CN108410831A

CN108410831A - Ketone acid reductase, gene, engineering bacteria and the application in synthesis of chiral fragrance 2- hydroxy acids

Info

Publication number: CN108410831A
Application number: CN201810239718.4A
Authority: CN
Inventors: 薛亚平; 郑裕国; 王闯; 柳志强
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2018-03-22
Filing date: 2018-03-22
Publication date: 2018-08-17
Anticipated expiration: 2038-03-22
Also published as: CN113355367A; CN113355299A; CN113355299B; CN113355367B; CN108410831B

Abstract

The invention discloses a kind of ketone acid reductase, gene, engineering bacteria and the applications in fragrant 2 hydroxy acids of synthesis of chiral, and the ketone acid reduction enzyme amino acid sequence is shown in one of SEQ ID NO.4, SEQ ID NO.8 or SEQ ID NO.10；The present invention provides a kind of efficient ketone acid reductase from lactic acid leukonid, can be catalyzed 2 ketone acid of wide spectrum aromatic series, and be promoted to 400mM by 100mM by the substrate useful load of substrate of benzoylformic acid.Three enzyme of the single bacterium double-mass model series connection redox established using ketone acid reductase, 2 hydroxy acid dehydrogenases and glucose dehydrogenase cascades system, it is chiral fragrant (R) 2 hydroxy acid that the efficient deracemization of fragrant 2 hydroxy acids of most of racemics, which can be catalyzed, and yield and e.e. values are all higher than 99%.It is finally applied to racemization 300mM o-chloromandelic acids and prepares optical voidness (R) o-chloromandelic acid, yield is up to 83.8g/ (Ld).

Description

Ketone acid reductase, gene, engineering bacteria and the application in synthesis of chiral fragrance 2- hydroxy acids

(1) technical field

The ketone acid reductase gene that the present invention relates to a kind of from lactic acid leukonid (Leuconostoc lactis), Codase, carrier, engineering bacteria and the application in synthesis of chiral fragrance 2- hydroxy acids.

(2) background technology

Chiral 2- hydroxy acids are a kind of in carboxyl (- COOH) side C1 compound for having hydroxyl (- OH) to replace, and distribution is extensively It is general, property is active, be produce drug and fine chemicals important chiral building block, chemical industry, medicine and other fields have it is important Application value.Wherein, more representative is the analog derivative containing phenyl ring in structure, and such as (R)-mandelic acid is production The key intermediate of semisynthetic penicillin, cephalosporin, antineoplastic and slimming drugs, while being important chiral resolving agent； (R)-o-chloromandelic acid is the key that synthesis antithrombotic (clopidogrel) chiral building block, has the very high market demand and economy Benefit；(R) -4- chloro mandelic acids are the synthesis precursors of the mandelic acidamide series bactericidal agent (mandipropamid) of first commercialization； (R) -4- hydroxymandelic acids can be used for producing D-HPG, and then synthesize a variety of broad-spectrum antibiotics, such as amoxycillin Penicillin and amoxycillin cephalo etc..

Just because of chiral 2- hydroxy acids in medical and field of fine chemical important application so that how to obtain optically pure Chiral 2- hydroxy acids become research hotspot all the time.Traditionally, the method for preparing optical homochiral 2- hydroxy acids depends on Chemical kinetics is split, that is, suitable chiral selectors is selected to convert racemic mixture to diastereoisomeric salt, changes two The physical property of kind configuration, then carries out subsequent separation and Extraction again.However, there are many flaws for this method, it is such as maximum to receive Rate be only 50%, optical purity is low, resolution reagent is expensive, severe reaction conditions and pollution environment the problems such as.In recent years, biology was urged Change method obtains more next due to its advantage such as mild, environmental-friendly with higher stereoselectivity and catalytic activity, reaction condition More research and development is used for the efficient production of optical homochiral 2- hydroxy acids.Wherein, more representative includes enzyme process Racemization is gone in fractionation, nitrilase method, asymmetric reduction and redox cascade.

1. Enzymatic Resolution, Enzymatic Resolution prepares optical homochiral 2- hydroxy acids and belongs to Kinetic Resolution, typically refers to utilize life A kind of configuration in object enzyme process degradation selectivity racemic 2- hydroxy acids and obtain another target configuration.It remains biological enzyme reaction Advantage, but it is maximum the disadvantage is that theoretical yield only has 50%.It is obtained in addition, also having and splitting o-chloromandelic acid ester using lipase Then the carboxylate of single configuration obtains optical voidness o-chloromandelic acid by the method for hydrolysis, but this method complex steps, no Suitable for industrial applications.

2. nitrilase method, nitrilase is a kind of important hydrolase, can be catalyzed one step of nitrile substrate and be converted into phase The carboxylic acid answered has good catalysis characteristics, therefore prepares optical homochiral 2- using nitrilase living things catalysis 2- hydroxyl nitriles Hydroxy acid is widely studied.However, since this method needs to use a large amount of hypertoxic hydrogen cyanide (HCN) in catalytic process, from And lead in practical application that there are certain danger and operation difficulties.

