GB2051053A - Process for the preparation of D- alpha -Aminoacids - Google Patents

Abstract

D- alpha -Aminoacids are prepared by the hydrolysis of a 5-substituted hydantoin, a corresponding hydantoic acid or a mixture thereof using an enzyme obtained by cultivating the novel microorganism Pseudomonas sp. HM-40 (CBS 259.79).

Description

SPECIFICATION Process for the preparation of D-a-aminoacids This invention relates to a process for the preparation of D-a-aminoacids by the enzymatic hydrolysis of 5-substituted hydantoins, corresponding hydantoic acids or mixtures thereof.

Preferred D-a-aminoacids which may be obtained in accordance with the process of the present invention are D-phenylalanine, D-methionine, D-phenylglycine or D-p-hydroxyphenylglycine, which are particularly usefui as medicaments or as intermediates in the preparation of products which can themselves be used in the fields of pharmacy or agriculture but numerous D-aminoacids, e.g. D-leucine, D-valine and D-tryptophan, can be obtained using the process.

The present invention provides a process for the preparation of a D--aminoacid which comprises the hydrolysis of a 5-substituted hydantoin, a corresponding hydantoic acid or a mixture thereof, using an enzyme prepared by cultivation of the microorganism Pseudomonas sp. HM-40 (CBS 259.79). The hydrolysis of a 5-substituted hydantoin is preferred.

The 5-substituted hydantoin used as starting material in the process of the present invention is preferably substituted in the 5-position by a straight-or branched-chain alkyl radical containing 1 to 4 carbon atoms which is optionally substituted by an alkyithio radical, the alkyl moiety of which contains 1 to 4 carbon atoms in a straight- or branched-chain, a cyano radical or an aryl, preferably phenyl, radical which is optionally substituted by one or more hydroxy groups, or substituted in the 5-position by an aryl, preferably phenyl, radical which is optionally substituted by or more hydroxy groups.

Corresponding hydantoic acids may also be used as starting materials.

The enzyme used in the process according to the present invention is obtained by cultivating in a suitable medium the microorganism Pseudomonas sp. HM-40 which is identified more completely below.

The microorganism of which the culture, under appropriate conditions, provides the enzyme used in the process according to the invention must be considered as a new species belonging to the genus Pseudomonas.

It was isolated from a sample of mud, taken in Commentary (France), in accordance with the usual methods for the isolation of microorganisms. A sample of the strain was deposited at the Centraal- bureau voor Schimmelcultures in Baarn (the Netherlands), where it was registered under the number CBS 259.79. That laboratory is authorised to dispsense the microorganism strain to any person legally having knowledge of the present document.

The microorganism HM--40 is a Pseudomonas of which certain characteristics do not correspond to those of the species described in "Bergey's Manual of Determinative Bacteriology, 8th edition". It is designated by the name Pseudomonas sp. HM-40.

The cultural and morphological characteristics of Pseudomonas sp. HM-40 are as follows: 1 ) Appearance of cultures on various media a) "Trypticase soy agar" medium at 30 OC After-24 hours of incubation: very small colonies.

After 48 hours of incubation: larger colonies (about 1. to 1.5 mm in diameter) which have distinct edges and are semi-transparent.

After 4 days of incubation: larger colonies (about 4 mm in diameter) which are opaque and mucoid.

b) Nutrient broth at 30 0C In test tubes (16 x 1 60 mm) containing 5 cc of medium, development is apparent after 24 hours, but it is not very abundant. Development increases over the following days. There is the formation of a uniform turbidity with a sediment which adheres fairly strongly to the bottom of the tubes. Carbon dioxide (10%) does not increase development.

c) Various solid media On nutrient agar and on "Brain Heart Infusion Agar", the development and the appearance of the cultures are identical to those which have been described for the "Trypticase soy agar" medium.

d) Various liquid media On "Brain Heart Infusion Agar", water containing peptone, water containing tryptone, and nutrient broth with yeast extract, the development and the appearance of the cultures are identical to those which have been described for nutrient broth. On "Trypticase Soy Broth" and on water containing peptone and 0.2% w/v of glucose, development is a little weaker.

