WO2000023598A1

WO2000023598A1 - Aminoacylase and its use in the production of d-aminoacids

Info

Publication number: WO2000023598A1
Application number: PCT/GB1999/003458
Authority: WO
Inventors: Stephen John Clifford Taylor; Robert Christopher Brown
Original assignee: Chirotech Technology Limited
Priority date: 1998-10-20
Filing date: 1999-10-20
Publication date: 2000-04-27
Also published as: CA2347079A1; JP2002527110A; EP1121446A1; AU6222799A; KR20010075649A

Abstract

An isolated enzyme capable of hydrolysing N-acetyl-D-tryptophan at a substrate concentration of 10 g/l and which exhibits faster conversion of (R)-N-acetyl-2-thienylalanine than of (R)-N-acetyl-4-chlorophenylalanine. This enzyme is useful for preparing D-aminoacids.

Description

AMINOACYLASE AND ITS USE IN THE PRODUCπON OF D-AMLNO ACIDS Field of the Invention

This invention relates to an enzyme having D-aminoacylase activity and to its use in the production of D-aminoacids, by resolving a racemic mixture of N-acyl aminoacids and deprotecting optically-enriched N-acyl aminoacids. Background of the Invention

D-Aminoacids are commercially important intermediates in the production of various pesticides, antibiotics and other pharmaceuticals. For example, phenylglycine and p-hydroxyphenylglycine are used in the synthesis of semi-synthetic penicillins and cephalosporins. There is also much demand for novel D-aminoacids as building blocks for new drug substances.

D-Aminoacids may be accessed by physical separation, for example by crystallisation of salts, or by asymmetric chemocatalysis by way of hydrogenation of an enamide precursor. Chemocatalysis provides a general method ofbroad applicability, e.g. for unnatural aminoacids, but requires subsequent N-deacylation for which conventional chemical hydrolysis often results in partial racemisation of the product. There are biocatalytic methods also, for example by the hydrolysis of hydantoins using a D-specific hydantoinase. However, the resulting D-carbamoyl aminoacid still requires enzymic or chemical deprotection to the aminoacid.

The production ofL-aminoacids by means of an L-specific aminoacylase-catalysed hydrolysis of the racemic N-acetylaminoacid is a technology that is well established. This uses the enzyme from Aspergillus oryzae and has been operated on a commercial basis at very large scale, to produce L-methionine, L-valine and L-phenylalanine. Such a large- scale technology does not exist for production ofD-aminoacids, although D-aminoacylase activity has been identified in several microbial strains of Pseudomonas, Streptomyces and Alcaligenes. See Sugie and Suzuki, Agric. Biol. Chem. 44:1089-1095 (1980); Daicel Chemical Industries, JP 64-5488 (1989); Moriguchi and Ideta, Appl. Env. Microbiol. 54: 2767-2770 (1988); Sakai et al, Agric. Biol. Chem. 54: 841-844 (1990); Sakai et al, J. Ferm. Bioeng.71:79-82 (1991); Sakai etal, Appl. Env. Microbiol.57: 2540-2543 (1991); Yang etal, Appl. Env. Microbiol. 57: 1259-1260 (1991); andKamedae/ /, Nature 169: 1016 (1952). The enzymes from these strains were isolated and characterised. It should in theory be relatively easy to use such strains in whole-cell form for the resolution or deprotection of N-acetyl aminoacids, but the cells were also shown to contain L-aminoacylases, thus reducing the stereoselectivity. In addition, the low levels of activity, even after growth on inducing media, make purification and use of the enzyme from the whole-cell unattractive economically. A solution was foreseen by way of cloning the enzymes, and this has been reported recently for an Alcaligenes species D-aminoacylase, though it would not be expected that such an enzyme would work at any higher substrate concentration, nor differ significantly in its substrate specificity from the wild-type enzyme. See Moriguchi et al, Biosci. Biotech. Biochem. 57 (7): 1149-1152 (1993); Wakayama et al, Biosci. Biotech. Biochem.59(11): 2115-2119(1995); and Wakayama etal, Prot. Express. Pur.7: 395-399 (1996).

