DE102004059376A1 - GDH mutant with improved chemical stability - Google Patents

Abstract

The invention relates to glucose dehydrogenase having an amino acid sequence which corresponds to the amino acid sequence of a Bacillus subtilis GDH comprising a glutamic acid residue at position 96, with the difference that the glutamic acid residue at position 96 is replaced by a polar or hydrophobic amino acid or glycine.

Description

The The invention relates to a mutant of the enzyme glucose dehydrogenase (GDH) of the species Bacillus subtilis with high process stability in biotransformations, their preparation and use for the regeneration of NADH, or NADPH redox cofactors in enzymatically catalyzed reduction reactions.

optical active hydroxy compounds are valuable synthetic building blocks, eg. As in the manufacture of pharmaceutical agents or agrochemicals. They therefore have an important economic importance. These connections are often difficult to produce by classical chemical methods. The required optical purities for pharmaceutical applications or agrochemicals are difficult to achieve in this way. Therefore, the production of chiral compounds is increasing Dimensions biotechnological Method applied. Especially enzymes, the carbonyl compounds can reduce because of their high enantioselectivity increased use.

enzymes from the class of oxidoreductases used to make chiral Compounds used by reduction of prochiral carbonyl compounds are, by definition, with the Collective term carbonyl reductases (hereinafter "CR") is designated. In the majority of cases the product of a CR reaction an alcohol. But it is also possible the product of a CR reaction is an amine. Among the collective term Carbonyl reductases summarized enzyme classes are among other aldehyde dehydrogenases (hereinafter "ADH"), aldo-ketoreductases ("AKR"), aldehyde reductases, Glycerol dehydrogenases and fatty acid synthase (referred to as "FAS"), but also aminotransferases or amino acid dehydrogenases (eg, threonine dehydrogenase) are to be counted among the CRs. this broad spectrum of reducing enzymes is common to them Electrons for the reduction of the carbonyl compound from redox cofactors in their reduced form, usually NADH or NADPH. These redox cofactors are used in the Reaction stoichiometric consumed, d. H. You need to either stoichiometric be used or regenerated by oxidation of a cosubstrate become. The stoichiometric Use of NADH or NADPH is due to the high price of this Uneconomical connections. This disadvantage can be caused by the so-called. Cofactor regeneration can be bypassed. Prerequisite for this is a cheap reducing agent and a cofactor reducing enzyme. First the efficient and cheap regeneration of the redox cofactor allows the technical use of biocatalytic reduction processes.

Various options for cofactor regeneration are known: when using a CR the class of ADH's is the cofactor regeneration by oxidation of a cheap alcohol (Cosubstrate), e.g. As isopropanol, possible. So it will be the stereoselective Obtained reduction of the substrate and the cofactor regeneration of an enzyme. It owns ADH's in general the disadvantage that their pH optimum reduction and Oxidation are very different, so the overall reaction only proceeds at a reduced rate, which affects the space-time yields. So z. For example the ADH from Rhodococcus erythropolis has a pH optimum for the reduction from 5.5, for the oxidation, on the other hand, of 9.5 (Zelinski et al., J. Biotechnol. (1994) 33: 283-292). additional Disadvantages are that not all ADH's react with isopropanol and also at the wished high space-time yields great Amounts of isopropanol must be used, or large quantities incurred in acetone, the reaction product of Isopropanoloxidation. As is known, Both compounds are enzyme damaging in high concentrations. Finally, can Setting a reaction equilibrium will cause quantitative reaction sales can not be achieved, resulting in reduced yields and similar physico-chemical properties of starting material (the substrate of CR reaction) and product often lead to difficulties in product isolation leads.

at Non-ADH CRs undergo cofactor regeneration due to a lack of cosubstrate in principle in a different way. Known is the simultaneous use of the CR with a second enzyme. A CR reduces the substrate stereoselectively to the desired Product. The regeneration of the spent NADH or NRDPH cofactor is accomplished by a second enzyme. In principle, each is Enzyme suitable for cofactor regeneration, which in an enzymatic Reaction of a substrate oxidized and simultaneously NRD to NADH, or NADP reduced to NADPH. However, one will use an enzyme that a cheap as possible Substrate oxidized. Enzymes that can be used for cofactor regeneration are z. B. formate dehydrogenase (FDH), glutamate dehydrogenase, glycerol dehydrogenase and glucose dehydrogenase (GDH).

In whole cell biotransformations, cofactor regeneration may also be due to the cellular matter change. A well-known example of this is biotransformations of the baker's yeast Saccharomyces cerevisiae. However, not every microorganism is suitable for it. Disadvantages of these biotransformations are the generally low space-time yields and the requirement for an intact cellular metabolism.

Under The known cofactor-regenerating enzymes are FDHs only for the regeneration of the NADH cofactor. Disadvantage of the FDH is the known low specific activity of the enzyme, giving low space-time yields conditionally. Furthermore is to be expected that formic acid (the cosubstrate) or their Salts in large Concentrations enzyme damaging to act on the CR. FDH mutants with specificity for NRDPH have been described the use on an industrial scale is of little use because of its low activity.

glutamate dehydrogenase is suitable for regenerating NADPH, glutamic acid being α-ketoglutarate is oxidized. about their use for cofactor regeneration is poorly understood.

GDH oxidizes glucose to δ-gluconolactone, that in watery solution continue to gluconic acid dehydrated. Advantages of GDH, especially the enzymes of the genus Bacillus, are a high specific activity of the enzyme as well as the ability to Both NRDH and NADPH regenerate cofactors. Thus, high space-time yields and a broad use of the enzyme in conjunction with virtually all CRs guaranteed. Besides that is Glucose as Cosubstrat a cheap reducing agent. Disadvantage of known GDH's is the bad chemical stability of the enzyme, what the use greater Requires quantities of enzyme.

The most widely used GDH for cofactor regeneration is a GDH from Bacillus megaterium (reviewed in a review by Kataoka et al., Appl. Microbiol. Biotechnol. (2003) 62: 437-445). GDH from B. megaterium is used for biotransformations ( EP 0967271 ). The enzyme is a tetramer of four identical subunits. Its low thermal stability is known (Makino et al., J. Biol. Chem. (1989) 264: 6381-6385). Some stabilization is achieved by high NaCl concentrations in the reaction buffer. The gene sequence of the Bacillus megaterium GDH and mutants of this GDH which have higher temperature stability are disclosed in Makino et al., J. Biol. Chem. (1989) 264: 6381-6385. However, these mutants are clearly inferior in their kinetic properties to the wild-type enzyme (Nagao et al., FEBS Lett. (1989) 253, 113-116).

