EP1534849A2 - Verwendung von malat dehydrogenase für die regenerierung von nadh - Google Patents

Verwendung von malat dehydrogenase für die regenerierung von nadh

Info

Publication number
EP1534849A2
EP1534849A2 EP03793686A EP03793686A EP1534849A2 EP 1534849 A2 EP1534849 A2 EP 1534849A2 EP 03793686 A EP03793686 A EP 03793686A EP 03793686 A EP03793686 A EP 03793686A EP 1534849 A2 EP1534849 A2 EP 1534849A2
Authority
EP
European Patent Office
Prior art keywords
dehydrogenase
nad
enzymatic transformation
malate dehydrogenase
pyruvate
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Withdrawn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
EP03793686A
Other languages
English (en)
French (fr)
Inventor
Shukrallah Naamnieh
Werner Hummel
Harald GRÖGER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Evonik Operations GmbH
Original Assignee
Degussa GmbH
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Degussa GmbH filed Critical Degussa GmbH
Publication of EP1534849A2 publication Critical patent/EP1534849A2/de
Withdrawn legal-status Critical Current

Links

Classifications

    • 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.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
    • 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
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
    • C12N9/0018—Phenylalanine dehydrogenase (1.4.1.20)
    • 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
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/222—Phenylalanine
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/36—Dinucleotides, e.g. nicotineamide-adenine dinucleotide phosphate
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids

