EP0625205A1 - Synthese chirale effectuee au moyen d'enzymes modifiees - Google Patents

Synthese chirale effectuee au moyen d'enzymes modifiees

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Publication number
EP0625205A1
EP0625205A1 EP93902488A EP93902488A EP0625205A1 EP 0625205 A1 EP0625205 A1 EP 0625205A1 EP 93902488 A EP93902488 A EP 93902488A EP 93902488 A EP93902488 A EP 93902488A EP 0625205 A1 EP0625205 A1 EP 0625205A1
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EP
European Patent Office
Prior art keywords
enzyme
dna
region
sequence
loop
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93902488A
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German (de)
English (en)
Inventor
Helen Margaret Wilks
Joseph John Holbrook
Keith William Hart
Ayman Elhawrani
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Euroapi UK Ltd
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Genzyme Ltd
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Filing date
Publication date
Priority claimed from GB929202033A external-priority patent/GB9202033D0/en
Priority claimed from GB929204702A external-priority patent/GB9204702D0/en
Application filed by Genzyme Ltd filed Critical Genzyme Ltd
Publication of EP0625205A1 publication Critical patent/EP0625205A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids

Definitions

  • This invention relates to chiral synthesis; more particularly, it relates to the modification of enzymes to facilitate such synthesis.
  • Enzymes are biological catalysts which are specific both in terms of chemical activity and substrate structure, and it is this specificity which has been exploited in a variety of commercial applications. Although many such activities are known, it may be desirable to change the range of substrates that are suitable for catalysis and/or to change the efficiency of a given catalysis for a particular type of enzyme. Given a type of enzyme with known key elements vis-a-vis substrate preference and hence activity, it may be possible purposefully to change those elements to bring about desired modifications and hence to expand the potential industrial utility of a particular enzyme.
  • Enzyme activity is primarily controlled by the amino acid composition especially in certain important functional areas of the enzyme, altering these amino acids is known to change activity and may be achieved by the use of either specific or non-specific techniques.
  • a neutralising amino acid may facilitate the catalysis of a substrate with an altered charge and this could be regarded as a predictable alteration, although no result may ever be predicted with total certainty, especially where the tertiary structures of enzymes are not as precisely known as would be necessary for complete confidence.
  • this would prove an almost infinite task and so it is often convenient initially to make a "macro-change" and then to "fine tune" with discrete changes.
  • site-directed mutagenesis involving the introduction of a neutralizing charge into the correct region of the active site alters substrate specificity allowing the enzyme to take on the activity that would be expected of a malate dehydrogenase.
  • Such specific mutations may be considered predictable in gross terms, but are very unlikely to be the ultimate refinement in increasing specificity towards such a substrate.
  • substrates such as those with increased alkyl chain lengths, phenyl residues or heterocyclic additions, predictions of site- specific changes are unlikely to be reliable.
  • lactate dehydrogenase for example, to make use of the known loop region forming part of the active site.
  • the loop region may be exchanged for a larger or smaller section of loop region from a similar enzyme. This may be expected to allow some variation in substrate specificity and relative catalytic efficiency, while retaining the typical activity.
  • specific amino acid residues may be targeted for further change.
  • the present invention relates to a method for modifying the specificity and/or efficiency of an enzyme, while retaining its catalytic activity, characterised in that it comprises: selecting an enzyme, the tertiary structure of which is substantially known or deduced; identifying at least one specificity and/or efficiency-related region; identifying or constructing unique restriction sites bounding the identified region in the DNA coding therefor; generating a DNA sequence which corresponds to at least a portion of the identified region, except that the nucleotides of.at least one codon are randomized, or selecting as a substitute for at least a portion of the identified region an alternative such region, which may itself be similarly randomized; using the generated or substitute DNA sequence to replace the original such sequence; expressing the DNA including the generated or substitute DNA sequence; and selecting for a desired modification so that the DNA coding therefor may be isolated.
