Mutants of formate dehydrogenase from Candida boidinii, new gene sequences encoding these and use of the new formate dehydrogenases

The invention relates to new mutants of formate dehydrogenase from Candida boidinii, new gene sequences encoding these and use of the new formate dehydrogenases. The wild type FDH used hitherto in the industrial process for preparing amino acids becomes inactive after a certain time. New mutants of this wild type have been produced by targeted mutagenesis of a recombinant FDH from E. coli. The new mutants are preferably used in an enzymatic process for preparing chiral compounds.

FIELD OF THE INVENTION
 The present invention relates to mutants of formate dehydrogenase from
 Candida boidinii (DSM 32195). The invention also relates to new gene
 sequences encoding these mutants and use of the formate dehydrogenases
 according to the invention in a process for preparing chiral compounds.
 DISCUSSION OF THE PRIOR ART
 To prepare L-amino acids, biocatalysts, inter alia, have been successfully
 used. One approach to the problem is to convert prochiral alpha-ketoacids
 by reductive amination. The amino acid dehydrogenases used for this
 purpose require stoichiometric amounts of NADH or NADPH as a coenzyme in
 order to convert the alpha-ketoacids. These coenzymes are very expensive
 and make the process mentioned above economically non-viable for use on an
 industrial scale.
 One possibility of avoiding high costs due to the coenzyme comprises
 regenerating the coenzyme in situ. NAD-dependent formate dehydrogenase
 from the yeast Candida boidinii is currently used, inter alia, in the
 enzyme reactor for coenzyme regeneration on an industrial scale.
 ##STR1##
 In situ regeneration of NADH with NAD-dependent formate dehydrogenase
 during the reductive amination of trimethyl pyruvate to give
 L-tert-leucine (Bommarius et al. Tetrahedron Asymmetry 1995, 6,
 2851-2888).
 A disadvantage of using FDH from Candida boidinii in a production process
 is the necessity of having to continue to add FDH during the process,
 since it becomes inactive as a result of lack of stability. This
 inactivation can be affected by a variety of factors:
 pH
 temperature
 mechanical stress
 ionic strength of and type of ion in the substrate solution
 traces of heavy metals
 oxidation of sulfhydryl groups by oxygen in the air
 cross-linking due to thiol/disulfide exchange.
 Tishkov et al. showed that targeted mutation of recombinant FDH from
 Pseudomonas sp. 101 could increase its stability towards mercury salts,
 whereas, however, the thermal stability was lowered by mutagenesis
 (Biochem, Biophys. Res. Commun. 1993, 192, 976-981).
 Sakai et al. elucidated the gene sequence of FDH from the methylotrophic
 yeast Candida boidinii (J. Bacteriol. 1997, 179, 4480-4485). The protein
 sequence derived agreed 100% with the amino acid sequence of the basic
 recombinant FDH from Candida boidinii in this work.
 SUMMARY OF THE INVENTION
 In view of the prior art outlined and discussed above, it was also the
 object of the present invention to modify the FDH from Candida boidinii
 used in the industrial process in such a way that this has greater
 resistance to oxidation than recombinant FDH and the wild type and thus
 make costly and complicated post-addition of FDH during the process
 unnecessary.
 Specifically, the invention is directed to stable mutants of rec-FDH from
 Candida boidinii having a higher level of stability to aggregation and
 oxidation than rec-FDH and the wild type enzyme. In these mutants one or
 more of the sulfur-containing amino acids in the rec-FDH are replaced by
 non sulfur containing amino acids. In particular those mutants wherein at
 least one of the cysteines at positions 23 and 262 is replaced by an amino
 acid selected from the group consisting of by serine, alanine or valine.
 The invention also includes the novel genes that encode these new mutants,
 in particular those genes, the DNA of which are set forth in sequences
 1,3,5,7,9,11,13,15,17,19,21,23,25,27 and 29 hereof. The invention also
 includes the process of converting alpha keto acids into the corresponding
 chiral alpha amino acids in the presence of these mutants. Suitably this
 process is one wherein the conversion is carried out in the further
 presence of NAD.sup.+.H.sub.2 O, preferably one wherein the conversion is
 carried out in the further presence of leucine dehydrogenase. Also
 included in the scope of the present invention is the process of preparing
 these mutant genes by means of targeted mutagenesis.