Definitions

  • the present invention relates to a process for the preparation of enantiomerically enriched organic compounds.
  • the present invention relates to an enzymatically operating process, in which, in a coupled enzymatic reaction system, NAD(P)H is consumed by one enzyme for the preparation of the organic compound and the NAD(P)H is simultaneously regenerated by a second enzyme system.
  • reaction system which operates according to the invention in this manner and an advantageous whole cell catalyst or suitable plasmids are also proposed.
  • optically active organic compounds e.g. alcohols and amino acids
  • biocatalytic route is increasingly gaining importance.
  • the coupled use of two dehydrogenases with cofactor regeneration has emerged, inter alia, as a route for the large-scale industrial synthesis of these compounds, in particular alcohols and amino acids (DE19753350, EP118750) .
  • the biocatalysts efficiently employed in an aqueous medium furthermore have the advantage that in contrast to a large number of synthetic metal-containing catalysts, the use of metal-containing starting substances, in particular those which contain heavy metals and are therefore toxic, can be dispensed with.
  • the use of expensive and furthermore hazardous reducing agents, such as, for example, borane, in the case of asymmetric reduction can also be dispensed with.
  • the FDH e.g. from Candida boidinii
  • the FDH has the disadvantage that the specific activity of this enzyme class at 4-8 ⁇ /mg is very low. This necessitates the use of a large amount of expensive enzyme with recycling which is difficult from the apparatus point of view if a process designed in this way is to be carried out advantageously under economic aspects on an industrial scale.
  • Malate dehydrogenase (MDH) , called “malic enzyme” catalyses the oxidative decarboxylation of malate to pyruvate.
  • malate dehydrogenases from various organisms are known, thus, inter alia, from higher animals, plants and microorganisms. A distinction is made between four types of malate dehydrogenases, which are classified into the enzyme classes E.C. 1.1.1.37 to E.C. 1.1.1.40
  • NAD and/or NADP is required as a cofactor, depending on the type of malate dehydrogenase.
  • malate dehydrogenase On the basis of the irreversibility of the oxidative decarboxylation reaction of L-malic acid to pyruvate, the use of malate dehydrogenase is also appropriate in the systems described above in respect of a favourable cofactor regeneration.
  • the use of malate dehydrogenase for regeneration of NAD is described, for example, by Suye et al. in a work from 1992 (S.-I. Suye, M. Kawagoe, S. Inuta, Can . J. Chem . Eng. 1992, 70, 306-312) .
  • NADH regeneration by means of malate dehydrogenase is used here for reductive amination of pyruvate by means of an alanine dehydrogenase, NADH being consumed.
  • the pyruvate is formed by oxidative decarboxylation from L-malic acid, and is immediately consumed again in the following step by alanine dehydrogenase.
  • a concentration of pyruvate in the reaction solution is therefore avoided, whereby the problem of any inhibitions of the enzymes involved by the presence of amounts of pyruvate in the stoichiometric range is also eliminated. Nevertheless, the system described is limited exclusively to the production of alanine (see also S.-I. Suye, iecent Res . Devel . Ferment . Bioeng. 1998, 2, 55-64) .
  • the object of the present invention was therefore to provide a further process for the preparation of chiral organic compounds, such as amino acids or alcohols, which can be obtained by a coupled enzymatic reaction system as described above, using malate dehydrogenase, which is not limited to the preparation of one substance.
  • chiral organic compounds such as amino acids or alcohols
  • malate dehydrogenase which is not limited to the preparation of one substance.
  • Claims 2 to 7 relate to preferred embodiments.
  • Claims 8 and 9 protect a reaction system according to the invention and a correspondingly operating whole cell catalyst.
  • Claim 10 protects preferred plasmids.
  • Enantiomerically enriched alcohols or amino acids are advantageously prepared with the process according to the invention.
  • inexpensive alcohol dehydrogenase or amino acid dehydrogenase which are known universally are possible as enzymes for the first enzymatic transformation.
  • the expert is in principle free in the choice thereof, which is made according to the nature of the substrate spectrum, stability and rate of conversion of the enzyme in question.
  • Known enzymes of this origin are described in K. Drauz, H. Waldmann (eds.), Enzyme Catalysis in Organic Synthesis, volume III, Wiley-VCH, Weinheim, 2002, chapter 15. The use of the alcohol dehydrogenase from the organisms
  • Rhodococcus erythropolis S-ADH
  • Lactobacillus k&fir R- ADH
  • ADH from R . erythropolis J. Peters, T. Zelinski, M.- R. Kula, Purification and characterization of a novel carbonyl reductase silated from Rhodococcus erythropolis, J. Biotechnol. 1994, 33, 283-292
  • ADH from Lactabacillus kefir C. W. Bradshaw, W. Hummel, C.-H. Wong, Lactobacillus kefir Alcohol Dehydrogenase: A Useful Catalyst for Synthesis, J. Org. Chem.
  • leucine dehydrogenases or phenylalanine dehydrogenases (A. Bommarius in: Enzyme Catalysis in Organic Synthesis (eds.: K. Drauz, H. Waldmann) , volume III, Wiley-VCH, Weinheim, 2002, chapter 15.3).
  • Malate dehydrogenases are also familiar to the expert (lit. see above or the thesis by S. Naamnieh, University of Dusseldorf, in preparation) .
  • the expert will choose the dehydrogenase which can be employed most efficiently for his purpose.
  • those malate dehydrogenases which regenerate the NAD(P)H in an extent such that no bottleneck arises for the course of the reaction of the other enzyme employed.
  • the known malate dehydrogenase from E. coli K12 is preferred in this connection.
  • Gene isolation and cloning are described in S. Naamnieh' s thesis, University of Dusseldorf, in preparation p. 70 et seq.
  • the process according to the invention can be carried out in purely aqueous solution.