  • a dehydrogenase in particular an ⁇ -hydroxy acid dehydrogenase, such as lactate dehydrogenase.
  • a dehydrogenase in particular an ⁇ -hydroxy acid dehydrogenase, such as lactate dehydrogenase.
  • it is the loop region of the enzyme which is identified initially as being specificity and/or efficiency-related.
  • the randomized DNA is generated by means of an inosine triphosphate PCR method or a spiked oligonucleotide method or a PCR assembly method, all of which will be discussed in more detail below. If a substitute is to be selected for at least a portion of the region of interest, it is often based on a corresponding sequence from a similar enzyme.
  • the original DNA sequence is replaced by the generated or substitute DNA sequence, it is cloned into a plasmid or phage vector and transformed into a bacterium or virus for expression. Thereafter, a screen may be used to select for a desired modification. Taking L-lactate dehydrogenase as an example, positions 101 and 102 are particularly appropriate for randomization.
  • the present invention also relates to the use of such modified enzymes particularly in the production of chiral products. Often, such processes involve the use of a cof ctor recycling system.
  • One example is the reduction of 2-oxo-4- phenyl-propanoic acid characterised in that it comprises the use of L-lactate dehydrogenase which has been modified in the loop region by the present method and another is the reduction of 4- methyl-2-oxo-3-pentenoic acid characterised in that it comprises the use of MVS/GG obtainable by the present method.
  • Another approach for obtaining suitable enzyme catalysts is to modify the structure of an existing enzyme to improve its catalysis for a particular substrate.
  • This approach of so-called "enzyme engineering”, which is in its very early stages has great potential for the preparation of catalysts for the synthesis of homochiral molecules. The importance of these molecules in the synthesis of single isomer pharmaceuticals and agrochemicals is well recognised.
  • enzyme engineering may be calculated for a 300 residue protein of 20 amino acids as 10 390 possible sequences. The vast majority of these sequences cannot have been explored for biological function. It may be suggested that a typical large protein of 300 amino acids residues cannot represent a global optimum for any biological function, but at best is an assembly of empirically optimised 25-35 amino acid domains. Thus, enzyme engineering should be capable of improving a large frame-work for any particular target function.
  • Random mutagenesis of existing proteins is also limited in its ability to produce radically altered proteins by problems of sampling all the possible variants.
  • the genetic code is very resistant to change. Not only are codons redundant at the third position, but also amino acid residues with similar properties are coded by similar sequences and thus resistant to sparse mutagenesis. For example: (i) a codon having a T at the second position always codes for an amino acid residue having a hydrophobic side chain; (ii) the codons for aspartate and glutamate differ only at the third position.
  • Fig. 1 depicts the active site of lactate dehydrogenase.
  • the rate-limiting step in lactate dehydrogenase catalysis is the rate at which this loop may sweep through a viscous solvent to close onto the upper surface of helix ⁇ 2G.
  • the rate-limiting step is largely independent of the sequence of amino acids on the "upper jaw” and since the chemical step is much faster than the shape change, the lactate dehydrogenase system has the advantage that the loop sequence may be easily varied to achieve different substrate specificities without much danger that the chemical step will become rate- limiting.
  • An object of the present invention was to modify an already useful, but substrate-restricted enzyme, S lactate dehydrogenase, to provide an improved catalyst for reduction of the ⁇ -keto group in acids larger than the natural substrate, pyruvate.
  • the substrates of interest contain bulky aromatic groups.
  • thermophilic lactate dehydrogenase isolated from Bacillus stearothermophilus. which has been cloned and expressed in Escherichia coii.
  • This enzyme has been one of the most thoroughly characterised protein frameworks (Dunn, C. R. , et al, Philos. Trans. R. Soc. London Ser. B, 1991, 332, 184) , including the study of inhibition, substrate interaction and genetic manipulation.