 As a result of modifying the recombinant formate dehydrogenase from Candida
 boidinii by means of targeted mutagenesis, it has been possible in a very
 advantageous, and nevertheless surprising manner, to generate mutants
 which are not sensitive to aggregation and oxidation. These are unlike
 rec-FDH and the wild type enzyme, and thus to enable a longer working
 lifetime for this enzyme in a production process. Surprisingly, other
 advantageous properties of FDH, such as e.g. catalytic activity,
 conformational stability, thermal stability, etc. are only marginally
 affected so the new advantage is not negated by introducing different
 additional disadvantages. This could not have been predicted since, in
 such a complex molecule, even the smallest modification frequently leads
 to the complete loss of activity of the enzyme.
 The recombinant formate dehydrogenase being considered is preferably
 modified in such a way that the sulfur-containing amino acids in the
 enzyme are replaced, independently, and separately or together, by amino
 acids which do not contain sulfur.
 The cysteine units at positions 23 and 262 in FDH appear to be the
 particularly preferred targets of targeted mutation. Targeted mutagenesis
 may take place either at only one of these positions or at both. The
 sulfur-containing amino acids at positions 23 and/or 262 are
 advantageously replaced, independently, and separately or together, by
 amino acids without a sulfhydryl group. Replacement with serine, alanine
 or valine is particularly preferred.
 The success of this modification, at the time when the invention was
 discovered, was neither predictable nor obvious, for the reasons given
 above.
 The enzymes with improved stability encoded by the new gene sequences are
 preferably used in an enzymatic process for preparing chiral compounds,
 including the type mentioned at the beginning.
 Enzymes with formate dehydrogenase activity according to the invention can
 advantageously be produced by means of targeted mutagenesis on the basis
 of the recombinant FDH gene and expressed in E. coli. Working with
 recombinant FDH offers the advantage that a standardised gene sequence and
 thus a standardised gene product is present, in which mutations can be
 produced. In order to be able effectively to compare the effects of the
 mutation in the mutants with the wild type enzyme, however, it is a
 critical advantage to be able to start from a standardised enzyme. There
 are probably several isoforms of the enzyme present in Candida boidinii
 itself and these are difficult to separate preparatively. In any case the
 wild enzyme exhibits microheterogeneities at the protein level.
 In addition, all the advantages of Escherichia coli which are known to a
 person skilled in the art and relate to the parent organism, such as
 multiplication and expression, can be used.
 DESCRIPTION OF THE PREFERRED EMBODIMENTS
 The gene and amino acid sequences according to the invention can be
 prepared by biochemical and microbiological methods, which are known per
 se.
 Thus, genomic DNA from Candida boidinii can be obtained by cultivation,
 lysis and precipitation using Ferbeyre et al.'s method (Bio Techniques
 1993, 14. 386) The FDH gene can then be amplified by means of a polymerase
 chain reaction (PCR). The primers that were required were derived from
 protein sequence data. The FDH gene obtained was ligated in a cloning
 vector and transformed in E. coli. After isolating the recombinant plasmid
 DNA from the E. coli cells using a commercially available preparation kit
 (e.g. Qiagen Plasmid Tip 20), both DNA strands were sequenced. The
 sequence is shown in Sequences 31 and 32.
 The recombinant plasmid DNA also acts as a template for PCR-promoted
 mutagenesis using Ho et al.'s method (Gene, 1989, 77, 52-59). The primers
 used contain the modified codon (in brackets) for replacement at the
 corresponding amino acid position: C23 (TGT bp 67-69) for S23 (TCT); C262
 (TGT. bp 784-786) for V262 (GTT) or A262 (GCT). The amplified, mutated FDH
 genes were cloned in expression vector pBTac2 (Boehringer) (FIG. 2) and
 expressed in E. coli. The mutants were obtained from cells in the form of
 a crude cell-free extract by lysing the cultivated E. coli.
