JP2002527110A - Aminoacylase and its use in the production of D-amino acids - Google Patents

Abstract

  (57) [Summary] An isolated enzyme capable of hydrolyzing N-acetyl-D-tryptophan at a substrate concentration of 10 g / l, which is faster than the conversion of (R) -N-acetyl-4-chlorophenylalanine. An isolated enzyme showing conversion of -acetyl-2-thienylalanine. This enzyme is useful for preparing D-amino acids.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an enzyme having D-aminoacylase activity, a D-amino acid by resolution of a racemic mixture of N-acylamino acids and deprotection of optically concentrated N-acylamino acids. Its use in the manufacture of

BACKGROUND OF THE INVENTION D-amino acids are industrially important intermediates in the manufacture of various pesticides, antibiotics and other medicaments. For example, phenylglycine and p-hydroxyphenylglycine have been used in the synthesis of semi-synthetic penicillins and cephalosporins. In addition, there is a great need for new D-amino acids as building blocks for new drug substances. D-amino acids can be obtained, for example, by salt crystallization or by physical resolution with an asymmetric chemical catalyst via hydrogenation of an enamide precursor. Chemical catalysis offers a general method of broad application, for example for artificial amino acids, which subsequently requires N-deacylation, which in conventional chemical hydrolysis often results in partial racemisation of the product. I do. For example, there is a biocatalytic method by hydrolysis of hydantoin using a D-specific hydantoinase. However, the resulting D-carbamoyl amino acids require further enzymatic or chemical deprotection to the amino acids.

The production of L-amino acids using L-specific aminoacylase-catalyzed hydrolysis of racemic N-acetylamino acids is a well-established technique. It uses enzymes from Aspergillus oryzae and has been used on a very large scale on an industrial basis to produce L-methionine, L-valine and L-phenylalanine. D-aminoacylase activity was determined by Pseudomonas
monas), Streptomyces (Streptomyces) and alkaline jeans (Alcaligen)
es), but no such large-scale technology exists for the production of D-amino acids. Sugie and Suzuki, Agric. Biol. C
hem. 44: 1089-1095 (1980); Daicel Chemical Industries, JP 64-5488 (1989); M
origuchi 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 et al, Appl.Env.Microbiol. 57: 2540-2543 (1991);
Yang et al, Appl.Env.Microbiol. 57: 1259-1260 (1991); and Kameda et al,
See Nature 169: 1016 (1952).

[0004] Enzymes from these strains have been isolated and characterized. The use of such a strain in whole cell form for the resolution or deprotection of N-acetylamino acids
Although relatively simple in theory, it has also been shown that cells contain L-aminoacylase, thereby reducing structure selectivity. In addition, the low levels of activity, even after growth on induction media, make the purification and use of whole cell-derived enzymes economically unattractive. A solution is expected by cloning the enzyme, which has recently been reported for D-aminoacylase of the alkaline jeans species, but the fact that such an enzyme works at any high substrate concentration does not disclose its substrate specificity. It is not expected to be significantly different from the wild-type enzyme. Moriguchi et
al, Biosci. Biotech.Biochem. 57 (7): 1149-1152 (1993); Wakayama et al, B
iosci.Biotech.Biochem.59 (11): 2115-2119 (1995); and Wakayama et al, Pr.
ot. Express. Pur. 7: 395-399 (1996).

[0005] Alkaline jeans xylosoxydans subsp.
), This enzyme obtained from xylosoxidans ("Alkaline Jeans A-6"), NC
IMB10771 does not hydrolyze N-acetyl-D-tryptophan. The activity of D-aminoacylase is inhibited by very low concentrations of 2 mM D-phenylalanine and N-acetyl-D-alloisoleucine by up to 37% and 40%. This suggests that the enzyme is susceptible to severe product and substrate inhibition. US-A-5206162 is Alcaligenes fae
calris), CCRC14817. EP-A-089605 (published after the first priority date claimed for this application)
7 discloses D-aminoacylase, IFO12806, obtained from Amycolatopsis orientalis.