  • the physical stability of the enzyme, especially to thermal denaturation, makes it an ideal candidate for demonstrating the features of redesign which would be generally applicable to ⁇ -hydroxy acid dehydrogenases, for example.
  • the modification of wild-type enzymes presents a significant challenge because, even in the case of a protein with considerable literature knowledge, the results may be unexpected and surprising. Thus, redesign of even well- studied enzymes is of limited predictability.
  • the enzymes of particular interest at present are ⁇ -hydroxy acid dehydrogenases, which catalyse the NADH/NADPH dependent reduction of a keto group in an ⁇ -position to a carboxylic acid, or, alternatively, the reverse reaction where the ⁇ -hydroxy group is oxidised to the ketone.
  • Attempts to modify the enzyme lactate dehydrogenase to expand the natural substrate specificity to allow an increased reaction rate with larger substrates with various functional groups has led to the present unpredictable observations. Although it may be possible to prepare substrates and corresponding chiral products of interest by chemical synthesis, followed by wild-type enzyme reduction, such an approach may not be attractive and it may be that preparation via a redesigned protein framework may provide a more rational and cost effective approach.
  • the alteration of the enzyme has demonstrated that the activity towards the natural substrate may be so dramatically reduced that completely different substrate selectivity is produced.
  • This may not be a requirement of a biotransformation catalyst, where the enzyme is presented with only one substrate species for reduction, but, when a mixture of potential substrates is present, such as may occur in a biological sample, this may be essential for achievement of selective conversion or the determination of one particular chemical species.
  • This alteration in substrate specificity could also be advantageous in a biotransformation using whole cells where the intended substrate is necessarily contaminated with other entities which could also be transformed.
  • Enzymes capable of reducing such substrates would be of particular value in the field of synthetic chemistry where an ⁇ -keto compound could be converted stereospecifically to the corresponding secondary alcohol.
  • the production of individual optical isomers of secondary alcohols is especially valuable in the manufacture of optical isomers of pharmaceuticals and drug intermediaries.
  • the feature of thermophilicity which may be obtained with some ⁇ -hydroxy acid dehydrogenases is valuable as it enables the enzymic reactions to be carried out at relatively high temperature where a rate acceleration may exist and the enzymes are inherently stable.
  • These enzymes may also be suitable for incorporation into determinations of the levels of particular substrates obtained in biological samples under certain disease states.
  • NAD dependent dehydrogenases
  • This system numbers amino acids in ascending order extending from the N terminus.
  • conserved residues such as glycine at positions 30 and 33, tyrosine at position 85, arginine at position 109, serine at position 163 and aspartic acid at position 168.
  • any given NAD dependant dehydrogenase natural or subject to mutation, there are regions of sequence which are homologous with the amino acid sequence of the numbering convention.
  • An important aspect of this convention is that any amino acid change in an NAD dependent dehydrogenase may be accurately described.
  • thermophilic lactate dehydrogenase fr Bacillus stearother ophilus The DNA sequence of the thermophilic lactate dehydrogenase fr Bacillus stearother ophilus.
  • the substrate recognition site is carried in part by a mobile loop of polypeptide chain, conventionally numbered 98 to 110. This sequence is contiguous but traditionally omits a residue 103.
  • the loop region is also involved in substrate selection and for that reason was the particular object for the present enzyme engineering study.
  • lactate dehydrogenase distinguishes different substrates
  • the mechanism by which lactate dehydrogenase distinguishes different substrates is the ability of the substrate to fit into a proton-impermeable, fixed-sized internal vacuole which is formed when the mobile surface polypeptide loop closes down onto the protein surface. Not only is loop closure only possible over suitably small and singly negatively charged substrates, but also the loop closure triggers catalysis through the arginine 109 residue. The variation in composition and length of this mobile loop region is the immediate object.
  • a particular gene for wild- , type Bacillus stearothermophilus lactate dehydrogenase was chosen where the amino acids alanine at positions 235 and 236 had been changed for glycines.