2) Morphology a) Microscopic examination without staining After 24 hours oF culture in a liquid medium at 370C: cells having a length of 0.5 to 5 F in shuttle shaped clusters.

The microorganism is mobile and the mobility is more pronounced when incubation is carried out at 24 to 300 C.

b) Microscopic examination after staining The microorganism is Gram negative: the central part of the cell body is frequently more weakly coloured.

3) Optimum temperature for development: 240C: good development 300C: very good development 370C: mediocre development 420C: weak development 4) Cultural and biochemical characteristics: a) Respiratory type: obligate aerobe b) Oxidase: positive c) Catalase: positive d) "Mac Conkey Agar": abundant culture e) "Salmonella Shigella Agar": not very abundant culture f) "Cetrimide Agar": no development g) Urease (in Ferguson's medium): positive h) Indole production: negative i) Methyl red test: negative j) Voges-Proskauer test: negative k) Use of citrates (Simmons): positive after 48 hours; alkalinisation I) Liquefaction of gelatin: negative m) Oxidation-fermentation test (Hugh and Leifson): oxidising n) Formation of acids from carbohydrates: glucose: positive lactose: positive arabinose: positive maltose: positive mannitol: positive sucrose: positive xylose: positive o) Reduction of nitrates: positive p) Reduction of nitrates: negative q) Denitrification: negative r) Formation of pigment: negative s) Formation of H2S: on a "Triple Sugar Iron Agar" medium: very slightly positive lead subacetate paper: traces of H2S t) Haemolysis (horse blood): negative u) Litmus milk: no change v) p-galactosidase: positive w) Arginine dihydrolase: negative x) Lysine decarboxylase: negative y) Ornithine decarboxylase: negative When the Pseudomonas sp. Hum 40 strain is cultivated in conventional culture media, the enzyme which can convert the hydantoins into the corresponding D-a-aminoacdis, forms in the cells of the microorganism and to a lesser extent in the culture medium.

Pseudomonas sp. HM-40 can be cultivated by any method of aerobic cultication. The culture medium must contain sources of assimilable carbon, nitrogen and inorganic substances, e.g. mineral salts, required for the development of the HM-40 strain. Enzyme production can be improved if a 5 substituted hydantoin is included in the culture medium as a source of nitrogen. The hydantoin of methionine is generally used for this purpose, but other hydantoins, such as the hydantoin of valine, the hydantoin of leucine, the hydantoin of isoleucine or 5-(3-cyanopropyl) hydantoin, can advantageously be used.

Sources of assimilable carbon which can be used are carbohydrates, such as glucose, sucrose, lactose or maltose, in the pure form or provided in the form of complex residues, such as molasses and milk serum; alcohols or organic acids can also be used. Certain animal or vegetable oils, such as lard oil or soya bean oil, can replace these various sources of carbon or can be added thereto.

Suitable sources of assimilable nitrogen are extremely varied. They can be very simple chemical substances, such as inorganic or organic ammonium salts or urea. They can also be provided by complex substances which mainly contain nitrogen in protein form.

Amongst the inorganic constituents added, some can have a buffer or neutralising effect, such as alkali metal or alkaline earth metal phosphates or calcium carbonate or magnesium carbonate. Others contribute to the ionic equilibrium required for development of the HM-40 strain, such as alkali metal and alkaline earth metal chlorides and sulphates or salts of zinc, cobalt, iron, copper and manganese.

The pH of the fermentation medium at the start of the culture must be between 4 and 9 and preferably between 6 and 8. The optimum temperature for the fermentation is between 25 and 350C, but satisfactory production is still obtained at temperatures between 20 and 370C.

The aeration of the fermentation can vary between fairly wide limits. However, it has been found that aerations of 0.3 to 3 litres of air per litre of broth per minute are particularly suitable. The maximum yield is obtained after 10 to 72 hours of culture, this time depending on the medium used.