3. asymmetric reduction, not right by prochiral 2- ketone acids using the reductase with stereoselectivity in microbial body Title is reduced to optical voidness 2- hydroxy acids.The advantages of method is theoretical yield height, easy to operate etc.；The disadvantage is that reaction process generally requires Coenzyme is added, coenzyme is expensive, greatly increases production cost, is unfavorable for industrial applications.In addition, relative to racemic chemical combination Object, prochiral 2- keto acid substrates are not easy acquisition or expensive, limit its application.

4. redox cascade go racemization, using with enantio-selectivity 2- hydroxy acid dehydrogenases, ketone acid reductase and Glucose dehydrogenase cascade catalysis racemic 2- hydroxy acids are converted into single configuration product.The method saves the time, saves middle product Extraction or purifying, it is also possible to reduce reversible reaction and be carried out to substrate direction, therefore be that most valuable chirality 2- hydroxy acids obtain Take method.Meanwhile technology is co-expressed by polygenes, three enzyme coexpression systems can be successfully built for racemic 2- hydroxy acids Efficient deracemization.

When application redox cascade goes the strategy of racemization to prepare optical homochiral 2- hydroxy acids, ketone acid reductase is found Activity limits whole catalytic efficiency, causes substrate useful load and yield relatively low.Therefore, finding has high activity and bottom The ketone acid reductase of object tolerance co-expresses catalyst system and catalyzing for three enzymes, to realizing that the efficient deracemization of racemic 2- hydroxy acids has There is significant meaning.

Ketone acid reductase is a kind of important oxidoreducing enzyme, and it is corresponding that can be catalyzed prochirality ketone acid asymmetric reduction Carboxylic acid, while NADH (nicotinamide adenine dinucleotide) or NADPH (nicotinamide-adenine dinucleotide phosphate) being needed to make Co-factor participates in reaction.In biocatalysis field, biological enzyme often has specific catalysis substrate, single enzyme logical as catalyst Chang Buhui has catalytic activity simultaneously to aliphatic and aromatic compound.The ketone acid reductase excavated in this research belongs to 2- Dehydrogenation pantoic acid -2- reductases (2-dehydropantoate 2-reductase), the substrate that is catalyzed reported is fat Compounds of group, but it is chiral fragrance 2- hydroxy acids to have been found that it can efficiently be catalyzed numerous aromatic series 2- keto acid transformations in testing, This is for realizing that the efficient deracemization of racemic fragrance 2- hydroxy acids has important application value.

(3) invention content

It is an object of the present invention to provide a kind of efficient ketone acids deriving from lactic acid leukonid (Leuconostoc lactis) Reductase gene, codase, carrier, engineering bacteria and its application in synthesis of chiral fragrance 2- hydroxy acids.The ketone acid excavated is also Protoenzyme can be catalyzed wide spectrum aromatic series 2- ketone acids, and have higher substrate useful load and catalytic efficiency.

The technical solution adopted by the present invention is：

The present invention provides a kind of ketone acid reductase, and the ketone acid reduction enzyme amino acid sequence is SEQ ID NO.4, SEQ ID Shown in one of NO.8 or SEQ ID NO.10, the more preferably described ketone acid reduction enzyme amino acid sequence is shown in SEQ ID NO.4.This Invention is to derive from the ketone acid reductase LeKAR (amino acid of Leuconostoc mesenteroides (Leuconostoc mesenteroides) Sequence is SEQ ID NO.2, nucleotides sequence is classified as SEQ ID NO.1) it is template, 5 kinds of amino are filtered out from ncbi database Acid sequence is the ketone acid of SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10 or SEQ ID NO.12 Reductase is respectively derived from lactic acid leukonid (Leuconostoc lactis), leuconostoc pseudomesenteroides (Leuconostoc Pseudomesenteroides), Leuconostoc mesenteroides (Leuconostoc mesenteroides), Klebsiella oxytoca (Klebsiella oxytoca) and Salmonella enteritidis (Salmonella enterica), amino acid sequence homology difference 84%, 78%, 74%, 49% and 49%, verified only SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.10 Ketone acid reductase has catalytic activity.

It is any that one or is carried out more to amino acid sequence shown in SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.10 The polypeptide fragment or its variant that missing, insertion or the replacement processing of a amino acid obtain, as long as it has with the amino acid sequence 95% or more homology, all belongs to the scope of protection of the present invention.

The present invention also provides a kind of encoding gene of the ketone acid reductase, the nucleotides sequence of the encoding gene is classified as Shown in one of SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.9.It is any to SEQ ID NO.3, SEQ ID NO.7, SEQ Nucleotide sequence shown in ID NO.9 carries out the nucleotides sequence that substitution, missing or the insertion process of one or more nucleotide obtain Row all belong to the scope of protection of the present invention as long as it has 90% or more homology with the nucleotide sequence.

