WO2006015885A1 - Biocatalyseurs derives d'aminoacide deshydrogenase - Google Patents

Biocatalyseurs derives d'aminoacide deshydrogenase Download PDF

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WO2006015885A1
WO2006015885A1 PCT/EP2005/008863 EP2005008863W WO2006015885A1 WO 2006015885 A1 WO2006015885 A1 WO 2006015885A1 EP 2005008863 W EP2005008863 W EP 2005008863W WO 2006015885 A1 WO2006015885 A1 WO 2006015885A1
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substituted
amino acid
unsubstituted
radical
saturated
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PCT/EP2005/008863
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English (en)
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Paul Engel
Francesca Paradisi
Oliver Mccrohan
Anita Maguire
Stuart Gerard Collins
Patricia Busca
Daria Giacomini
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University College Dublin, National University Of Ireland, Dublin
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids

Definitions

  • the present invention relates to amino acid dehydrogenase-derived biocatalysts designed by site-directed mutagenesis to produce chirally pure, non-natural L- ⁇ - amino acids and to allow kinetic resolution of racemic DL- ⁇ -amino acid mixtures, to provide chirally pure, non-natural D- ⁇ -amino acids.
  • ⁇ -amino acid means an entity in which the amino group and the COOH group are both attached to the same carbon atom.
  • natural ⁇ -amino acids is taken as those which normally occur in human or other mammal organisms, in vivo, either as a constituent of their proteins (L- alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L- phenylalanine, L-proline, L-serine, L-tyrosine, L-threonine, L-tryptophan and L- valine) or else as mainstream metabolites (e.g.
  • Enantiopure drugs constitute an increasing proportion (35% of drugs on the market in 2000) of pharmaceuticals and there is substantial commercial interest in methods for asymmetric synthesis [R.M. Williams et al, Synthesis of Optically Active Amino Acids, VoI 7 of Organic Chemistry Series; Pergamon Press, Oxford, 1989; and Duthaler, R.O., Tetrahedron, 1994, 50, 1539-1650].
  • Non-natural ⁇ -amino acids, in enantiopure form are of considerable interest in the synthesis of alkaloids, peptides and other compounds with therapeutic application e.g. in HIV-protease inhibitors [A. S. Bommarius et al Tetrahedron: Asymm., 1995, 6, 2851-2888].
  • Non-natural ⁇ -amino acids are increasingly in demand by the pharmaceutical industry for peptidomimetic and other single-enantiomer drugs [P.P. Taylor et al, Trends in Biotechnology, 1998, 16, 412-418]. They are also in demand as precursors to ligands for asymmetric synthesis [A.S. Bommarras et al, Tetrahedron: Asymm., 1995, 6, 2851-2888]. While some enantiopure non-natural ⁇ -amino acids are commercially available, they are expensive.
  • biocatalysts the application of biological species such as microbial cells or enzymes derived therefrom to catalyse organic reactions
  • biocatalysts exhibit high regio-, chemo- and stereo- selectivity making them superior to chemical catalysts for asymmetric synthesis
  • biocatalysts are ideal energy-efficient, environmentally acceptable reagents, as virtually all reactions proceed under mild conditions and avoid the use of toxic reagents and disposal of byproducts.
  • biocatalysts offer a. good opportunity to prepare industrially useful chiral compounds [A.L. Margolin, Enzyme Microb. Technol., 1993, 15, 266-280; K. Mori, Synlett, 1995, 11, 1097-1109; R.N. Patel, Adv. Appl. Microbiol, 1997, 43, 91-140; and R.N. Patel, Adv. Appl. Microbiol, 2000, 47, 33-78].
  • Phenylalanine dehydrogenase (PheDH) nevertheless has been employed for the enantioselective synthesis of L-2-amino-4-phenylbutanoic acid with excellent enantiopurity by supplying the homologous appropriate ⁇ -keto acid [E. Santaniello et al, Chem. Rev., 1992, 92, 1071-1140; Y. Asano et al, J. Org. Chem., 1990, 55, 5567; and CW. Bradshaw et al, Bioorg. Chem., 1991, 19, 29].
  • Site-directed mutagenesis allows alteration of the amino acid residues surrounding the substrate-binding pocket of the enzyme to alter the size, shape and polarity of the pocket.
  • Seah et al [FEBS Letters 1995, 370, 93-96] undertook site-directed mutagenesis on PheDH and reported in 1995 that the resulting mutant enzymes displayed reduced activity for L-phenylalanine compared to the wild type enzyme and enhanced activity towards other natural ⁇ -amino acid substrates, indicating that the substrate profile of the enzyme was varied by the mutations.
  • Enhanced discrimination between phenylalanine and tyrosine in another set of engineered enzymes was subsequently reported [Biochemistry 2002, 41, 11390-11397].
  • the present invention concerns the hypothesis that engineered amino acid dehydrogenase mutants, for example PheDH mutants, might prove useful as biocatalysts for the asymmetric synthesis of a wide range of non-natural ⁇ -amino acids, such as phenylalanine analogues.
  • leucine, valine and phenylalanine dehydrogenases showed amino acid sequence homology to GIuDH; b) that the 3-D structure in general reflects strong similarities in linear primary sequence; c) that the high-resolution structure of GIuDH provides an unambiguous structural interpretation of the basis of its amino acid specificity led to the prediction that our 3-D insight allowed us to interpret the basis of specificity also in other amino acid, dehydrogenases [K. L. Britton et al, J. MoI. Biol. (1993) 234, 938-945]. This in turn allowed specification of ways in which to alter amino acid specificity by mutating key amino acid residues in the active-site region. A number of these key residue positions were listed in US Patent No. 5,798,234, which is co-owned by one of the present inventors.
  • mutant amino acid dehydrogenases may be successfully made and purified with the same ease as the normal 'wild-type' enzyme.
  • Similar protein engineering work has of course been carried out with many other enzymes, but each enzyme is unique and the general view that site-directed mutagenesis is a legitimate and feasible strategy cannot be unconditionally extrapolated; each case has to be tested and established).
  • GIuDH, VaIDH and PheDH amino acid dehydrogenase starting points
  • the present invention shows that engineered amino acid dehydrogenase biocatalysts can, surprisingly, handle a wide range of non-natural ⁇ -amino acids, often with high efficiency and always with perfect retention of the absolute discrimination between the L- and D- series of ⁇ -amino acids, the former being substrates, the latter being in some cases inhibitors but never substrates [P. Busca et al, Org.
  • biocatalysts may be used for quantitative conversion of an ⁇ - ketoacid, achiral at the ⁇ -position, to the corresponding non-natural L- ⁇ -amino acid and vice versa, namely, for quantitative conversion of a non-natural L- ⁇ -amino acid to the corresponding ⁇ -ketoacid, achiral at the ⁇ -position.