As the enzyme used in the hydrolysis process of the invention is produced mainly in the cells, the process of the invention to prepare the D-a-aminoacid can be carried out using cells of the microorganism in the medium used to cultivate them, or using the cells isolated from the culture medium, e.g. by centrifugation; the isolated cells can be used as an aqueous suspension thereof, or after precipitation by acetone or lyophilisation; they can also be used in the form of an enzymatic cell extract obtained by grinding or ultrasonic treatment.

The process of the invention to prepare the D-c-aminoacid is generally carried out in an aqueous medium at a pH of from 6 to 9 and preferably at about 7.8. If appropriate, the pH is kept in the region of this value by adding an alkaline aqueous solution, preferably a sodium hydroxide solution.

The temperature of the reaction medium for the hydrolysis is generally from 20 to 550C and preferably about 37 OC.

The initial concentration of a hydantoin in the reaction medium depends on its solubility. It is generally about 5% (w/v), but it can be more than 10% (w/v).

The reaction medium for the hydrolysis can contain surface-active agents, antioxidants, and/or coenzymes which can assist the formation of the D-a-aminoacid and thus enable the yield to be improved.

The duration of the reaction is generally from 2 hours to 4 days. In fact, the reaction is generally continued until no further D-a'-aminoacid is produced.

In the conversion of the hydantoin into the D-a-aminoacid, a corresponding D-hydantoic acid can be formed as an intermediate which, under the action of the enzymatic system, is converted into the Da-aminoacid. The process according to the invention can thus be carried out either on a hydantoin, or on a hydantoic acid, or on a mixture thereof.

The process of the invention is preferably used to prepare the aminoacids D-phenylalanine, Dmethionine, D-phenylglycine or D-p-hydroxyphenylglycine. Other a-aminoacids which may be prepared by the process of the present invention are D-leucine, D-valine, D-tryptophan and D-2-amino-5-cyanovaleric acid.

A racemic 5-substituted hydantoin is generally used as starting material in the process of the present invention and selective hydrolysis of the D-5-substituted hydantoin in addition to producing the desired D-a-aminoacid leaves the L-5-substituted hydantoin in the reaction medium. However, the HM-40 strain favours the racemisation, in situ, of the L-hydantoin, so that the racemic hydantoin employed can be totally converted into the D-a-aminoacid.

The D-a-aminoacid obtained in accordance with the process of the present invention can be isolated from the reaction medium in accordance with the known methods for the separation of aminoacids, generally after clarification of the reaction medium by centrifugation and filtration on an ion exchange resin. Precipitation of the aminoacid at its isoelectric point is generally carried out. By the expression "known methods" as used in this Specification and the accompanying claims is meant methods heretofore used or described in the chemical literature.

The following Examples illustrate the invention.

EXAMPLE 1 a) Preparation of the enzymatic composition A culture medium having the following composition is prepared: glucose 20 g/litre monopotassium phosphate 3 g/litre dipotassium phosphate 7 g/litre magnesium sulphate 0.7 g/litre manganese sulphate 0.02 g/litre ferrous sulphate 002 g/litre sodium chloride 1 litre hydantoin of D,L-methionine 3 g/litre The medium is divided up into 250 cc flasks, at a rate of 50 cc per flask, and is sterilised by heating for 1 5 minutes at 1 200 C. After inoculation with the Pseudomonas sp. HM-40 strain, kept on a selective agar medium, the flasks are stirred (250 rpm) for 48 hours at 300C.

The production culture is prepared in a 2 litre fermenter dontaining the above culture medium (1 litre) and an anti-foaming agent (1 cc). Inoculation is carried out with the contents of one of the flasks, prepared as described above. The culture is developed at 300C for 48 hours, whilst stirring with a stirrer rotating at 500 rpm and aerating with sterile air at a rate of 1 litre/minute.

When the development of the culture is complete, the cell materials are separated off by centrifugation and then suspended in distilled water (50 cc). The suspension obtained contains 7.6 g of solids per 100 cc.

b) Hydrolysis of the hydantoin ofD,L-phenylalanine The hydantoin of D,L-phenylalanine (5 g) is added to distilled water (200 cc). After heating in order to dissolve part of the hydantoin, the mixture is cooled to 370C. The pH is adjusted to 7.8 by adding a 1 N aqueous solution of sodium hydroxide. The cell suspension obtained above (25 cc) is then added.

