according to the prior art.

The method according to the invention for the enzymatic preparation of an optically active amino acid from a corresponding amino acid amide is characterized in that the amino acid amide is contacted with a biocatalyst having amino acid amide racemase activity in combination with stereoselective D- or L -aminopeptidase activity.

The applicant has thus succeeded in producing, by enzymatic route and with 100% utilization of both enantiomers of an amino acid amide, a D-amino acid or an L-amino acid with a high degree of optical purity (more than 98% enantiomeric excess) as required. . See scheme II.

Scheme II: D, L-amino acid amide amino acid amide amino acid amide racemase racemase + + l-ami nopept i dase D-ami nopept idase

L-amino acid D-amino acid

The advantage of this method is that it is possible to stereoselectively prepare an optically pure D or L amino acid from mixtures of enantiomers, including racemic mixtures, of an amino acid amide, but also from optically pure D- or L-amino acid amide. hetaminoacid amide is completely converted into the desired amino acid.

In particular, the combination of a biocatalyst with D-aminopeptidase activity and a biocatalyst with comparatively high amino acid amide racemase activity, and preferably one biocatalyst with both comparatively high activities, provides a suitable preparation method for optically pure D-amino acids non-optically pure D or L-amino acid amides. The same applies mutatis mutandis for the combination of an L-aminopeptidase and an amino acid amide racemase preferably in one biocatalyst with comparatively high activities, as regards the preparation of optically pure L-amino acids from the above raw materials.

An example of an enzyme system, in which the combination of amino acid amide racemase activity and aminopeptidase occurs / is the applicant's mutant of Pseudomonas putida ATCC12633 (deposit no .....).

Expression of the inducible D-aminopeptidase activity is by the resulting mutant on toes in a culture medium in which a D-amino acid amide / preferably D-phenylglycinamide (whether or not as N source) is present.

The combination of amino acid amide racemase activity and L-aminopeptidase activity can be obtained by growing the deposited Pseudomonas putida mutant in the absence of a D-amino acid amide, thereby not expressing the D-aminopeptidase activity.

The combination of amino acid amide racemase activity and D-aminopeptidase activity can be obtained, for example, by preparing the L-aminopeptidase negative mutant of the deposited Pseudomonas putida mutant by means of said genetic engineering techniques and cultivating them in the presence of a D-amino acid amide (whether or not as N- source). It is also possible to obtain purified fractions by means of enzyme purification with D-aminopeptidase activity on the one hand and amino acid amide racemase activity on the other. Combination of these fractions then yields the desired biocatalyst for the preparation of optically pure D-amino acids.

Where reference is made in this application to one biocatalyst in which combined enzyme activity is present, this is of course also understood to include a mixture of biocatalysts / each separately containing one of the enzyme activities to be combined.

When carrying out the process according to the invention, depending on whether the desired product is an optically pure D- or L-amino acid, an amino acid amide (in optically pure form or an optionally racemic mixture of its enantiomers) contacts a biocatalyst which, in the case of an optically pure D-amino acid as desired product, contains a D-aminopeptidase activity in combination with an amino acid amide racemase activity / respectively in the case of an optically pure L-amino acid as desired product / an L-aminopeptidase activity contains, in addition to the said amino acid amide, racemase activity.

The contact of the amino acid amide with the biocatalyst preferably takes place in an aqueous medium at a temperature between 0 and 60 nc, in particular between 20 and 40 nc, and at a pH value between 6 and 12, in particular between 7.5. and 9.5. Outside these areas, the activity and / or stability of the biocatalyst is generally insufficient for a satisfactory yield. The biocatalyst can be activated or stabilized, if necessary / in a known manner, for example by adding a metal compound, such as potassium, magnesium, manganese or zinc.

If desired, organic solvents can also be added to the reaction mixture to increase the solubility of the reactants. For example, the following solvents can be used for this purpose: alcohols, such as ethanol, isopropanol or t-butanol, or organic solvents such as N, N-dimethylformamide, dimethylsulfoxide or hexamethylphosphorus triamide. Alternatively, the reaction can be carried out in a two-phase system, using two immiscible solvents or a full organic phase of a hydrophobic, water-saturated, organic solvent.

The biocatalyst used in the reaction can be both a purified enzyme and a crude enzyme solution, as a (permeabilized) microbial cell, which possesses the desired activity or optionally a homogenate of cells with such activity. Also, the biocatalyst used can be immobilized or chemically modified to ensure that good stability and reactivity of the biocatalyst is maintained under the reaction conditions.

The weight ratio of the biocatalyst to the substrate can vary widely between wide limits depending on the specific activity of the enzyme fraction * microbial cells used as biocatalyst, and depending on the ratio of the different enzyme activities in the biocatalyst, in particular the ratio between the amino acid amide racemase activity and the D ofL-aminopeptidase activity. For example, even a variation in the weight ratio between the biocatalyst and the substrate can occur from 4: 1 to 1: 500.