SUMMARY OF THE INVENTION The present invention has been accomplished after screening for D-aminoacylase activity performed in a collection of bacteria, and from this screening, several have been identified.
It was identified as having an aminoacylase. Five of these strains were used for genomic DNA preparation. Next, oligonucleotide primers were designed by examining known literature sequences and used in PCR experiments to obtain a 1.4 kb fragment having D-aminoacylase activity. PTr
It was subcloned into the c99C expression vector. The recombinant plasmid containing the D-aminoacylase fragment was then transformed into E. coli DH5 for overexpression. After fermentation of the host, cells with good D-aminoacylase activity were obtained.

[0007] The sequence of the enzyme has been shown to have six differences from the published sequence of the known cloned alkaline jeans D-aminoacylase. These are Se
from r ² Ala; from Gln ³ Glu; from ^{Ala 14 Val;} Arg from ^{Gly 126;} Arg from ^{Gly 240;} and ^was Lys from ^{Glu 242.} These differences, individually or in combination, clearly make significant and surprising differences in the properties of the enzyme. For example, the novel enzyme hydrolyzes N-acetyl-D-tryptophan, whereas the published enzyme does not. Surprisingly,
This enzyme is active at high substrate concentrations. The published literature only shows examples of low substrate concentrations of about 20 mM. At these concentrations, mass production efficiency is low, which increases the cost of recovering the product and reduces the economic viability of the process. Thus, it has surprisingly been found that the enzyme is effective on 100 g / l substrate and shows good activity even at 200 g / l. For the deprotection of some (D) -N-acetylamino acids, a high volumetric efficiency of about 100 g / l is effective. This makes it possible to develop economical methods.

[0008] More generally, the isolated enzyme according to the invention is capable of hydrolyzing N-acetyl-D-tryptophan at a substrate concentration of 10 g / l. That is, the desired activity can be obtained even at a predetermined concentration or a higher concentration. In addition, unlike the enzymes disclosed in US-A-5206162 and EP-A-0896057, it shows the potential to convert (R) -N-acetyl-2-thienylalanine and converts it to (R) It also shows the potential to convert faster than -N-acetyl-4-chlorophenylalanine.

DESCRIPTION OF THE INVENTION In general, the substrate used in the present invention may be part of a mixture of (L)-and (D) -N-acylamino acids, apart from (D)- N-acyl amino acids may be enantiomerically enriched, for example, may be essentially optically pure. The novel enzymes can be used to produce natural and artificial amino acids. One class of the latter is aryl / heteroaryl substituted amino acids.

[0010] In some instances, the enzyme may be subject to substrate inhibition, especially when using hydrophobic substrates. In these cases, for example with N-acetyl-D-stilalanine or N-acetyl-D-2-naphthylalanine, high substrate concentrations can only lead to conversion to low levels of the product, There is no effective reaction for mass production. However, this effect can be overcome by simple means of adding the substrate in several batches throughout the biotransformation, and if kept at low concentrations, a high product accumulation is possible. For example, when the enzyme is exposed to> 20 g / l of N-acetyl-D-2-naphthylalanine, the hydrolysis of the substrate is insignificant. However, the enzyme effectively hydrolyzes 15 g / l and, with several additions of substrate, makes it possible to accumulate about 75 g / l of D-2-naphthylalanine.

[0011] The enzyme can be used in whole cell or isolated form. If desired, they may be fixed by methods known to those skilled in the art. The enzyme may be produced from the deposited microorganism (details are given below). Alternatively, it may be produced by recombinant technology. Using the DNA and amino acid sequences provided herein, one of skill in the art can readily construct fragments or variants of the genes and enzymes disclosed herein. Those fragments and variants that retain the activity of the exemplified enzymes are within the scope of the invention. Also, due to the duplication of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed herein.
Creating these other DNA sequences, encoding the same or similar enzymes,
It is well within the skill of the artisan. These DNA sequences are within the scope of the present invention. As used herein, an “essentially identical” sequence refers to a sequence that has amino acid substitutions, deletions, additions, or insertions that do not significantly affect activity.
Fragments that retain activity are also included within this definition.

The gene of the present invention can be isolated by a known method and can be introduced into a wide variety of microbial hosts. When a gene is expressed, directly or indirectly,
Enzymes are produced and maintained intracellularly. The gene may be introduced into a microbial host by a suitable vector. A wide variety of methods are available for introducing a gene into a microbial host under conditions that permit stable retention and expression of the gene. DNA constructs include transcriptional and translational control signals for expression of the gene, the gene under its control and a DNA sequence homologous to the sequence of the host organism (which results in integration), and / or function in the host. Replication system (which results in integration and proper maintenance)
May be included.