  • a water-soluble organic solvent to the aqueous solution in order e.g. to optimize the reaction in respect of poorly water-soluble substrates.
  • Possible such solvents are, in particular, ethylene glycol, DME or glycerol .
  • multi-phase, in particular two-phase systems comprising an aqueous phase can furthermore also serve as the solvent mixture for the process according to the invention.
  • the use of certain solvents which are not water- soluble has already proved itself here (DE10233107) .
  • the statements made there in this respect also apply here accordingly.
  • the expert in principle has a free choice of the temperature present during the reaction. He preferably directs himself towards obtaining a highest possible yield of product in the highest possible purity in the shortest possible time.
  • the enzymes employed should be sufficiently stable under the temperatures employed and the reaction should proceed with the highest possible enantioselectivity.
  • temperatures of 100°C it is entirely possible for temperatures of 100°C to represent the upper limit of the temperature range in the reaction. -15°C is certainly appropriate as the lower limit in aqueous systems.
  • a temperature interval between 10 and 60, particularly preferably between 20 and 40 2 C is advantageously to be established.
  • the pH during the reaction is determined by the expert from the enzyme stabilities and conversion rates and is adjusted accordingly for the process according to the invention.
  • the optimum pH is > 10.
  • the range preferred for enzymes from pH 5 to 11 is chosen.
  • the invention also provides a coupled enzymatic reaction system for the preparation of enantiomerically enriched organic compounds, comprising a first enzymatic transformation of an organic substrate, NAD(P)H being consumed, and the regeneration of the NAD(P)H in a second enzymatic transformation by a malate dehydrogenase, with oxidation of L-malic acid to pyruvate and C0 2 , wherein the pyruvate formed from the second enzymatic transformation is not employed as the substrate in the first enzymatic transformation.
  • a coupled enzymatic reaction system for the preparation of enantiomerically enriched organic compounds, comprising a first enzymatic transformation of an organic substrate, NAD(P)H being consumed, and the regeneration of the NAD(P)H in a second enzymatic transformation by a malate dehydrogenase, with oxidation of L-malic acid to pyruvate and C0 2 , wherein the pyruvate formed from the second
  • the reaction system is advantageously employed, for example, in a stirred tank, a cascade of stirred tanks or in membrane reactors, which can be operated both in batch operation and continuously.
  • membrane reactor is understood as meaning any reaction vessel in which the catalyst is enclosed in a reactor, while low molecular weight substances are fed to the reactor or can leave it.
  • the membrane here can be integrated directly into the reaction space or incorporated outside in a separate filtration module, in which the reaction solution flows continuously or intermittently through the filtration module and the retained product is recycled into the reactor. Suitable embodiments are described, inter alia, in W098/22415 and in Wandrey et al. in Yearbook 1998,
  • the present invention also provides whole cell catalysts comprising a cloned gene for a first enzyme for transformation of an organic substrate and a cloned gene for a malate dehydrogenase, these being capable of preparation of an enantiomerically enriched organic compound in a first enzymatic transformation, NAD(P)H being consumed, and of allowing the regeneration of the NAD(P)H to take place in a second enzymatic transformation by malate dehydrogenase, with oxidation of L-malic acid to pyruvate and C0 2 , wherein the pyruvate formed from the second enzymatic transformation is not employed as the substrate in the first enzymatic transformation.
  • the whole cell catalyst according to the invention preferably has an enzyme (polypeptide) with amino acid or alcohol dehydrogenase activity and one with malate dehydrogenase activity, originating in particular from the organisms mentioned above.
  • Microorganisms which can be used are in principle all the organisms possible to the expert for this purpose, such as e.g. yeasts, such as Hansenula polymorpha, Pichia sp . and Saccharomyces cerevisiae, prokaryotes, such as E. coli and Bacillus subtilis or eukaryotes, such as mammalian cells and insect cells.
  • E. coli strains are preferably to be used for this purpose. The following are very particularly preferred: E. coli XL1 Blue, NM 522, JM101, JM109, JM105, RR1, DH5 ⁇ , TOP 10 " or HB101.
  • An organism as mentioned in DE10155928 is preferably employed as the host organism.
  • the advantage of such an organism is the simultaneous expression of both polypeptide systems, which means that only one rec-organism has to be cultured for the reaction according to the invention.
  • the corresponding coding nucleic acid sequences can be accommodated on different plasmids with different numbers of copies and/or promoters of different potency can be used for an expression of the nucleic acid sequences of different intensity.
  • the whole cell catalyst according to the invention optionally further metabolizes the pyruvate formed in the reaction according to the invention, as it may uses it as a nutrient source.
  • Whole cell catalysts designed in this manner mean that the pyruvate is not obtained as a by-product of the reaction and therefore also does not have to be separated off from the chiral product actually desired in further process steps.
  • the preparation of the whole cell catalyst can in principle be carried out by measures known to the expert (Sambrook, J.; Fritsch, E. F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York; Balbas, P. and Bolivar, F. (1990) , Design and construction of expression plasmid vectors in E. coli, Methods Enzymol. 185, 14-37; Rodriguez, R.L. and Denhardt, D. T (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 205-225, Butterworth, Stoneham) .