The invention further relates to the recombinant vector and recombination engineering bacteria of the encoding gene of ketone acid reductase structure, institutes It is one of following to state recombination engineering bacteria：(1) ketone acid reduction enzyme coding gene is imported what host strain obtained；(2) by ketone acid It restores enzyme coding gene, 2- hydroxy acid dehydrogenations enzyme coding gene and glucose dehydrogenase encoding gene and imports what host strain obtained.

In addition, the application the present invention also provides a kind of ketone acid reductase in catalytically synthesizing chiral fragrance 2- hydroxy acids.

Application process one：When recombination engineering bacteria is that ketone acid reduction enzyme coding gene is imported host strain to obtain, institute The application process stated is：The wet thallus that engineering bacterium fermentation culture to restore enzyme coding gene containing ketone acid obtains is after ultrasonication Ketone acid reductase supernatant and encoding gene containing glucose dehydrogenase (nucleotides sequence is classified as shown in SEQ ID NO.15) work Glucose dehydrogenase supernatant of the wet thallus that journey fermented and cultured obtains after ultrasonication is catalyst, using benzoylformic acid the bottom of as Object, with NAD⁺For coenzyme, using glucose as cosubstrate, with the KH of 100mM, pH7.0₂PO₄-K₂HPO₄Buffer solution is that reaction is situated between Matter reacts under the conditions of 35 DEG C, 700rpm, after the reaction was complete, is contained the reaction solution of (R)-mandelic acid；The ketone acid reductase Supernatant dosage is calculated as 800U/mL buffer solutions with ketone acid reductase enzyme activity, and the glucose dehydrogenase supernatant dosage is with grape Glucocorticoid dehydrogenase enzyme activity is calculated as 800U/mL buffer solutions, and the glucose dosage is calculated as 200~800mM, the bottom with buffer solution volume Object dosage is calculated as 100~400mM, NAD with buffer solution volume⁺Dosage is calculated as 0.5mM with buffer solution volume.

Further, catalyst is prepared as follows in the method one：The engineering of enzyme coding gene will be restored containing ketone acid Bacterium is inoculated into the LB liquid medium containing 50 μ g/mL kanamycins, 8~10h of shaken cultivation under the conditions of 37 DEG C, 150rpm, Obtain seed liquor；Seed liquor is linked into the LB Liquid Cultures containing 50 μ g/mL kanamycins by the inoculum concentration of 2% (volumetric concentration) In base, shaken cultivation is to OD under the conditions of 37 DEG C, 150rpm₆₀₀Reach 0.4~0.8 (preferably 0.6), IPTG is added to final concentration For 0.1mM, wet thallus is collected by centrifugation and with brine two in 10~12h of shaken cultivation under the conditions of 28 DEG C, 150rpm It is secondary to get resting cell；Resting cell is resuspended in KH₂PO₄-K₂HPO₄In buffer solution (100mM, pH7.0), under condition of ice bath Ultrasonication 20min (broken power 40W, work 1s, stops 1s), broken liquid centrifuges 10min under 4 DEG C, 12000rpm, collects ketone Sour reductase supernatant.The glucose dehydrogenase supernatant liquid and preparation method thereof is the same as ketone acid reductase supernatant.

Application process one of the present invention is to utilize the interior reductase with stereoselectivity of microbial body by prochiral 2- ketone Sour asymmetric reduction is optical voidness 2- hydroxy acids.

Application process two：When recombination engineering bacteria is that ketone acid is restored enzyme coding gene, 2- hydroxy acid dehydrogenases coding base When cause and glucose dehydrogenase encoding gene import host strain acquisition jointly, the application process is：To contain ketone acid reductase The fermented culture of the engineering bacteria of (preferably LlKAR), 2- hydroxy acid dehydrogenases (HADH) and glucose dehydrogenase (GDH) encoding gene obtains The wet thallus obtained is catalyst, using racemic fragrance 2- hydroxy acids as substrate, the substrate supplemented by glucose, with the slow of pH6.0~8.0 Fliud flushing (the preferably KH of pH7.0₂PO₄-K₂HPO₄Buffer solution) it is that reaction medium constitutes reaction system, at 20~45 DEG C (preferably 30 DEG C), after the reaction was complete under the conditions of 700rpm, obtain the conversion fluid containing fragrant (the R) -2- hydroxy acids of optical voidness.

Further, the racemic fragrance 2- hydroxy acids are one of following：Mandelic acid 1a；2- fluorine mandelic acids 1b；4- fluorine almonds Sour 1c；2,4- difluoro mandelic acids 1d；3,5- difluoro mandelic acids 1e；2- chloro mandelic acids 1f；3- chloro mandelic acids 1g；4- chloro mandelic acids 1h；2- bromine mandelic acids 1i；3- bromine mandelic acids 1j；4- bromine mandelic acids 1k；4- methyl-mandelic acids 1l；4- trifluoromethyl mandelic acids 1m； 3- hydroxymandelic acids 1n；4- hydroxymandelic acids 1o；4- methoxv mandelic acids 1p；3 methoxy 4 hydroxymandelic acid 1q；3- hydroxyls Base -4- methyl-mandelic acids 1r；3- hydroxyl -4- trifluoromethyl mandelic acids 1s；3- methyl -4- methoxv mandelic acid 1t, preferably 1a- 1m, letter is only numbered, without meaning.