This enzyme, obtained from Alcaligenes xylosoxydans subsp. xylosoxydans ( Alcaligenes A-6^M), NCUvffi 10771, does not hydrolyse N-acetyl-D-tryptophan. It is reported that the activity of D-aminoacylase is inhibited by 37% and 40% by D- phenylalanine and N-acetyl-D-alloisoleucine at a very low concentration of 2mM. This suggests that the enzyme is susceptible to severe product and substrate inhibition.

US-A-5206162 discloses a D-aminoacylase obtained from Alcaligenes faecalis, CCRC 14817. EP-A-0896057 (published after the first priority date claimed for this Application) discloses a D-aminoacylase obtained from Amycolatopsis orientalis, IFO 12806. Summary of the Invention

The present invention was made following a screen for D-aminoacylase activity performed on a collection of bacteria, and from this screen several were identified as having a D-aminoacylase. Five of these strains were used for genomic DNA preparation. It was then possible, by examining a known literature sequence, to design oligonucleotide primers, and use these in PCR experiments to generate a 1.4kb fragment possessing D- aminoacylase activity. The recombinant fragment was sub-cloned into pTrc99C expression vector. The recombinant plasmid carrying the D-amino acylase fragment was then transformed in to E. coli DH5 for over-expression. After fermentation of the host, cells were obtained which had good D-aminoacylase activity. Sequencing of the enzyme showed that it had six differences to the published sequence of the known cloned Alcaligenes D-aminoacylase. These were Ser ² to Ala; Gin³ to Glu; Ala¹⁴ to Val; Gly^12β to Arg; Gly²⁴⁰ to Arg; and Glu²⁴² to Lys. These differences, individually or in combination, apparently bring about notable and surprising differences in the properties of the enzyme. For example, the novel enzyme will hydrolyse N-acetyl-D-tryptophan, whereas the published enzyme does not. Surprisingly, this enzyme is active at high substrate concentration; the published literature gives only examples of low substrate concentration, in the region of 20mM. At these concentrations, the volume efficiency is low, which increases the cost of recovering the product and reduces the economic viability of the process. Thus, it was surprising to find that the enzyme is effective at lOOg/l of substrate; even at 200g/l good activity was demonstrated. It is useful at high volume efficiency, of about lOOg/l, for the deprotection of several (D)-N- acetylaminoacids. This allows an economical process to be developed.

More generally, an isolated enzyme according to the present invention is capable of hydrolysing N-acetyl-D-tryptophan at a substrate concentration of 10 g 1. Thus, it is capable of the desired activity at the given concentration, and also at higher concentrations. In addition, unlike the enzymes disclosed in US- A-5206162 and in EP-A- 0896057, it exhibits the ability to convert (R)-N-acetyl-2-thienylalanine, and also to convert it faster than (R)-N-acetyl-4-chlorophenylalanine. Description of the Invention

In general terms, the substrate used in the invention may be part of a mixture of the (L)- and (D)-N-acylaminoacids. Alternatively, the (D)-N-acylaminoacid may be enantiomerically enriched, e.g. essentially optically pure.

The novel enzyme may be used to produce natural and unnatural aminoacids. One class of the latter is aryl/heteroaryl-substituted aminoacids.

In some instances, particularly with substrates of a hydrophobic nature, the enzyme may suffer from substrate inhibition. In these cases, for example with N-acetyl-D- styrylalanine or N-acetyl-D-2-naphthylalanine, a high substrate concentration may merely lead to a low conversion to product, so that a volume efficient reaction is not possible. However, this effect can be overcome by the simple expedient of adding the substrate in several batches over the course of thebiotransformation, and, if kept at low concentration, a high product accumulation is possible. For example, if the enzyme is exposed to >20g/l of N-acetyl-D-2-naphthylalanine, substrate hydrolysis is poor. However, the enzyme will hydrolyse 15 g/1 efficiently and, by making several additions of the substrate, it is possible to accumulate about 75 g/1 of D-2-naphthylalanine.

The enzyme may be used in whole cell or isolated form. It may be immobilised, if desired, by methods known to those of ordinary skill in the art.