  • the invention also provides plasmids containing gene constructs in which the gene for a malate dehydrogenase and a gene for an enzyme for transformation of an organic substrate with consumption of NAD(P)H are present.
  • Possible plasmids or vectors of origin are in principle all the embodiments available to the expert for this purpose.
  • Such plasmids and vectors can be found e.g. in Studier and colleagues (Studier, W. F. ; Rosenberg A. H. ; Dunn J. J. ; Dubendroff J. W. ; (1990), Use of the T7 RNA polymerase to direct expression of cloned genes, Methods Enzymol.
  • Plasmids with which the gene construct containing the nucleic acids according to the invention can be cloned into the host organism in a very preferred manner are: pUC18 (Roche Biochemicals) , pKK-177-3H (Roche Biochemicals) , pBTac2 (Roche Biochemicals), pKK223-3 (Amersham Pharmacia Biotech) , pKK-233-3 (Stratagene) or pET ⁇ Novagen) .
  • the plasmid pkk/phe/mali (fig. 5) is advantageous in this connection.
  • polypeptides in question of the process according to the invention can be used in the free form as homogeneously purified compounds or as an enzyme prepared by a recombinant method. These polypeptides can furthermore also be employed as a constituent of an intact guest organism or in combination with the broken-down cell mass of the host organism, which has been purified to any desired extent.
  • the use of the enzymes in immobilized form is also possible (Sharma B. P.; Bailey L. F. and Messing R. A.
  • the immobilization is advantageously carried out by lyophilization (Paradkar, V. M.; Dordick, J. S. (1994), Aqueous-Like Activity of ⁇ - Chymotrypsin Dissolved in Nearly Anhydrous Organic
  • Lyophilization in the presence of surface-active substances such as Aerosol OT or polyvinylpyrrolidone or polyethylene glycol (PEG) or Brij 52 (diethylene glycol monocetyl ether) (Kamiya, N.; Okazaki, S.-Y.; Goto, M. (1997), Surfactant-horseradish peroxidase complex catalytically active in anhydrous benzene, Biotechnol. Tech. 11, 375-378), is very particularly preferred.
  • Aerosol OT polyvinylpyrrolidone or polyethylene glycol (PEG) or Brij 52 (diethylene glycol monocetyl ether)
  • PEG polyethylene glycol
  • Brij 52 diethylene glycol monocetyl ether
  • Immobilization on Eupergit ® in particular Eupergit C ® and Eupergit 250L ® (Rohm) (Eupergit .RTM. C, a carrier for immobilization of enzymes of industrial potential. Katchalski-Katzir, E.; Kraemer, D. M. Journal of Molecular Catalysis B: Enzymatic (2000), 10(1-3), 157-176), is extremely preferred. Immobilization on Ni-NTA in combination with the polypeptide supplemented with the His tag (hexa-histidine) is likewise preferred (Purification of proteins using polyhistidine affinity tags. Bornhorst, Joshua A.; Falke, Joseph J. Methods in Enzymology (2000), 326, 245-254).
  • CLEC Cofactor-bound cross-linked enzyme crystals
  • the process according to the invention can be carried out such that the MDH from E. coli is coupled with an NAD-dependent leucine dehydrogenase (LeuDH from Bacillus cereus; Sigma) .
  • LeuDH catalyses the reductive amination of aliphatic keto acids, such as, for example, ketoisocaproate, to the corresponding L-amino acids, such as. L-leucine, with consumption of NADH (equation (1) ) .
  • Table 1 Comparison of the formation of L-leucine (HPLC) in a coupled batch with coenzyme regeneration by MDH and FDH. In each case 10 mM ketoisocaproate and for the regeneration by malate dehydrogenase (MDH) 100 mM L-malate or by formate dehydrogenase (FDH) 100 mM formate were employed.
  • MDH malate dehydrogenase
  • FDH formate dehydrogenase
  • the use of alcohol dehydrogenases in combination with malate dehydrogenases was also investigated.
  • the MDH from E. coli is coupled with an NAD-dependent S-specific alcohol dehydrogenase from Rhodococcus erythropolis (RE-ADH;
  • Enantiomerically enriched or enantiomer-enriched describes the fact that one optical antipode is present in a mixture with its other to >50%.
  • the structures shown relate to the two possible enantiomers, and if more than one stereo-centre is present in the molecule they relate to all the possible diastereomers and, in respect of a diastereomer, to the two possible enantiomers of the compound in question which fall under this.
  • Candida boidinii is deposited under number
  • Fig. 2 shows a membrane reactor with dead-end filtration.
  • the substrate 1 is transferred via a pump 2 into the reactor space 3, which contains a membrane 5.
  • the reactor space which is operated with a stirrer, are, in addition to the solvent, the catalyst 4, the product 6 and unreacted substrate 1.
  • Low molecular weight 6 is chiefly filtered off via the membrane 5.
  • Fig. 3 shows a membrane reactor with cross-flow filtration.
  • the substrate 7 is transferred here via the pump 8 into the stirred reactor space, in which is also solvent, catalyst 9 and product 14.
  • a solvent flow which leads via a heat exchanger 12, which may be present, into the cross-flow filtration cell 15 is established via the pump 16.
  • the low molecular weight product 14 is separated off here via the membrane 13.
  • High molecular weight catalyst 9 is then passed back with the solvent flow, if appropriate via a heat exchanger 12 again, if appropriate via the valve 11, into the reactor 10.
  • the purification of the recMDH from E. coli crude extracts was carried out in accordance with purification protocol (Stols L. , and Donnelly M. I. (1997). Production of succinic acid through overexpression of NAD (+) -dependent malic enzyme in an Escherichia coli mutant. Appl Environ Microbiol 63: 2695- 701.) .
  • the rec-bacteria cells were first broken down by disintegration with glass beads (breakdown buffer Tris/HCl 100 mM pH 7.5). Thereafter, a purification step by a Q- Sepharose was carried out.
  • Km values lie in a low range of ⁇ 1 mM, and they show that the two substrates are recognized by the enzyme with a good affinity. The two values suggest that the MDH can be used for the regeneration of NADH.
  • MDH shows a maximum activity at relatively high pH values of 11 and higher. Nevertheless, the drop in activity at lower pH values is relatively small, thus 72% activity is still present at pH 8.0, and 67% activity still at pH 7.0. - Optimum temperature