  • X is a substituted or unsubstituted, saturated or unsaturated C 0-3 (optionally Co-Oalkylene radical, optionally containing a heteroatom (for example, O, S and/or N); and
  • R is a substituted or unsubstituted, branched or unbranched, saturated or unsaturated, lower alkyl of Cj -8 (optionally C] -6 ), preferably Ci -5 or Ci -3 or C 3-5 ; a substituted or unsubstituted aryl radical; a substituted or unsubstituted fused aryl radical; a substituted or unsubstituted, saturated or unsaturated heterocyclic radical; a substituted or unsubstituted, saturated or unsaturated fused heterocyclic radical; or a substituted or unsubstituted cycloalkyl radical of C 5-8 (optionally C 5-6 ), with the proviso that, when X is a C 2 saturated alkylene radical, R is not selected from the group consisting of an unsubstituted phenyl radical, an unsubstituted C 1-2 saturated alkyl radical or a C 1 saturated alkyl radical substituted with -CONHNH 2
  • an ammonia source for example NADH or NADPH but other analogue coenzyme molecules able to satisfy the coenzyme specificity e.g. deamino NADH, reduced acetyl pyridine adenine dinucleotide etc. may also be employed) in a suitable reaction solvent, under reaction conditions sufficient to form the non-natural L- ⁇ -amino acid.
  • an appropriate reduced coenzyme for example NADH or NADPH but other analogue coenzyme molecules able to satisfy the coenzyme specificity e.g. deamino NADH, reduced acetyl pyridine adenine dinucleotide etc. may also be employed
  • a suitable reaction solvent under reaction conditions sufficient to form the non-natural L- ⁇ -amino acid.
  • X may be substituted with a carbon-based substituent such as a C 1-5 (optionally C 1-3 ) alkyl, alkenyl or alkynyl group.
  • X may be substituted with a non- carbon based substituent (by non-carbon based, is meant substituents containing no carbon atoms such as halide (such as fluoride or chloride), -OH, -NH 2 , -SH or -NO 2 , as well as, substituents containing carbon atoms but linked by at least one non-carbon atom to the rest of the moiety such as C 1-5 , optionally C 1-3, alkoxy, alkenyloxy and alkynyloxy such as-OCH 3 or C 1-5 , optionally C 1-3; thioalkyl, thioalkenyl and thioalkynyl such as-SCH 3 .
  • R is a substituted or unsubstituted, branched or unbranched, saturated or unsaturated, lower alkyl of C 1-8 (optionally C 1-6 ), preferably Cj -5 or C 1-3 or C 3- s ;
  • R may be substituted with a non-carbon based substituent (by non-carbon based, is meant substituents containing no carbon atoms such as halide (such as fluoride or chloride), -OH, -NH 2 , -NHNH 2 , -CO, -SH or -NO 2 , as well as, substituents containing carbon atoms but linked by at least one non-carbon atom to the rest of the moiety such as C 1- 5 , optionally Ci -3, alkoxy, alkenyloxy and alkynyloxy such as-OCH 3 or C 1-5 , optionally Ci -3) thioalkyl, thioalkenyl and thioalkynyl such as-SCH 3 .
  • R is a substituted or unsubstituted aryl radical; a substituted or unsubstituted fused aryl radical; a substituted or unsubstituted, saturated or unsaturated heterocyclic radical; a substituted or unsubstituted, saturated or unsaturated fused heterocyclic radical; or a substituted or unsubstituted cycloalkyl radical of C 5-8 (optionally C 5-6 ).
  • R is a substituted or unsubstituted, simple or fused aryl radical, preferably a substituted or unsubstituted phenyl radical or a substituted or unsubstituted naphthyl radical, optionally a substituted phenyl or naphthyl radical.
  • IfR is phenyl, it maybe substituted at one or more of ortho, meta or para positions (optionally the para position) with aldehyde; nitrile; nitro; halo, for example fluoro or chloro; lower C 1-5 alkyl, for example Ci -3 alkyl; lower C 1-5 alkoxy, for example C 1-3 alkoxy; lower Ci -5 haloalkyl such as C] -3 haloalkyl, including, but not limited to, lower C 1-5 perhaloalkyl such as C 1-3 perhaloalkyl; lower C 1-5 haloalkoxy such as Ci -3 haloalkoxy, including, but not limited to, lower C 1-5 perhaloalkoxy such as C 1-3 perhaloalkoxy; hydroxy or a mixture thereof.
  • R is a substituted or unsubstituted heterocyclic radical optionally selected from substituted or unsubstituted furan, pyran, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, furazan, pyrrolidine, pyrroline, imidazolidine, imidazoline, pyrazolidine, pyrazoline, piperidine, piperazine, morpholine or thiophene.
  • R may be substituted with a carbon- .
  • R may be substituted with a non-carbon based substituent (by non- carbon based, is meant substituents containing no carbon atoms such as halide (such as fluoride or chloride), -OH, -NH 2 , -SH or -NO 2 , as well as, substituents containing carbon atoms but linked by at least one non-carbon atom to the rest of the moiety such as C 1-5 , optionally C 1-3) alkoxy, alkenyloxy and alkynyloxy such as-OCH 3 or C 1-5 , optionally C 1-3; thioalkyl, thioalkenyl and thioalkynyl such as-SCH 3 .
  • halide such as fluoride or chloride
  • R is a substituted or unsubstituted fused heterocyclic radical optionally selected from substituted or unsubstituted benzofuran, isobenzofuran, indole, isoindole, benzothiophene, benzo[c]thiophene, benzimidazole, purine, indazole, benzoxazole, benzisoxazole, benzothiazole, quinoline, isoquinoline, quinoxaline, quinazoline or cinnoline.
  • R may be substituted with a carbon-based substituent such as a C] -5 (optionally Cj -3 ) alkyl, alkenyl or alkynyl group.
  • R may be substituted with a non-carbon based substituent (by non- carbon based, is meant substituents containing no carbon atoms such as halide (such as fluoride or chloride), -OH, -NH 2 , -SH or -NO 2 , as well as, substituents containing carbon atoms but linked by at least one non-carbon atom to the rest of the moiety such as Cj- 5 , optionally Q -3; alkoxy, alkenyloxy and alkynyloxy such as-OCH 3 or Q- 5 , optionally C 1-3j thioalkyl, thioalkenyl and thioalkynyl such as-SCH 3 .