The volume of the reaction mixture is adjusted to 250 cc and the pH is adjusted to 7.8.

The reaction mixture is placed under a nitrogen atmosphere; its ternperature.is370C. ThepH is kept at 7.8 by gradually adding a 1 N aqueous solution of sodium hydroxide. After moderate stirring for 24 hours, all the hydantoin has been solubilised.

The reaction mixture is clarified by centrifugation and then passed through an ion exchange resin (Dowex 50 resin, H+ form), elution being carried out with a 2N-solution-of ammonia. The alkaline fractions are concentrated to dryness. D-phenylalanine (3.98 g) is thus obtained (yield 92.5%) and has the following characteristics: [aj20-2920 +20 (c= 1;water) N = 8.63% (theory 8.48%) Potentiometric titre: 93.3%.

The optical purity of the D-phenylalanine obtained is more than 93%.

EXAMPLE 2 The hydantoin of D,L-methionine (4 g) and a cell suspension (8 cc) containing 0.6 g of solids (and prepared under the conditions described in Example 1) are added to distilled water (150 cc).

The pH is adjusted to 7.8 by adding a 1 N aqueous solution of sodium hydroxide. The volume of the reaction mixture is adjusted to 200 cc by adding distilled water. The reaction is continued for 24 hours at 370C, the pH being kept at 7.8 by gradually adding a 1 N aqueous solution of sodium hydroxide.

The D-methionine obtained is determined in the reaction mixture in accordance with the method of Stein and Moore; its concentration is 17 g/litre, which corresponds to a degree of conversion of 100%, relative to the hydantoin of D,L-methionine employed.

The reaction mixture is clarified by centrifugation and then passed through an ion exchange resin (Dowex 50 resin, H+ form). D-Methionine (2.7 g) is thus obtained (yield 8056, relative to the Dmethionine determined in the reaciton mixture) and has the following characteristics: [α]D30 = - 1930 (c = 1; 5N hydrochloric acid) N =9.36-9.42% (theory: 9.39%).

The optical purity of the D-methionine obtained is of the order of 90%.

EXAMPLE 3 a) Preparation of the enzymatic composition A culture medium having the following composition is prepared: glucose 20 g/litre yeast extract (DIFCO) 10 g/litre Bacto peptone (DIFCO) 10 g/litre The medium is divided up into 250 cc flasks, at a rate of 50 cc per flask, and is sterilised by heating for 1 5 minutes at 1 200 C. After inoculation with the Pseudomonas sp. HM-40 strain, the flasks are stirred (250 rpm) for 24 hours at 300C.

The contents of one flask are used to inoculate a two litre fermenter containing the above culture medium (1 litre). The culture is developed for 24 hours at 300C, whilst stirring with a stirrer rotating at 500 rpm and aerating with sterile air at a rate of 1 iitre/minute.

When the development of the culture is complete, the cell materials are separated off by centrifugation and suspended in distilled water (100 cc): this suspension contains 8.9 g of cell solids.

b) Enzymatic hydrolysis of the hydantoin of methionine The hydantoin of D,L-methionine (5 g) is dissolved in distilled water (200 cc). The pH is adjusted te 7.5 by adding a decinormal aqueous solution of sodium hydroxide, and the cell suspension obtained above (20 cc) is then added. The volume is adjusted to 250 cc; the reaction mixture is placed under a nitrogen atmosphere and kept at 370C.

After a reaction time of 5 hours, the methionine present in the medium is determined by chromatography in accordance with the method of Stein and Moore. The hydantoinase activity of the cell suspension (number of y mols of methionine produced per mg of cell solids per hour of enzymatic reaction) is calculated; it is 4.5.

After a reaction time of 22 hours, the reaction medium is clarified by centrifugation. The methionine formed is isolated by passing the mixture through an ion exchange resin (DOWEX 50, H+ form), elution being carried out with a 2N solution of ammonia.

