Classifications

• • 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
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
• • 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
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic

Definitions

• the present invention relates to a process for the production of primary alcohols from aldehydes.
• the reduction is accomplished by cofactor-dependent oxidoreductases, again regenerating the cofactor by a second enzymatic system.
• the production of primary alcohols is of interest to the food industry due to the use of these products as aroma chemicals in direct form or after conversion to corresponding ester compounds.
• a preferred preparation is the recovery of the primary alcohols by reduction of aldehydes. This could be done in principle by the use of non-natural chemical reducing agents according to numerous existing literature regulations, for example by the use of metal hydrides. However, only “naturally identical” but not “natural” primary alcohols would be obtained in this way. However, the extraction of natural primary alcohols is of particular importance for the food industry. One way to produce them is in the enzymatic reduction of aldehydes.
• Substrate concentration of 0.05 g per L reaction volume is in the presence of Botyris cinerea strains of G. Bock et al. Bock et al., Z. Lebensm. Unters. Forsch. 1988, 186, 33-35). The highest yields were 0.019 and 0.025 g per L reaction volume.
• Phenylethanol with a product concentration of 12.6 g / L, corresponding to an amount of about 100 mM (D. Stark, T. Münch, B. Sonnleitner, IW Marison, U. von Stockar, Biotechnol., Prog. 514-523).
• Alcohol dehydrogenase from Rhodococcus erythropolis is used after an activity for trans-2-hexenal has been found for these enzymes.
• the cofactor regeneration was carried out with a formate dehydrogenase after this cofactor regenerating enzyme was used successfully in many cases in the enzymatic reduction of ketones (M.R. KuIa, U. Kragl, Dehydrogenases in the Synthesis of Chiral Compounds in: Stereoselective Biocatalysis (ed. RN Patel), Marcel Dekker, New York, 2000, chapter 28, p. 839f.).
• M.R. KuIa U. Kragl
• the object of the present invention was therefore the development of a fast, simple, inexpensive and effective enzymatic process for the preparation of primary alcohols from aldehydes.
• the space / time yield should be improved over the known methods of the prior art.
• this requires working at high substrate concentrations with correspondingly good yields in a robust one
• Claim 1 relates to a method according to the invention, in which a rec-whole-cell catalyst is used.
• Claim 2 comprises a preferred embodiment of this method.
• Claim 3 is directed to the use of the free enzymes for the purpose of the invention. Claims 3 to 9 protect preferred embodiments of the method according to the invention,
• recombinant (rec-) whole-cell catalyst containing an alcohol dehydrogenase and an enzyme capable of cofactor regeneration from the group of glucose dehydrogenases or malate dehydrogenases.
• Alcohol dehydrogenase with an enzyme capable of cofactor regeneration from the group of glucose dehydrogenases or malate dehydrogenases as isolated enzymes, in particular taking into account the non-satisfactory yields in the same application the formate dehydrogenase (see also example 4 comparative example).
• the reaction takes place at high substrate concentrations of> 150 mM, in particular> 250 mM and very preferably> 500 mM of aldehyde.
• Substrate concentrations of> 150 mM are to be understood as meaning that> 150 mM of the substrate are reacted per starting volume of aqueous solvent (including buffer system) using the method shown
• Concentration in the reaction mixture can actually be achieved, or whether a total of the starting volume of aqueous solvent, a substrate concentration of> 150 mM is reacted.
• the variant in which a substrate concentration of> 150 mM etc. of aldehyde is actually provided for the reaction is very particularly preferred.
• concentrations referred to here refer to concentrations of the substrate (aldehyde) actually obtained in the reaction mixture based on the starting volume of aqueous solvent, irrespective of when in the course of the incubation time of an all-cell catalyst or isolated enzymes (in purified or partially purified form or as crude extract) these Initial concentration is reached. It is only reached at least once.
• the aldehyde When using a whole-cell catalyst, the aldehyde can be used in the form of a "baten" approach directly at the beginning of a whole-cell approach at these concentrations, or it can first be a whole-cell catalyst to a certain optical density are used before the aldehyde is added however, in the approach, preferably at least once in the batch, a concentration of substrate of> 150 inM, etc. during the conversion of the substrate to the desired alcohol This applies mutatis mutandis to higher concentrations.