  • halide such as fluoride or chloride
  • substituents containing carbon atoms such as halide (such as fluoride or chloride), -OH, -NH 2 , -SH or -NO 2 , as well as, substituents containing carbon atoms but linked by
  • R is a substituted or unsubstituted, saturated or unsaturated, cycloCs-s alkyl radical such as cyclopentyl or cyclohexyl.
  • R may be substituted with a carbon-based substituent such as a C 1-5 (optionally C 1-3 ) alkyl, alkenyl or alkynyl group.
  • R may be substituted with a non-carbon based substituent (by non-carbon based, is meant substituents containing no carbon atoms such as halide (such as fluoride or chloride), -OH, -NH 2 , -SH or -NO 2 , as well as, substituents containing carbon atoms but linked by at least one non-carbon atom to the rest of the moiety such as C 1-5 , optionally C 1-3, alkoxy, alkenyloxy and alkynyloxy such as-OCH 3 or Cj -5 , optionally Ci -3 ,thioalkyl, thioalkenyl and thioalkynyl such as- SCH 3 .
  • halide such as fluoride or chloride
  • substituents containing carbon atoms such as halide (such as fluoride or chloride), -OH, -NH 2 , -SH or -NO 2 , as well as, substituents containing carbon atoms but linked
  • the ammonia source is selected from ammonia or an ammonium salt.
  • the ammonium salt is selected from ammonium sulphate, ammonium esters (such as ammonium formate and ammonium acetate) and ammonium halides, for example, ammonium chloride.
  • the source of an appropriate reduced coenzyme is either the appropriate reduced coenzyme itself or, alternatively, the corresponding oxidised coenzyme and means for converting the corresponding oxidised coenzyme into the appropriate reduced coenzyme.
  • the converting means might comprise ethanol and an alcohol dehydrogenase. If such a converting means is used, reduction of the ethanol to ethanal, in turn, converts the oxidised coenzyme into the corresponding reduced coenzyme.
  • the extent of conversion of an ⁇ -ketoacid substrate to the L- ⁇ -amino acid product is limited by the stoichiometry of reaction, so that x millimoles of amino acid, for instance, cannot be quantitatively formed from x millimoles of ⁇ -ketoacid unless x millimoles of reduced coenzyme and x millimoles of ammonium ions (as ammonia or preferably as an ammonium salt) are supplied over the timecourse of the reaction.
  • the requisite supply of reduced coenzyme may be accomplished in two ways.
  • the required x millimoles of reduced coenzyme may be supplied in total at the outset; alternatively and preferably in view of the high commercial cost of the reduced coenzyme, the reaction mixture may be supplemented with the components of the aforementioned converting means.
  • reaction mixture will require a clear stoichiometric excess of a reduced coenzyme such as NADH and ammonium ions over the starting ketoacid.
  • a stoichiometric excess of ammonium ions is still required, but the coenzyme may be supplied, for example either as NADH or as NAD + , in a greatly reduced, catalytic amount.
  • the mutant amino acid dehydrogenase useful in the processes of the first, second and third aspects of the invention is selected from a mutant of glutamate dehydrogenase, phenylalanine dehydrogenase, leucine dehydrogenase and valine dehydrogenase, preferably phenylalanine dehydrogenase.
  • the mutant is prepared by site-directed mutagenesis of the corresponding wild type enzyme by following, for example, the teaching of US Patent No. 5,798,234, the contents of which are incorporated herein by reference.
  • Nl 45 mutant amino acid dehydrogenases favour substrates with a hydrophobic substitution at the para position of an aryl radical, if an aryl radical is present.
  • mutant phenylalanine dehydrogenase derived from Bacillus sphaericus and having the following amino acid sequence:
  • mutant amino acid dehydrogenase of the further aspect of the invention favours bulky substrates, as will be observed hereinafter.
  • the above-mentioned mutant enzymes(s) may also be used in the opposite direction of reaction, starting with a racemic mixture of a non-natural ⁇ -amino acid, to effect quantitative removal of the L-enantiomer. This leaves the enantiomerically pure D- ⁇ -amino acid, readily separable from other reaction products.
  • X is a substituted or unsubstituted, saturated or unsaturated Co -3 , optionally Co-i,alkylene radical, optionally containing a heteroatom (for example, O, S and N); and
  • R is a substituted or unsubstituted, branched or unbranched, saturated or unsaturated, lower alkyl of C 1-8 (optionally C 1-6 ), preferably C 1 ⁇ ; a substituted or unsubstituted aryl radical; a substituted or unsubstituted fused aryl radical;a substituted or unsubstituted, saturated or unsaturated heterocyclic radical; a substituted or unsubstituted, saturated or unsaturated fused heterocyclic radical; or a substituted or unsubstituted cycloalkyl radical of C 5-8, with the proviso that, when X is a C 2 saturated alkylene radical, R is not selected from the group consisting of an unsubstituted phenyl radical, an unsubstituted C 1-2 saturated alkyl radical or a Cjsaturated alkyl radical substituted with -CONHNH 2
  • a mutant amino acid dehydrogenase and a source of an appropriate oxidised coenzyme NAD + or NADP but optionally other analogue coenzyme molecules able to satisfy the coenzyme specificity e.g. deamino NAD + , acetyl pyridine adenine dinucleotide etc.
  • NAD + or NADP an appropriate oxidised coenzyme
  • suitable reaction solvent under reaction conditions sufficient to convert the L- ⁇ -amino acid of the racemic non-natural ⁇ -amino acid into an ⁇ -ketoacid; and leaving the unreacted D- ⁇ -amino acid.
  • the extent of conversion of an L- ⁇ -amino acid substrate to the ⁇ -ketoacid product is limited by the stoichiometry of reaction, so that x millimoles of ⁇ -ketoacid, for instance, cannot be quantitatively formed from x millimoles of amino acid unless x millimoles of oxidised coenzyme and x millimoles of water are supplied over the timecourse of the reaction.
  • the requisite supply of oxidised coenzyme may be accomplished in two ways.
  • the required x millimoles of oxidised coenzyme such as NAD + may be supplied either in total at the outset or over the timecourse of the reaction.
  • the requisite water supply will normally be ensured by either using a reaction solvent comprising water or by using a reaction solvent which contains water and which optionally has a predominantly aqueous environment. It will be appreciated by those skilled in the art that pure water is 55.6M. Thus, in a reaction system containing 10 or 20% water, the water would still be present in a vast stoichiometric excess. In fact, even in a reaction system containing 1% water, the water would still be present in a substantial stoichiometric excess over any likely working concentration of the amino acid substrate.
  • reaction mixture will require a clear stoichiometric excess of an oxidised coenzyme such as NAD + or NADP + over the starting amino acid.
  • an oxidised coenzyme such as NAD + or NADP +
  • a stoichiometric excess of water is still required.
  • the reaction solvent comprises the water.