After evaporating the eluate to dryness and further drying of the residue, D-methionine (3.9 g) is obtained (yield: 91%) and has the following characteristics: N = 9.40zÓ (theory: 9.39%) [a9]20 x 230 (c = 1; 5N hydrochloric acid) Potentiometric titre: 98%.

EXAMPLE 4 Enzymatic hydrolysis of the hydantoin of para-hydroxyphenylglycine An aqueous suspension (100 cc) containing 8.9 g of cell solids is prepared under the conditions described in Example 3 (a).

The hydantoin of p-hydroxyphenylglycine (5 g) is dissolved in distlled water (250 cc). The pH is adjusted to 7.5 by adding a decinormal aqueous solution of sodium hydroxide, and the cell suspension (20 cc) is added. The volume is adjusted to 250 cc; the reaction mixture is placed under a nitrogen atmosphere and kept at 370C.

After a reaction time of 5 hours, the p-hydroxyphenylglycine is determined. The hydantoinase activity is 1.8,us of aminoacid formed per mg of cell solids per hour of reaciton time.

After a reaction time of 22 hours, the p-hydroxyphenylglycine is isolated by passing the mixture through an ion exchange resin (DOWEX 50, H+ form). D-p-Hydroxyphenylglycine (3.78 g), which is homogeneous when subjected to chromatography, is thus obtained (yield 87%) and has the following characteristics: N = 7.6 ,6 (theory: 8.4%) [a]D0 = I 350C (c = 1; 5N hydrochloric acid) Potentiometric titre: 110%.

EXAMPLE 5 The Pseudomonas sp. HM-40 strain is cultivated under the following conditions: Composition of the culture medium glucose 20 g/litre dipotassium phosphate 7 g/litre monopotassium phosphate 3 g/litre magnesium sulphate 0.7 g/litre manganese sulphate 0.02 g/litre ferrous sulphate 0.02 g/litre sodium chloride 1 g/litre hydantoin of D,L-methionine 3 g/litre yeast extract 2 g/litre Precultures A first preculture is prepared, over 24 hours, in a 250 cc flask containing the culture medium (50 cc). A second preculture, inoculated with the contents of the first preculture flask, is prepared, also over 24 hours, in a 2 litre fermenter containing the culture medium (1 litre).

Production culture The contents of the 2 litre fermenter are used to incoulate a 7.5 litre fermenter containing the culture medium (5 litres). The culture is developed for 24 hours at 370C whilst aerating with sterile air at a rate of 5 litres per minute and stirring with a stirrer rotating at 500 rpm. The biomass obtained under these conditions represents 3.8 g of solids per litre of culture medium.

Enzymatic reaction When the development of the culture is complete, the cell materials are isolated from the culture medium by centrifugation and suspended in distilled water (250 cc).

The hydantoin of D,L-phenylglycine (10 g) is suspended in distilled water (400 cc) and the pH is adjusted to 7.5 by adding a decinormal aqueous solution of.sodium hydroxide. The cell suspension obtained as described above (50 cc) is added and the volume of the mixture is made up to 500 cc by adding water. The mixture is then left under a nitrogen atmosphere at 370C for 17 hours, the pH being kept at 7.5.

After a reaction time of 1 7 hours, the reaction mixture is clarified by centrifugation, concentrated by evaporation to a fifth of its volume and acidified to pH 1 by adding concentrated hydrochloric acid.

The precipitate formed is filtered off and dried. Chromatographic analysis shows that the precipitate is the hydantoic acid of phenylglycine, which has the following characteristics: N = 13.9% (theory: 14.4%) [aj20 1370 (e = 1; 1% w/v ammonia solution) Yield of the D-hydantoic acid obtained: 6.3%, relative to the hydantoin employed.

The mother liquors are concentrated again and then brought to pH 5 by adding aqueous sodium hydroxide solution; the precipitate of D-phenylglycine obtained is filtered off, washed with water and ethyl alcohol and then dried. D-Phenylglycine (7.6 g) is thus obtained (yield: 88%) and has the following characteristics: N = 9.2% (theory: 9.3%) [a] 20 = -1480 (c = 1; 5N hydrochloric acid).