Around with GDH from B. megaterium for cofactor regeneration in biotransformations To achieve high space-time yields are usually complex process steps used as two-phase systems. Often then can not be quantitative Sales of the reactant can be achieved more. In addition, then often prepares the Separation of unreacted educt Difficulty of product isolation. Also, a reuse of the GDH is not known, the cost-effectiveness biotransformations.

One biotransformed product of great economic value Meaning is (S) -4-chloro-3-hydroxybutyric acid ethyl ester (S-CHBE), the industrially used as a synthesis building block for the production of so-called statins becomes. Statins are a class of pharmacological agents that used as a cholesterol-lowering drug. you are of tremendous medical importance in prevention and treatment the one leading to heart attack Arteriosclerosis. S-CHBE is technically transformed into biotransformations 4Cl-ACE produced. As a chlorinated secondary alcohol has S-CHBE a high chemical reactivity and S-CHBE is for the rapid irreversible inactivation of those involved in biotransformation Enzymes, especially the cofactor-regenerating GDH responsible.

task Therefore, it is the object of the present invention to provide a GDH which does not have the disadvantages of the prior art and in particular an increased chemical stability against alpha-halogenated carbonyl compounds or alpha-halogenated alcohols having.

One example for such a compound is S-CHBE.

A Another object of the invention is a GDH with a comparison increased to the prior art stability across from solvents which are used in a biotransformation available to put. examples for such solvents are n-butyl acetate, which is a non-water-miscible solvent in two-phase biotransformations, as well as methyl tertiary butyl ether (MTBE) as a solvent for product extraction from aqueous Biotransformationsansätzen.

The Task is solved by a GDH with an amino acid sequence, the amino acid sequence of a GDH of Bacillus subtilis comprising a glutamic acid residue at position 96 corresponds with the difference that the glutamic acid residue at position 96 against a nonpolar or hydrophobic amino acid or glycine is exchanged.

The amino acid sequence a Bacillus according to the invention subtilis GDH mutant comprising an alanine residue at position 96 preferably SEQ. ID. NO: 1.

Prefers is in the GDH according to the invention at position 96 a amino acid selected from the group glycine, alanine, valine, leucine, isoleucine, proline, methionine, Phenylalanine, tyrosine, and tryptophan.

Especially preferred is in the GDH according to the invention at position 96 a Amino acid selected from the group glycine, alanine, valine, leucine and isoleucine.

Especially Preferably located in the GDH according to the invention at position 96 the hydrophobic amino acid Alanine.

The GDHs according to the invention, in the following also called GDH mutants, are characterized mainly by a surprise high chemical stability against (S) -4-chloro-3-hydroxybutyric acid ethyl ester (S-CHBE). About that In addition, the GDH mutants possess a high stability across from solvents such as n-butyl acetate and MTBE that often be used in biotransformations. Finally, the GDH mutants exhibit not the disadvantage of a greatly decreased enzymatic activity in comparison to the wild-type enzyme, as in similar ones Enzymes from the genus Bacillus megaterium observed.

The GDH mutants according to the invention Due to their high process stability, they are suitable for biotransformations with high yields Product and product concentration, for two-phase systems and reusing the biocatalyst. The invention serves therefore to improve the profitability of biotransformations.

The The invention further relates to a gene encoding a GDH according to the invention. A preferred embodiment such a gene is characterized by SEQ. ID. NO 2.

Genes according to the invention are also all variants due to the degenerate genetic code coding for genes a GDH according to the invention.

One gene of the invention let yourself produce from a GDH gene from B. subtilis. The gene may be from B. subtilis genomic DNA isolated by methods known per se, such as PCR, and into an expression vector be cloned. This is the production of the enzyme of the invention and its genetic variants in recombinant form. For example the gene can be obtained from the B. subtilis strain LK5 DSM 13487 (US Pat after the Budapest contract with the DSMZ German collection for microorganisms and Zellkulturen GmbH, Braunschweig).

The GDH wild-type enzyme from the strain Bacillus subtilis LK5 is characterized by the protein sequence given in SEQ ID NO: 3 and the coding therefor Gene sequence (SEQ ID NO: 4). The protein sequence of SEQ ID NO: 3 is 99% identical to that described in Lampel et al., J. Bacteriol. (1986) 166: 238-243 disclosed protein sequence of the GDH from Bacillus subtilis, or 83% identical to the protein sequence of the GDH from Bacillus megaterium.

In In the present application all homology values refer to Results obtained with the computer program "Wisconsin Package Version 10.3, Accelrys Inc. ". The homology determination was carried out by comparison of the protein sequences with the subprogram "gap". Here are the default parameters "gap creation penalty 8 "and" gap extension penalty 2 "used.

Out the crystal structure of a B. megaterium GDH (Yamamoto et al., J. Biol. Biochem. (2001) 129: 303-312) it is known that the enzymatic active GDH tetramer by hydrophobic interaction between the individual Subunits is stabilized. An enhancement of the hydrophobic interaction, z. B. by a NaCl concentration of 1-2 M or by addition of glycerol to a limited extent the increase the thermal stability. About one Influence on chemical inactivation of the enzyme by compounds Like S-CHBE, no predictions can be made from this observation to meet.

In Makino et al., J. Biol. Chem. (1989) 264: 6381-6385 discloses gene mutations revealed that to an increase the temperature stability GDH from B. megaterium. One of these mutations replaced the negatively charged amino acid glutamic acid in position 96 by an uncharged amino acid, such as Alanine or glycine. The increased temperature stability in this Mutant is explained by that the negative charge of glutamic acid to an electrostatic rejection the individual subunits of the GDH enzyme leads, especially at higher temperatures and pH values leads to dissociation and inactivation of the enzyme. The introduction an uncharged amino acid reduces the electrostatic repulsion and increases the thermal stability of the enzyme. From these observations, no statements can be made about the change the chemical stability derived. The expert expects rather no connection between thermal and chemical stability, since the enzyme inactivation by a compound like S-CHBE not by affecting ionic or hydrophobic interactions, but by chemical Modification of reactive amino acid residues in the GDH protein.