Further, in two reaction system of the application process, Final substrate concentrations 20-300mM, the auxiliary concentration of substrate is 10-300mM, the catalyst amount are calculated as 4-20g/L with wet thallus dry weight.

Further, engineering bacteria described in application process two of the present invention is by ketone acid reductase (preferably LlKAR), 2- hydroxy acid dehydrogenations Enzyme (HADH) and the encoding gene of glucose dehydrogenase (GDH) import structure in host strain E.coli BL21 (DE3) jointly and At；The encoding gene nucleotides sequence of the 2- hydroxy acid dehydrogenases is classified as shown in SEQ ID NO.13, the glucose dehydrogenase Encoding gene nucleotides sequence is classified as shown in SEQ ID NO.15.

Specifically, reductase containing ketone acid (LlKAR) of the present invention, 2- hydroxy acid dehydrogenases (HADH) and glucose dehydrogenase (GDH) engineering bacteria of encoding gene is built as follows：

(1) structure E.coli BL21 (DE3)/pET28b-LlKAR bacterial strains

Ketone acid reductase LlKAR nucleotide sequences are connected into expression plasmid pET28b, recombinant plasmid pET28b- is obtained LlKAR is transformed into E.coli BL21 (DE3), structure recombinant bacterium E.coli BL21 (DE3)/pET28b-LlKAR.

(2) structure E.coli BL21 (DE3)/pCDFDuet-LlKAR-GDH bacterial strains

It will be from the glucose dehydrogenase of Exiguobacterium sibiricum (GDH) gene (nucleotide sequence SEQ ID NO.15) and LlKAR genes successively connect with expression vector pCDFDuet-1, obtain recombinant plasmid pCDFDuet- LlKAR-GDH is transformed into E.coli BL21 (DE3), structure recombinant bacterium E.coli BL21 (DE3)/pCDFDuet-LlKAR- GDH。

(3) structure E.coli BL21 (DE3)/pET28b-HADH bacterial strains

It will be from 2- hydroxy acid dehydrogenases (HADH) gene (the nucleotide sequence SEQ of Pseudomonas aeruginosa ID NO.13) through synthesizing corresponding nucleotide sequence in vitro and being connected into expression plasmid pET28b, obtain recombinant plasmid PET28b-HADH is transformed into E.coli BL21 (DE3), structure recombinant bacterium E.coli BL21 (DE3)/pET28b-HADH.

(4) structure E.coli BL21 (DE3)/pET28b-HADH/pCDFDuet-LlKAR-GDH bacterial strains

Respectively from recombinant bacterium E.coli BL21 (DE3)/pET28b-HADH and E.coli BL21 (DE3)/pCDFDuet- Plasmid pET28b-HADH and pCDFDuet-LlKAR-GDH are extracted in LlKAR-GDH, by 1:After 1 molar concentration rate mixing, altogether With being transformed into E.coli BL21 (DE3), it is coated on the dual anti-LB tablets containing 50 μ g/mL kanamycins and 50 μ g/mL streptomysins On, it screens while recombinant bacterium E.coli BL21 (DE3)/pET28b-HADH/ containing HADH, LlKAR and GDH gene PCDFDuet-LlKAR-GDH is abbreviated as E.coli (HADH-LlKAR-GDH), to build three enzyme of single bacterium double-mass model series connection oxygen Change reduction cascade system.

The reaction mechanism of three enzyme of single bacterium double-mass model series connection redox cascade system is：With racemic fragrance 2- hydroxy acids It is 2- ketone acids using (S) -2- hydroxy acids in the HAHD asymmetric oxidation substrates with S- stereoselectivities for substrate, consumption Co-factor FMN can self-regeneration；Recycle the ketone acid reductase (preferably LlKAR) with R- stereoselectivities by the 2- ketone of generation Sour asymmetric reduction is (R) -2- hydroxy acids, finally obtains optical voidness (R) -2- hydroxy acids, coenzyme NAD H, which may be implemented, in GDH therein has The circular regeneration of effect, without adding exogenous coenzyme.Specific reaction mechanism in figure as shown in Figure 1, list representative The substrate of 20 kinds of racemic fragrance 2- hydroxy acids, three enzyme coexpression systems catalysis of the invention includes but not limited to this 20 kinds of racemic virtues Fragrant 2- hydroxy acids.