The enzyme may be produced from the deposited organism (details given below). Alternatively, it may be produced by recombinant technology.

Using the DNA and amino-acid sequence provided herein, a person skilled in the art can readily construct fragments or mutations of the genes and enzymes disclosed herein. These fragments and mutations, which retain the activity of the exemplified enzyme, are within the scope of the present invention. Also, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino-acid sequences disclosed herein. It is well within the skill of one of ordinary skill in the art to create these alternative DNA sequences encoding the same, or similar, enzymes. These DNA sequences are within the scope of the present invention. As used herein, reference to "essentially the same" sequence refers to sequences which have amino-acid substitutions, deletions, additions or insertions which do not materially affect activity. Fragments retaining activity are also included in this definition.

The genes of this invention can be isolated by known procedures and can be introduced into a wide variety of microbial hosts. Expression of the gene results, directly or indirectly, in the intracellular production and maintenance of the enzyme. The gene may be introduced via a suitable vector into a microbial host.

A wide variety of ways are available for introducing the gene into the microorganism host under conditions which allow for stable maintenance and expression of the gene. A DNA construct may include the transcriptional and translational regulatory signals for expression of the gene, the gene under their regulatory control and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur. In the direction of transcription, namely in the 5' to 3' direction of the coding or sense sequence, the construct can involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 5' or 3* of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon(s), the polyadenylation signal sequence, if any, and the terminator region. This sequence as a double strand may be used by itself for transformation of a microorganism host, but will usually be included with a DNA sequence involving a marker.

The gene can be introduced between the transcriptional/translational initiation and termination regions, so as to be under the regulatory control of the initiation region. This construct can be included in a plasmid, which could include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host. In addition, one or more markers may be present, as described above. Where integration is desired, the plasmid will desirably include a sequence homologous with the host genome.

The transformants can be isolated in accordance with conventional ways, usually employing a selection technique, which allows for selection of the desired organism as against unmodified organisms or transferring organisms, when present. The transformants then can be tested for activity.

Suitable host cells include prokaryotes and eukaryotes. An example is E. coli.

The following Examples illustrate the invention. Example 1 Production of D-Aminoacylase

Genomic DNA was prepared from 5 Alcaligenes strains held in the Chirotech culture collection; CMC3352, 3353, 2916, 3378, 3823. From these genomic preparations PCR was carried out to amplify the D-aminoacylase reported by Wakayama et al (1995), supra. Primers were synthesised according to the published sequence of the dan gene from Alcaligenes A-6. The 5' PCR primer in SEQ ID NO. 1 ; the 3' PCR primer is SEQ ID NO. 2.

A 1.4 kb PCR fragment was amplified from strains CMC 3352 and 3353. These fragments were then cloned into the PCR cloning vector from Stratagene, pCR-script and transformed into E. coli. Resultant clones were analysed by restriction mapping to ascertain the presence of a 1.4kb acylase fragment. Clones harbouring this fragment were sequenced to verify that the putative acylase showed homology to the reported sequence. DNA sequence analysis show the majority of cloned fragments to include SEQ ID NO. 3. The deduced aminoacid sequence is given below as SEQ ID NO. 4. The residues of the recombinant D-acylase differ from the published sequence as follows; Ser ² to Ala; Gin³ to Glu; Ala¹⁴ to Val; ; Gly¹²⁶ to Arg; Gly²⁴⁰ to Arg; Glu²⁴² to Lys.

The recombinant fragment was sub-cloned into pTrc99C expression vector via the 5' Ncόl and 3' BamYΩ. engineered restriction sites. The recombinant plasmid carrying the D-amino acylase fragment was transformed into E. coli DH5 for over-expression.

The recombinant cells, E. coli strain CMC 4406, have been deposited at NCIMB, 23 St. Machar Drive, Aberdeen AB243RY, Scotland. The accession number is NCIMB 40965. The recombinant cells were grown by fermentation in a medium containing glucose, peptides and salts. The seed culture was inoculated from plates, and incubated overnight in TSB medium containing 0.1 g/1 ampicillin at 37 °C.