  • the source of an appropriate oxidised coenzyme is the oxidised coenzyme itself.
  • the source of an appropriate oxidised coenzyme is the corresponding reduced coenzyme, together with means for converting the corresponding reduced coenzyme into the appropriate oxidised coenzyme.
  • a converting means might comprise diaphorase and a source of oxygen, each in an amount sufficient to convert, for example, NADH/NADPH to NAD + /NADP + .
  • X may be substituted with a carbon-based substituent such as a C] -5 (optionally C 1-3 ) alkyl, alkenyl or alkynyl group.
  • X may be substituted with a non- carbon based substituent (by non-carbon based, is meant substituents containing no carbon atoms such as halide (such as fluoride or chloride), -OH, -NH 2 , -SH or -NO 2 , as well as, substituents containing carbon atoms but linked by at least one non-carbon atom to the rest of the moiety such as C] -5 , optionally C 1-3 , alkoxy, alkenyloxy and alkynyloxy such as-OCH 3 or Ci -55 optionally C 1-3i thioalkyl, thioalkenyl and thioalkynyl such as-SCH 3 .
  • R When R is a substituted or unsubstituted, branched or unbranched, saturated or unsaturated, lower alkyl of C 1-8 (optionally Q -6 ), preferably C ]-5 or C 1-3 or C 3-5 , R may be substituted with a non-carbon based substituent (by non-carbon based, is meant substituents containing no carbon atoms such as halide (such as fluoride or chloride), -OH, -NH 2 , -NHNH 2 , -CO, -SH or -NO 2 , as well as, substituents containing carbon atoms but linked by at least one non-carbon atom to the rest of the moiety such as C 1- 5 , optionally C 1-3; alkoxy, alkenyloxy and alkynyloxy such as-OCH 3 or C 1-5 , optionally C 1-3j thioalkyl, thioalkenyl and thioalkynyl such as-SCH 3 .
  • R is a substituted or unsubstituted aryl radical; a substituted or unsubstituted fused aryl radical; a substituted or unsubstituted, saturated or unsaturated heterocyclic radical; a substituted or unsubstituted, saturated or unsaturated fused heterocyclic radical; or a substituted or unsubstituted cycloalkyl radical of C 5-8 (optionally C 5-6 ).
  • R is a substituted or unsubstituted, simple or fused aryl radical, preferably a substituted or unsubstituted phenyl radical or a substituted or unsubstituted haphthyl radical, optionally a substituted phenyl or naphthyl radical.
  • R is phenyl R may be substituted at one or more of ortho, meta or para positions (optionally, the para position) with aldehyde; nitrile; nitro; halo, for example fluoro or chloro; lower C 1-5 alkyl, for example Ci -3 alkyl; lower Ci -5 alkoxy, for example C 1-3 alkoxy; lower C 1-5 haloalkyl such as C 1-3 haloalkyl, including, but not limited to, lower C 1-5 perhaloalkyl such as C 1-3 perhaloalkyl; lower C 1-5 haloalkoxy such as Ci -3 haloalkoxy, including, but not limited to, lower C 1-5 perhaloalkoxy such as C 1-3 perhaloalkoxy; hydroxy or a mixture thereof.
  • halo for example fluoro or chloro
  • lower C 1-5 alkyl for example Ci -3 alkyl
  • lower Ci -5 alkoxy for example C 1-3 alkoxy
  • R is a substituted or unsubstituted heterocyclic radical, optionally selected from substituted or unsubstituted furan, pyran, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, furazan, pyrrolidine, pyrroline, imidazolidine, imidazoline, pyrazolidine, pyrazoline, piperidine, piperazine, morpholine or thiophene.
  • R may be substituted with a carbon- based substituent such as a C] -5 (optionally Cj -3 ) alkyl, alkenyl or alkynyl group.
  • R maybe substituted with a non-carbon based substituent (by non- carbon based, is meant substituents containing no carbon atoms such as halide (such as fluoride or chloride), -OH, -NH 2 , -SH or -NO 2 , as well as, substituents containing carbon atoms but linked by at least one non-carbon atom to the rest of the moiety such as Ci- 5 , optionally Cj -3 , alkoxy, alkenyloxy and alkynyloxy such as-OCH 3 or C 1-5 , optionally C 1-3, thioalkyl, thioalkenyl and thioalkynyl such as-SCH 3 .
  • R is a substituted or unsubstituted fused heterocyclic radical optionally selected from substituted or unsubstituted benzofuran, isobenzofuran, indole, isoindole, benzothiophene, benzo[c]thiophene, benzimidazole, purine, indazole, benzoxazole, benzisoxazole, benzothiazole, quinoline, isoquinoline, quinoxaline, quinazoline or cinnoline.
  • R may be substituted with a carbon-based substituent such as a C 1-5 (optionally Cj -3 ) alkyl, alkenyl or alkynyl group.
  • R may be substituted with a non-carbon based substituent (by non- carbon based, is meant substituents containing no carbon atoms such as halide (such as fluoride or chloride), -OH, -NH 2 , -SH or -NO 2 , as well as, substituents containing carbon atoms but linked by at least one non-carbon atom to the rest of the moiety such as C 1-5 , optionally C 1-3 , alkoxy, alkenyloxy and alkynyloxy such as-OCH 3 or C 1-5 , optionally C 1-3> thioalkyl, thioalkenyl and thioalkynyl such as-SCH 3 .
  • halide such as fluoride or chloride
  • substituents containing carbon atoms such as halide (such as fluoride or chloride), -OH, -NH 2 , -SH or -NO 2 , as well as, substituents containing carbon atoms but linked by
  • R is a substituted or unsubstituted, saturated or unsaturated, cycloC 5-8 alkyl radical such as cyclopentyl or cyclohexyl.
  • R may be substituted with a carbon-based substituent such as a Cj -5 (optionally C 1-3 ) alkyl, alkenyl or alkynyl group.
  • R may be substituted with a non-carbon based substituent (by non-carbon based, is meant substituents containing no carbon atoms such as halide (such as fluoride or chloride), -OH, -NH 2 , -SH or -NO 2 , as well as, substituents containing carbon atoms but linked by at least one non-carbon atom to the rest of the moiety such as C 1-5 , optionally C 1-3> alkoxy, alkenyloxy and alkynyloxy such as-OCH 3 or C 1-5 , optionally C 1-3j thioalkyl, thioalkenyl and thioalkynyl such as- SCH 3 .
  • halide such as fluoride or chloride
  • substituents containing carbon atoms such as halide (such as fluoride or chloride), -OH, -NH 2 , -SH or -NO 2 , as well as, substituents containing carbon atoms but linked by
  • the reaction solvent comprises water and the water is an aqueous buffer.
