NO147452B - HYDROLASE PREPARATION FOR THE PREPARATION OF D-PHENYLGYLINE BY ENZYMATIC CONVERSION OF D, L-PHENYL HYDANTOIN - Google Patents

Description

The present invention relates to a hydrolase preparation for the production of D-phenylglycine by enzymatic conversion of D,L-phenylhydantoin and subsequent cleavage of the carbamyl residue from the formed D-carbamyl-phenylglycine, and the special feature of the hydrolase preparation according to the invention is that the preparation is obtained from microorganisms of the genus Pseudomonas which can use hydantoins as the sole source of nitrogen, preferably from the strain CBS 145.75 or CBS 146.75.

The present invention thus relates to enzyme complexes

which is capable of converting a racemic hydantoin into an optically active amino acid derivative, as special microorganisms are used during the hydrolysis that use hydantoin as a nitrogen source. The amino acid derivative (D-carbamyl-phenylglycine) can then be converted to the desired D-phenylglycine in a manner known per se.

A small number of amino acids, especially phenylglycine and p-hydroxyphenylglycine, are important starting compounds for the production of compounds with wide application in the pharmaceutical industry.

A number of attempts have previously been made to produce D-amino acids, but none of these methods could be carried out on an industrial scale.

The chemical methods that have been used to date for the separation of optical isomers are very expensive and give lower yields. They are based on the use of camphor sulphonic acid.

Another method involves the selective hydrolysis of D-acylamino acid with an acylase enzyme. However, the D-acylases are relatively rare and always impure with regard to L-acylase, and this makes the method difficult and leads to obtaining a product with poor optical purity.

The enzyme preparation according to the present invention

•on the other hand, allows the preparation of a simple stereoisomeric form of an amino acid from a racemic compound. A method for the enzymatic separation of certain D,L-amino acids has already been previously proposed in the Norwegian patent document 141.689. This method mainly comprises the step of subjecting the racemic form of hydantoin compounds to the general formula

in which

R is methyl or isopropyl for an enzymatic hydrolysis by means of dihydropyrimidinase extracted from calf liver.

The hydrolysis takes place according to the scheme.

after which the carbamyl residue can be cleaved from the carbamyl derivative to obtain a desired D-phenylglycine.

The enzymatic separation of racemic compounds can thus be carried out with hydrolases provided by microorganisms of the genus Pseudomonas which can use hydantoin as the only nitrogen source.

The aim is therefore to first produce the specific enzyme that is formed during the cultivation of microorganisms of the genus Pseudomonas, with which the hydantoin is hydrolysed into derivatives of the D-amino acid, as the enzyme can be used directly as a bacterial suspension or culture medium, or can be extracted from the cells and culture medium.

The test for the selective hydrolysis of hydantoin derivatives to the D-amino acid derivative using the enzyme preparation was carried out in the following way:

Behind the terial strains, isolated from soil, plants, waste of various types, etc., as well as strains from bacterial collections, were inoculated from slanted agar in 250 ml flasks containing 50 ml of the following culture medium:

After 20 to 24 hours of incubation (orbital stirring) at 30°C, 30 ml flasks containing 100 ml of the same medium were incubated with 5 ml of the above pre-culture.

After 18 to 22 hours of further incubation, the enzymatic reaction with the resting cells was carried out in test tubes with 10 ml of phosphate buffer, 0.07 M, pH = 8.5 with 20 micromol/ml 5-(D,L)- phenylhydantoin, 1 ml of the bacterial suspension was added (dry weight approximately 50 mg per ml). After 30 min. incubation at 30°C, the reaction with p-dimethylaminobenzaldehyde was carried out for the quantitative determination of the carbamyl derivative thus formed (J. Biol. Chem., 238, 3325

(1963). The strains that were used are reproduced in table 1.

Those of them designated with the numbers 942 and 945 have been deposited in the Centraalbureau voor Schimmelcultures where they have been given the designations CBS 145.75 respectively. 146.75.

The bacterial strains listed in Table 1 grow well in all common laboratory media. They are all able to use phenyl-hydantoin as a sole nitrogen source. Growth takes place at a temperature of from 5 to 40°C with an optimum temperature between 25 and 30°C.

With regard to the classification, the scheme in Bergey's Manual, 7th edition was followed along with suggestions by Lysenko, J.

Gen. Microbiol. 25, 379 (1961) and Stanier, Palleroni, Doroff, J. Gen. Microbiol. 43, 159-271 (1966).

It has been found that during the enzymatic hydrolysis of D-hydantoin at alkaline pH (7-10) a non-enzymatic racemization of the remaining L-hydantoin takes place. For this reason, and as a consequence of the continuous removal of the D-hydantoin by the enzymatic hydrolysis, all the carbamyl derivative will be obtained in the D form at the end of the reaction.

The identity of D(-)-N-carbamylphenylglycine was shown after crystallization of the reaction product, on the basis of IR, NMR, mass spectra and elemental analysis.

The specific rotation is JdJ^ = -137° (c = 1% in IN NH.OH)

D 4

corresponding to that reproduced in the literature.

The rate of racemization of L-hydantoin is a function of temperature and pH and is all the greater at higher temperature and more basic pH. However, by working at a pH in the vicinity of 7.5, the racemization rate is sufficiently high not to limit the reaction rate.

The temperature of the enzymatic reaction can be kept between 10 and 60°C, but for practical reasons the temperature between 25 and 40°C is preferred.

The hydrolysis of the D-phenyl-hydantoin takes place not only in the presence of microorganisms that have been cultivated or in the presence of intact cells thereof, but also in extracts of the above-mentioned microorganisms. The microorganisms can be cultivated, e.g. in a liquid nutrient medium to achieve an accumulation of hydrolase in the cells and the DL-hydantoins can then be added to the culture. The enzymatic reaction can also be carried out with so-called resting cells. In this case, the bacterial cells are recovered from the culture medium, washed and suspended in a suitable buffered medium to which the racemic hydantoin is added.