  • the optimum temperature of the MDH is approx. 55°C (fig. 4).
  • the MDH from E. coli is coupled with an NAD-dependent leucine dehydrogenase (LeuDH from Bacillus cereus; Sigma) .
  • LeuDH catalyses the reductive amination of aliphatic keto acids, such as, for example, ketoisocaproate, to the corresponding L-amino acids, such as L-leucine, with consumption of NADH.
  • Test batch (1 ml in total; unless stated otherwise the concentration of the stock solution is stated in parentheses) :
  • test batch is incubated at 30°C, and after 0, 10, 30, 60 and 120 min samples are taken (50 ⁇ l; Eppendorf reaction vessels) and heated for 3 min at 95°C to stop the reaction.
  • Denatured protein is separated off by centrifugation for 10 min at 13,000 rp (Eppendorf bench centrifuge) and the supernatant is analysed by means of HPLC, after derivatization with ortho-phthalaldehyde (OPA) .
  • OPA ortho-phthalaldehyde
  • the MDH from E. coli (expression strain: E. coli derivative JM 105) is coupled with an NAD-dependent S-specific alcohol dehydrogenase from Rhodococcus erythropolis (RE-ADH) .
  • the usability of the MDH is tested here via the reduction of a ketone (p-Cl-acetophenone) .
  • Test batch (1 ml in total; unless stated otherwise the concentration of the stock solution is stated in parentheses) :
  • test batch is incubated at 30°C, and after 0, 10, 20, 30 and 60 min samples (50 ⁇ l) are taken, 100 ⁇ l ethyl acetate are added and the upper phase is analysed by means of gas chromatography for the formation of the alcohol p- Cl-phenylethanol.
  • primers with integrated restriction cleavage sites and a codon for the ribosomal binding site were constructed.
  • the PCR fragment was cloned into the recombinant recPhe-pKK-223-3 expression vector after the
  • One cycle consists of:
  • the new construct of the recombinant plasmid (fig. 5) was transformed into competent E. coli cells JM 105 or HB 101.
  • the recombinant strain HB101 shows a malate dehydrogenase activity of 100 U/ml and likewise a PheDH activity of 130 U/ml.
  • the activity of the two enzymes in the recombinant strain JM 105 is clearly higher and is at ⁇ 600 U/ml for malate dehydrogenase and ⁇ 1,200 U/ml for the PheDH.
  • the two recombinant strains were cultured in a 10 1 fermenter and the activity of the two enzymes was determined.
  • Table 5 Determination of the activity of the expressed enzymes in a 10 1 fermenter with LB medium as batch fermentation.
  • the two heterologously expressed enzymes show the best stability properties under different conditions. It was of interest to determine the optimum properties for both enzymes in the same system. The following experiments were carried out with a view to this fact.
  • the breakdown buffer also influenced the activities. It was to be seen here that the suitable breakdown buffer was different for the individual enzymes.
  • the Kpi buffer was more suitable for the PheDH than for the malate dehydrogenase, but since the decrease in activity of the malate dehydrogenase in the Kpi buffer was relatively low, the recombinant cells continued to be broken down in this buffer after the heterologous expression.
  • the duration of the breakdown was also investigated and the critical point for stability of the enzymes during this operation was determined.
  • the optimum duration of breakdown can be obtained from these data (fig. 9 - Stability determination of the PheDH and the malate dehydrogenase after various breakdown times by means of ultrasound.
  • the cells were cooled intermediately for 30 seconds after a 60 and 30 second treatment.
  • the following suspension was used for this experiment:
  • Table 7 Protein determination of the two enzymes expressed, PheDH and malate dehydrogenase, after variation of the breakdown time. The cells were cooled intermediately for 30 seconds after a 60 and 30 second treatment.
  • the K M values were determined for the substrate and the coenzyme of the malate dehydrogenase.
  • the K M values were determined on homogeneous or partly purified rec-malate dehydrogenase samples.
  • Important factors for a coupled reaction are the optimum pH and the heat stability of the two enzymes. In addition to the stability of the enzymes, further factors play a role, such as e.g. the influence of the various substrates on the enzymes .
  • a kinetics study was conducted and the pH-dependency of the synthesis was determined. The increase in the activity at an increasing pH can be seen from fig. 10, a pH of 8.0, in which although the two enzymes do not show the highest activity, the coenzyme remains, stable, being chosen for the synthesis for coenzyme stability reasons (fig. 10 - Optimum pH of malate dehydrogenase and PheDH. The activity of the two enzymes increases as the pH increases. The measurements were carried out with partly purified enzyme. For the phenylalanine dehydrogenase, the reductive amination was measured) .
  • a second factor for the coupled enzyme reaction is the suitable temperature at which the two enzymes remain stable for a relatively long period of time.
  • a further experiment was therefore carried out to determine the optimum temperature (fig. 11 - Optimum temperature. The measurements were carried out at pH 8.5 and in 0.1 M HEPES buffer) .
  • the optimum temperature of both enzymes is 50°C. At 30°C the activity measured is only 60%.
  • the malate dehydrogenase is stable at 45°C for a relatively long period of time.
  • the syntheses were carried out at 30°C, so that it was possible to ensure the stability of both enzymes and of the coenzyme over a relatively long period of time.
  • Enzymes achieve their optimum activity in the particular suitable buffer.
  • the two enzymes were tested with two different buffers in each case in a reaction batch (table 9).
  • Table 9 Comparison of the PheDH and malate dehydrogenase activity in various reaction buffers. The activities are to be seen in per cent of the optimum.
  • the conversion was carried out at 30°C and with 1 g of recombinant E. coli cells.
  • the phenylalanine formed was detected by means of HPLC (fig. 6) .