Therefore is the finding that the introduction an inventively defined Mutation in the GDH enzyme from B. subtilis the chemical stability of this Enzyme drastically increased, without simultaneously stabilizing by NaCl concentrations of 1-2 M is required, surprisingly. The mutation according to the invention, which in the GDH from Bacillus subtilis to an increased chemical stability leads, causes mutations of the GDH from Bacillus megaterium only one increase the temperature stability.

The Particularly preferred GDH mutant according to the invention, in which a glutamic acid at position 96 is replaced by alanine, referred to as GDBS-E96A. The gene sequence of this mutant is specified by the sequences shown in SEQ ID NO: 2 listed DNS sequence or the deduced amino acid sequence (SEQ ID NO: 1).

unexpectedly cause the mutations of the invention in GDBS-E96A a drastic increase the chemical stability across from enzyme-damaging Compounds like S-CHBE.

The GDBS-E96A according to the invention is characterized by a high process stability in biotransformations from such. B. the enzymatic synthesis of S-CHBE from 4Cl-ACE.

The GDH according to the invention, z. B. the GDBS-E96A protein can be prepared by methods known per se in recombinant form. For this purpose, a GDH according to the invention Gene, preferably the GDBS-E96A gene, into a suitable expression vector cloned, transformed into a production host, transformed Cells are cultivated, wherein the GDH according to the invention either constitutively or produced after induction of gene expression. Furthermore can in the same production host in addition to the GDH invention one or more CRs are produced in recombinant form.

The The invention thus also encompasses expression vectors which are recombinant Production of a GDH according to the invention, preferably the GDH GDBS-E96A are suitable. Where desired themselves with the expression vectors according to the invention also produce one or more CRs in a host organism.

The GDH enzyme produced in this way can then be directly in the cells be used by the production host or after digestion the cells as protein extract or after appropriate workup by z. For example, column chromatography as a purified protein.

For enzyme production Bacterial and eukaryotic expression systems are suitable. Host organisms for the enzyme production are preferably selected from Escherichia coli, strains of Genus Bacillus, yeasts such as Pichia pastoris, Hansenula polymorpha or Saccharomyces cerevisiae as well as fungi such as Aspergillus or Neurospora, but they are not on the mentioned host organisms limited.

To the preferred expression systems include E. coli, Bacillus, Pichia pastoris, S. cerevisiae, Hansenula polymorpha or Aspergillus.

Especially preferred expression systems for the production of the GDH enzyme are E. coli, Pichia pastoris and S. cerevisiae.

The thus produced GDH protein according to the invention, preferably GDBS-E96A, either in whole cells or as a cell extract or as a purified protein, can be used in biotransformations for the regeneration of NADH and NADPH cofactors. It is characterized by the fact that it is after a Treatment with S-CHBE up to a concentration of 10% (v / v) (corresponding to a concentration of 710 mM) in an aqueous medium at pH 7.0 and 30 ° C for 2.5 h less than 30% of its activity in Compared to the untreated enzyme loses. By contrast, the wild-type GDH (corresponding to the protein sequence shown in SEQ ID NO: 3) is already 100% inactive after treatment with 3 (v / v) S-CHBE for 30 min at pH 7.0 and 30 ° C. This inventively high chemical stability is the basis for the use of the GDH mutant in biotransformations.

In a preferred embodiment invention, the GDH mutant according to the invention is characterized in that that after treatment with S-CHBE up to a concentration of 10% (v / v) in an aqueous Medium at pH 7.0 and 30 ° C for 2.5 h less than 20% of its activity compared to untreated Enzyme loses.

In a particularly preferred embodiment invention, the GDH mutant is characterized in that it after treatment with S-CHBE up to a concentration of 10% (v / v) in an aqueous medium at pH 7.0 and 30 ° C for 2.5 h less than 10% of its activity compared to untreated Enzyme loses.

Further the GDH mutant according to the invention is distinguished by a high solvent stability against n-butyl acetate and MTBE. In a preferred embodiment of the invention is the GDH mutant characterized by being after treatment in water solution in a two-phase mixture with constant mixing to phase separation Prevent with 20% (v / v) of either solvent n-butyl acetate or MTBE for 2 h at 30 ° C and pH 7.0, does not lose more than 20% of its activity.

In a particularly preferred embodiment loses the GDH mutant under these conditions no more than 10% of its activity.

The Application of the GDH according to the invention however, is not limited to biotransformations only. It is also used in other applications. Are known z. B. the use in biosensors, z. For glucose determination. The enzyme according to the invention but can also for the production of gluconic acid, or their salts, by Oxidation of glucose, can be used. In addition, the invention includes also the use of the GDH mutant according to the invention for the oxidation other sugars, such as B. 2-deoxyglucose or D-glucosamine.

Biotransformation compositions according to the invention comprise a GDH mutant according to the invention, a CR enzyme, a redox cofactor selected from NAD, NADH, NADP or NADPH, a reducing agent, preferably Glucose or 2-deoxyglucose and a substrate to be reduced (Educt) for the CR enzyme selected from the class of carbonyl compounds.

The Composition according to the invention is used in a process for the preparation of chiral alcohols or chiral amines, wherein the GDH invention, the cost-effective regeneration of NADH or NADPH cofactors, which in the enantioselective Reduction of the reactant can be consumed by the CR.

The The invention therefore also relates to processes in which a GDH according to the invention for Cofactor regeneration is used. The input quantity of the GDH in relation to the CR enzyme is not specified. The biotransformation becomes preferably carried out in a buffered, aqueous medium. It but it is also possible to carry out the biotransformation in a two-phase mixture, consisting from a buffered, watery Phase and a second, not water-miscible phase, preferably an organic solvent.

Especially The invention also includes methods in which the GDH due their high chemical stability, either alone or together with the CR, from the reaction mixture be recovered can for a reuse with the goal, the profitability of the procedure to increase. The recovery refers to processes where the reaction product in a conventional manner after completion of the reaction by extraction with one in the watery one Phase insoluble solvent is extracted and the enzyme with the aqueous phase for reuse recovered becomes. The extraction can be continuous or discontinuous respectively.