In the direction of transcription, ie, 5 ′ to 3 ′ of the coding or sense sequence, the construct may, in some cases, include a transcriptional regulatory region and a promoter (the regulatory region is
May be at either the 5 'or 3' of the promoter), a ribosome binding site, an initiation codon, a structural gene having an open reading frame with the initiation codon in phase, a stop codon, and in some cases a polyadenylation signal. It can include a sequence and a termination region. This sequence as a duplex can itself be used to transform a microbial host, but is usually
Included with NA sequence. Genes can be introduced between the transcription / translation initiation and termination regions to be placed under regulatory control of the initiation region. This construct can be included in a plasmid, and the plasmid can include at least one replication system, but can include one or more, in which case one replication system provides for cloning during construction of the plasmid. And a second replication system is required for functionalization in the final host. In addition, one or more markers may be present as described above. If integration is desired, the plasmid desirably contains sequences homologous to the host genome.

The transformed individual is isolated in accordance with conventional practice, usually using selection methods that allow the selection of the desired organism from unmodified organisms or, if present, mutant organisms. Can be. Transformants can then be tested for activity. Suitable host cells include prokaryotes and eukaryotes. One example is E. coli. The following examples illustrate the invention.

EXAMPLE 1 Production of D-Aminoacylase Five Alkaline Jeans Strains Stored in the Chirotec Culture Collection
Genomic DNA from CMC3352, 3353, 2916, 3378, 3823
Was prepared. PCR was performed from these genomic preparations, and Wakayama et al (1995),
The previously reported D-aminoacylase was amplified. Alkaline jeans A-6
Primers were synthesized according to the published sequence of the derived dan gene. The 5 'PCR primer is SEQ ID NO: 1, and the 3' PCR primer is SEQ ID NO: 2. A 1.4 kb PCR fragment was amplified from strains CMC3352 and 3353. These fragments were then cloned into a PCR cloning vector from Stratagene, pCR-script, and transformed into E. coli. Resulting clones were analyzed by restriction mapping to confirm the presence of the 1.4 kb acylase fragment. Clones with this fragment were sequenced and confirmed that the putative acylase showed homology to the reported sequence.
Analysis of the DNA sequence indicates that the majority of the cloned fragment contains SEQ ID NO: 3.

The deduced amino acid sequence is shown below as SEQ ID NO: 4. Of recombinant D-acylase
Residues differ from the published sequence as follows. ; Ser²To Ala;
Gln³To Glu; Ala¹⁴Is Val; Gly¹²⁶Is Arg; Gly ²⁴⁰ To Arg; Glu²⁴²Is Lys. The recombinant fragment was inserted into the pTrc99C expression vector with 5'NcoI and 3 '
Subcloned with the 'BamHI treated restriction site. D-amino reed
E. coli D for overexpressing a recombinant plasmid having a lase fragment
H5 was transformed. The recombinant cell, E. coli strain CMC4406, was transferred to NCIMB, 23 St. Machar Driv
e, Aberdeen AB24 3RY, deposited in Scotland. The accession number is NCIMB 40965
It is.

[0017] Recombinant cells were grown by fermentation in a medium containing glucose, peptides and salts. Seed cultures are inoculated from the plate and TS containing 0.1 g / l ampicillin
Incubate overnight at 37 ° C. in B medium. Inoculum (5ml, OD5.0) _{a, KH 2 PO 4 8 K 2} HPO 4 7 (NH 4) 2 SO 4 1 MgSO 4 .7H 2 O 1 yeast extract 15 trace element solution 1Ml.L ^-1 glucose 10 Polypropylene Glycol was grown in 1.5 l medium containing ¹ ml.l- ¹ hicase SF15 (units gl- ¹ unless otherwise indicated). ;

The trace element solution (unit G.L ^-1 if not indicated _{otherwise) CaCl 2 .2H 2 O 3.6 CoCl} 2 .6H 2 O 2.4 CuCl 2 .2H 2 O 0.85 FeCl 3 it is made of _{.6H 2 O 5.4 H 3 BO 4} 0.3 HCl 333ml.l -1 MnCl 2 .4H 2 O 2.0 Na 2 MoO 4 .2H 2 O 4.8 ZnO 2.0.