 The advantage of the new enzymes is obvious from stability tests. The
 inactivation of recombinant FDH from Candida boidinii, of FDH-C23S, of
 FDH-C23S/C262A, of FDH-C23S/C262V and of FDH-C262V were measured in a
 comparative trial and their inactivation half-lives were determined. The
 results are given in Table 1.
 TABLE 1
 Enzyme Half-life (h)
 recFDH &lt;21
 FDH-C23S/C262A &gt;750
 FDH-C23S &gt;750
 FDH-C23S/C262V 160
 FDH-C262V 21
 The improvement in stability is obvious from the increase in half-lives of
 the mutants FDH-C23S/C262A, FDH-C23S and FDH-C23S/C262V. The notation
 FDH-C23S means that, in the formate dehydrogenase being considered,
 cysteine (C) has been replaced by serine (3) at position 23 in the protein
 sequence. In the same way, the expression FDH-C262V is understood to
 indicate that cysteine at position 262 has been replaced by valine.
 The expression rec-FDH is understood to represent the recombinant formate
 dehydrogenase which can be obtained by cloning and expressing the gene
 from Candida boidinii in E. coli in accordance with the description given
 below.
 Heterogeneous FDH, which can be obtained from Candida boidinii, is called
 the wild type enzyme.
 The mutations of the present invention are set forth in detail in the
 sequences attached to this application and made part thereof. They can be
 summarised as follows.
 TABLE 2
 Amino Amino
 Abbreviated DNA at DNA at Acid at Acid at
 name codon 23 Seq # codon 262 Seq # codon 23 Seq # codon 262 Seq #
 Rec-FDH TGT 31 TGT 31 Cys 32 Cys 32
 C23s TCT 1 TGT 1 Ser 1,2 Cys 1,2
 C23v GTT 3 TGT 3 Val 3,4 Cys 3,4
 C23a GCT 5 TGT 5 Ala 5,6 Cys 5,6
 C262s TGT 7 TCT 7 Cys 7,8 Ser 7,8
 C262v TGT 9 GTT 9 Cys 9,10 Val 9,10
 C262a TGT 11 GCT 11 Cys 11,12 Ala 11,12
 C23s/c262s TCT 13 TCT 13 Ser 13,14 Ser 13,14
 C23s/c262a TCT 15 GCT 15 Ser 15,16 Ala 15,16
 C23s/c262v TCT 17 GTT 17 Ser 17,18 Val 17,18
 C23a/c262s GCT 19 TCT 19 Ala 19,20 Ser 19,20
 C23a/c262a GCT 21 GCT 21 Ala 21,22 Ala 21,22
 C23a/c262v GCT 23 GTT 23 Ala 23,24 Val 23,24
 C23v/c262v GTT 25 GTT 25 Val 25,26 Val 25,26
 C23v/c262a GTT 27 CGT 27 Val 27,28 Ala 27,28
 C23v/c262s GTT 29 TCT 29 Val 29,30 Ser 29,30
 The DNAs at the above codons are illustrative not limiting. The degenerated
 base triplets encoding the same amino acids are within the scope of this
 invention. The following examples are intended to illustrate the
 invention.

EXAMPLES
 Example 1
 Preparing Genomic DNA from Candida boidinii
 Preparing genomic DNA from the yeast was performed using a modified form of
 Ferbeyre et al.'s method (Bio Techniques 1993, 14, 386). The Candida
 boidinii cells were cultivated in 200 ml of YEPD medium at 30.degree. C.
 and 200 rpm up to the time of the late logarithmic growth phase and then
 harvested by centrifuging (10 min, 15.degree. C., 5000 rpm, GSA rotor).
 Under these conditions, about 2.0 g of moist cell material were produced
 per 100 ml of culture. The cells were washed once with 10 mM citrate
 phosphate buffer, pH 7.5, and then resuspended in 10 ml of lysis buffer. 1
 mg of protease [Qiagen] and 200 units of lyticase from Arthrobacter luteus
 [Sigma] per ml of lysis buffer were added to the cell suspension. The
 suspension was incubated for 60 min at 37.degree. C. and then extracted
 with the same volume (vol.) of phenol/chloroform/isoamyl alcohol (PCl).