The method according to the invention also comprises all CR enzymes which can reduce a carbonyl compound as the starting material of a biotransformation, wherein NADH or NADPH ver as reducing agent ver is needed.

preferred CRs are all enzymes that are a carbonyl compound to an alcohol, especially a chiral alcohol, can reduce.

Especially preferred are CRs from the group of ADHs, AKRs, glycerol dehydrogenases, Aldehyde reductases and fatty acid synthases.

Especially preferred are ADH's from the genera Lactobacillus, Rhodococcus, Thermoanaerobacter (or Thermoanaerobium), as well as AKRs, glycerol dehydrogenases, aldehyde reductases and fatty acid synthases from yeasts or filamentous Mushrooms.

The GDH according to the invention and the CR can in the process according to the invention either completely be used purified or partially purified or in cells contained (whole cell method) are used. This excludes the possibilities one that the GDH and the CR separated or together in the Cells (co-expression) are included. The cells used can native, permeabilized or lysed. The quantities used the CR and the GDH according to the invention in the method according to the invention are not fixed, but the person skilled in the art is familiar with that with one as possible low enzyme use at high space-time yield a possible quantitative conversion of the substrate to the product is to take place.

When Substrates of the biotransformations in which the GDH according to the invention the Regeneration of NADH or NADPH can be general Carbonyl compounds are used, preferably those with 3 bis 40 C atoms.

In a preferred embodiment of the process according to the invention, prochiral carbonyl compounds of the general formula (I) R1-C (O) -R2 (I) used, where
R1 and R2 are independently selected from the group consisting of C1-C20 alkyl, C3-C20 cycloalkyl, C5-C20 aryl, C1-C20 heteroaryl, C2-C20 alkenyl, C5-C20 aralkyl, C5-C20 Alkylaryl or R1 and R2 together can form a ring
and R1 and R2 may optionally be independently substituted with one or more Z radicals, wherein
Z is selected from the group consisting of fluorine, chlorine, bromine, iodine, -CN, -NO2, -NO, -NR3OR3, -CHO, SO3H, -COOH or -R3 and
R3 is hydrogen or may have the meaning of R1 and
in R 1 and R 2, optionally independently of one another, one or more methylene groups may be replaced by identical or different groups Y, where
Y is selected from the group consisting of -CR3 = CR3-, -C = C-, -C (O) -, -C (O) O-, -OC (O) -, -C (O) OC (O) -, -O-, -OO-, -CR3 = N-, -C (O) -NR3-, N = N-, -NR3-NR3-, -NR3-O-, -NR3-, -P (O ) (OR3) O-, -OP (O) (R3) O-, -P (R3) -, P (O) (R3) -, -S-, -SS-, -S (O) -, - S (O) 2-, -S (O) NR3-, -S (O) (OR3) O-, -Si (R3) 2-, Si (R3) 2O-, -Si (R3) (OR3) - , -OSi (R3) 2O-, -OSi (R3) 2-or -Si (R3) 2OSi (R3) 2-.

preferred C5-C20-aryl or C1-C20-heteroaryl radicals for R1 and R2 are in particular selected from the group comprising -phenyl, -naphthyl, -indolyl, -benzofuranyl, Thiophenyl, pyrrolyl, pyridinyl, imidazolyl, oxazolyl, isoxazolyl, -Furanyl, or -Thiazolyl.

The Compounds of the general formula (I) can in general also be described as Salts are used.

Especially Preferred compounds of general formula (I) are selected from the class of α-ketoesters, β-ketoesters, γ-ketoesters, α-diketones, β-diketones, γ-diketones, δ-diketones, α-haloketones, β-haloketones, αα-dihalo ketones, α-alkoxy ketones, α-acyloxy ketones , β-alkoxy ketones, α-alkene ketones, Alkene ketones, αα -dialkoxyketones, ββ-dialkoxyketones, Aryl ketones, heteroaryl ketones.

Particularly suitable compounds of the general formula (I) are methyl 3-oxo-butanoate, ethyl 3-oxo-butanoate, methyl 4-chloro-3-oxo-butanoate, ethyl 4-chloro-3-oxo-butanoate, Methyl 3-oxo-pentanoate, ethyl 3-oxo-pentanoate, 1-chloro-propan-2-one, 1,1-dichloro-propan-2-one, 3-oxo-butan-2-one, 2,6-dimethyl hexane-3,5-dione, 2,7-dimethyl-hexane-3,6-dione, 2,4-hexanedione, 2,4-pentanedione, tert-butyl 3-oxobutanoate, 2,5-hexanedione, 4-trimethylsilyl 3-butyn-2-one, 4-triisopropylsilyl-3-butyn-2-one, 1-chloro-4-trimethylsilyl-3-butyn-2-one, 1-chloro-4-triisopropylsilyl-3-butyne-2 or 1-chloro-butyn-2-one, 1-chlorobutan-3-one, butanone, pentan-2-one, hexan-2-one, heptan-2-one, octan-2-one, 3-pentene 2-one, 4,4-dimethoxybutan-2-one, 1-acetoxypropan-2-one, cyclopent-2-en-1-one, methyl 3-oxotetradecanoate, methyl 3-oxododecanoate, 1,1-dimethoxy-4-methyl 3-pentanone, 2-oxo-propionic acid ethyl ester, 1,4-dichlorobutanone, acetophenone, 3-methylbutanone, 1-benzyloxy-propan-2-one or 2-methylcyclopentanone.

The Compounds of the general formula (I) are used in the process according to the invention in an amount of 1% to 50% based on the total volume of a each reaction batch used, preferably from 3% to 30%, in particular from 5% to 20%.

The Reaction mixture should generally have a pH of 5 to 9, preferably from 6 to 8.

The aqueous Phase (reaction medium) contains prefers a buffer, in particular a potassium phosphate / potassium hydrogen phosphate, Tris (hydroxymethyl) aminomethane / HCl or triethanolamine / HCl buffer with a pH of 5 to 10, preferably a pH of 6 to 9. The buffer concentration should be from 5 mM to 150 mM.