The pH was adjusted between 6.9 and 7.2 by addition of a NaOH solution and the temperature was maintained at 30 ° C. IPTG (0.24 g / l) was added after inoculation. After 24 hours, the biomass reached an OD of 34. Cells were then collected by centrifugation, stored at -15 ° C, and then used for biotransformation when desired.

Example 2 Deprotection of (D) -N-acetyl- (1-bromovinyl) alanine KH ₂ PO ₄ (0.8 g, 10 mmol) was added to water (8
00 ml). (D) -N-acetyl- (1-bromovinyl) alanine (100 g
, 0.42 mol, ９５95% ee _R ) was added and the pH was adjusted to 8.0 with NaOH (46-48%). The temperature of the jacketed vessel was raised to 40 ° C. and the solution was stirred for 10 minutes while maintaining the pH at 8. D-aminoacylase enzyme whole cell (
9 g) was added in one portion and the reaction mixture was stirred at 40 ° C. while maintaining the pH at 8.0 by continued addition of NaOH. The reaction was measured by chiral GC as follows. : 0.5 ml of reaction mixture was collected and acidified to pH 2.0 with concentrated HCl. The aqueous phase was extracted with EtOAc, which was dried (MgSO ₄ ), filtered and
Treated with .1 ml of TMS-diazomethane. The derived product was converted to chiral GC (Chr
ompack Chirasil L-Val, 25 m, 20 psiHe, 60 ° C. for 10 minutes, 5 ° C./min to 200 ° C. for 10 minutes, FID detection). After 1 hour, the ee of the substrate was reduced to 68%, 24% after 2 hours and 7% after 22 hours. The reaction mixture was then acidified to pH 2.0 using concentrated HCl, then filtered through a pad of celite and washed with EtOAc (3 × 300 ml).

The solution was then adjusted to pH 6.5 with NaOH (46-48%) and concentrated under reduced pressure until about 200 ml of solution remained. A white solid was collected from the solution, then filtered off and washed with acetone. This provided the product (D)-(1-bromovinyl) alanine as a pure white solid (60 g, ee _R > 99%). The enantiomeric excess was determined by HPLC (25 cm Chirobiotic T column, 70:30 MeOH:
Water, 1 ml / min, 210 nm). Other amino acids were determined by the same method or using a variation on the MeOH: water mobile phase composition.

Example 3 Deprotection of (D) -N-acetylpropargylglycine KH ₂ PO ₄ (2.1 g) was dissolved in water (2.5 l) in a 2 liter jacketed vessel. (D) -N-acetylpropargylglycine (232 g, 1.50 mol,
９５95% ee _R ) was added and the pH was adjusted to 8.0 with NaOH (46% -48%). The temperature of the jacketed vessel was raised to 40 ° C. and the solution was stirred for 10 minutes while maintaining the pH at 8. All cells of the D-aminoacylase enzyme (7 g) were added in one portion and the reaction mixture was stirred at 40 ° C. while maintaining the pH at 8.0 by subsequently adding NaOH. 88 hours later, the remaining ee _{(R) -N-Ac}
Was 25.8%, only slightly lower than at 16 hours.
Another 3% / wt cells were added and 104 hours later the ee _{(R) -N-Ac} was 17.8%. Assuming 99% ee, the product conversion was assumed to be 85% and worked up the biotransformation. Work-up and isolation as in Example 2 gave a brown solid (271 g), which was slurried in MeOH for 10 minutes and an uncontaminated white solid (171 g) of (R) -propargylglycine with> 99% ee. ).