  • the byproduct of the above-recited method of the second aspect of the invention namely, the ⁇ -ketoacid may, by sequential operation of the method of the first aspect of the invention, be converted to the L- ⁇ -amino acid, so that the biocatalyst has in effect resolved the racemic mixture into its two components.
  • This combined strategy putting together the two processes illustrated above, gives access to both pure enantiomers starting from the racemic ⁇ -amino acid. Given that the racemic mixture is often the most readily accessible synthetic product, this combined process, yielding two high-value products, is very attractive commercially.
  • biocatalyst may be successfully immobilised on a solid support via adsorption, entrapment or cross-linking on Celite (Trade Mark), alginate, chitosan -or a mixture of the last two- silica gel or agarose beads, both for enhanced stability and for ease of separation from a reaction mixture and reuse [S. S. Betigeri et al, Biomaterials (2002) 23, 3627; S. Soni et al, Journal of Applied Polymer Science (2001) 82, 1299; and W. Limbut et al, Biosensors and Bioelectronics (2004) 19, 813].
  • biocatalyst may be used in the presence of miscible organic solvents (short chain alcohols such as methanol, miscible ketones such as acetone, nitriles such as acetonitrile, miscible ethers, amides, alkyl sulfoxides, dioxane, tetrahydrofuran), facilitating reaction with substrates poorly soluble in purely aqueous reaction media.
  • miscible organic solvents short chain alcohols such as methanol, miscible ketones such as acetone, nitriles such as acetonitrile, miscible ethers, amides, alkyl sulfoxides, dioxane, tetrahydrofuran
  • miscible organic solvents short chain alcohols such as methanol, miscible ketones such as acetone, nitriles such as acetonitrile, miscible ethers, amides, alkyl sulfoxides, dioxan
  • Fig. 1 HPLC separation of 4-Cl-phenyl pyruvate (3) + DL-4-Cl-Phe (A) and HPLC chromatogram of the reductive animation of 4-Cl-phenyl pyruvate (3) with the mutant amino acid dehydrogenase N 145 A (B).
  • Splitting patterns in 1 H spectra are designated as s (singlet), bs (broad singlet), bd (broad doublet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), ABq (AB quartet) and (m) multiplet. Coupling constants are quoted in Hz.
  • assignments are made from 13 C DEPT spectra run in the DEPT-90 and DEPT-135 modes and with the aid of COSY and HETCOR experiments in some cases.
  • Mass spectra were measured on a Kratos Profile spectrometer in electron impact (E.I.) mode with an ionisation voltage of 7OeV.
  • the one used herein is the phenylalanine dehydrogenase of Bacillus sphaericus [S. Y. K Seah et al, FEBS Letters (1995) 370, 93-96] but it will be appreciated by those skilled in the art that the invention is not so limited.
  • the DNA coding sequence for the enzyme in question is inserted into a cloning site in a suitable expression vector. This is a plasmid which provides an inducible promoter, allowing high-level production of the enzyme to be initiated as desired. It also contains a drug resistance gene so that the presence of the plasmid in a bacterial host population may be maintained by the selective pressure of including the drug in the growth medium.
  • mutant biocatalysts amino acid sidechains in the protein structure are targeted by studying the protein 3-D structure and selecting those -most .likely to affect the fit of the substrate to its binding pocket on the protein surface.
  • clostridial GIuDH this can be done by reference to the published high-resolution structures [P. J. Baker et al, Proteins (1992) 12, 75-86; and Stillman, TJ. et al, (1993) J. MoI. Biol. 234, 1131-1139].
  • PheDH it was done initially [S. Y. K Seah et al, FEBS Letters (1995) 370, 93-96; and S.Y.K.
  • mutations may be introduced in a random way by using chemical mutagens, error-prone PCR and/or 'directed evolution', involving a nutritional selective pressure.
  • mutants with desirable properties may be identified by activity screening of colony blots from Petri dishes.
  • Such mutations may be at the positions listed above but not necessarily so since alterations elsewhere may produce small structural movements that affect catalytic performance.
  • catalysis requires not only adequate binding of the substrate but also appropriate orientation in relation to catalytic groups, so that a small change in geometry may have large effects on catalytic rate.
  • a colony of cells may be used to inoculate a 5 or 10 ml culture which, after approximately 12 hours growth at 37 0 C, is used as the inoculum for a 1 litre growth.
  • IPTG is added to the 1 litre culture under sterile conditions in mid-log phase - i.e. after about 9 hours, but the choice of timing may be varied to optimise yields of enzyme.
  • the cells produce 20-40% of their total cell protein as the desired enzyme (this may be approximately assessed by the intensity of Coomassie Blue staining on SDS-PAGE gels), and a 1 litre culture may yield 20 - 150 mg of pure enzyme (typically 40 - 80 mg) following purification from the cells.
  • Growth can also be successfully scaled up e.g. to 20 litres in fermenter culture, and the use of a fed batch approach permits a large increase in cell density and hence an increase of up to 50-fold in the yield of biocatalyst per litre of vessel capacity.
  • stationary phase typically about 18 hr.
  • the cell paste is resuspended in a limited volume of liquid (aqueous buffer at pH 7-8) so that the cells in the resultant slurry may be broken open e.g. by sonic disintegration or by shear forces in an instrument such as the Manton-Gaulin homogeniser.
  • the released soluble material is separated from cell walls and other solid debris e.g. by centrifugation.
  • the resulting crude extract may be an adequate source of biocatalyst without further treatment but, if required, the extract is further processed by standard column chromatographic procedures to yield homogeneous biocatalyst protein.
  • Celite/purified enzyme The purified enzyme (0.6 mg/ml) was taken straight from the eluate of the Procion Red P3BN chromatography column in potassium phosphate buffer (20 mM, pH 7.9) containing NaCl (0.5 M), [Y.K. Seah, K.L. Britton, PJ. Baker, D.W. Rice, Y. Asano, P.C. Engel, FEBS Lett. 1995, 370, 93] without precipitating with ammonium sulphate. 2 ml of this protein solution was added to Celite (1 mg) [M. Persson, E. Wehtje, P. Adlercreutz, Biotechnol. Lett. 2000, 22, 1571]. The preparation was dried for approx. 18 hr under vacuum and then stored at 4 0 C. This form of the biocatalyst was used for all the reactions carried out in presence of organic solvents (see results).
  • Alginate beads/whole cells A solution of alginate 2% was prepared by dissolving 100 mg of alginate in 5 mL of H 2 O under vigorous stirring. A volume of 1 mL of cell paste was added to the alginate solution and the mixture was then polymerised by dropping it with a syringe into a solution of CaCl 2 (1 %). The beads form immediately under these conditions. They were left curing for 30 min and stored at 4 0 C.