  • the mutation where alanines at 235,236 are replaced by glycines has been combined with three mutations in the mobile polypeptide loop (residues 98-112) , namely glycine 102 by methionine, lysine 103 by valine and proline 105 by serine (MVS/GG) .
  • This new enzyme construction was evaluated for activity towards longer substrates, in particular an unsaturated branched substrate 4-methyl-2-oxo-3-pentenoic acid, which is reduced to the following alcohol:
  • the method used to make new loop constructions was to insert restriction enzyme sites at either end of the DNA coding for the loop region. These new restriction sites which are unique within the DNA coding for the enzyme, are cleaved and then religated with synthetic DNA designed to code for the required new loop region.
  • One of the restriction sites introduced was for Sacll near amino acid 97.
  • the construction of the Sac II restriction site required that the wild type coding sequence for cysteine 97 was changed to threonine.
  • the Xbal site retained the wild-type amino acid sequence with arginine at 109, but did result in the creation of an Mlul site close to threonine 108.
  • the new Mlul site was used to advantage as it was destroyed in transformants and thus enabled easy distinction thereof from the wild-type gene.
  • novel loops were introduced, two shorter by 3 amino acids and one longer by 4 amino acids.
  • the new enzymes generated in this manner were evaluated against a range of experimental substrates to determine the effect of the loop exchanges.
  • the introduction of the new loop sequences further alters the substrate specificity of the enzyme reducing the turnover of the natural substrate from that of the wild type enzyme.
  • the three new loop enzymes retained most of the wild type catalytic potential towards the 2-oxo-4-phenyl propanoic acid as shown by turnover and Km and, in the example of the longer loop and second shorter loop version, resulted in an increase in turnover.
  • the alteration in specificity of the enzyme from pyruvate to 2-oxo-4-phenyl propanoic acid renders the new enzyme suitable for the determination of the concentration of 2-oxo-4- phenyl propanoic acid, often termed phenyl pyruvate in clinical chemistry nomenclature, especially from body fluids, such as blood and urine.
  • Phenyl pyruvate levels are normally low, but rise to significant levels with the increase in phenylalanine concentration, which is associated with the genetic disease phenylketonuria (Langenbeck et al. , J. Inher. Metab. Dis. , 4., 1981, 69) . It is also possible that the phenyl pyruvate reductase or phenyl lactate dehydrogenase enzyme could be used in conjugation with phenylalanine dehydrogenase, a current method of determining the phenylketonuria level such that interference from phenyl pyruvate could be negated, thereby enhancing the sensitivity of the phenylalanine-based method.
  • the construct having the restriction sites at either end of the loop region may be used to produce a series of dehydrogenases having loops of variable length and variable sequence.
  • the random mutagenesis may be generated by use of spiked oligonucleotides at specific positions and on different length loops or, alternatively, by the incorporation of inosine triphosphate in a polymerase chain reaction (PCR) that randomises either the entire loop region or specific residues. Both of these techniques have been employed to prepare mutant libraries using the restriction sites engineered into the DNA coding for the loop region of LDH.
  • a further PCR method was used to generate a random combinational DNA library of specific positions of the loop region. This technique was specifically targeted to positions 101 and 102 as these are involved in defining enzyme substrate specificity.
  • the PCR was initially used to generate 300 & 800 base pair fragments that had complementary overlapping ends. These primary products which had random sequences incorporated in the overlap, were then primed on each other and extended to yield an LDH hybrid gene. A second PCR with two outer primers annealing at non-overlapping ends was finally used to amplify the LDH product.
  • primer 2 with an EcoRI site enables the cloning of gene product into a number of vectors.
  • the screen is based on the work of Katzen and Schimkel (PNAS, 54., 1218) and relies on the ability of a colony expressing an enzyme with specificity to oxidise the required substrate and to reduce NAD + to NADH.