• aldehyde component all aldehydes can be used.
• the aldehyde component used can be subsumed under the following general structural formula
• R denotes (C 1 -C 2 O) -alkyl, (C 2 -C 20 ) -alkenyl
• one of the preferred enzymes to be selected is an alcohol dehydrogenase.
• the skilled person is free in the choice of alcohol dehydrogenase.
• preferred alcohol dehydrogenases are alcohol dehydrogenases from a Lactobacillus strain, in particular from Lactobacillus kefir and Lactobacillus brevis, or alcohol dehydrogenases from a Rhodococcus strain, in particular from Rhodococcus erythropolis and Rhodococcus ruber, or alcohol dehydrogenases from an Arthrobacter strain, in particular from Arthrobacter paraffineus ,
• Preferred dehydrogenases for cofactor generation have been glucose dehydrogenases, preferably a glucose dehydrogenase from Bacillus, Thermoplasma and Pseudomonas strains.
• Glucose dehydrogenases are described, for example, in A. Bommarius in: Enzyme Catalysis in Organic Synthesis (Ed .: K. Drauz, H. Waldmann), Volume III, Wiley-VCH, Weinheim, 2002, Chapter 15.3.
• Malate dehydrogenases are well known to those skilled in the art (S.-I. Suye, M. Kawagoe, S. Inuta, Can. J. Chem. Eng 1992, 70, 306-312; S.-I. Suye, Recent Res. Devel. Ferment Bioeng 1998, 1, 55-64, Dissertation S. Naamnieh, University of Dusseldorf, W02004 / 022764). Again, the skilled person will select the most efficient dehydrogenase for his purpose. In principle, those malate dehydrogenases are preferred which regenerate the NAD (P) H to such an extent that no bottleneck occurs for the reaction of the other enzyme used.
• P NAD
• Malate enzyme is preferably a "malic enzyme” from Sulfolobus, Clostridium, Bacillus and Pseudomonas strains and from E. coli used. Most preferably, the known malate dehydrogenase from E. coli K12 is in this context. Gene isolation and cloning are described in S. Naamnieh, Dissertation, University of Dusseldorf, p. 7Off.
• the addition of the aldehyde can be done in any manner.
• the aldehyde is added in the total amount from the beginning ("batch” approach) or alternatively metered in.
• a continuous addition (“continuous feed-in process”) can also be used. These procedures are well known to the person skilled in the art and are used analogously in the present case.
• a "recombinant whole-cell catalyst” is to be understood as meaning a cell in which at least one recombinant gene is expressed or expressed - ie at least one recombinant protein is present which has the inventive conversion (reduction of the aldehyde and / or regeneration of the cofactor) can catalyze.
• the recombinant proteins are not limited to being present in living or non-living whole-cell catalysts, but may be in any active form.
• active form is meant the ability of the recombinant protein to catalyze an enzymatic reaction.
• the recombinant protein is distributed exclusively in free form over the cytosol of the cell and not in the form of inclusion bodies.
• the cell is or was able to express an alcohol dehydrogenase and a dehydrogenase capable of cofactor regeneration.
• microorganisms for the whole cell catalyst containing an alcohol dehydrogenase and for cofactor regeneration Enabled enzyme, all known cells are suitable.
• microorganisms in this regard are organisms such as yeasts such as Hansenula polymorpha, Pichia sp., Saccharomyces cerevisiae, prokaryotes such as E. coli, Bacillus subtilis or eukaryotes such as mammalian cells, insect cells or
• E. coli strains are to be used for this purpose. Most particularly preferred are: E.
• Plasmids with which the gene construct comprising the nucleic acid according to the invention is preferably cloned into the host organism are likewise known to the person skilled in the art (see also PCT / EP03 / 07148, see below).