  • Chitosan beads/whole cells A solution of chitosan 2% was prepared by dissolving 100 mg of chitosan in 5 mL of H 2 O acidified with CH 3 COOH 1% under vigorous stirring at 30°C. A volume of 1 mL of cell paste was added to the chitosan solution and the mixture was then polymerised by dropping it with a syringe into a solution of tripolyphosphate (0.1 M). The beads form immediately under these conditions. They are left curing for 10 min, rinsed with potassium phosphate (50 mM, pH: 8.0) and stored at 4 0 C. .
  • thermodynamics are represented by an equilibrium constant which is likely to be similar for most ⁇ -amino acids and has been accurately determined in the case of the GIuDH reaction [P. C. Engel & K. Dalziel, Biochem. J., 105 (1967) 691-695].
  • a crucial feature in relation to pH dependence is the fact that a proton is produced in the oxidative deamination (or consumed in the reductive animation).
  • the cofactor used in the oxidative deamination (for example, NAD + ) and in the reductive amination (for example, NADH) can be used in sub- stoichiometric (possibly catalytic) amounts to minimise the cost of the process.
  • oxidative deamination for example, NAD +
  • NADH reductive amination
  • Several recycling systems may be used as long as the appropriate additional substrate is added to the reaction mixture.
  • the recycling system adopted must be stable under the same reaction conditions as the mutant amino acid dehydrogenase enzyme.
  • enzymes used to recycle NAD + /NADP + to NADH/NADPH are alcohol dehydrogenases from different sources which convert NAD + /NADP + and ethanol to NADH/NADPH and ethanal [K.F.
  • NADH oxidases (NOX) which use molecular oxygen to re- oxidise the cofactor [W. Hummel et al, Org. Lett. (2003) 20, 3649]. Similar reactions may be used to recycle other suitable oxidised coenzymes to their corresponding reduced coenzymes, and vice versa.
  • a reaction mixture would be prepared as follows: 1 mmol of ⁇ -ketoacid, KCl 1 mmol (this is an essential activator for PheDH), ammonium salt 4mmol, NADH 1.2-4 mmol and Tris buffer pH 8.5 to a final volume of 10 mL (the ammonium salt and NAD + both need to be in stoichiometric excess over the ⁇ -ketoacid to be converted).
  • a reaction mixture was prepared as follows: 1 mmol of ⁇ -ketoacid, KCl 1 mmol, ammonium salt 4mmol, NAD + 20 ⁇ mol, EDTA 10 ⁇ mol, ethanol 0.5mL and Tris buffer pH 8.5 to a final volume of 10 mL (the ammonium salt needs to be in stoichiometric excess over the ⁇ -ketoacid_to be converted; the coenzyme is present in a catalytic amount since NADH is recycled, and for the same reason may be supplied either as NADH or as NAD + , the latter being preferable on grounds of cost; EtOH is present at high concentration in order to drive the recycling reaction).
  • the reaction was started by adding lmg alcohol dehydrogenase (ADH) (663 U/mg) and a suitable amount of the chosen biocatalyst (50 ⁇ g wild-type PheDH, 60 ⁇ g N145A, 10 ⁇ g N145V or 30 ⁇ g N145L).
  • ADH alcohol dehydrogenase
  • reaction mixture was incubated at 25°C in an orbital shaker incubator and the formation of the amino acid was monitored by chiral HPLC (CHIROBIOTIC T column) over a period of 24 hours (the reaction could be performed at any temperature between 15 and 40 0 C depending on the stability of the substrates, higher temperature will lead to an increase rate but may be deleterious for sensitive substrates; if necessary the timecourse of the reaction may be decreased by increasing the amount of biocatalyst added).
  • chiral HPLC CHIROBIOTIC T column
  • the same reaction can be performed also by adding beads of chitosan or alginate prepared as described above.
  • NAD + which either must be supplied in stoichiometric excess or else, preferably, is constantly replenished by recycling NADH to NAD + with bacterial diaphorase from Tliernius aquaticus [C. Logan et al, J Biol Chem. 2000, 275, 30019].
  • reaction would be performed at pH 10.5 (carbonate/bicarbonate buffer 0.1 M or similar; glycine buffer is preferably not used in order to avoid subsequent problems in separation).
  • pH optimum for enzyme activity the equilibrium of the reaction, but also the stability of reaction components.
  • pH 10.5 is good both for activity of this enzyme and for pulling the reaction in the desired direction, but it is less desirable in terms of coenzyme stability. Without recycling one is forced to go for the equilibrium advantage of the higher pH. With recycling, which works much better, one can employ the lower pH which stabilises the coenzyme, still knowing that the reaction will be driven to completion.
  • the reaction mixture would contain 5-10 mM of the DL- ⁇ -amino acid, at least a 2- fold molar excess of NAD + , 10OmM KCl.
  • the reaction would be started by adding a suitable amount of the appropriate biocatalyst and monitored either by spectrophotometric measurement of the production of NADH or by chiral HPLC, both as taught above. On page22 and in Table 6,the use of chiral HPLC (the
  • CHIROBIOTIC T column is mentioned.
  • the solvent system is an isocratic elution with 30% methanol in water, and the same system is used both for following the formation and consumption of amino acids.
  • the reaction is performed at pH 9.5 (ethanolamine-HCl, 20 mL, 50 mM).
  • the reaction mixture contains 5-10 mM of the DL- ⁇ -amino acid, 1 mM NAD + , 100 mM KCl and 0.1 mg DCPIP (dichlorophenol indophenol).
  • the reaction is started by adding a suitable amount of the appropriate biocatalyst, 0.1 mg diaphorase and monitored by chiral HPLC as reported above. Although high pHs will favour the equilibrium of the reaction as described above, the stability of NAD + needs to be considered: for this reason the pH is lowered to a maximum of 9.5.
  • the reaction is carried out in a vessel with a large surface area shaking or stirring to maximise the exchange with molecular oxygen. We carried out the reaction at room temperature (15-20 0 C) but in principle higher temperature up to 30°C could be used.
  • the separation of the end product from the reaction mixture can be achieved by ion-exchange chromatography [Y. Asano et al, Agric.Biol. Chem. (1987) 51, 2035], crystallisation or preparative HPLC.
  • the separation of the end product from the reaction mixture can additionally be achieved with the use of trapping agents such as functionalised silica.
  • the ⁇ -keto esters (20)-(25) were obtained in excellent yields.
  • the Grignard reagents were prepared in Et 2 O instead of THF to avoid the Wurtz coupling side reaction [K. V. Baker et al, J. Org. Chem., 1991, 45, 698-703]. While this approach gave the ⁇ -keto esters in high yields, the recovery of the acids after hydrolysis was poor, which can be attributed to a number of side reactions.