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
EP03793686A 2002-09-03 2003-08-05 Verwendung von malat dehydrogenase für die regenerierung von nadh Withdrawn EP1534849A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10240603 2002-09-03
DE10240603A DE10240603A1 (de) 2002-09-03 2002-09-03 Verwendung von Malat-Dehydrogenase zur NADH-Regenerierung
PCT/EP2003/008631 WO2004022764A2 (en) 2002-09-03 2003-08-05 Use of malate dehydrogenase for nadh regeneration

Publications (1)

Publication Number Publication Date
EP1534849A2 true EP1534849A2 (de) 2005-06-01

Family

ID=31502323

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03793686A Withdrawn EP1534849A2 (de) 2002-09-03 2003-08-05 Verwendung von malat dehydrogenase für die regenerierung von nadh

Country Status (6)

Country Link
EP (1) EP1534849A2 (de)
JP (1) JP2005537017A (de)
AU (1) AU2003255368A1 (de)
CA (1) CA2497499A1 (de)
DE (1) DE10240603A1 (de)
WO (1) WO2004022764A2 (de)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10313972A1 (de) * 2003-03-27 2004-10-21 Degussa Ag Gekoppeltes cofaktorabhängiges enzymatisches Reaktionssystem
DE102004008445A1 (de) 2004-02-19 2005-09-08 Degussa Ag Verfahren zur Herstellung von L-Aminosäuren aus D-Aminosäuren
DE102004014280A1 (de) * 2004-03-22 2005-10-27 Degussa Ag Verfahren zur Herstellung von optisch aktiven Aminosäuren mittels eines Ganzzellkatalysators
DE102004028407A1 (de) * 2004-06-14 2005-12-29 Degussa Ag Herstellung optisch aktiver Alkohole mit Hilfe von Ganzzellkatalysatoren
DE102004038054A1 (de) * 2004-08-05 2006-03-16 Maxens Gmbh Verfahren zur Herstellung primärer Alkohole
TW201343623A (zh) 2012-02-07 2013-11-01 Annikki Gmbh 使氧化還原輔因子經酶催化再生之方法
WO2013117251A1 (de) 2012-02-07 2013-08-15 Annikki Gmbh Verfahren zur enzymatischen regenerierung von redoxkofaktoren