Biocatalysts which can be used in the process of the invention can be obtained from both plant and animal cells, and preferably from microorganisms such as bacteria, yeasts and fungi. Some examples of suitable genera of microorganisms for use as a biocatalyst in the above process are: Al caligenes, Arthrobacter, Aspergillus, Bacillus, Cryptococcus, Flavobacterium, Mycobacterium, Nocardia, Pseudomonas, Rhodococcus or Staphylococcus.

To find a biocatalyst with the appropriate enzyme activity, one can use mutagenesis and screening techniques that can be readily determined for the person skilled in the art, as well as the process for the preparation of biocatalysts described in NL-A-88.01864 of Applicant. For an overview of these techniques see for example. Enzyme Microb. Technol. £ (1984) p. 3-10 and 9_ (1987) pp. 194-213. However, this does not have to be limited to the above-mentioned genera. Combinations of microorganisms and possible new mutants thereof are also possible.

Preferably, the combined desired activities are present in one micro-organism in sufficiently high activity with respect to each other. Cells of such a microorganism (optionally permeabilized) can then be used as a biocatalyst.

The method of the invention is suitable for preparing optically pure D or L amino acids, such as the D or L enantiomer of aliphatic amino acids, for example valine, leucine alanine, or of aromatic amino acids, for example phenylglycine, para-hydroxyphenylglycine and phenylalanine but of course the invention is not limited thereto.

The invention is now further elucidated by the following experiments, without, however, being limited thereto.

Experiment I;

The mutant of Pseudomonas putidaATCC 12633 (Deposit No. .....) prepared by the applicant was grown overnight in a YCB (10 g / l) medium with D-phenylglycinamide as N source (1 g / l). After harvesting the cells by centrifugation and washing, 120 mg each was set wet-weight with a 5 ml 1% (w / v) solution of resp. D- and L-phenylglycine amide and D- and L-phenylglycine as substrate. The reaction was carried out with shaking in a vial at 37 ° C and pH 8.5 for 20 hours.

The different reactions gave the following results:

Substrate Biocatalyst Conversion rates to D-phenylglycine and L-phenylglycine D-phenylglycinamide present 77% 14% L-phenylglycinamide present 7% 89% D-phenylglycinamide absent <1% <1% L-phenylglycinamide absent <1% <1% D-phenylglycine present - <1% L-phenylglycine present <1% -

The above-mentioned results, which are calculated after analysis of the obtained reaction mixtures by means of chiral HPLC show that this biocatalyst possesses in addition to the prepared D-aminopeptidase activity an amino acid amide racemase activity for phenylglycinamide. Namely, racemization does not occur at the level of the produced amino acid. Only with the corresponding amide as substrate (both the D- and the L-phenylglycinamide), both D-phenylglycine and L-phenylglycine are formed in the simultaneous presence of the (constitutive) L-aminopeptidase and the (inducible) D-aminopeptidase .

Experiment II:

The mutant of Pseudomonas putida prepared by the applicant

ATCC 12633 (depot no .....) was grown overnight at 28 nc in a YCB

medium (10 g / l) with D-phenylglycinamide as N source (1 g / l). After harvesting the cells by centrifugation and washing, the cells were disintegrated using French press, after which a so-called centrifugation was carried out by centrifugation. cell-free extract was obtained.

Since many reactions / comparable to the racemization of amino acid amide, such as racemization of S-containing amino acids and, for example, transaminase reactions, have pyridoxal 5-phosphate (PLP) as a cofactor, this was also investigated for the racemization of amino acid amides.

In case the amino acid amide racemase would indeed be PLP dependent, the reaction must be inhibited by a PLP-dependent reactions specific inhibitor such as hydroxylamine.

The reactions were performed for 16 hours with D-phenylglycinamide 5 ml 1% (w / v) as a substrate at 37 oc, pH 8.5 and 0.5 ml of the cell-free extract as a biocatalyst. The following results were obtained:

Hydroxylamine sulfate Reaction products 0.2 mmol / ml D-phenylglycine L-phenylglycine absent + +++ 10 μί + ++ 50 μl + +

Additional PLP increases these amino acid amide racemase activity in these racemization reactions.

Indeed, these results demonstrate that the racemization of amino acid amides is a PLP-dependent reaction, which can be inhibited with hydroxylamine.

Experiment III:

The mutant of Pseudomonas putida ATCC 12653 (deposit no .....) prepared by the applicant was grown overnight at 28 bq in a 30

Itr. bioreactor (MBR), pH 7.1, with a medium content of 20 ltr. without a D-amino acid amide (e.g., D-phenylglycinamide) being added to this medium. The biocatalyst cultured in this way therefore has only L-aminopeptidase and amino acid amide racemase activity and no D-aminopeptidase activity. After harvesting these cells by successively applying ultrafiltration, centrifugation, washing and freeze drying, these cells were used as a biocatalyst - in different proportions to the substrate - in the enzymatic reactions described below. As a substrate, 50 ml of a 2% (w / v) D, L-phenylglycinamide solution, pH 8.6 was used. The reactions were run at 40 ac.