• Such plasmids and vectors may, for. B. Studier and coworkers (Studier, WF, Rosenberg AH, thin JJ, Dubendroff JW, (1990), Use of the T7 RNA polymerase to direct expression of cloned genes, Methods Enzymol 185, 61-89) or the leaflets of the Companies Novagen, Promega, New England Biolabs, Clontech or Gibco BRL. Further preferred plasmids and vectors can be found in: Glover, D.M. (1985), DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd. , Oxford; Rodriguez, R.L. and Denhardt, D. T (eds)
• Plasmids with which the gene constructs comprising the nucleic acid sequences of interest can be cloned into the host organism are or are based on: pUC18 / 19 (Roche Biochemicals), pKK-177-3H (Roche Biochemicals), pBTac2 (Roche Biochemicals ), pKK223-3 (Amersham Pharmacia Biotech), pKK-233-3 (Stratagene) or pET (Novagen).
• the whole-cell catalyst is preferably pretreated prior to its use in such a way that the permeability of the cell membrane for the substrates and products is increased compared to the intact system.
• a process in which the whole-cell catalyst is pretreated for example, by freezing and / or treatment with an organic solvent, in particular toluene.
• Whole-cell catalyst containing an alcohol dehydrogenase from R. erythropolis or Lactobacillus kefir and a malate dehydrogenase are also recombinant whole cell catalysts with an alcohol dehydrogenase from R. erythropolis or Lactobacillus kefir and a glucose dehydrogenase from Thermoplasma acidophilum or Bacillus subtilis are suitable in all possible combinations.
• the two particularly preferred recombinant whole-cell catalysts are those described in the experimental section.
• the concentration of this recombinant whole-cell catalyst is preferably at most 75 g / L, in a further preferred embodiment up to 50 g / L, extremely preferably at up to 25 g / L and more preferably at up to 15 g / L, where g refers to g of wet biomass (BFM).
• reaction temperatures which are suitable in particular for the recombinant whole-cell catalyst used.
• a particularly suitable reaction temperature is to be regarded as a reaction temperature which is from 10 to 90 ° C., preferably from 15 to 50 ° C., and particularly preferably from 20 to 35 ° C.
• the reaction can be carried out both at a fixed pH, and under variation of the pH in a pH interval.
• the pH is chosen in particular taking into account the needs of the host organism used or the isolated enzymes used.
• the reaction is preferably carried out at a pH which is at pH 5 to 9, preferably pH 6 to 8 and particularly preferably pH 6.5 to 7.5.
• the reaction of the substrate used to the desired product is preferably carried out in cell culture using a suitable recombinant whole-cell catalyst.
• a suitable nutrient medium is used.
• the media suitable for the host cells are well known and commercially available.
• the cell cultures may also be supplemented with conventional additives, e.g. Antibiotics, growth promoting agents, e.g. Serums (fetal calf serum, etc.) and similar known additives.
• the conversion of the aldehyde to the desired primary alcohol is carried out without addition of an organic solvent.
• an organic solvent preferably those which are water-soluble, to the water additive necessary for carrying out the reaction.
• further organic solvents preferably those which are water-soluble, to the water additive necessary for carrying out the reaction.
• water-soluble organic solvents such as alcohols, in particular methanol or ethanol, or ethers, such as THF or dioxane understood.
• a cell suspension of the suitable recombinant whole-cell catalyst wherein the aldehyde used in the cell suspension.
• the aldehyde used in the cell suspension may also be present as a suspension, or in the form of an emulsion or solution in the cell suspension.
• the corresponding cofactor is added in suitable amounts in a preferred embodiment.
• the cofactor amounts added are in the range of 0 ' .00001 to 0.1 equivalents, preferably 0.0001 to 0.01 and more preferably 0.0001 and
• an "external" cofactor additive can be dispensed with or such an “external” cofactor additive can be used in the amount range below 0.0005 equivalents.
• the recombinant whole-cell catalyst or the isolated enzymes and the substrate are initially introduced in the chosen solvent system.
• a specific amount of the cofactor required for this purpose for example NADH or NADPH or their oxidized forms NAD + and NADP +
• NADH or NADPH or their oxidized forms NAD + and NADP + can then be added to the reaction mixture.
• the order of addition is variable.
• the work-up of the reaction mixture takes place according to methods known in the art.
• the biomass can be easily separated from the product by filtration or centrifugation.
• the alcohol obtained can then be isolated by conventional methods (eg extraction, distillation, crystallization).
• the subject method can also be carried out continuously.
• the reaction is carried out in a so-called enzyme membrane reactor in which high molecular weight substances - the enzymes or biomass - are retained behind an ultrafiltration membrane and low molecular weight substances - such as the amino acids produced - can pass through the membrane.