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61128895A (ja) * 1984-11-26 1986-06-16 Takara Shuzo Co Ltd 補酵素再生反応を利用した有用物質の製造方法
JPH01285193A (ja) * 1988-05-12 1989-11-16 Daicel Chem Ind Ltd Ｄ−アスパラギン酸の製造方法
EP0385415B1 (de) * 1989-02-28 1995-06-07 Mitsubishi Petrochemical Co., Ltd. Verfahren zur Herstellung von NADH-Oxydase
FI980551A7 (fi) * 1998-03-11 1999-09-12 Valtion Teknillinen Transformoidut mikro-organismit, joilla on parannettuja ominaisuuksia
JP2000236883A (ja) * 1998-12-21 2000-09-05 Daicel Chem Ind Ltd 新規なカルボニル還元酵素、該酵素の製造方法、該酵素をコードするｄｎａおよびこれを利用したアルコールの製造方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2004022764A2 *

Also Published As

Publication number Publication date
WO2004022764A2 (en) 2004-03-18
CA2497499A1 (en) 2004-03-18
WO2004022764A3 (en) 2004-10-21
AU2003255368A8 (en) 2004-03-29
JP2005537017A (ja) 2005-12-08
DE10240603A1 (de) 2004-03-11
WO2004022764A9 (en) 2005-04-14
AU2003255368A1 (en) 2004-03-29

Similar Documents

Publication Publication Date Title
Van Der Donk et al. Recent developments in pyridine nucleotide regeneration
Chaparro‐Riggers et al. Comparison of three enoate reductases and their potential use for biotransformations
US12060584B2 (en) D-amino acid oxidase mutants and uses thereof in preparing L-glufosinate
US20090203096A1 (en) Process for Production of Optically Active Alcohol
JP4757804B2 (ja) 新規カルボニル還元酵素、その遺伝子、およびその利用法
EP1499716B1 (de) Adh aus rhodococcus erythropolis
US9273332B2 (en) Method for production of L-amino acid
CN111019982B (zh) 一种利用羟基酸脱氢酶制备l-草铵膦的方法
WO2004022764A2 (en) Use of malate dehydrogenase for nadh regeneration
JP2007529234A (ja) 光学活性アミノ酸をホールセル触媒を用いて製造する方法
US20080145904A1 (en) Method For Producing Primary Alcohols
WO2007099764A1 (ja) 新規カルボニル還元酵素、その遺伝子、およびそれらを利用した光学活性アルコールの製造方法
JP4688313B2 (ja) 新規なエノン還元酵素、その製造方法、およびこれを利用したα，β−不飽和ケトンの炭素−炭素２重結合を選択的に還元する方法
US12480148B2 (en) Method for preparing (s)-1,2,3,4-tetrahydroisoquinoline-1 carboxylic acid and derivatives thereof
US7247465B2 (en) Screening process for hydantoin racemases
JP4729919B2 (ja) 微生物の培養方法及び光学活性カルボン酸の製造方法
JP4270918B2 (ja) 新規カルボニル還元酵素及びこれをコードする遺伝子、ならびにこれらを利用した光学活性アルコールの製造方法
CA2629216A1 (en) Process for preparing 1,1,1-trifluoroisopropanol predominantly comprising one enantiomer
AU2020103435A4 (en) Method for preparing (s)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid and derivatives thereof
WO2007028729A1 (en) Nocardia globerula alcohol dehydrogenase and use thereof
JP2008502334A (ja) 全菌体触媒を用いる光学活性アルコールの製法
WO2024010785A1 (en) Ketoreductase enzymes for the synthesis of 1,3-diol substituted indanes
CN121320288A (zh) 一种亮氨酸脱氢酶突变体及其应用
CN118406724A (zh) 一种四酶级联生物催化制备苯乳酸的方法
JP2007274901A (ja) 光学活性プロパルギルアルコールの製造方法

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20050126

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20060301