In addition, can the watery Phase also contain magnesium ions, for example in the form of added MgCl2 in a concentration of 0.2 mM to 10 mM, preferably 0.5 mM to 2 mM based on the amount of water used. Besides can the watery Phase further salts, such as NaCl, preferably in one Concentration of 50-200 mM, and other additives such as dimethyl sulfoxide, glycerol, Glycol, ethylene glycol or known enzyme-stabilizing sugars, such as trehalose, sorbitol or mannitol.

When Coenzyme can NADP, NADPH, NAD, NADH or salts thereof. The concentration to coenzyme in the aqueous Phase is preferably from 0.01 mM to 0.25 mM, more preferably from 0.02 mM to 0.1 mM.

The Temperature of the reaction mixture is preferably from 0 ° C to 70 ° C, especially preferably from 20 ° C up to 50 ° C.

in the During the reaction, further reducing agent, preferably in the form of glucose, or educt in particular in the form of carbonyl compounds of general formula (I). The addition can be in each case continuously (as so-called "feed") or else in portions (so-called "batchwise").

in the inventive method the pH of the batch becomes continuous due to the oxidation of glucose to gluconic acid what is corrected by counter-titration with a base. The pH of the reaction mixture is kept constant by addition a base, selected from sodium hydroxide solution, potassium hydroxide solution, ammonia (so-called. "pH-stat" method familiar to those skilled in the art) or but by adding calcium carbonate, from the acid gaseous Carbon dioxide is released.

ever the type and amount of alcohol dehydrogenase used and the used compound of general formula (I) is the reaction time between 30 minutes and 200 hours, preferably 6 hours to 60 hours.

The Extraction of the product is carried out according to methods known per se, preferably with a water immiscible, organic Solvent. The extraction may be discontinuous (batchwise) or continuous respectively. As known to those skilled in the process, temperature and Pressure adjusted so that optimal extraction of the product from the watery Phase guaranteed is.

When organic solvents are all water immiscible solvents suitable, the formed secondary Alcohol from the watery Phase can isolate.

Prefers become organic solvents, selected from the group of esters and / or ethers used.

Especially preferred are ethyl acetate, methyl acetate, propyl acetate, isopropyl acetate, Buhylacetat, Tertbutylacetat, diethyl ether, diisopropyl ether, dibutyl ether and MTBE or mixtures thereof.

Especially preferred solvents are MTBE, ethyl acetate and butyl acetate.

To the separation of the organic extraction phase, this is preferably worked up by distillation, with an enrichment of the reaction product achieved and the partial to complete separation of by-products from the extraction solvent is effected and this are used again for extraction can.

By Purification of the organic extraction solution containing the crude product, for example by means of fine distillation, to obtain the desired end product. The final product is typically characterized by yields> 90% and an enantiomeric excess ee> 90%, preferred by yields> 95 % and ee> 97%.

The inventive method also includes the reuse of an aqueous, enzyme-containing reaction medium for the conversion of carbonyl compounds to chiral alcohols. The resulting high space-time performances and by repeated Use of the enzyme solution Low enzyme consumption per mole of product enable cost-effective implementation of carbonyl compounds to chiral alcohols using of enzymes.

The GDH mutant according to the invention GDBS-E96A is characterized by an unexpectedly high chemical stability, the was not expected by the prior art. Surprisingly revealed to the person skilled in the art including the inventive, chemically stable, GDH mutant GDBS-E96A for cofactor regeneration the advantages shown in the enzyme-catalyzed preparation chiral alcohols, starting from prochiral carbonyl compounds.

The Invention is described by the following examples:

1st example:

Isolation and cloning of the GDH gene from genomic B. subtilis DNA.

The B. subtilis strain LK5 was used. Genomic DNA from Bacillus subtilis was isolated by methods known per se (Makino et al., J. Biol. Chem. (1989) 264: 6381-6385). The GDH gene was isolated by PCR with Pfu DNA polymerase (Stratagene) according to the manufacturer's instructions with genomic DNA from Bacillus subtilis LK5 and the primers gdbs-a (SEQ ID NO: 5) and gdbs-b (SEQ ID NO: 6 ) amplified. The primers were derived from the published sequence (Lampel et al., J. Bacteriol. (1986) 166: 238-243) and had the following sequences:
Oligo gdbs-a:
5'-GCAGAATTCATGTATCCGGATTTAAAAGG-3 'SEQ ID NO: 5
Oligo gdbs-b:
5'-CACGCGGCCGCTTAACCGCGGCCTGCCTGG-3 'SEQ ID NO: 6

primer gdbs-a contained at the 5'-end an Eco RI site, primer gdbs-b contained a distress I at the 5 'end Interface. In the PCR reaction of genomic Bacillus subtilis DNA with the primers gdbs-a and gdbs-b resulted in a DNA fragment of 780 bp size.

Vector pGDBSpic for expression in Pichia pastoris:

The 780 bp DNA fragment of the B. subtilis GDH produced by the PCR reaction was cut with Eco RI and Not I and cloned into the Eco RI and Not I cut and dephosphorylated vector pPIC3 (Invitrogen). The vector pGDBSpic ( 1 ). Sequencing of the cloned GDH gene revealed the DNA sequence set forth in SEQ ID NO: 4. The expression vector pGDBSpic is suitable for the expression of the GDH gene in the yeast Pichia pastoris.

Vector pGDHyexl for expression in Saccharomyces cerevisiae:

The GDH gene was excised from vector pGDBSpic with Eco RI and Not I and isolated. The resulting 780 bp fragment was cloned into the Not I and Eco RI cut and dephosphorylated S. cerevisiae expression vector pYEXL (Clontech). This gave rise to the expression vector pGDHy exl ( 2 ). In pGDHyexl the GDH gene is under the control of the PGK promoter (phosphoglycerate kinase gene). The URA3 selection marker gene complements uracil auxotrophy in transformed S. cerevisiae strains. Vector pGDHyexl is suitable as an autonomously replicating vector for the expression of the GDH gene in S. cerevisiae.