The inoculum (5ml, OD 5.0) was grown in 1.51 of the following medium which contained (amounts in g.l^"1 unless otherwise indicated): KHTO 8

K₂HPO₄ 7

(NHJjSO* 1

MgSO . 7H₂O 1

Yeast Extract 15 Trace elements solution lml.l^"1

Glucose 10

Polypropylene glycol lml..^"1

Hycase SF 15

The trace elements solution consisted of (amounts in g.1^"1 unless otherwise indicated):

CaCl₂.2H₂O 3.6

CuCl₂. 2H₂O 0.85

FeClj. 6H₂O 5.4 HaBO₄ 0.3

HC1 333ml.--¹

MnCl₂. 4H₂O 2.0

Na,MoO₄. 2H,0 4.8

ZnO 2.0 The pH was controlled between 6.9 and 7.2 by the addition ofNaOH solution, and the temperature maintained at 30°C. IPTG (0.24g/l) was added after inoculation. After 24 hrs, the biomass had reached an OD of 34. Cells were then harvested by centrifugation and stored at -15°C, then used in biotransformations as required. Example 2 Deprotection of (D)-N-Acetyl-(l-bromovinyl)alanine

KH_jPO₄ (0.8g, lOmmol) was dissolved in water (800 ml) in a 2 litre jacketed vessel. (D)-N-acetyl-(l-bromovmyl)alanine (lOOg, 0.42 mol, -95% ee was added and the pH adjusted to 8.0 using NaOH (46-48%). The temperature of thejacketed vessel was raised to 40°C and the solution stirred for 10 minutes while maintaining the pH at 8. The D-aminoacylase enzyme whole cells (9g) were added in one portion and the reaction mixture stirred at 40°C while maintaining the pH at 8.0 by subsequent additions ofNaOH. The reaction was monitored by chiral GC as follows: 0.5 ml of the reaction mixture was taken and acidified to pH 2.0 with cone. HC1. The aqueous was extracted with EtOAc which was dried (MgSO₄) and filtered and treated with OJml of TMS-diazomethane. The derivatised product was assayed by chiral GC (Chrompack Chirasil L-Nal, 25m, 20psi He, 60°C for 10 mins, 5°C/min to 200°C, holding for 10 minutes, FID detection). After 1 hour the ee of the substrate had decreased to 68%, after 2 hours it was 24% and after 22 hours was 7%. The reaction mixture was then acidified to pH 2.0 using concentrated HC1 solution, then filtered through a celite pad, and washed with EtOAc (3 x 300ml). The solution was then adjusted to pH 6.5 using ΝaOH (46-48%) and concentrated under reduced pressure until about 200 ml of solution remained. A white solid crashed out of solution and was filtered off and washed with acetone. This gave the product (D)- (l-bromovinyl)alanine as a clean white solid (60g, ee_R>99 %). The enantiomeric excess was measured by HPLC (25cm Chirobiotic T column, 70:30 MeOH:water, lml/min, 21 Onm). Other aminoacids were determined by the same method or with variations in the MeOH:water mobile phase composition. Example 3 Deprotection of (D)-Ν-acetylpropargylglycine

KH_;2PO₄ (2Jg) was dissolved in water (2.51) in a 2 litre jacketed vessel. The (D)- N-acetylpropargylglycine (232g, 1.50mol, -95% eβjj was added and the pH adjusted to 8.0 using NaOH (46-48%). The temperature of the jacketed vessel was raised to 40°C and the solution stirred for 10 minutes while maintaining the pH at 8. The D-aminoacylase enzyme whole cells (7g) were added in one portion and the reaction mixture stirred at 40°C while maintaining the pH at 8.0 by subsequent additions of NaOH. After 88 hours the remaining ee^-n-^ was 25.8%, only marginally lower than that at 16 hours. Another 3%/wt of cells were added and after 104 hrs the ee^--,.^ was 17.8%. The biotransformation was worked up since assuming 99% ee of product conversion was 85%. Work-up and isolation as for Example 2 gave a brownish solid (271g) which was slurried in MeOH for 10 minutes to give a clean white solid (171g) of >99% ee (R)- propargylglycine. Example 4 Deprotection of (D)-N-acetyl-2-furylaIanine