  • the reduced coenzyme then reduces phenazine metasulphate which in turn reduces nitroblue tetrazolium to form an insoluble blue dye.
  • the mutant DNA is transformed into competent Ej_ coli cells and is stored on agar plates containing 15% glycerol and ampicillin at -80°C. Obtaining electro-competent cells with high transformation rates has produced rates of 10 6 per ⁇ g of DNA, a rate which produces a sufficiently representative population of mutant colonies for screening. Copies of this plate are made using a velvet replicator and the copies grown up overnight.
  • the E_ s _ coli LDH activity is removed by incubation of the filter paper at 67°C for 30 minutes, the activity of the wild-type enzyme is not lost until 45 minutes at this temperature.)
  • the copies are then screened against a range of substrates and individual colonies may be compared. Each master plate is screened at least three times to ensure conditions are ideal in each case.
  • a 54-mer oligonucleotide was used to direct mutagenesis to introduce unique restriction sites (SacII and Xbal) at either end of the active site loop (amino acids 98-110) using the wild- type template (Barstow loc. cit) .
  • the mutagenic oligonucleotide was:
  • Mutants were identified by making mini-preps and restricting with SacII and Xbal. Mutant mini-preps were restricted with EcoRI and Xhol and the small fragment was sub- cloned into PKK223-3 containing Ala235Gly, Ala236Gly mutant LDH from which the small EcoRI/Xhol fragment had been removed (Wilks et al. Biochemistry, 1990, 2_9_, 8587) . The resulting plasmid
  • Single-stranded oligos were made such that the oligos were only different to the wild-type sequence at positions encoding amino acids 101 and 102 where each one of the bases A, T, ' C, G has an equal chance of being inserted. (Oligo mix 101,102 forward.)
  • An Mlul restriction site which is present in the wild-type template is destroyed by change of the third codon position of amino acid 108 from an ACG to an ACT without altering threonine as the amino acid being coded. The absence of the Mlul site enables verification that the mutants have been generated and to select against wild-type sequences.
  • a DNA primer which has 14 base homology to olio mix 101,102 forward was used to make the complementary strand (oligo mix 101,102 reverse) using a Klenow reaction.
  • Single-stranded library oligos were used with primer 1 and 5ng of wild-type template in order to generate a 300 base pair product with 25 cycles of PCR (94°C, for 1 minute, 55°C for 1 minute, 72°C for 2 minutes) .
  • Double-stranded Klenow oligos were used with primer 2 and 5ng of wild-type template to generate an 800 base pair product which overlaps the 300 base pair product. (PCR conditions as in 4.)
  • double-stranded oligo as primer in 5 is very important in ensuring that both the 300 and 800 base pair products are made and primed using mutant oligos and that the wild-type sequence at position 101 and 102 is not copied. 6.
  • 20ng of the 300 base pair product and 60 ng of the 800 base pair product were mixed without primers and thermocycled seven times in order to join the fragments (94°C for 2 minutes, 55°C for 1 minute, 72°C for 4 minutes) .
  • primers 1 and 2 were added, and the product amplified for twenty cycles (94°C for 1.5 minutes, 55°C for 1 minute, 72*C for 2.5 minutes).
  • the 1 kb PCR product was then gel purified, digested with EcoRI, and gel purified again before ligation into EcoRI-cut PUC18 plasmid vector and transformation into E ⁇ coli. 9. Recombinant colonies were selected for by IPTG and X-Gal insertional inactivation.
  • Each pair of overlapping oligonucleotides (20 ⁇ M of each) were subjected to 30 cycles of annealing and extension (94°C for 1 minute, cool to 45°C for 2 minutes, 45°C for 1 minute, heat to 72°C in 1 minute, 72°C for 1 minute in 50 ⁇ l containing 0.05 M KC1, lOmM Tris pH 8.3, 1.5 mM MgCl 2 , 0.01% gelatin), 200 ⁇ M of each dNTP and 2.5 units TAQ DNA polymerase) .