In addition, it is possible to use preparations containing the hydrolases, such as e.g. extracts or concentrates thereof, crude or purified hydrolase preparations obtained from cells of the above-mentioned microorganisms. Finally, crude or purified hydrolases can be used, which are immobilized by combinations with macromolecular compounds.

The following examples illustrate the invention.

EXAMPLE 1

To 100 ml of a liquid culture in the above medium of the strain Pseudomonas sp. 942 in a 500 ml flask is added after 24 hours of incubation (orbital stirring) at 30°C, 100 ml of a phosphate buffer (0.14 M) pH = 8.5, which contained 30 micromol/ml 5-(D,1) -phenylhydantoin.

After a further 5 hours of incubation, under the same conditions, the amount of N-carbamylphenylglycine thus formed was determined.

From 530 mg of 5-(D,L)-phenylhydantoin, 525 mg of N-carbamylphenylglycine were formed, corresponding to a yield of approximately 90%.

EXAMPLE 2

A culture liquid was prepared, with the above composition and containing 1 g per liter of 5-(D,L)-methylhydantoin.

The pH was adjusted to 7.2 with soda and the medium was distributed in 50 ml portions in 250 ml flasks. After sterilization at 110°C for 30 min. the flasks were inoculated with a culture of Pseudomonas sp. 942 from agar slants containing the same medium

with 2% agar (DIFCO) and incubated at 30°C for 20 hours with orbital agitation (220 rpm).

With this pre-culture (optical density at 550 nm: 0.400: dilution 1:10) 5 ml were inoculated into 500 ml flasks containing 100 ml of the same medium and the culture was incubated at 30°C with orbital agitation (220 rpm . minute) for 18 hours (last phase of exponential growth). The cells were then collected by centrifugation (500 g, 20 months) and washed 3 times in buffered physiological salt solution and finally resuspended in a phosphate salt buffer pH = 8.5, 0.07 M with resting cells.

For the enzymatic hydrolysis, 64 ml of the reaction mixture composed of: 260 mg bacteria (dry weight) and 20 micromol/ml D,L-phenylhydantoin ( = 3.52 mg/ml). At different time intervals, the product from the hydrolysis, i.e. D-carbamylphenylglycine, was determined with the colorimetric method indicated in J. Biol. Chem. 238, 3325 (1963 at 438 nm). The figure shows the results, as a percentage of theoretical yield of the resulting carbamyl derivative.

On the abscissa is the time in hours and on the ordinate is the percentage formed of D (-)-N-carbamylf enylglycine..

EXAMPLE 3

A semi-synthetic medium with the following composition was produced.

The medium was distributed in 50 ml portions in 250 ml flasks and in 100 ml portions in 500 ml flasks and sterilized for 30 min. at 110°C.

For the pre-culture, 250 ml flasks were inoculated from agar slants with a culture of Pseudomonas sp. 942 strain and incubated as in Example 2 for 24 hours at 30°C.

From this culture (optical density at 550 nm: 0.245: dilution 1:10) 5 ml were inoculated into 500 ml flasks. After a 19 hour incubation at 30°C, as above, the quiescent cells were prepared as in Example 2.

Enzymatic hydrolysis was carried out at 30°C in a reaction mixture containing in 24 ml buffer 280 mg bacteria (on a dry weight basis) and 20 micromol/ml 5-(D,L)-phenylhydantoin. After 5, 10 and 15 min. the amount of the carbamyl derivative thus formed was determined.

EXAMPLE 4

Bacterial cells were prepared from the strain Pseudomonas sp. 942 as described in Example 2. A suspension of them (42 mg per ml of dry cells) in a phosphate salt buffer, 0.1 M, pH = 8 was subjected to mechanical crushing using a "Manto-Gaulin" homogenizer working at a pressure of 850 kg/cm 2 at a temperature below 24°C.

The cell mass was separated from the extract by centrifugation (25,000 g for 30 min.).

To 950 ml of phosphate salt buffer, pH = 7.7, containing 2.12 g of 5-(D,L)-phenylhydantoin was added 50 ml of the extract containing 8,500 U of the enzyme. (1U is the amount of enzyme which, in a phosphate buffer, pH =7.7 at 30°C, containing 12 micromol/ml 5-(D,L)-phenylhydantoin, converts 1 micromol/ml of the substrate within 60 min.) .

After 1 hour at 30°C, 1.7 g of D-carbamyl derivative corresponding to approximately 80% total hydrolysis had formed.

EXAMPLE 5

As in example 4, 7 ml of a preparation of the extract that had been purified approximately 7 times was added to 993 ml of substrate. After 1 hour at 30°C, 1.7 g of D-carbamyl derivative corresponding to approximately 80% of total hydrolysis had been formed.

EXAMPLE 6

Under the same conditions as in example 2, after 30 min. at 30°C, with the strain Pseudomonas sp. 945,

from 352 mg of 5-(D,L)-phenylhydantoin, obtained 220 mg of N-carbamylphenyl-glycine, corresponding to approximately 57% of the theoretical yield.

Claims (1)

Hydrolase preparation for use in the production of D-phenylglycine by enzymatic conversion of D,L-phenylhydantoin and subsequent cleavage of the carbamyl residue from the formed D-carbamyl-phenylglycine, characterized in that the preparation is produced by cultivating microorganisms from the genus Pseudomonas and hydantoins can also be used as the only nitrogen source, preferably the strain CBS 145.75 or CBS 146.75.