(D)-N-Acetyl-2-fiιrylalanine (18g, ee_R>99%) was added to water (200ml) containing lOmmole ofKiy^ and the temperature of thejacketed vessel raised to 40°C. The suspension was then adjusted to pH 8.0 with NaOH and stirred for 10 minutes. 0.72g (4% wt) of D-aminoacylase whole cells were then added and the reaction stirred vigorously while maintaining the pH at 8.0 by subsequent addition ofNaOH. The reaction was followed by chiral GC by the method described above, and was complete after 2 hours. The aqueous was the acidified to pH 2.0 with cone. HCl, and filtered through a Celite pad. The filtrate was then washed with EtOAc and adjusted to pH 7 with NaOH. The solution was the treated with Na₂CO₃ (2 equivalents assuming 100% conversion), cooled to ~10°C and then a solution of Fmoc-OSu (1 equivalent) in THF (300ml) added. After work-up the product was isolated as a white solid and recrystallised from methanol/water to give 18.4g of >99% ee (D)-2-furylalanine. Example 5 Deprotection of (D)-N-acet lallylglycine

(D)-N-Acetylallylglycine (40g, eeg 89%) was added to water (200ml) containing lOmmole of 13-y ^ and the temperature of the jacketed vessel raised to 40°C. The suspension was then adjusted to pH 8.0 with NaOH and stirred for 10 minutes. 1.0g (2.5% wt) of D-aminoacylase whole cells were then added and the reaction stirred vigorously while maintaining the pH at 8.0 by subsequent addition of NaOH. After 3 hours HPLC showed 30% conversion. After 16 hours the reaction had not gone any further and a further 2.5% wt of whole cells were added. After 60 hours the reaction still had not progressed past 30% and the solution was diluted to 10% by the addition of water. After another 2 hours, the reaction had reached 44% conversion, and after 88 hours 82% conversion. Example 6 Deprotection of (D)-N-acetyl-2-naphthylaIanine

(D)-N-Acetyl-2-naphthylalanine(0.75g) was added to 50ml Tris buffer (0.1 M,pH 7.5, 30 °C). The suspension was then adjusted to pH 8.0 with NaOH and stirred for 10 minutes. A crude lysate of D-aminoacylase (3 ml, 165U, lU=hydrolysis of lμmol of N- acetyl-D-tryptophan/min at 25°C, pH 7.5, 0.1M Tris buffer) was then added and the reaction stirred while maintaining the pH at 8.0 by subsequent addition of NaOH. More substrate (0.75g) and enzyme (165U) was added at 22, 46, 96, 173 and 218 hours. The final conversion by HPLC was 95% by peak area. Example 7 Deprotection of (D)-N-acetyI-3-pyridylalanine

(D)-N-Acetyl-3-ρyridylalanine (50g) was added to 750ml Tris buffer (0.1 M, pH 7.5, 30°C). The suspension was then adjusted to pH 8.0 with NaOH and stirred for 10 minutes. A crude lysate of D-aminoacylase (20 ml, 1100U, 1 U=hydrolysis of 1 μmol of N- acetyl-D-tryptophan/min at 25°C, pH 7.5, 0.1M Tris buffer) was then added and the reaction stirred while maintaining the pH at 8.0 by subsequent addition of NaOH. After 15 hours the reaction had reached about 80% conversion as measured by HPLC peak area. Examples 8 to 20 D-Acylase Reactions

Table 1 reports D-acylase reactions using a range of unnatural (R)-N-Ac- phenylalanine and (R)-N-Ac-alanine derivatives, and (R)-N-Ac-4-fluorophenylglycine.