 After centrifuging for 30 min at RT and 12000 rpm in a SS34 rotor, the
 aqueous phase was removed and again extracted with PCl when a large
 interphase appeared. The DNA was then precipitated in the aqueous phase
 with 1/10 vol. of 3 M sodium acetate, pH 5.2, and 2 vol. of ice-cold
 ethanol (abs.), placed on ice for 5 min, wound onto a glass rod and dried
 under the sterile bank. After drying, the DNA was dissolved overnight in 5
 ml of TE, pH 7.5, at 4.degree. C. The RNA in the DNA preparation was
 digested by adding 100 .mu.g of RNAse'A per ml of solution and incubating
 for 60 min at 37.degree. C. with slight shaking on a horizontal shaker (40
 rpm). Then the RNAseA was precipitated by extracting with one vol. of PCI
 and the aqueous phase was extracted once with one vol. of CI in order to
 remove traces of phenol. After centrifuging, the DNA was precipitated from
 the aqueous phase at RT using 1/10 vol. of 3M sodium acetate, pH 5.2, and
 0.7 vol. of 2-propanol. wound onto a glass rod and dried in the same way
 as before. The genomic DNA (gDNA) was dissolved overnight in 2:5 ml of TE,
 pH 7.5 at 4.degree. C.
 The size distribution of the gDNA was then analysed in a 0.5% strength
 agarose gel and quantified and qualified by determining the OD260nm and
 OD280nm.
 High molecular weight DNA which was clean enough for most microbiological
 applications could be obtained in good yield (700 pg of genomic DNA per g
 of moist cell material) using this method.

Composition of the media and buffers used:
 YEPD medium: 1% (w/v) yeast extract
 2% (w/v) peptone
 2% (w/v) glucose
 Lysis buffer: 10 mM citrate phosphate pH 7.5
 1 M sorbitol
 100 mM EDTA
 1% (w/v) SDS
 1% (v/v) .beta.-mercaptoethanol
 TE pH 7.5: 10 mM Tris-HCl, pH 7.5
 1 mM EDTA
 PCl phenol/chloroform/isoamyl/alcohol
 (25:24:1)
 Cl chloroform/isoamyl alcohol (24:1)
 Example 2
 Amplifying the FDH Gene Using PCR Starting from Genomic DNA
 All PCR batches were covered with a layer of 50-100 .mu.l of light mineral
 oil [Sigma] and PCR was performed using an automatic DNA thermal cycler
 [Robocycler, Stratagene] in accordance with the following programme:
 PCR programme:
 2 min. denaturation at 94.degree. C. (1 x at start of programme)
 1 min denaturation at 94.degree. C.
 1.5 min. annealing of primer at 46-60.degree. C. (depending on the melting
 point of the primer)
 1.5 min. extension at 72.degree. C. (to extend primer by means of Taq
 polymerase) cyclic repetition of last three steps (25-30 x)
 10 min. extension at 72.degree. C. to ensure that all the amplified
 fragments are fully extended.
 The PCR mixture contains.
 100 ng of gDNA
 20 pmol of primer N-TermF3
 20 pmol of primer C-TermR5
 0.2 mM each of dNTPs
 0.5 .mu.l of Taq polymerase (Boehringer)
 10 .mu.l of buffer 10x (Boehringer)
 ad 100 .mu.l with dist. water
 Annealing temperature: 48.degree. C., 35 cycles
 The PCR fragment was ligated using Sure Clone kits (Pharmacia, Freiburg) in
 the vector pUC18. Hanahan's method (J. Mol. Biol. 1983, 166, 557) was used
 for transformation. 2 .mu.l of ligation mixture vector pUC-FDH were added
 to 100 .mu.l of competent E coli XL1 blue cells.