Further, catalyst is prepared as follows in the method two：To contain ketone acid reductase, 2- hydroxy acid dehydrogenases and The engineering bacteria of glucose dehydrogenase encoding gene is inoculated into be trained containing the LB liquid of 50 μ g/mL kanamycins and 50 μ g/mL streptomysins It supports in base, 8~10h of shaken cultivation under the conditions of 37 DEG C, 150rpm, obtains seed liquor；By seed liquor by 2% (volumetric concentration) Inoculum concentration is linked into the LB liquid medium containing 50 μ g/mL kanamycins and 50 μ g/mL streptomysins, in 37 DEG C, 150rpm items Shaken cultivation is to OD under part₆₀₀Reach 0.4~0.8 (preferably 0.6), IPTG to final concentration of 0.1mM is added, in 28 DEG C, 150rpm Under the conditions of 10~12h of shaken cultivation, wet thallus is collected by centrifugation and with brine twice to get wet thallus.

The recombinant bacterium E.coli (HADH-LlKAR-GDH) is finally further used in catalysis high concentration o-chloromandelic acid Deracemization.Reaction is using the resting cell of recombinant bacterium E.coli (HADH-LlKAR-GDH) as catalyst, with racemic neighbour's chlorine Mandelic acid is substrate, the substrate supplemented by glucose, with KH₂PO₄-K₂HPO₄Buffer solution is reaction medium.3.0M is used in reaction process NaOH automatically control pH 7.0, the conversion fluid of (R)-o-chloromandelic acid is contained after the reaction was complete.

Compared with prior art, the beneficial effects are mainly as follows：

(1) present invention provides a kind of efficient ketone acid reduction from lactic acid leukonid (Leuconostoc lactis) Enzyme (preferably LlKAR), can be catalyzed wide spectrum aromatic series 2- ketone acids, and using benzoylformic acid as the substrate useful load of substrate by 100mM is promoted to 400mM.Utilize ketone acid reductase (preferably LlKAR), 2- hydroxy acid dehydrogenases (HADH) and glucose dehydrogenase (GDH) three enzyme of the single bacterium double-mass model series connection redox established cascades system, can be catalyzed most of racemic fragrance 2- hydroxy acids Efficient deracemization is chiral fragrant (R) -2- hydroxy acids, and yield and e.e. values are all higher than 99%.It is finally applied to racemization 300mM O-chloromandelic acid prepares optical voidness (R)-o-chloromandelic acid, and yield is up to 83.8g/ (Ld).

(2) three enzyme of the single bacterium double-mass model series connection oxidation of ketone acid reductase (preferably LlKAR), HADH and GDH structures is utilized also Simple conjuncted system can be used for the efficient deracemization of the chiral fragrance 2- hydroxy acids of racemic, prepare the light with higher utility value Learn pure fragrant (R) -2- hydroxy acids.The reaction has at low cost, environmentally protective, simple for process, high catalytic efficiency, without adding external source Advantages, the industrial prospects such as property coenzyme are wide.

(4) it illustrates

Fig. 1 is the reaction mechanism that the series connection redox of three enzyme of single bacterium double-mass model cascades system.

Fig. 2 is that ketone acid reductase LeKAR, LlKAR, LmKAR and KoKAR substrate loads amount comparing analysis.

Fig. 3 is that recombinant bacterium E.coli BL21 (DE3)/pET28b-HADH/pCDFDuet-LlKAR-GDH builds schematic diagram.

Fig. 4 is the protein electrophoresis analysis of HADH, LlKAR and GDH coexpression, and swimming lane M is standard protein molecular weight marker, Swimming lane 1 is recombinant bacterium E.coli BL21 (the DE3)/pCDFDuet-LlKAR-GDH not induced, and swimming lane 2 is to be induced through IPTG Recombinant bacterium E.coli BL21 (DE3)/pCDFDuet-LlKAR-GDH, swimming lane 3 are the recombinant bacterium E.coli BL21 not induced (DE3)/pET28b-HADH/pCDFDuet-LlKAR-GDH, swimming lane 4 are the recombinant bacterium E.coli BL21 induced through IPTG (DE3)/pET28b-HADH/pCDFDuet-LlKAR-GDH。

Fig. 5 is that E.coli (HADH-LeKAR-GDH) catalysis 200mM o-chloromandelic acids go racemization process.

Fig. 6 is that E.coli (HADH-LlKAR-GDH) catalysis 200mM o-chloromandelic acids go racemization process.

Fig. 7 is that E.coli (HADH-LlKAR-GDH) catalysis 300mM o-chloromandelic acids go racemization process.