Table 1

^b All reactions carried

of reaction. ^d Isolated yield. * Conversion in crude biotransformation reaction mixture. ^f%e.e. rose from 90% in N-acetyl substrate to 96.5% in aminoacid. ^β Reaction done using whole cells. TAZ = 4-ThiazoylAlanine Comparative Testing

In order to evaluate the properties of the novel enzyme and known D-acylases, comparative experiments were run. The results are reported in Table 2. In each set of 3 results, the respective enzymes were those deposited as IFO 12806 (104470) and CCRC 14817 (104476), and that of Example 1 (D-Ace). The results (U/U) show that, when appropriate corrections have been made, the novel enzyme converts certain unnatural aminoacids, e.g. (R)-N-Ac-thienylalanine, faster than CCRC 14817, although the rate is slower for (R)-N-Ac-4-chlorophenylalanine. Example 21 Whole Cell Immobilisation Whole cells of E. coli CMC4406 containing recombinant D-acylase were immobilised on a reactive soluble polymer (RSP). The RSP was prepared by reaction of polyethyleneimine (0.8g) with aqueous 25% w/v glutaraldehyde (1.6ml), to a total volume of 20 ml H₂O. The RSP was then mixed with 1 Og of cells resuspended in 20ml H₂O. This was stirred vigorously for 30 minutes, after which the immobilised cells, having the consistency of foam rubber, were recovered by filtration. The final product (20g) had a specific activity of 20.55 U/g and the recovery of activity was 43% of the whole cells used in the immobilisation. 1 Unit of activity is defined as the hydrolysis of 1 μmol/min N-Ac- D-tryptophan to D-tryptophan measured at a substrate concentration of lOmM at 25 °C, pH7.5. Example 22 Whole Cell Immobilisation

An aqueous suspension of cells ofE. coli CMC4406 (lOg in 20ml of water) was mixed thoroughly with 0.8g PEI, before the addition of 1.6ml of 25% w/v glutaraldehyde. Stirring of this mixture resulted in the formation of bead-like aggregates which were recovered by filtration. The final product (13.5g) had a specific activity of 20.39U/g and yielded 25% of the starting activity. Table 2

Enz loading

U/ml Sub Loading % Time Activity Activity Activity

Substrate Enzyme Dilution (N-Ac-DL-Trp) (mM) Conversion (Hrs) (U/U) (U/g) (U/ml)

(+/-)-N-Ac-4-Fluoro-Phe-Gly 104470 2 0.005 10 2 30 2 0 0

(+/-)-N-Ac-4-Fluoro-Phe-Gly 104476 2 0.115 10 22.4 30 1 2 0

(+/-)-N-Ac-4-Fluoro-Phe-Gly D-Ace 200 0.2775 10 7.6 30 0 20 8

(R)-N-Ac-Thienyl Ala 104470 2 0.005 10 0.3 30 - - -

(R)-N-Ac-Thienyl Ala 104476 2 0.115 10 26.6 30 1 3 0

(R)-N-Ac-Thienyl Ala D-Ace 200 0.2775 10 65 6 7 840 361

(R)-N-Ac-Naphthyl Ala 104470 2 0.005 50 1.5 22 11 1 0

(R)-N-AoNaphthyl Ala 104476 2 0.115 50 2.4 22 1 2 0

(R)-N-Ac-Naphthyl Ala D-Ace 200 0.2775 50 1.7 22 0 30 13

(R)-N-AoStyryl Ala 104470 2 0.005 50 1.8 22 14 1 0

(R)-N-Ac-Styryl Ala 104476 2 0.115 50 1 22 - - -

(R)-N-Ac-Styryl Ala D-Ace 200 0.2775 50 3.5 22 0 62 27

(R)-N-Ac-4-Chloro-Phe-Ala 104470 2 0.005 50 0.3 22 - - -

(R)-N-Ac-4-Chloro-P e-Ala 104476 2 0.115 50 7.1 22 2 5 1

(R)-N-Ac-4-Chloro-Phe-Ala D-Ace 200 0.2775 50 26 22 4 458 197

N-Ac-DL-Leu 104470 2 0.005 20 29 4 483 48 5

N-Ac-DL-Leu 104476 10 0.023 20 31 4 112 258 26

N-Ac-DL-Leu D-Ace 5000 0.0111 20 13 4 98 12,597 5,417

N-Ac-DL-Met 104470 2 0.005 20 21 4 350 35 4

N-Ac-DL-Met 104476 10 0.023 20 17 4 62 142 14

N-Ac-DL-Met D-Ace 5000 0.0111 20 7 4 53 6.873 2.917

Table 2 (contd.)