Integrative Vector pGDHnts for the Expression of GDH in S. cerevisiae:

vector pGDHyexl was cut with Nde I and the linearized vector subsequently treated with Pfu DNA polymerase to the overhanging, single-stranded DNA ends fill. This was followed by digestion with Hind III. It was created next to one 2.4 kb also a 6.6 kb DNA fragment, in addition to the GDH expression cassette also contained the URA3 selection marker gene. The 6.6 kb gene fragment was by preparative Gel electrophoresis purified and cut in with Hpa I and Hind III and dephosphorylated vector pNTS cloned.

Vector pNTS ( 3 ) contained a 980 bp DNA fragment from the gene region of the repetitive ribosomal rDNA from S. cerevisiae. The NTS gene fragment was isolated by PCR with the Taq DNA polymerase (Qiagen) with the primers ntsA (SEQ ID NO: 7) and ntsB (SEQ ID NO: 8) from S. cerevisiae genomic DNA. The primers were selected from the published sequence (gene database file SCNTS1.gb p1, primer ntsA: ab bp 11th primer ntsB: ab bp 990, in reverse-complementary orientation) and had the following sequences:
Primer ntsA:
5'-GCGGGATCCGGCGCGCCATCCGGGTAAAAACATG-3 'SEQ ID NO: 7
Primer ntsB:
5'-GCGATCGATGGCGCGCCTATGCTAAATCCCATAAC-3 'SEQ ID NO: 8

primer ntsA contained at the 5'-end a Bam HI interface, followed by an Asc I interface. Primer ntsB contained at the 5 'end a Cla I interface, followed by an Asc I interface.

The PCR fragment formed during the PCR reaction was cut with Bam HI and Cla I and cloned into the Bam HI and Cla I cut and dephosphorylated Bluescript vector (Stratagene), whereby the vector pNTS (FIG. 3 ) originated.

Vector pecGDBS for expression in Escherichia coli:

Plasmid DNA of the expression vector pGDBSpic was used to generate the GDH gene with the primers gdbs-c (SEQ ID NO: 9) and gdbs-d (SEQ ID NO: 10) in a PCR reaction with the Pfu DNA polymerase manufacture. The primers had the following sequences:
Oligo gdbs-c:
5'-GAATTCGGTCTCAAATGTATCCGGATTTAAAAGG-3 'SEQ ID NO: 9
Oligo gdbs-d:
5'-GTAAAGCTTGGTCTCTGCGCTTTAACCGCGGCCTGCCTGGA-3 'SEQ ID NO: 10

primer gdbs-c contained at the 5'-end an Eco RI interface followed by a Bsa I interface. The primer gdbs-d contained at the 5'-end a Hind III site followed by a Bsa I site.

In the PCR reaction of pGDBSpic plasmid DNA with the primers gdbs-c and gdbs-d, a DNA fragment of 780 bp size was generated. The DNA fragment was cut with Bsa I and cloned into the Bsa I-cut and dephosphorylated vector pASK-IBA3 (so-called "Strep-tag" protein production system from IBA, Institute for Bioanalytics) to give the vector pecGDBS ( 4 ). Sequencing of the cloned GDH gene revealed the DNA sequence set forth in SEQ ID NO: 4. The expression vector pecGDBS is suitable for expression of the GDH gene in E. coli.

2.Example:

Production of the mutant GDBS-E96A

For targeted mutagenesis (so-called "site-directed" mutagenesis) of the GDH gene, cloned into the expression vector pecGDBS, the "Quick Change" mutagenesis kit from Stratagene was used. As primers for mutagenesis, GDmut1 (SEQ ID NO: 11) and GDmut2 (SEQ ID NO: 12) were used. The primers had the following sequences:
GDmut1:
5'-AATGCCGGTCTTGCAAATCCTGTGCCATCTC-3 'SEQ ID NO: 11
GDmut2:
5'-GAGATGGCACAGGATTTGCAAGACCGGCATT-3 'SEQ ID NO: 12

The mutagenesis was carried out according to the manufacturer's instructions. The resulting vector with the mutant GDH gene was identified as pecGDBS-E96A ( 5 ) designated. The successful introduction of the mutation was verified by DNA sequencing. The DNA sequence of the GDH mutant GDBS-E96A is listed in SEQ ID NO: 2. The protein sequence derived from SEQ ID NO: 2 is listed in SEQ ID NO: 1. The expression vector pecGDBS-E96A is suitable for expression of the GDH gene in E. coli.

Autonomously replicating Vector pGDBS-E96Ayex1 for expression of the GDH mutant in S. cerevisiae:

By PCR reaction with the primers gdbs-a (SEQ ID NO: 5) and gdbs-b (SEQ ID NO: 6, see Example 1), pecGDBS-E96A plasmid DNA and Pfu DNA polymerase, the mutated GDH- Gene was prepared as a 780 bp fragment, cut with Eco RI and Not I and cloned into the Eco RI and Not I cut and dephosphorylated vector pYEXL (see Example 1). The correct DNA sequence of the mutant GDH gene was verified by sequencing. The result was the expression vector pGDBS-E96Ayexl ( 6 ) suitable for expression of the mutated GDH gene in S. cerevisiae.

Integrative vector pGDBS-e96aNTS for expression of the GDH mutant in S. cerevisiae:

From the vector pGDBS-E96Ayexl the GDH gene was isolated with Eco RI and Bgl II as a 780 bp gene fragment. From the integrative vector pGDHnts, the wild-type GDH gene was excised by digestion with Eco RI and Bgl II, the 9.7 kb vector fragment isolated and dephosphorylated. The 780 bp gene fragment of the GDH mutant was cloned into the vector fragment. The integrative expression vector pGDe96aNTS ( 7 ), which is suitable for the expression of the GDH mutant in S. cerevisiae.

3rd example:

Expression of GDH genes in E. coli

used the expression vectors were pecGDBS and pecGDBS-E96A. Plasmid DNA was transformed according to methods known per se into the E. coli strain Top 10F '(Invitrogen). As control served E. coli Top 10F ', transformed with the pASK-IBA3 empty vector. One clone each was selected and in a shake flask cultivation according to the manufacturer of the pASK-IBA3 vector (IBA Institut for bioanalytics). From the E. coli strains was in LBamp medium (10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl, 0.1 mg / ml ampicillin) produced a preculture (cultivation at 37 ° C and 120 rpm over Night). 2 ml of each preculture were used as inoculum of a main culture of 100 ml LBamp medium (300 ml Erlenmeyer flask). The main cultures were at 30 ° C and shaken 180 rpm, until a cell density OD600 of 0.5 was reached. Then the Inducer Anhydrotetracycline (IBA Institute of Bioanalytics, final concentration 2 μg / ml) added and a further 3 h at 30 ° C. and shaken 180 rpm.