(5) specific implementation mode

With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in This：

Embodiment 1：Screen efficient ketone acid reductase LlKAR

With the amino acid sequence of known ketone acid reductase LeKAR, (shown in SEQ ID NO.2, corresponding nucleotides sequence is classified as SEQ Shown in ID NO.1) it is that template compares through NCBI-Blastp and obtains 5 kinds of potential ketone acids reduction enzyme sequences online, respectively LlKAR (shown in amino acid sequence SEQ ID NO.4, nucleotides sequence is classified as shown in SEQ ID NO.3), LpKAR (amino acid sequences Shown in SEQ ID NO.6, nucleotides sequence is classified as shown in SEQ ID NO.5), LmKAR (shown in amino acid sequence SEQ ID NO.8, Nucleotides sequence is classified as shown in SEQ ID NO.7), (shown in amino acid sequence SEQ ID NO.10, nucleotides sequence is classified as SEQ to KoKAR Shown in ID NO.9) and SnKAR (shown in amino acid sequence SEQ ID NO.12, nucleotides sequence is classified as shown in SEQ ID NO.11), Amino acid sequence homology is respectively 84%, 78%, 74%, 49% and 49%.Subsequently through synthesizing corresponding nucleotides sequence in vitro It arranges and is connected into expression plasmid pET28b, obtain 6 kinds of recombinant plasmids pET28b-LeKAR, pET28b-LlKAR, pET28b- LpKAR, pET28b-LmKAR, pET28b-KoKAR and pET28b-SnKAR are transformed into respectively in E.coli BL21 (DE3), are applied For cloth on the LB tablets containing 50 μ g/mL kanamycins, screening obtains 6 kinds of recombinant bacterium E.coli BL21 (DE3)/pET28b- LeKAR、E.coli BL21(DE3)/pET28b-LlKAR、E.coli BL21(DE3)/pET28b-LpKAR、E.coli BL21 (DE3)/pET28b-LmKAR, E.coli BL21 (DE3)/pET28b-KoKAR and E.coli BL21 (DE3)/pET28b- SnKAR。

By 6 kinds of ketone acid reductase recombinant bacteriums and glucose glucocorticoid dehydrogenase recombinant bacterium E.coli BL21 (DE3)/pET28b-GDH (GDH gene nucleotide series are shown in SEQ ID NO.15, shown in amino acid sequence SEQ ID NO.16) is inoculated into respectively to be contained In the LB liquid medium of 50 μ g/mL kanamycins, 8~10h of shaken cultivation under the conditions of 37 DEG C, 150rpm obtains seed liquor； By seed liquor by 2% (volumetric concentration) inoculum concentration access containing 50 μ g/mL kanamycins LB liquid medium in, in 37 DEG C, Shaken cultivation is to OD under the conditions of 150rpm₆₀₀Reach 0.4~0.8 (preferably 0.6), IPTG is added to final concentration of 0.1mM, in 28 DEG C, 10~12h of shaken cultivation under the conditions of 150rpm, be collected by centrifugation wet thallus and with brine twice, that is, respectively obtain 4 The resting cell of kind ketone acid reductase recombinant bacterium and glucose glucocorticoid dehydrogenase recombinant bacterium.Resting cell is resuspended in KH₂PO₄-K₂HPO₄ In buffer solution (100mM, pH7.0), ultrasonication 20min (broken power 40W, work 1s, stops 1s), is crushed liquid under condition of ice bath 10min is centrifuged under 4 DEG C, 12000rpm, it is corresponding crude enzyme liquid to collect supernatant.

The enzyme activity determination of 6 kinds of ketone acid reductases carries out in 1mL reaction systems, and system includes KH₂PO₄-K₂HPO₄Buffer solution (100mM, pH7.0), benzoylformic acid (10mM), NADH (5mM) and suitable thick enzyme (0.2 μ g).Reaction system and crude enzyme liquid point After incubating 5min not at 35 DEG C, 2min is reacted under the conditions of 35 DEG C, 700rpm, is terminated and is reacted with HCl (6.0M).Sample pass through from Chiral HPLC is carried out after the heart (12000rpm, 2min), 2 times of ultra-pure water dilution, 0.22 μm of membrane filtration.Unit enzyme activity defines (1U)：Under standard conditions, it is a unit enzyme activity that per min, catalysis benzoylformic acid, which generates the enzyme amount needed for 1 μm of ol product,.Specific enzyme activity Unit：The thick enzymes of kU/mg.The results show that the ratio of 6 kinds of ketone acid reductases LeKAR, LlKAR, LpKAR, LmKAR, KoKAR and SnKAR Enzyme activity is respectively 1.11kU/mg, 3.71kU/mg, 0kU/mg, 3.37kU/mg, 2.89kU/mg and 0kU/mg, wherein only 4 kinds Ketone acid reductase LeKAR, LlKAR, LmKAR and KoKAR have catalytic activity, and LlKAR has highest specific enzyme activity.

The detection of enantiomeric excess value (e.e.) carries out in 1mL reaction systems, and system includes KH₂PO₄-K₂HPO₄Buffering The thick enzyme of liquid (100mM, pH7.0), benzoylformic acid (10mM), NADH (15mM) and suitable concentration.Under the conditions of 35 DEG C, 700rpm It is reacted with HCl (6.0M) terminations after reacting 10h.Sample dilutes 2 times, 0.22 μm of film through centrifugation (12000rpm, 2min), ultra-pure water Chiral HPLC is carried out after filtering.The results show that 4 kinds of ketone acid reductases LeKAR, LlKAR, LmKAR and KoKAR are catalyzed benzene second The e.e. values that ketone acid generates (R)-mandelic acid are all higher than 99%.