 Example 3
 Preparing Mutants FDH-C23S
 The point mutants of FDH were produced on the basis of the cloned FDH gene
 (pUC-FDH) (see example 1) using Ho et al.'s method (Gene 1989, 77, 52-59).
 The following "internal" oligonucleotide primers, which contained both the
 mutations, were used:
 internal primer for introducing C23S mutation:
 S23sense, 5'-TTTTCAGTAGMCCATATAA-3' (SEQ ID NO:33)
 S23antisense: 5'-TATATGGTTCTACTGAAAAT-3' (SEQ ID NO:34)
 The following oligonucleotide primers were used as "external" primers:
 PUC181S: 5'-CGCGCGTTTCGGTGATGACG-3' (SEQ ID NO:35)
 C-TermR5/Pstl: 5'-CTGCAGTTATTTCTTATCGTGTTTACCGTA-3' (SEQ ID NO:36)
 N-TermF3IEcoRl: 5'-GAATTCATGAAGATTGTCTTAGTTCTTTAT-3' (SEQ ID NO:37)
 1. Preparation of individual fragments:
 Mixture A: Preparing SER23S1-CTERMR5/Pstl --fragments (1.0 kb)
 100 ng pUC-FDH (1.1 kbFDH-EcoRl/Pstl in pUC18)
 30 pmol of primer SER23SI
 30 pmol of primer CTERMR5/Pstl
 1.5 .mu.I of Pfu-polymerase (2.5 U/.mu.l)
 1/10 vol polymerase buffer 10x
 0.2 mM each of dNTP
 ad 100 .mu.l with dist. water
 Annealing temperature: 46.degree. C., 30 cycles
 Mixture B: Preparing PUC18SI-SER23AS1 -fragments (500 bp)
 100 ng of pUC-FDH (see above)
 30 pmol of primer PUC 18SI
 30 pmol of primer SER23ASI
 1.5 1 .mu.l of Pfu-polymerase (see above)
 1/10 vol of poiymerase buffer 10x
 0.2 mM each of dNTP
 ad 100 .mu.l with dist. water
 Annealing temperature: 44.degree. C., 30 cycles.
 After the PCR programme, the mixtures were separated in a preparative
 agarose gel (1%), the bands were cut out, isolated by using Jetsorb gel
 extraction kits (Genomed), the concentrations were estimated in an
 analytical agarose gel (reference material: 1 .mu.g of kb-ladder, Gibco)
 and used as a template in fusion PCR with overlapping fragments.
 2. Fusion PCR for preparing the complete FDH-C23S gene (1.1 kb)
 150 ng of SER23S1-CTERMR5/Pstl fragment
 90 ng of PUC18SI -SER23ASI fragment
 20 pmol pfimer NTERMF3/EcoRl
 20 pmol of primer CTERMR5/Pstl
 1.5 .mu.l of Pfu-polymerase (2.5 U/.mu.l)
 1/10 vol of polymerase buffer 10x
 0.2 mM each of dNTP
 ad 100 .mu.l with dist. water
 Annealing-T.: 46.degree. C. 30 cycles
 The PCR mixture was used directly in A-tailing (see below).
 3. Cloning the PCR products in pMOS blue:
 Cloning was performed with pMOS blue T-vector kits (Amersham)
 a ) A-tailing
 100 .mu.l of fusion PCR mixture were extracted with 1 vol of
 chloroform/isoamyl alcohol (Cl) (24:1).
 25 .mu.l of aqueous phase=1/4 of the PCR mixture
 1.8 .mu.l 10 x buffer (see Amersham instruction sheet)
 1.8 .mu.l dNTP-Mix (see Amersham instruction sheet)
 8.5 .mu.l A-tailing buffer (see Amersham instruction sheet)
 0.5 .mu.l of Tth-DNA-polymerase
 ad 85 .mu.l with dist. water
 15 min at 70.degree. C.
 extracted 1 x with Cl
 After isolating the PCR fragment containing the FDH-C23S gene by agarose
 gel electrophoresis and isolation of the PCR fragment using Jet-Sorb
 (Genomed), the fragment was ligated in the vector pMOS blue.