Were used for the GDH-production analysis in each case 1 of the E. ml coli cells in a conventional manner with a so-called. "French ^® Press" high-pressure homogenizer (SLM-AMINCO) open and rpm a cell extract by centrifugation (15 min 15000, Sorvall SS34 rotor Aliquots of the cell extracts were used to photometrically determine GDH activity, while in the control strain no GDH activity could be measured, the specific GDH activity in cell extracts was 34.8 U / mg protein for the wild-type enzyme and 30.8 U The mutant GDBS-E96A (equivalent to 88.5% of the specific activity of the wild-type GDH) has the same enzymatic properties as GDB-E96A from B. subtilis and has unexpectedly better enzymatic properties than the GDB-E96A mutant analogous mutant from Bacillus megaterium, which only had about 30% of the activity of the wild-type enzyme (Nagao et al., FEBS Lett. (1989) 253, 113-116).

spectrophotometric Determination of GDH activity:

The 1 ml volume for the photometric determination of GDH activity was composed of measurement buffer (0.1 M potassium phosphate, pH 7.0, 0.1 M NaCl), 10 mg / ml glucose, 2 mM NADP and GDH-containing cell extract , Measurement temperature was 25 ° C. The reaction was started by adding the glucose (from a 40X concentrated stock solution) and the absorbance increase due to the formation of NADPH measured at a wavelength of 340 nm (extinction coefficient of NADPH: ε = 0.63 × 10 ⁴ l × mol ^-1 × cm ^-1 ). One unit of GDH activity is defined as the formation of 1 μmol NADPH / min under assay conditions.

to Determination of the specific activity was the protein concentration the cell extracts in a conventional manner with the so-called. "BioRad Protein Assay "the Company BioRad determined.

4th example:

Expression of GDH genes in the yeast S. cerevisiae

starting strain was the S. cerevisiae strain CM3260 (uracil, histidine, tryptophan auxotrophy, described in Dancis et al., Cell (1994) 76: 393-402). The transformed Expression construct was pGDe96ants. pGDe96ants contained the mutant GDH gene GDBS-E96A under control of the PGK promoter. selection marker was the URA3 gene (uracil selection). The vector was before the transformation cut with Asc I to release the expression cassette. The transformation of the yeast strain CM3260 took place after the Professional common Lithium acetate method (Gietz and Woods, Biotechniques (2001) 30: 816-831).

Transformants were isolated and tested for GDH activity by shake flask culture. For this purpose, in each case 50 ml of YPD-CSL medium (10 g / l yeast extract, 20 g / l peptone, 20 g / l glucose, 20 g / l corn spring water, "Corn Steep Liquor") was inoculated with one transformant and four days at 28 The cells were incubated with the " ^French® Press" and the cell extracts were analyzed for GDH activity, as described in the 3rd example for E. coli, and 140 rpm (Infors shaker). From the comparison of about 50 transformants tested, the strain GD34 emerged as the best GDH producer. The specific GDH activity in crude extracts (determined as described in Example 3) was 1 U / mg protein. In the CM3260 control strain no GDH activity could be measured.

5th example:

Chemical stability of the GDH mutant GDBS-E96A

Compared became the GDH activity in cell extracts from the culture in E. coli (see 3rd example) after pretreatment with the enzyme-damaging product S-CHBE, the formed in the biotransformation of 4Cl-ACE. It was also the stability against the solvents investigated n-butyl acetate and MTBE.

In In a first test, the same enzyme activities of wild type GDH and mutant were observed GDBS-E96A in measuring buffer (see 3rd example) at 30 ° C and pH Treated with S-CHBE at a concentration of 3 (v / v) and 7.0 the GDH activity determined after different incubation times. Table 1 shows that the wild-type enzyme was completely inactive after 30 min. The mutant GDBS-E96A, on the other hand, was still closed after incubation for 120 min at 30 ° C more than 90% active.

Table 1: Kinetics of inactivation of wild type GDH and GDH mutant GDBS-E96A by preincubation (30 ° C, pH 7.0) with 3 (v / v) S-CHBE

In In a second test, the GDH mutant was treated with increasing amounts of S-CHBE incubated (dose-dependent Inactivation) and then the GDH activity certainly. As shown in Tab. 2, under reaction conditions (30 ° C, pH 7.0) after 2.5 h incubation with S-CHBE in concentrations up to 10 (v / v) (equivalent to a 0.71M concentration) is not significant Inactivation of the enzyme was observed.

Table 2: Dose-dependent inactivation of the GDH mutant GDBS-E96A by preincubation (2.5 h 30 ° C, pH 7.0) with increasing amounts of S-CHBE

In In a third test the same enzyme activities of wild type GDH and mutant were observed GDBS-E96A in measuring buffer (see 3rd example) at 30 ° C and pH 7.0 with increasing amounts of n-butyl acetate or MTBE incubated (dose-dependent inactivation) and subsequently the GDH activity certainly. As shown in Tab. 3, under reaction conditions (30 ° C, pH 7.0) after 2.5 h incubation with n-butyl acetate or MTBE in concentrations to 20 (v / v) no significant inactivation (n-butyl acetate), resp. an inactivation <20 % (MTBE) of the GDH mutant GDBS-E96A, while the wild-type GDH 50% (n-butyl acetate), or up to 90% (MTBE) was inactivated.

Table 3: Dose-dependent inactivation of the wild-type GDH and the GDH mutant GDBS-E96A by preincubation (2 h 30 ° C, pH 7.0) with increasing amounts of n-butyl acetate or MTBE

These Results mean a significant and state of the art unanticipated improvement in the chemical stability of the GDH mutant GDBS-E96A opposite the wild-type enzyme.