The conversion percentage was determined from the amount of ammonia formed, which was formed by the enzymatic hydrolysis of the amino acid amide into the corresponding amino acid. This determination of the ammonia concentration in the reaction medium was carried out with the aid of an selective ammonia electrode (Orion).

Biocatalyst E / S Ratio Reaction Time Conversion Rate Amg Hours of Substrate (Amino Acid Amide) 100 1:10 24 52% 50 53% 72 58% 500 1: 2 24 53% 50 57% 72 63% 2000 2: 1 24 65% 50 74% 72 80%

Although these results clearly show that the amino acid amide racemase activity is the limiting enzymatic activity in the hydrolysis of the amino acid amide, it does show that the enzymatic racemization of the amino acid amide as substrate for the reaction can yield a conversion percentage higher than 50%, In addition, chiral TLC analyzes showed that only L-phenylglycine was formed and no D-phenylglycine was formed, which makes this preparation method extremely suitable for the preparation of optically pure L-phenylglycine.

Experiment IV:

Since the preparation method described in Experiment III requires a long reaction time to arrive at the conversion percentage obtained, induction of the D-aminopeptidase activity could possibly occur during this long reaction time, since D-phenylglycinamide also contains D-phenylglycinamide in the reaction mixture. . To prevent this, the same reaction was carried out in the presence of a protein synthesis inhibitor, in this case tetracycline, at a concentration that is completely inhibitory (10 Mg / ml). The reaction was performed at 40 ° C, pH 8.15 and using a 2% (w / v) racemic D, L-phenylglycinamide mixture (40 ml) as a substrate. Thus, the following results were obtained.

Biocatalyst E / S Ratio Reaction Time Conversion Rate Mg Hours of Substrate 403 1: 2 17.5 52% 47.5 56% 72 58% 1600 2: 1 17.5 58% 47.5 69% 72 75%

Since the conversion rates now obtained are at the same level levels as in Experiment III, where no enzyme synthesis inhibitor was added to the reaction mixture, it must be concluded that due to the presence of an amino acid amide racemase, the aforementioned conversion rates are above 50%.

Experiment V:

Since it should also be possible to obtain L-phenylglycine as product starting from optically pure D-phenylglycinamide as substrate, in the presence of a biocatalyst with amino acid amide racemase activity combined with L-aminopeptidase activity, this was done in an analogous experiment (compare experiments III and IV) were carried out. The cells of the Pseudomonas putida ATCC 12633 prepared in the same way as in experiment III were used as a biocatalyst (300 mg) in a 30 ml 1% (w / v) aqueous solution of D-phenylglycinamide. The reaction was carried out at 40 Ken pH 9.0. The conversion percentage was again determined by measuring the amount of ammonia produced using an ion-selective ammonia electrode. The following results were obtained.

Response time Conversion rate hours of the substrate 17 9% 43 26%

This experiment again provides evidence for the enzymatic racemization of an amino acid amide, in this case the optically pure D-phenylglycinamide, with simultaneous hydrolysis of the formed L-phenylglycinamide to optically pure L-phenylglycine.

Experiment VI;

To determine the substrate specificity of the amino acid amide racemasete, both two aromatic and two aliphatic D-amino acid amides were used as the substrate (5 ml 1% (w / v) aqueous solution). Freeze-dried cells were used as the biocatalyst, obtained in the same manner as described in Experiments III, IV and V. The reaction proceeded at 38 ** C and a pH of 8.1 for 17 hours. D-phenylglycinamide and D-homophenylalaninamide were taken as aromatic amino acid amides and D-valinamide and D-leucinamide as aliphatic amino acid amides. As reaction products, only the corresponding L-amino acids were formed in all cases, which was analyzed using chiral TLC. hit is evidence of the broad substrate specificity of the amino acid amide racemase, which has a racemizing enzyme activity for both aromatic amino acid amides and aliphatic amino acid amides.

Claims (5)

1. Method for the racemization of an amino acid amide with the general formula:

wherein R represents an unsubstituted or substituted alkyl or aryl group / characterized in that the amino acid amide is contacted with a biocatalyst having amino acid amide acase activity. 2. Process according to claim 1, characterized in that the amino acid amide is also contacted with a biocatalyst with L-aminopeptidase activity, respectively. D-aminopeptidase activity and an L-amino acid resp. prepare a D-amino acid. A method according to any one of claims 1-2, characterized in that the amino acid amide is contacted with an enzyme preparation obtained from a mutant of Pseudomonas putida ATCC 12633. 4. Process according to claim 3, characterized in that the amino acid amide is contacted with the mutant of Pseudomonas putida ATCC 12633 deposited under number ......... 5. Procedure as described in the description and / or the examples.