Further comparative analysis of 4 kinds of ketone acid reductase LeKAR, LlKAR, LmKAR and KoKAR under high concentration of substrate exists It is carried out in 10mL reaction systems, system includes KH₂PO₄-K₂HPO₄Buffer solution (100mM, pH7.0), benzoylformic acid (selection 100mM With two kinds of concentration of 400mM), glucose (concentration is 2 times of benzoylformic acid, i.e., respectively 200mM and 800mM), NAD⁺ (0.5mM), KAR (ketone acid reductase, 800U/mL) and GDH (glucose dehydrogenase, 800U/mL).In 35 DEG C, 700rpm conditions After lower reaction 3h reaction is terminated with HCl (6.0M).Sample dilutes 2 times, 0.22 μm through centrifugation (12000rpm, 2min), ultra-pure water Chiral HPLC is carried out after membrane filtration.PH is automatically controlled 7.0 with the NaOH of 3.0M in reaction process.The results are shown in Figure 2, Conversion ratios of 4 kinds of ketone acid reductase LeKAR, LlKAR, LmKAR and KoKAR when being catalyzed 100mM benzoylformic acid asymmetric reductions It is all higher than 99% with e.e. values；E.e. values when being catalyzed 400m benzoylformic acid asymmetric reductions are all higher than 99%, but conversion ratio Respectively 65.8%, 99.2%, 75.5% and 86.4%.Only LlKAR can be catalyzed the acetophenone of 100mM and 400mM simultaneously Acid is fully converted to (R)-mandelic acid, and conversion ratio and e.e. values are all higher than 99%, therefore select specific enzyme activity and substrate useful load highest LlKAR for building three enzymes coexpression recombinant bacterium E.coli BL21 (DE3)/pET28b-HADH/pCDFDuet-LlKAR- GDH。

Chiral HPLC method is as follows：Reverse phase chiral column (model C hirobioticTM R250 × 4.6mm, Sigma, USA), mobile phase is 0.5% ammonium hydroxide:CH₃OH(10:90, v/v), Detection wavelength 215nm, 3 μ L of sample size.

E.e. the computational methods being worth：Ee (%)=(R-S)/(R+S) × 100%.R indicates (R) -2- after reaction in formula The concentration of hydroxy acid, S indicate the concentration of (S) -2- hydroxy acids after reaction.

Embodiment 2：Build recombinant bacterium E.coli BL21 (DE3)/pET28b-HADH/pCDFDuet-LlKAR-GDH

The glucose dehydrogenase (GDH) of Exiguobacterium sibiricum (WP_012369122.1) will be derived from Gene (nucleotides sequence is classified as shown in SEQ ID NO.15, shown in amino acid sequence SEQ ID NO.16) is corresponding through external synthesis Nucleotide sequence is simultaneously connected into expression plasmid pET28b, is obtained recombinant plasmid pET28b-GDH, is transformed into E.coli BL21 (DE3) it in, is coated on the LB tablets containing 50 μ g/mL kanamycins, screening recombinant bacterium E.coli BL21 (DE3)/pET28b- GDH。

By seamless Cloning Kit (II, Vazyme Biotech Co., Ltd), by GDH and preferably LlKAR nucleotide sequence successively connect with expression vector pCDFDuet-1, obtain recombinant plasmid pCDFDuet-LlKAR- GDH is transformed into E.coli BL21 (DE3), is coated on the LB tablets containing 50 μ g/mL streptomysins, screening recombinant bacterium E.coli BL21(DE3)/pCDFDuet-LlKAR-GDH。

2- hydroxy acid dehydrogenases (HADH) gene (core of Pseudomonas aeruginosa (AGM49308.1) will be derived from Nucleotide sequence is shown in SEQ ID NO.13, shown in amino acid sequence SEQ ID NO.14) through synthesizing corresponding nucleotide in vitro Sequence is simultaneously connected into expression plasmid pET28b, obtains recombinant plasmid pET28b-HADH, is transformed into E.coli BL21 (DE3) In, it is coated on the LB tablets containing 50 μ g/mL kanamycins, screening recombinant bacterium E.coli BL21 (DE3)/pET28b-HADH.

Respectively from recombinant bacterium E.coli BL21 (DE3)/pET28b-HADH and E.coli BL21 (DE3)/pCDFDuet- Plasmid pET28b-HADH and pCDFDuet-LlKAR-GDH are extracted in LlKAR-GDH, by 1:After 1 molar concentration rate mixing, altogether With being transformed into E.coli BL21 (DE3), it is coated on the dual anti-LB tablets containing 50 μ g/mL kanamycins and 50 μ g/mL streptomysins On, it screens while recombinant bacterium E.coli BL21 (DE3)/pET28b-HADH/ containing HADH, LlKAR and GDH gene PCDFDuet-LlKAR-GDH is abbreviated as E.coli (HADH-LlKAR-GDH).Same method builds recombinant bacterium E.coli (HADH-LeKAR-GDH)。

The structure of recombinant plasmid and recombinant bacterium is as shown in Figure 3.