6th example:

whole cell biotransformation with GDH-producing yeast cells

For biotransformations the recombinant S. cerevisiae strain FasB-his6 was used. The strain FasB-his6 is derived from the GDH-producing strain GD34. It is characterized by an increased FAS CR activity and is suitable for the production of S-CHBE and other chiral β-hydroxy esters, as in the usual Yeast Strains observable CR activities, which lead to R-CHBE, in naturally lower activity available. The increase in expression of FAS activity was According to the state of the art, achieved by overexpression the so-called Fas1 subunit of yeast fatty acid synthase (described in Wenz et al., Nucleic Acids Res. (2001) 29: 4625-4632).

The cultivation of the yeast strain FasB-his6 was carried out in shake flasks. 6 × 250 ml of YPD-CSL medium, each in 1 liter Erlenmeyer flasks, were inoculated with the strain FasB-his6. Cultivation was by incubation for four days on an orbital shaker (Infors) at 28 ° C and 120 rpm. Cells were either directly reused for biotransformation or frozen at -20 ° C.

One Biotransformation approach was carried out using 1.5 L of FasB-his6 Cells from shake flask cultivation centrifuged and in reaction buffer (0.1 M potassium phosphate, pH 7.0, 0.1 M NaCl, 2 M glucose) were added so that the final volume 200 ml. The cell density OD600 was determined. Usually the total OD600 in the batch was 30,000. The cells were in one thermostated reaction vessel, heated to 25 ° C. and with a magnetic stirrer touched. NADP was added at a concentration of 10 μM. The reaction vessel was over one pH electrode and a sodium hydroxide solution (10 M stock solution) supporting burette with a titrator (Titroline alpha, Schott), so that the biotransformation under pH stat conditions at a constant pH of 7.5 could be. The substrate 4Cl-ACE, dissolved in n-butyl acetate, became continuously added.

In two series of experiments were either 12 g (batch A) or 18 g (batch B) of the substrate 4Cl-ACE mixed with n-butyl acetate, so that the Final volume was 40 ml. The substrate solution was then each over a Duration of 16-20 h continuously added. The final concentration of the dosed Substrate A in the batch A was 5% (w / v), in batch B 7.5% (w / v).

The Reaction was stopped after 24 h. For workup was the entire Biotransformation approach three times with an equal volume of ethyl acetate extracted, the ethyl acetate phases combined and in a rotary evaporator the solvent away. The thus obtained S-CHBE crude fraction was known per se Manner analyzed by chiral gas chromatography. In both approaches was achieved a quantitative conversion of 4Cl-ACE used. The by gas chromatography certain S-CHBE content of the crude fractions or enantiomeric excess ee is listed in Tab. 4.

Table 4 Production of S-CHBE by whole-cell biotransformation with GDH-overexpressing yeast cells

7. Example:

Biotransformation with cell extracts

The Cultivation of the E. coli strain pecGDBS-E96A in shake flasks and extraction of the GDH enzyme was carried out as described in Example 3. The ADH out Lactobacillus brevis (LB-ADH) was commercially available (Jülich Fine Chemicals GmbH).

One Biotransformation batch of 100 ml final volume contained beside reaction buffer (0.1 M potassium phosphate, pH 6.5, 0.1 M NaCl, 1 mM magnesium sulfate, 2 M glucose) GDBS-E96A GDH and LB-ADH, each in 15 U / ml dosage, 50 μM NADP and 12.9 g of methyl acetoacetate (ACMe) in a Dosage of 12.9% (w / v). The approach was in a thermostatable Reaction vessel submitted, tempered to 30 ° C. and with a magnetic stirrer touched. The reaction vessel was over one pH electrode and a caustic soda solution (10 M stock solution) promotional burette connected to a titrator (Schott), so that the biotransformation be carried out under pH-stat conditions at a constant pH of 6.5 could.

The Reaction was stopped after 21 h. For workup was the entire Biotransformation approach three times with an equal volume of ethyl acetate extracted, the ethyl acetate phases combined and in a rotary evaporator the solvent away. The thus obtained (R) -3-hydroxybutyric acid methyl ester (R-HBMe) crude fraction was analyzed by chiral gas chromatography. It became one achieved quantitative sales of the used ACMe. The by gas chromatography certain R-HBMe content of the crude fractions was> 99%, the enantiomeric excess ee was 100%.

It follows a sequence listing according to WIPO St. 25. This can from the official publication platform downloaded from the DPMA.

Claims (10)

Glucose dehydrogenase with an amino acid sequence that of the amino acid sequence a GDH of Bacillus subtilis comprising a glutamic acid residue at position 96 corresponds with the difference that the glutamic acid residue at position 96 against a nonpolar or hydrophobic amino acid or Glycine is exchanged. Glucose dehydrogenase according to claim 1, characterized that the amino acid sequence of Bacillus subtilis GDH comprising a glutamic acid residue at position 96 SEQ. ID. NO: 1 corresponds. Glucose dehydrogenase according to claim 1 or 2, characterized characterized in that the glutamic acid residue at position 96 against an amino acid selected from the group glycine, alanine, valine, leucine, isoleucine, proline, Methionine, phenylalanine, tyrosine, and tryptophan. Glucose dehydrogenase according to claim 3, characterized that the glutamic acid residue at position 96 against an amino acid selected from the group amino acid selected is replaced by glycine, alanine, valine, leucine and isoleucine is. Glucose dehydrogenase according to claim 3, characterized that the glutamic acid residue at position 96 against the amino acid Alanine is exchanged. Glucose dehydrogenase according to one of claims 1 to 5, characterized in that in a treatment with 10 (v / v) S-CHBE for 2.5 hours at 30 ° C, pH 7, a decrease <30 %, preferably <20 % particularly preferred <10 % of their enzymatic activity compared to the untreated enzyme shows. Gene coding for a glucose dehydrogenase according to a the claims 1 to 7. Process for the preparation of chiral alcohols or chiral amines characterized by a carbonyl reductase characterized in that a glucose dehydrogenase according to one of claims 1 to 7 is used for the regeneration of NADH or NADPH cofactors. The method of claim 9, wherein the carbonyl reductase selected is from the group of the Alkohldehydrogenasen, the Aldo Ketoreduktasen, the Glycerol dehydrogenases, aldehyde reductases and fatty acid synthases. Process according to claim 8 or 9 for the preparation a chiral alcohol of the general formula (I).