• enzyme membrane reactor in which high molecular weight substances - the enzymes or biomass - are retained behind an ultrafiltration membrane and low molecular weight substances - such as the amino acids produced - can pass through the membrane.
• Particularly suitable (C 1 -C 20 ) -alkyl radicals are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl , Dodecyl with all its binding isomers.
• a (C 1 -C 8 ) -alkyl radical is analogous to one in which 1 to 8 carbon atoms are present in the chain.
• (C 2 -C 20) alkynyl group refers to a (Ci-C 20) -alkyl radical as described above with at least one C ⁇ C triple bond.
• the radical (C 1 -C 2 O) -alkoxy corresponds to the radical (C 1 -C 20 ) -alkyl, with the proviso that it is bonded to the molecule via an oxygen atom. The same applies to a (C 1 -C 8 ) -alkoxy radical.
• (C 2 -C 20 ) alkoxyalkyl residues in which the alkyl chain is interrupted by at least one oxygen function, whereby two oxygen atoms can not be linked together.
• the number of Carbon atoms indicate the total number of carbon atoms in the rest.
• radicals just described may be monosubstituted or polysubstituted by halogens and / or N, O, P, S, Si atom-containing radicals.
• halogens and / or N, O, P, S, Si atom-containing radicals.
• alkyl radicals of the abovementioned type which have one or more of these heteroatoms in their chain or which are bonded to the molecule via one of these heteroatoms.
• (C 3 -C 8 ) -cycloalkyl is meant cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl radicals, etc. These may be with one or more halogens and / or N, O, P, S, Si atom-containing radicals be substituted and / or have N, 0, P, S atoms in the ring, such as. 1-, 2-, 3-, 4-piperidyl, 1-, 2-, 3-pyrrolidinyl, 2-, 3-tetrahydrofuryl, 2-, 3-, 4-morpholinyl.
• a (C 3 -C 8 ) -cycloalkyl (C 1 -C 20 ) -alkyl radical denotes a cycloalkyl radical as described above which is bonded to the molecule via an alkyl radical as specified above.
• (Ci-C 8 ) -Acyloxy in the context of the invention means an alkyl radical as defined above with max. 8 C atoms, which is bound to the molecule via a COO function.
• (C 1 -C 8 ) acyl means in the context of the invention an alkyl radical as defined above with max. 8 C atoms, which is bound to the molecule via a CO function.
• a (C 6 -C 8 ) -aryl radical is meant an aromatic radical having 6 to 18 C atoms.
• these include compounds such as phenyl, naphthyl, anthryl, phenanthryl, biphenyl radicals or systems of the type described above, such as, for example, indenyl systems which are optionally substituted by (C 1 -C 8 ) -alkyl, (C 1 -C 8 ) -alkyl.
• C 8 ) - alkoxy, NR 1 R 2 , (Ci-C 8 ) acyl, (Ci-C 8 ) acyloxy may be substituted.
• a (C 7 -C 26 ) aralkyl radical is a (C 6 -C 18 ) -aryl radical bonded to the molecule via a (C 1 -C 8 ) -alkyl radical.
• a (C 3 -C 18 ) heteroaryl radical in the context of the invention denotes a five-, six- or seven-membered aromatic ring system of 3 to 18 C atoms, which
• Heteroatoms such. B. nitrogen, oxygen or sulfur in the ring.
• Particular examples of such heteroaromatics are radicals such as 1-, 2-, 3-furyl, such as 1-, 2-, 3-pyrrolyl, 1-, 2-, 3-thienyl, 2-, 3-, 4-pyridyl , 2-, 3-, 4-, 5-, 6-, 7-indolyl, 3-, 4-, 5-pyrazolyl, 2-, 4-, 5-imidazolyl, acridinyl, quinolinyl, phenanthridinyl, 2-, 4 , 5, 6-pyrimidinyl.
• a (C 4 -C 26 ) heteroaralkyl is meant a heteroaromatic system corresponding to the (C 7 -C 26 ) aralkyl radical.
• Halogens come fluorine, chlorine, bromine and iodine in question.