 Ligation in pMOS blue:
 50 ng of pMOS blue vecto
 120 ng of FDH-Ser23 - 3'dA
 0.5 .mu.l of ATP 10 mM
 1.0 .mu.l of ligase buffer
 0.5 .mu.l DTT 100 mM
 0.5 .mu.l of T4 DNA ligase (2-3 Weiss Units)
 ad 10 .mu.l with dist. water
 Incubation overnight at 16.degree. C.
 b) Transformation in MOS blue competent cells (according to Amersham
 Instructions, 1994)
 1 .mu.l of ligation mixture
 20 .mu.l of competent cells
 40 sec 42.degree. C.
 2 min on ice
 80 .mu.l of LB medium added, 1 h 37.degree. C.
 50 .mu.l plated out on LB with ampicillin and tetracyclin.
 4. Cloning the FDH-C23S gene in the expression vector PBTac2 (Boehringer)
 The plasmid DNA (pMOS-FDH-C23S) was isolated from the recombinant pMOS blue
 cells after multiplication in E. coli and the FDH-C23S-EcoRl/Pstl fragment
 (1.1 kb) was prepared by means of restriction digestion.
 Preparative EcoRl-Pstl digestion
 15 .mu.l of plasmid DNA (ca. 200 ng) pMOS-FDH-C23S
 3 .mu.l of buffer H (Boehringer)
 0.5 .mu.l of EcoRl (10U/.mu.l)
 0.5 .mu.l of Pstl (10U/.mu.l)
 11 .mu.l dist. water.
 2 h, 37.degree. C.
 The fragment was isolated using the method mentioned above.
 Ligation in pBTac2
 100 ng of pBTac2 were digested, as described above, with EcoRl and Pstl,
 and the linearised vector was isolated using the method mentioned above.
 The FDH-C23S-EcoRl /Pstl fragment (1.1 kb) was ligated in the open vector.
 The ligation mixture contained:
 45 ng of pBTac2-EcoRl/Pstl
 60 ng of FDH-C23S-EcoRl/Pstl
 1 .mu.l of ligase buffer 10x (Boehringer)
 0.5 .mu.l of T4 DNA ligase (Boehringer)
 ad 10 .mu.l with dist. water
 Incubated overnight at 16.degree. C.
 5. Transformation in E. coli JM 105
 For the transformation, 100 .mu.l of competent E. coli JM 105 cells were
 added to 5 .mu.l of ligation mixture and transformation was performed
 using Hanahan's method (see above).
 Example 4
 Expression of FDH-C23S in E. coli
 The recombinant wild type FDH gene and the FDH mutants were expressed on a
 200 ml scale in E. coli JM1 05 cells. 200 ml of LB medium were added to
 select 100 .mu.g of ampicillin per ml and inoculated in the ratio of 1:50
 with a preliminary culture which had been incubated overnight. The cells
 were cultivated at 37.degree. C. and 180 rpm on a reciprocating shaker and
 induced with 1 mM of IPTG at an optical density (OD 550 nm) of 0.6-0.8.
 The expression time was between 5.0 h (0.7 g of moist cell material) and
 20 h (1.25 g of moist cell material). The cells were harvested by
 centrifuging (10 min, GSA rotor, 10,000 rpm).

LB (Luria Bertani) medium: 1.0% Bacto - Tryptone
 0.5% Bacto - yeast extract
 1.0% NaCl adjusted to pH 7.5 with NaOH
 LBamp medium LB medium with 100 .mu.g/ml ampicillin
 Cell lysis took place mechanically using glass beads (diameter 0.3 mm). A
 30% strength E. coli cell suspension was prepared in the lysis buffer, to
 which was added twice the weight of glass beads. Lysis took place,
 depending on the volume, on a 1-2 ml scale in a vibratory mill [Retsch;
 Hummel and Kula (1989) J. Microbiol. Meth. 9, 210], in a SS34 centrifuge
 tube (10-20 ml), in a vortex [IKA] or in a disintegrator S (20-50 ml)
 [IMA] for a period of 20 min. The lysed product was centrifuged for 10 min
 at 10000 rpm and 4.degree. C., the cell-free supernatant liquid was
 removed and the glass beads and cell pellets were washed once with a
 volume of buffer equal to 1/4-1/2 the volume of cell suspension. After
 centrifuging a second time, the cell-free supernatant liquids were
 combined. The volume activity of the FDH-C23S in the crude cell-free
 extract was 10 U/ml

Lysis buffer: 100 mM of potassium phosphate buffer, pH 7.5
 10 drops of Ucolup/L buffer
 Example 5
 Determining the Stability of the FDH-C23S
 The crude cell-free extract of FDH-C23S was used to determine the half-life
 for deactivation.