Example 4 Deprotection of (D) -N-acetyl-2-furylalanine (D) -N-acetyl-2-furylalanine (18 g, ee _R > 99%) was added to 10 mmo.
1 KH ₂ PO ₄ in water (200 ml) and the temperature of the jacketed vessel was raised to 40 ° C. The suspension was then adjusted to pH 8.0 with NaOH and stirred for 10 minutes. 0.72 g (4% wt) of D-aminoacylase whole cells were then added,
The reaction was stirred vigorously while the pH was adjusted to 8.
It was kept at zero. After the reaction, chiral GC was performed by the above method, and the reaction was completed after 2 hours. The aqueous phase was acidified to pH 2.0 using concentrated HCl and filtered through a pad of celite. The filtrate was then washed with EtOAc and adjusted to pH 7 with NaOH. The solution was treated with Na ₂ CO ₃ (2 equivalents assuming 100% conversion), cooled to -10 ° C., and then a solution of Fmoc-OSu (1 equivalent) in THF (300 ml) was added. After workup, the product was isolated as a white solid and recrystallized from methanol / water to give 18.4 g of (D) -2-furylalanine with> 99% ee.

Example 5 Deprotection of (D) -N-acetylallylglycine (D) -N-acetylallylglycine (40 g, ee _R 89%) was added to 10 mmol of K
H ₂ PO ₄ was added to water (200 ml), and the temperature of the jacketed vessel was set to 40 ° C.
Raised to. The suspension was then adjusted to pH 8.0 with NaOH and stirred for 10 minutes.
1.0 g (2.5% wt) of D-aminoacylase whole cells were then added and the reaction was stirred vigorously while maintaining the pH at 8.0 by the subsequent addition of NaOH. After 3 hours, HPLC showed 30% conversion. After 16 hours, the reaction did not proceed any further and an additional 2.5% wt of whole cells was added. After 60 hours, the reaction is 3
With no progress above 0%, the solution was diluted to 10% by the addition of more water. 2 more
After hours, the reaction reached 44% conversion and after 88 hours, 82% conversion.

Example 6 Deprotection of (D) -N-acetyl-2-naphthylalanine (0.75 g) of (D) -N-acetyl-2-naphthylalanine was added to 50 ml of Tris buffer (0.1 M, pH 7.1). 5, 30 ° C.). The suspension is then poured with NaOH
The mixture was adjusted to H 8.0 and stirred for 10 minutes. D-aminoacylase (3 ml, 165 U
(1 U = hydrolysis of 1 μmol N-acetyl-D-tryptophan / min at 25 ° C., pH 7.5, 0.1 M Tris buffer) was then added and the reaction was stirred while NaOH was added. The pH was maintained at 8.0 by the addition. Further, the substrate (0.75 g) and the enzyme (165 U) were added to 22, 46, 96, 17
Added at 3 and 218 hours. 95% final conversion by HPLC in peak range
Met.

Example 7 Deprotection of (D) -N-acetyl-3-pyridylalanine (D) -N-acetyl-3-pyridylalanine (50 g) was added to 750 ml of Tris buffer (0.1 M, pH 7.5, 30 ° C.). The suspension is then brought to pH with NaOH.
Adjusted to 8.0 and stirred for 10 minutes. D-aminoacylase (20 ml, 1100
U, 1 U = hydrolysis of 1 μmol N-acetyl-D-tryptophan / min at 25 ° C., pH 7.5, 0.1 M Tris buffer), then stir the reaction,
Meanwhile, the pH was maintained at 8.0 by subsequent addition of NaOH. After 15 hours, the reaction had reached about 80% conversion as measured by the HPLC peak range.

Examples 8 to 20 D-Acylase Reaction Table 1 shows a series of synthetic (R) -N-Ac-phenylalanine and (R) -N-Ac-alanine derivatives and (R) -N-Ac-4. 1 shows a D-acylase reaction using -fluorophenylglycine.

[0028]

[Table 1]

^B : All reactions performed in NaOH / KH ₂ PO ₄ buffer. ^c ; time to reach completion / stop of the reaction. ^d ; isolation yield. ^e ; Conversion in crude biotransformation reaction mixture. ^f ;% ee increased from 90% for N-acetyl substrates to 96.5% for amino acids. ^g : Reaction performed with whole cells. TAZ = 4-thiazoylalanine

Comparative Tests To evaluate the properties of the novel enzyme and the known D-acylase, comparative experiments were performed. Table 2 shows the results. In each set of three results, each enzyme was IF
O12806 (104470) and CCRC14817 (104476) and Example 1 (D-Ace). The results (U / U) indicate that, with appropriate modifications, the novel enzyme may have some artificial amino acids, such as (R) -N-
It shows that Ac-thienylalanine converts faster than CCRC14817 (albeit at a slower rate than (R) -N-Ac-4-chlorophenylalanine).