• isolated enzyme is understood according to the invention to mean the use of alcohol dehydrogenase and of the enzyme capable of cofactor regeneration from the group of glucose dehydrogenases or malate dehydrogenases as isolated enzymes (in purified or partially purified form or as crude extract or in immobilized form).
• Malate dehydrogenase catalyzes the oxidative decarboxylation of malate to pyruvate.
• Many malate dehydrogenases from various organisms are known, including higher animals, plants, and microorganisms.There are four types of malate dehydrogenases that are known in the United States
• Enzyme classes EC 1.1.1.37 to EC 1.1.1.40 are classified (http://www.genome.ad.jp). Depending on the type of Malate dehydrogenase requires NAD and / or NADP as cofactor.
• the E. coli used in the examples was deposited by the applicant under the number DSM 14459 at DSMZ GmbH, Mascheroder Weg Ib, D-38124 Braunschweig on 24.08.01 under the Budapest Treaty.
• FIG. 1 shows the plasmid map of the plasmid pNO5c
• FIG. 2 shows the plasmid map of the plasmid pNO8c
• Figure 3 shows the plasmid map of plasmid pN014c
• This plasmid encodes Lactobacillus kefir alcohol dehydrogenase (Lactobacillus kefir alcohol dehydrogenase: a useful catalyst for synthesis, Bradshaw et al JOC 1992, 57 1532-6, Reduction of acetophenone to R (+) -phenylethanol by a novel alcohol dehydrogenase from Lactobacillus kefir Hummel W Ap Microbiol Biotech 1990, 34, 15-19).
• Lactobacillus kefir alcohol dehydrogenase Lactobacillus kefir alcohol dehydrogenase
• coli DSM14459 (pNO5c-1) was made chemically competent and transformed with the plasmid pNO8c ( Figure 2) which encodes the gene of a codon-optimized glucose dehydrogenase from Thermoplasma acidophilum (Bright, JR et al., 1993 Eur. J. Biochem 211: 549-554). Both genes are under the control of a rhamnose promoter (Stumpp, Tina, Wilms, Burkhard, Altenbuchner, Josef A new, L-rhamnose-inducible expression system for Escherichia coli BIOspektrum (2000), 6 (1), 33-36). Sequences and plasmid maps of pN05c and pNO8c are given below. Production of active cells
• E. coli DSM14459 (pN05c, pN08c) was incubated in 2 ml LB medium supplemented with antibiotics (50 ⁇ g / l ampicillin and 20 ⁇ g / ml chloramphenical) for 18 hours at 37 ° C, shaken (250 rpm).
• This culture was diluted 1: 100, shaken in fresh LB medium with rhamnose (2 g / 1) as inducer, added antibiotics (50 ug / l ampicillin and 20 ug / ml chloramphenical) and IMM ZnCl2 diluted and 18 hours at 30 0 C (250 rpm ), incubated.
• Cells were centrifuged (10000g, 10min, 4 0 C), discarded the supernatant and the cell pellet used directly or after storage at -2O 0 C in biotransformation experiments.
• Glucose dehydrogenase from Bacillus subtilis expressed in Escherichia coli Purification, characterization and comparison with glucose dehydrogenase from Bacillus megaterium. HiIt W; Pfleiderer G; Fortnagel P Biochimica et biophysica acta (1991 Jan 29), 1076 (2), 298-304). Alcohol dehydrogenase is under the control of a rhamnose promoter (Stumpp, Tina, Wilms, Burkhard, Altenbuchner, Josef A new, L-rhamnose-inducible expression system for Escherichia coli BIOspektrum (2000), 6 (1), 33-36). The sequence and plasmid map of pN014c is given below.
• E. coli DSM14459 (pN014c - Fig. 3) was incubated in 2 ml LB medium supplemented with antibiotics (50 ⁇ g / l ampicillin and 20 ⁇ g / ml chloramphenical) shaken for 18 hours at 37 ° C (250 rpm).
• This culture was diluted 1: 100 in fresh LB medium with rhamnose (2 g / l) as an inducer, antibiotics (50 ⁇ g / l ampicillin and 20 ⁇ g / ml chloramphenical) and ImM ZnCl2 and shaken for 18 hours at 30 0 C (250 rpm ), incubated.