  • the double-stranded DNA product was purified and then cut with Sacll and Xbal before ligating it into the plasmid pLDHrs cut with the same enzymes.
  • the ligated products were restricted with Mlul to cleave wild-type plasmid pLDHrs.
  • the DNA was purified, microdialysed and used to transform E ⁇ coli TG2 cells by electroporation. Transformed cells were selected for ampicillin resistance. Ten such colonies were picked and plasmid DNA purified from overnight cultures. The presence of mutant loops was confirmed by resistance to Mlul digestion.
  • LDH was eluted with 50 mM triethanolamine, pH 9.0, 0.3 M NaCl. The elutant was precipitated with 65% ammonium sulphate and then resuspended in and dialysed against 50 mM triethanolamine, pH 7.5. The protein was then loaded onto a Q- Sepharose Fast Flow column and eluted with a salt gradient. LDH eluted at a concentration of 0.25 M NaCl.
  • the double glycine mutant enzyme the first chromatography procedure with oxamate Sepharose was replaced by chromatography on Blue Sepharose -F3GA, otherwise the procedure was essentially the same. All proteins were judged to be greater than 98% pure from the intensity of Coomassie blue staining on an SDS Phast gel (Pharmacia) . The yield of protein was usually 0.2g/l of original broth.
  • MVS/GG (6 units ( ⁇ moles/minute/30°C) ) and yeast formate dehydrogenase (5 units) were added to a solution of 4- methyl-2-oxo-3-pentenoic acid (1.0 mM) in deoxygenated Tris buffer (5mM:pH 6.0; 80ml) containing NADH (0.02 mM) , sodium formate (3.1 mM) , fructose-1,6-bisphosphate (0.4 mM) and dithiothreitol (0.08 mM) .
  • the solution was stirred at room temperature (-20°C) under nitrogen for 5 days with periodic addition of 0.2 mM HCl to maintain pH in the range of 6.0 - 6.2.

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Abstract

L'invention décrit un procédé servant à modifier la spécificité et/ou l'efficacité d'une enzyme, tout en maintenant son activité catalytique et caractérisé par le fait qu'il ocmprend: la sélection d'une enzyme, dont la structure tertiaire est sensiblement connue ou déduite; l'identification d'au moins une région relative à la spécificité et/ou à l'efficacité; l'identification ou la construction de sites uniques de restriction limitant la région identifiée au codage d'ADN correspondant; la génération d'une séquence d'ADN correspondant à au moins une partie de la région identifiée, à l'exception du fait que les nucléotides d'au moins un codon sont randomisés, ou la sélection en tant que substitut d'au moins une partie de ladite région identifiée d'une région alternative similaire pouvant être elle-même randomisée similaire; l'utilisation de la séquence d'ADN générée ou substituée, afin de remplacer la séquence originale; l'expression de l'ADN comprenant la séquence d'ADN générée ou substituée; enfin, la sélection d'une modification souhaitée, de façon à pouvoir isoler le codage d'ADN correspondant.
EP93902488A 1992-01-30 1993-01-29 Synthese chirale effectuee au moyen d'enzymes modifiees Withdrawn EP0625205A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GB9202033 1992-01-30
GB929202033A GB9202033D0 (en) 1992-01-30 1992-01-30 Chiral synthesis
GB929204702A GB9204702D0 (en) 1992-03-04 1992-03-04 Chiral synthesis
GB9204702 1992-03-04
PCT/GB1993/000204 WO1993015208A1 (fr) 1992-01-30 1993-01-29 Synthese chirale effectuee au moyen d'enzymes modifiees

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WO1993015208A1 (fr) 1993-08-05
CA2127126A1 (fr) 1993-08-05
JPH07503371A (ja) 1995-04-13
AU674137B2 (en) 1996-12-12

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