 The test mixtures contained: 0.05-0.5 M NH.sub.4 -trimethyl pyruvate
 0.1-1 M L-tert. leucine
 2.7 M NH.sub.4 -formate
 0.5 U/ml FDH-C23S
 pH 6-9
 T=40.degree. C.
 The mixtures were incubated for 18 days. Samples for activity tests were
 taken each day. The semi-logarithmic plot of residual activity against
 time was a straight line, whose gradient gave the inactivation constant k.
 The half-life .quadrature. for inactivation was obtained from the
 relationship .quadrature.=In2/k.
 Example 6
 Synthesis of (S)-neopentylglycine
 31.53g (0.5 mole) ammonium formate and 20.89 g (125 mmole),
 2.about.-keto-4,4.about.dimethyl-pentanoic acid sodium salt are suspended
 in 400 ml of water, the pH value is adjusted with ammonia to pH 8.5 to
 provide a solution, the volume is adjusted then to 500 ml. Subsequently,
 71.7 mg (0.1 mmole) NAD.sup.+ H.sub.2 O cofactor as well as 2000 U of
 leucine dehydrogenase (LeUDH) and 2500 U of rec-formate dehydrogenase
 (rec-FDH) are added. The temperature is set to 28.degree. C. The reaction
 is gently stirred and the pH value is adjusted to 8.2 during the reaction
 by a pH stat unit. The completion point of the reaction is demonstrated by
 determining the degree of conversion with HPLC. The enzymes are separated
 via an ultra filter of pore size 10000 kDA and the solution is adjusted
 with ammonia to pH 9.5 subsequently, the solution is clarified with 2%
 active charcoal and the almost colourless solution is concentrated with a
 rotary evaporator, the amino acid is crystallized, separated via a filter
 funnel, washed three times with small amounts of ethanol and dried
 overnight under vacuum at 50.degree. C.
 Example 7
 Synthesis of (5)-3-methyl-isoleucine ((S)-3,3-dimethyl-norvalin)
 6.3 g (0.1 mole) ammonium formate and 1.67 g (10 mmoles),
 2-keto-3,3-dimethyl pentanoic acid sodium salt are suspended in 80 ml of
 water, the pH value is adjusted with ammonia to 8.2, so that the solids
 dissolve and the volume is adjusted to 100 ml. subsequently, 14.34 mg
 (0.02 mmole) NAD.sup.+ 3H.sub.2 O cofactor as well as 800 U of leucine
 dehydrogenase (LeUDH) and 500 U of rec-formate dehydrogenase (rec-FDH) are
 added. The temperature is set to 32.degree. C . During the reaction the
 system is gently stirred and the pH value is adjusted to 8.2 via a pH stat
 unit. The completion point of the reaction is sdemonstrated by determining
 the degree of conversion with HPLC. The reaction solution is adjusted to
 pH 9,5 with ammonia and subsequently clarified with 2% active charcoal.
 The almost colourless solution is concentrated with a rotary evaporator,
 the amino acid is crystallized, is separated via a filter funnel, washed
 three times with a little ethanol and dried overnight under vacuum at
 50.degree. C.
 Example 8
 Synthesis of (S) nornoneopentyiglycine ((S)-5,5-dimethyl norleucin)
 Reaction and isolation are carried out analogously to example 7. Substrate
 charged: 1.81 g (10 mmole) of 2-keto-S,5-dimethyl-hexanoic acid sodium
 salt.