• Cells were centrifuged (10000g, 10min, 4 ° C), the supernatant discarded and the cell pellet used directly, or after storage at -2O 0 C in biotransformation experiments.
• Example 1 Reduction of cinnamaldehyde in a 0.2 M solution using a whole-cell catalyst containing an alcohol dehydrogenase and glucose dehydrogenase
• Cinnamaldehyde (corresponding to a substrate concentration based on phosphate buffer used of 0.2 M) was added.
• the reaction mixture is stirred at room temperature, the pH being kept constant by addition of sodium hydroxide solution (2M NaOH) (to pH 6.5).
• Samples are taken at regular intervals and the conversion of cinnamic aldehyde to cinnamyl alcohol is determined by HPLC. Sales were 93% at 1 hour and 100% at 2 hours.
• Example 2 Reduction of cinnamaldehyde in a 0.5 M solution using a whole-cell catalyst containing an alcohol dehydrogenase and glucose dehydrogenase
• reaction mixture is stirred for 25 h at room temperature, the pH being kept constant by addition of sodium hydroxide solution (2M NaOH) (to pH 6.5).
• 2M NaOH sodium hydroxide solution
• Samples are taken at regular intervals and the conversion of cinnamic aldehyde to cinnamyl alcohol is determined by HPLC. Sales were 44% after 1 hour, 91% after 5h and 93% after 25h.
• Example 3 Reduction of cinnamaldehyde in a 1.5 M solution using a whole-cell catalyst containing an (R) -selective alcohol dehydrogenase
• Cinnamaldehyde (corresponding to a substrate concentration based on phosphate buffer used of 1.5 M) was added.
• the reaction mixture is stirred at room temperature, the pH being kept constant by addition of sodium hydroxide solution (5M NaOH). Samples are taken at regular intervals and the conversion of cinnamic aldehyde to cinnamyl alcohol is determined by HPLC. Sales were 15% after 1 hour, 58% after 5 hours and 98% after 23.5 hours.
• Example 4 (Comparative Example): Reaction of trans-2-hexenal at 100 mM using a formate dehydrogenase for cofactor regeneration
• reaction mixture consisting of trans-2-hexenal (100 mM) and NADH (3.5 mM, corresponding to 0.035 equivalents based on the aldehyde), sodium formate (455 mM, corresponding to 4.55 equivalents based on the aldehyde) at enzyme quantities of 20 U / mmol an (S) -ADH from R. erythropolis (expr in E. coli) and 20 U / mmol of a
• Formate dehydrogenase from Candida boidinii is allowed to stir at a reaction temperature of 30 0 C over a period of 72 hours in 1 mL of a phosphate buffer (100 mM, pH 7.0). During this period, samples are taken and the respective turnover is determined via HPLC. Sales were 7% after 5h and 16% after 72h.
• Example 5 Reaction of tra ⁇ s-2-hexenal at 100 mM using a glucose dehydrogenase for cofactor regeneration
• a reaction mixture consisting of trai-S-2-hexenal (100 mM) and NADH (1.4 mM, corresponding to 0.014 equivalents based on the aldehyde), glucose (300 mM, corresponding to 3 equivalents based on the aldehyde) at enzyme quantities of 20 U / mmol of an (S) -ADH from R. erythropolis (expr. in E. coli) and 150 U / mmol of a glucose dehydrogenase from Bacillus sp., Is allowed at a reaction temperature of 30 0 C over a period of 72 hours in 1 mL of a phosphate buffer (100 mM, pH 7.0).
• Example 6 Implementation of. trans-2-hexenal at 100 mM using a glucose dehydrogenase for cofactor regeneration
• a reaction mixture consisting of trans-2-hexenal (100 mM) and NADPH (1 mM, corresponding to 0.01 equivalent based on the aldehyde), magnesium chloride (5 mM), glucose (300 mM, corresponding to 3 equivalents based on the aldehyde) Enzyme amounts of 13 U / mmol of an (R) -ADH from L. kefir and 60 U / mmol of a glucose dehydrogenase from Thermoplasma acidophilum, is allowed at a
• Example 7 Reduction of trans-2-hexenal in a 0.5 M solution using a whole-cell catalyst containing an alcohol dehydrogenase and glucose dehydrogenase

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