N-Ac-DL-Phe 104470 10 0.001 20 5 5.25 317 32 3

N-Ac-DL-Phe 104476 20 0.0115 20 22 5.25 121 279 28

N-Ac-DL-Phe D-Ace 5000 0.0111 20 6 5.25 34 4,430 1,905

N-Ac-DL-Tφ 104470 2 0.005 25 16 23.5 57 6 1

N-AoDL-Tφ 104476 2 0.115 25 36 1 130 300 30

N-Ac-DL-Tφ D-Ace 5000 0.0111 25 13 2 244 31,492 13,542

N-Ac-DL-Val 104470 10 0.001 20 9 4 750 75 8

N-Ac-DL-Val 104476 10 0.023 20 38 4 138 317 32

N-Ac-DL-Val D-Ace 1000 0.0555 20 4 4 6 775 333

N-AoD-Phe 104470 10 0.001 10 7 5.25 222 22 2

N-Ac-D-Phe 104476 20 0.0115 10 14 5.25 39 89 9

N-AoD-Phe D-Ace 5000 0.0111 10 10 1 150 19,380 8,333

N-Ac-D-Tφ 104470 2 0.005 10 20 23.5 28 3 0 t

N-Ac-D-Trp 104476 2 0.115 10 36 2 26 60 6

N-Ac-D-Tφ D-Ace 5000 0.0111 10 14 1 210 27,132 11,667

N-Benzoyl-DL-Phe 104470 10 0.001 20 0.1 5.25 - - -

N-Benzoyl-DL-Phe 104476 10 0.023 20 42 5.25 116 267 27

N-Benzoyl-DL-Phe D-Ace 5000 0.0111 20 1.7 5.25 10 1.255 540

Claims

1. An isolated enzyme capable of hydrolysing N-acetyl-D-tryptophan at a substrate concentration of 10 g/1 and which exhibits faster conversion of (R)-N-acetyl-2- thienylalanine than of (R)-N-acetyl-4-chlorophenylalanine.

2. An isolated enzyme having the aminoacid sequence of SEQ ID NO: 4, or a fragment thereof capable of hydrolysing N-acetyl-D-tryptophan at a substrate concentration of 10 g/1.

3. An enzyme according to claim 1 or claim 2, wherein the substrate concentration is 30 g/1.

4. An enzyme according to claim 3, wherein the substrate concentration is 100 g/1.

5. An enzyme according to any preceding claim, in immobilised form.

6. An isolated polynucleotide encoding an enzyme according to claim 2.

7. A polynucleotide according to claim 6, having part or all of the sequence shown in SEQ ID No. 3.

8. A microorganism transformed to express an enzyme according to any of claims 1 to 5.

9. A microorganism having the characteristics of NCIMB 40965.

10. A method for producing an enzyme according to any of claims 1 to 5, which comprises culturing a microorganism according to claim 8 or claim 9.

11. A process for the preparation of a (D)-aminoacid, which comprises the conversion of a corresponding (D)-N-acylaminoacid using an enzyme according to any of claims 1 to

5 or a microorganism according to claim 8 or claim 9.

12. A process according to claim 11, wherein the concentration of the

N-acylaminoacid is at least 30g/l.

13. A process according to claim 11, wherein the concentration of the

N-acylaminoacid is at least 100 g/1.

14. A process according to any of claims 11 to 13, wherein the (D)-N-acylaminoacid is part of a mixture of the (L)- and (D)-N-acylaminoacids.

15. A process according to any of claims 11 to 13, wherein the (D)-N-acylaminoacid is enantiomerically enriched.

16. A process according to any of claims 11 to 13, wherein the (D)-N-aminoacid is essentially a single enantiomer.

17. A process according to claims 11 to 16, wherein the aminoacid is unnatural.

18. A process according to any of claims 11 to 15, wherein the substrate is hydrophobic, such that there is substrate inhibition, which comprises adding the substrate batchwise during the conversion.

19. A process according to any of claims 11 to 18, wherein the concentration of the accumulated D-aminoacid is at least 30 g/1.