EXAMPLE 21 Immobilization of Whole Cells Whole cells of E. coli CMC4406 containing recombinant D-acylase were immobilized on a reactive soluble polymer (RSP). RSP consists of polyethyleneimine (0.8 g)
A 25% w / v aqueous solution of glutaraldehyde based on the total volume of 20 ml of H ₂ O (1
.6 ml). Was mixed with 10g of cells resuspended in _{H 2} O in 20ml is then RSP. This was stirred vigorously for 30 minutes, after which the fixed cells with the consistency of cellular rubber were collected by filtration. Final product (20
g) had a specific activity of 20.55 U / g, and the activity recovery was 43% of the total cells used for the fixation. The activity of one unit is determined at 25 ° C., p
Defined as 1 μmol / min hydrolysis of N-Ac-D-tryptophan to D-tryptophan, measured at H7.5.

Example 22 Immobilization of whole cells 0.8 g of a cell suspension of E. coli CMC4406 (10 g in 20 ml of water)
After thorough mixing with PEI, 1.6 ml of 25% w / v glutaraldehyde was added. The mixture was stirred to form a bead-like aggregate, which was collected by filtration. The final product (13.5 g) had a specific activity of 20.39 U / g, yielding 25% of the starting activity.

[0033]

[Table 2]

[0034]

[Table 3]

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:05) C12N 15/00 ZNAA (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD) , RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE , DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ , TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW Cambridge Science Park, Cairo Tech Technology Limited F-term (reference) 4B024 AA01 AA07 BA11 BA71 CA02 DA05 GA19 4B050 CC03 DD02 EE10 LL01 LL05 4B064 AE03 CA02 CA19 CB03 CC10 CC24 DA01 DA11 4B065 AA12Y AA26 X AB01 AC14 BA01 BB12 CA17

Claims (19)

[Claims] 1. An isolated enzyme capable of hydrolyzing N-acetyl-D-tryptophan at a substrate concentration of 10 g / l, which is faster than the conversion of (R) -N-acetyl-4-chlorophenylalanine. An isolated enzyme showing the conversion of (R) -N-acetyl-2-thienylalanine. 2. An isolated enzyme having the amino acid sequence of SEQ ID NO: 4 or a fragment thereof, which is capable of hydrolyzing N-acetyl-D-tryptophan at a substrate concentration of 10 g / l. 3. The enzyme according to claim 1, wherein the substrate concentration is 30 g / l. 4. The enzyme according to claim 3, wherein the substrate concentration is 100 g / l. 5. An enzyme according to any one of the preceding claims in fixed form. 6. An isolated polynucleotide encoding the enzyme according to claim 2. 7. The method according to claim 6, which has part or all of the sequence shown in SEQ ID NO: 3.
The polynucleotide of any of the preceding claims. A microorganism transformed to express the enzyme according to any one of claims 1 to 5. 9. A microorganism having the characteristics of NCIMB 40965. 10. A method for producing the enzyme according to any one of claims 1 to 5, comprising culturing the microorganism according to claim 8 or 9. 11. A process for the preparation of (D) -amino acids, which comprises using an enzyme according to any one of claims 1 to 5 or a microorganism according to claim 8 or 9. (D) -N-acyl amino acid conversion. 12. The method according to claim 11, wherein the concentration of the N-acyl amino acid is at least 30 g / l. 13. The method according to claim 11, wherein the concentration of the N-acyl amino acid is at least 100 g / l. 14. The method according to any one of claims 11 to 13, wherein the (D) -N-acyl amino acid is part of a mixture of (L)-and (D) -N-acyl amino acids. 15. The method of claim 1, wherein the (D) -N-acyl amino acid is enantiomerically enriched.
14. The method according to any one of 1 to 13. 16. The method according to any one of claims 11 to 13, wherein the (D) -N-amino acid is essentially a single enantiomer. 17. The method according to claim 11, wherein the amino acid is artificial. 18. The method according to any one of claims 11 to 15, comprising adding the substrate batchwise during the conversion, wherein the substrate is hydrophobic, such that there is substrate inhibition. 19. The method according to claim 11, wherein the concentration of accumulated D-amino acids is at least 30 g / l.