EXAMPLE 6 The culture of Pseudomonas sp. HM-40 is carried out under the conditions of Example 5. After centrifugation, the cell materials are suspended in 0.1 M phosphate buffer at pH 7.8: this suspension is used as a catalyst for the hydrolysis of various hydantoin derivatives; the reaction conditions are as follows: the initial concentration of hydantoin is 20 g/litre in the 0.1 M phosphate buffer at pH 7.8; the biomass employed provides 6.5 g (calculated as solids) per litre of reaction mixture: the reaction temperature is 370C; the reaction time is 5 hours.

The aminoacid formed is determined in the reaction mixture and the enzymatic activity of the cell extract is calculated therefrom.

The activities measured in this way are expressed in the following Table in yM of aminoacid formed per mg of enzymatic solids per hour of reaction time.

TABLE

SUBSTRATE FORMULP OF ACTIVITY SUBSTRATE C1M/mg/hour Hydantoin of ,CO-NH D,L-methionine CH3-S-CH2-CH2 CH L of CO-NH D,L-phenylalanine O C -CH / CH,-CH 2.0 \NW-- CO Hydantoin of CHz CH-CH Hydantoin of 3CRCR2CRCIO 1.6 DL-leucine CH/ CH CO-NH Hydantoin tf 3 > CH-CH / l 1.0 D,L-valine CR NH-CO 3

SUBSTRATE FORMULA OF SUBSTRATE ACTIVITY M/mg/hour Hydantoin of D, L-tryptophan CO-NH #-CH2-CH 0.7 N NH-CO H Hydantoin of CO-NH D,L-phenylglycine #-CH 0.6 NH-CO CO-NH Hydantoin of D,L-para- HO-#-CH 0.6 hydroxyphenylglycine NH-CO Racemic 3- CO-NH cyanopropyl-hydantion CN-CH2-CH2-CH2-CH 3.5 NH-CO Hydantoin of D,L-meta- HO CO-NH hydroxyphenylglycine 0.7 #-CH NH-CO EXAMPLE 7 A cell suspension of Pseudomonas sp. HM-40 in phosphate buffer was obtained as described in Example 6.The suspension contains 6.5 g of solids in 100 cc; before use, the cells are disrupted by ultrasonic treatment under the following conditions: apparatus: PONS, equipped with a TC4C probe; ultrasonic freuqency: 20 kHz; volume treated: 1 5 cc; temperature kept in the region of OOC; treatment time: 10 minutes.

The treated suspension is used to catalyst the hydrolysis of the hydantoins of methionine, phenylalanine and phenylglycine under the experimental conditions described in Example 6. The activities measured are as follows:

ACTIVITY (M of aminoacid formed/hour/mg of SUBSTRATE enzymatic solids) Hydantoin of D,L-methionine 1.7 Hydantoin of D,L-phenylalanine 1.0 Hydantoin of D,Lphenylglycine 0.8 EXAMPLE 8 A cell suspension of Pseudomonas sp. HM-40 in water, obtained as described in Example 5 but containing 7.3 g of solids in 100 cc, was lyophilised, before use, under the following conditions: : The suspension (120 cc) is divided up into four 500 cc round-bottomed flasks, is frozen and then subjected to reduced pressure (0.5 x 10-2 mm Hg) for 16 hours. A lyophilised enzymatic powder (8.8 g) is obtained and is used under the conditions described in Example 6.

The activities measured are as follows:

ACTIVITY M of aminoacid formed/hour/mg SUBSTRATE of solids Hydantoin of D,L-methionine 2.4 Hydantoin of D,L-phenylglycine 0.4 Hydantoin of D,L-phenylalanine 1.5

Claims (27)

1. A process for the preparation of a D-a-aminoacid which comprises the hydrolysis of a 5substituted hydantoin, a corresponding hydantoic acid or a mixture thereof using an enzyme prepared by cultivation of the microorganism Pseudomonas sp. HM--40 (CBS 259.79).