  • Decarboxylation can occur during hydrolysis at high temperature or in basic media during the extraction process. Rapid decomposition can occur in polar solvents during concentration of the organic layer at reduced pressure giving an unidentifiable mixture by NMR.
  • ⁇ -Keto acids (3)-(6), (12) were prepared following the procedure described for (2). Precipitation using EtOAc:DCM 1:19 was used to purify the acids in each case.
  • N-acetylglycine AcONa, Ac 2 O, ⁇ Ih; 2. 3M HCl, ⁇ , 3h.
  • reaction time for the hydrolysis step was also reduced from a reported 24-48 hours to typically 3 hours again monitoring by TLC. Filtration of the resultant precipitate gave the ⁇ -keto acids in high yields and purity. Again 1 H and 13 C NMR showed that only the enol form of each compound (7)-(ll) was present in CD 3 OD or d 6 -DMSO.
  • the 2-chlorophenylpyruvate derivative (1) was synthesized as illustrated in Scheme 3. Based on earlier work describing acylation of organocadmium reagents with acid chlorides [J. Cason, Chem Rev., 1947, 40, 15-32; and D.A. Shirley, Org. Reactions, 1954, 8, 28-58], reaction of an organocadmium reagent with oxalyl chloride followed by hydrolysis was envisaged as a short synthetic route to the ⁇ -keto acid. However, while this route did produce the ⁇ -keto acid (1), this synthetic method is less satisfactory than the routes described above so it was not investigated further.
  • 2-Ketocaproic acid (13) is commercially available from Sigma and was included in this study as an acyclic derivative.
  • Each ⁇ -keto acid (4 mM) was dissolved to form a component of a reaction mixture containing NH 4 Cl (40OmM), KCl (10OmM), O.lmM NADH and Tris (5OmM).
  • the pH was adjusted to 8.0 by adding a suitable amount of HCl.
  • ImL of reaction mixture was incubated at 25°C.
  • the reaction was followed at 340nm over 1 minute after adding an appropriate amount of enzyme to achieve an optimally measurable reaction rate (between 0.01-0.03 min "1 ). Each of the reactions was carried out in duplicate and the average value is reported.
  • Example 1 Specificity for non-natural substrate resulting from a set of mutations created at position 145 of PheDH.
  • N145 Asn in position 145
  • N145A Asn in position 145
  • N145L a leucine
  • N 145Y valine
  • the non-natural ⁇ -keto acids were initially screened for activity in the reductive amination of the ⁇ -keto acid with the wild type PheDH and three different mutants (Nl 45 A, N145L and Nl 45V). The results are reported in Table 4. The activity is measured under standard conditions at 25 0 C [H. Hongwen & F. Xianqi, Gaodeng Xuexiao Huaxue Xuebao, 1988, 9, 966-968], by following the decrease in absorbance of NADH at 340nm (UV spectrophotometer, Gary 50) according to the reaction in Scheme 4.
  • each of the results in Table 4 is expressed both as a specific activity (a unit, U, of enzyme is defined as the amount of enzyme which converts one ⁇ mole of substrate per minute under standard conditions) and also normalized relative to activity with phenylpyruvate for each of the enzymes.
  • Example 2 Non-natural substrate specificity by a series of mutants, single and double, at various positions in the PheDH sequence.
  • Each ⁇ -keto acid ( ⁇ 0.5mM) was dissolved in a solution containing NH 4 Cl (40OmM), KCl (10OmM), ImM NADH and Tris (5OmM). The pH was adjusted to 8.0 by adding a suitable amount of HCl. The solution was filtered through a sterile filter Acrodisc ® 0.45 ⁇ m. ImL of reaction mixture was incubated at 25°C and an appropriate amount of enzyme (N 145 V, N 145 A or N145L - each gave similar results) was added to allow approximate completion of the reaction within about 40min. The formation of the corresponding ⁇ -amino acid was followed by loading 20 ⁇ L of the mixture onto a CHIROBIOTIC T, chiral HPLC column. The elution mixture was MeOH/H 2 O 70/30 at a flow rate of 1.OmL/min.
  • Figure 1 shows an example of HPLC outcome, in which all the peaks are clearly separated and the L enantiomer is the only one detectable.
  • PheDH derivatives in particular this has been exhaustively tested by monitoring reaction mixtures by chiral HPLC analysis. In no case was there any detectable production of the D-isomer of the ⁇ -amino acid (Fig. — l) ⁇
  • Fig. IA shows that chiral HPLC gives a good separation of the L- and D- enantiomers of the ⁇ -amino acid and separates both from other, early eluting components of a typical reaction mixture.
  • Fig. IB shows that when the reaction is carried out using the ⁇ -ketoacid as the starting substrate, a clean peak is seen at the position corresponding to the L- ⁇ — amino acid product and there is no trace of a similar peak in the D- ⁇ -position.
  • Some ⁇ -amino acids have more than one chiral centre. All except glycine exhibit chirality at the ⁇ -carbon atom, but others, such as threonine and isoleucine, contain a second chiral atom in their sidechain. The ability to distinguish the enantiomers at these positions will also be a very valuable property of these biocatalysts.
  • N145A offered the best affinity for 4-CF 3 -phenylpyruvate (9), while N145V and N145L represented the best choice for 4-F-phenylpyruvate (6) and 4-MeO- phenylpyruvate (8).
  • Figure 2 is the NMR spectrum of a reaction mixture after 24hrs for synthesis of the 4- F- ⁇ henylalanine. This spectrum is very similar to that reported in the literature for the authentic compound. The peaks corresponding to the aromatic hydrogens and the single hydrogen attached to the chiral carbon atom are clearly seen.
  • biocatalyst a) as the substantially purified protein; b) as a crude extract without purification; c) as whole cells without breakage or purification; d) as crude extract or as substantially purified protein immobilised on a solid support; e) as crude extract or as substantially purified protein or cells entrapped in (or cross-linked to) sol-gels (such as alginate, chitosan or agarose beads).
  • sol-gels such as alginate, chitosan or agarose beads
  • biocatalyst is so highly over-expressed in the bacterial cells that it comprises typically 25-40% of total soluble protein.
  • Whole cells with externally added substrates function very satisfactorily without any apparent problems resulting from permeability barriers.
  • the PheDH-derived biocatalysts were also very effective and stable after absorption onto Celite. Either of these modes of use allow very simple physical separation of the biocatalyst from the reaction mixture at the end of reaction, allowing repeated use of the biocatalyst preparation.
  • Tables 9 and 10 represent preliminary data for the oxidative deamination reaction of WT PheDH and mutants with aromatic substrate analogues of phenylalanine (Phe) substituted at position 4 (36-40 and 44-48), with increased chain length (L- homophenylalanine 43) or substituted at the ⁇ -carbon ( ⁇ -methyl-DL-Phe 51) and aliphatic ⁇ -amino acids corresponding to the ⁇ -keto -acids 12 (L-cyclohexylalanine 42 and DL-Cyclohexyl-alanine 50) and 13 (L-norleucine 41 and DL-norleucine 49) reported in Table 4 or with a smaller aliphatic ring (DL- cyclopentylalanine 52).
  • Table 9 Preliminary results for activity of WT and mutants at 0.2mM concentration of substrate (* 2.5 mM). Assays are performed at pH 10.4 in Gly/NaOH buffer by following spectrophotometrically the production of NADH at 340nm. In this table we are comparing relative activities (top numbers) and specific activities (bottom numbers) with pure L- ⁇ -amino acids.
  • Table 10 Preliminary results for activity of WT and mutants at 0.4mM concentration of racemic ⁇ -amino acids (* 10 mM). Where available, the relative activities (top numbers) refer to the activity shown in comparison to that of the corresponding L- ⁇ -amino acid (100). Reactions are performed at pH 10.4 in Gly/NaOH buffer by following spectrophotometrically the production of NADH at 340nm.
  • the wild type enzyme is able to catalyse the reductive animation of ⁇ -keto acids bearing 4-substituted aromatic groups, although it is necessary to distinguish between halogens and bulky non-polar groups: while the activity with 4-fluoro (6) and A- chloro (3) substitution is certainly satisfactory (114 and 62.3%), it drops substantially with 4-methyl (7) or 4-methoxy (8) (14.6 and 14.9%) and it is relatively poor with A- trifluoromethyl (9) (0.9%).
  • the activities of the mutants are remarkable: while the mutants show decreased activity for phenyl pyruvate compared to the WT enzyme, the ⁇ -amino acid substitution in the binding pocket of these enzymes results ill generally increased tolerance of substitution at the 4-position of the aromatic ring.
  • the 4-chloro substitution one of the most striking results is that for N145L (359% as compared with 62% for the wild-type enzyme).
  • this mutant shows much the poorest reference activity with phenyl pyruvate (only 22 U/mg). Thus 359% is only 79 U/mg which is in fact the lowest figure for the 4-chloro ⁇ -keto acid (3) with the four enzymes tested.
  • the improvement in catalytic activity is absolute, in that activities are not only higher in most cases than with phenyl pyruvate, but also uniformly much higher than the activity of the wild type enzyme with the same substrates (3.9-fold for N145L with 4- methoxy and 4.6-fold for N145A with 4-methyl).
  • the N 145 A mutant shows a remarkably high activity of 29.4 U/mg.
  • mutant enzymes have considerable potential to be employed as synthetic biocatalysts for the kinetic resolution of racemic non-natural ⁇ -amino acids to produce the D- ⁇ -amino acids.
  • the potential for use of the engineered enzymes as biocatalysts for the production of non-natural D- ⁇ -amino acids and/or non-L- ⁇ -amino acids is clear from this work.
  • the optional use of recycling systems for achieving quantitative conversion in both directions is an advantage in the production of non-natural D- ⁇ -amino acids and/or non-L- ⁇ -amino acids.

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Abstract

Les procédés de la présente invention permettent d'accéder aux deux séries énantiomères d'a-aminoacides non naturels. Selon un premier aspect de l'invention, on fait appel à un procédé de résolution d'a-aminoacides racémiques non naturels pour donner un a-céto-acide, achiral à la position a, et un D-a-aminoacide. Selon un second aspect de l'invention, on fait appel à un procédé d'élaboration d'un L-a-aminoacide non naturel à partir d'un a-céto-acide, achiral à la position a. De préférence, l'a-céto-acide achiral s'obtient grâce au procédé du premier aspect de l'invention. Tous les procédés emploient des aminoacides déshydrogénases mutantes. La présente invention montre que les aminoacides déshydrogénases mutantes peuvent, étonnamment, traiter une vaste gamme d'a-aminoacides non naturels, souvent avec une grande efficacité et toujours avec une parfaite rétention de la discrimination absolue entre les séries L et D d'a-aminoacides.
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CN103555683A (zh) * 2013-11-19 2014-02-05 南京博优康远生物医药科技有限公司 一种沙格列汀手性中间体的合成方法
US9273332B2 (en) 2006-10-12 2016-03-01 Kaneka Corporation Method for production of L-amino acid
CN110951705A (zh) * 2019-12-20 2020-04-03 中国科学院苏州生物医学工程技术研究所 胺脱氢酶突变体、酶制剂、重组载体、重组细胞及其制备方法和应用
WO2020133989A1 (fr) * 2018-12-28 2020-07-02 浙江工业大学 Mutant de déshydrogénase d'acide aminé et son utilisation dans la préparation de l-glufosinate
WO2022007881A1 (fr) * 2020-07-09 2022-01-13 四川利尔生物科技有限公司 Glutamate déshydrogénase modifiée et son utilisation
CN114196642A (zh) * 2021-11-10 2022-03-18 浙江大学杭州国际科创中心 谷氨酸脱氢酶变体及其在制备l-氨基酸中的应用

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WO2020133989A1 (fr) * 2018-12-28 2020-07-02 浙江工业大学 Mutant de déshydrogénase d'acide aminé et son utilisation dans la préparation de l-glufosinate
US11408016B2 (en) 2018-12-28 2022-08-09 Zhejiang University Of Technology Amino acid dehydrogenase mutant and application in synthesis of L-glufosinate-ammonium thereof
CN110951705A (zh) * 2019-12-20 2020-04-03 中国科学院苏州生物医学工程技术研究所 胺脱氢酶突变体、酶制剂、重组载体、重组细胞及其制备方法和应用
CN110951705B (zh) * 2019-12-20 2020-09-25 中国科学院苏州生物医学工程技术研究所 胺脱氢酶突变体、酶制剂、重组载体、重组细胞及其制备方法和应用
WO2022007881A1 (fr) * 2020-07-09 2022-01-13 四川利尔生物科技有限公司 Glutamate déshydrogénase modifiée et son utilisation
CN114196642A (zh) * 2021-11-10 2022-03-18 浙江大学杭州国际科创中心 谷氨酸脱氢酶变体及其在制备l-氨基酸中的应用
CN114196642B (zh) * 2021-11-10 2023-12-05 浙江大学杭州国际科创中心 谷氨酸脱氢酶变体及其在制备l-氨基酸中的应用

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