2. A process according to claim 1 which comprises the hydrolysis of a 5-substituted hydantoin.

3. A process according to claim 1 or 2 in which the 5-substituted hydantoin is a hydantoin substituted in the 5-position by a strai9ht- or branched-chain alkyl radical containing 1 to 4 carbon atoms which is optionally substituted by an alkylthio radical, the alkyl moiety of which contains 1 to 4 carbon atoms in a straight- or branched-chain, a cyano radical or an aryl radical which is optionally substituted by one or more hydroxy groups, or substituted in the 5-position by an aryl radical which is optionally substituted by one or more hydroxy groups.

4. A process according to any one of the preceding claims in which the enzyme is prepared by cultivating the microorganism aerobically in a culture medium containing assimilable source of carbon, nitrogen and inorganic substances, the culture medium including a 5-substituted hydantoin as a source of nitrogen.

5. A process according to claim 4 in which the 5-substituted hydantoin is the hydantoin of methionine.

6. A process according to claim 4 in which the 5-substituted hydantoin is the hydantoin of valine, leucine, or isoleucine or is 5-(3-cyanopropyl) hydantoin.

7. A process according to any one of the preceding claims in which the hydrolysis is carried out using cells of the microorganism in the medium used to cultivate them.

8. A process according to any one of claims 1 to 6 in which the hydrolysis is carried out in an aqueous suspension of cells isolated from the culture medium of the microorganism.

9. A process according to any one of claims 1 to 6 in which the hydrolysis is carried out using an enzymatic extract obtained by grinding or ultrasonic treatment of cells of the microorganism.

10. A process according to any one of the preceding claims in which the hydrolysis is carried out in an aqueous medium at a pH from 6 to 9.

11. A process according to any one of claims 1 to 9 in which the hydrolysis is carried out in an aqueous medium at pH of about 7.8.

12. A process according to claim 11 in which the pH of the aqueous medium is kept at about 7.8 by the addition, if necessary, of an aqueous solution of sodium hydroxide.

13. A process according to any one of the preceding claims in which the temperature of the reaction medium for the hydrolysis is from 20 to 550C.

14. A process according to any one of claims 1 to 12 in which the temperature of the reaction medium for the hydrolysis is about 370C.

1 5. A process according to any one of the preceding claims in which the initial concentration of the 5-substituted hydantoin is about 5% (w/v).

16 A process according to any one of the preceding claims in which the reaction medium for the hydrolysis also contains a.surface active agent, an antioxidant and/or a coenzyme to improve the yield of D-ex-aminoacid.

17. A process according to any one of the preceding claims in which the duration of the hydrolysis reaction is from 2 hours to 4 days.

18. A process according to any one of the preceding claims in which a racemic 5-substituted hydantoin is used and the L-5-substituted hydantoin left in the reaction medium after selective hydrolysis of the D-5-substituted hydantoin is racemised in situ by the microorganism and then hydrolysed to D-a-aminoacid.

19. A process according to any one of the preceding claims in which the aminoacid obtained is Dphenylalanine.

20. A process according to any one of claims 1 to 1 8 in which the aminoacid obtained is Dmethionine.

21. A process according to any one of claims 1 to 18 in which the aminoacid obtained is Dphenylglycine.

22. A process according to any one of claims 1 to 18 in which the aminoacid obtained is D-phydroxyphenylglycine.

23. A process according to any one of claims 1 to 1 8 in which the aminoacid obtained is Dleucine, D-valine, D-tryptophan or D-2-amino-5-cyanovaleric acid.

24. A process for the preparation of a D-a-aminoacid substantially as hereinbefore described.

25. A process for the preparation of a D-a-aminoacid substantially as hereinbefore described with especial reference to any one of Examples 1 to 8.

26. A D--aminoacid when prepared by the process claimed in any one of claims 1 to 25.

27. A culture of the microorganism Pseudomonas sp. HM-40 (CBS 259.79) in a synthetic culture medium containing a source of assimilable carbon, a source of assimilable nitrogen and inorganic substances, and substantially free from other microorganisms.