Embodiment 3：Induce the bis- enzyme coexpressions of recombinant bacterium E.coli BL21 (DE3)/pCDFDuet-LlKAR-GDH

Recombination bacillus coli E.coli BL21 (DE3)/pCDFDuet-LlKAR-GDH is inoculated into containing 50 μ g/mL strepto-s In the LB liquid medium of element, 8~10h of shaken cultivation under the conditions of 37 DEG C, 150rpm obtains seed liquor；Seed liquor is pressed 2% In the LB liquid medium of the inoculum concentration access containing 50 μ g/mL streptomysins of (volumetric concentration), vibrated under the conditions of 37 DEG C, 150rpm It cultivates to OD₆₀₀Reach 0.6, IPTG to final concentration of 0.1mM, 10~12h of shaken cultivation under the conditions of 28 DEG C, 150rpm be added, Wet thallus is collected by centrifugation and with brine twice to get E.coli BL21 (DE3)/pCDFDuet-LlKAR-GDH's Resting cell.E.coli BL21 (DE3)/pCDFDuet-LlKAR-GDH not induce carries out SDS-PAGE eggs as a contrast White electrophoretic analysis, the results are shown in Figure 4, it can be seen that respectively occurs one obviously near 32kDa and 28kDa after IPTG is induced Band, it is consistent with the theoretical protein molecular size range of LlKAR and GDH, it was demonstrated that recombinant bacterium E.coli BL21 (DE3)/ PCDFDuet-LlKAR-GDH is built successfully.

Embodiment 4：Induce recombinant bacterium E.coli (HADH-LlKAR-GDH) three enzyme coexpression

Recombination bacillus coli E.coli (HADH-LlKAR-GDH) is inoculated into containing 50 μ g/mL kanamycins and 50 μ g/mL In the LB liquid medium of streptomysin, 8~10h of shaken cultivation under the conditions of 37 DEG C, 150rpm obtains seed liquor；By seed liquor In the LB liquid medium for containing 50 μ g/mL kanamycins and 50 μ g/mL streptomysins by the inoculum concentration access of 2% (volumetric concentration), Shaken cultivation is to OD under the conditions of 37 DEG C, 150rpm₆₀₀Reach 0.6, IPTG to final concentration of 0.1mM be added, in 28 DEG C, Wet thallus is collected by centrifugation and with brine twice to get E.coli in 10~12h of shaken cultivation under the conditions of 150rpm (HADH-LlKAR-GDH) resting cell.E.coli (HADH-LlKAR-GDH) not induce carries out SDS- as a contrast PAGE protein electrophoresis is analyzed, and the results are shown in Figure 4, it can be seen that is divided near 41kDa, 32kDa and 28kDa after IPTG is induced Not Chu Xian an apparent band, it is consistent with the theoretical protein molecular size range of HADH, LlKAR and GDH, it was demonstrated that recombinant bacterium E.coli (HADH-LlKAR-GDH) is built successfully.Same method Prepare restructuring bacterium E.coli (HADH-LeKAR-GDH) tranquillization is thin Born of the same parents.

Embodiment 5：Recombinant bacterium E.coli (HADH-LlKAR-GDH) catalytic condition optimizes

By the method for embodiment 4, the resting cell of recombinant bacterium E.coli (HADH-LlKAR-GDH), design optimization are obtained Reaction system is 10mL：It is substrate to select the racemic o-chloromandelic acid of final concentration of 20mM, investigates cell concentration 4g/L, 8g/ L, 12g/L (with dry bacterium restatement), auxiliary substrate glucose concentration 10mM, 20mM, 30mM, reaction medium KH₂PO₄-K₂HPO₄Buffer solution PH 6.0,7.0,8.0,25 DEG C of temperature, 30 DEG C, influence of the factors such as 35 DEG C to reaction.HCl is used after reacting 2h under 700rpm (6.0M) terminates reaction, and sample uses the Chiral HPLC Method in embodiment 1 to be detected analysis, is to comment with target product yield Valence index, as a result such as following table：

Table 1

The result shows that recombinant bacterium E.coli (HADH-LlKAR-GDH) is catalyzed 20mM racemic fragrance 2- hydroxy acid deracemizations When, cell concentration is with the dry preferred 8g/L of bacterium restatement, the auxiliary preferred 20mM of concentration of substrate, the KH of the preferred pH7.0 of reaction medium₂PO₄- K₂HPO₄Buffer solution, preferably 30 DEG C of temperature.

Embodiment 6：Recombinant bacterium E.coli (HADH-LlKAR-GDH) is catalyzed the deracemization of racemic fragrance 2- hydroxy acids

Reaction carries out in the transformation system of 10mL, and system includes KH₂PO₄-K₂HPO₄It is buffer solution (100mM, pH7.0), outer Racemization fragrance 2- hydroxy acids (final concentration 20mM, structure are shown in Fig. 1 and table 2), glucose (final concentration 20mM) are obtained by embodiment 4 Three enzymes coexpression recombinant bacterium (dosage is calculated as 8g/L with dry cell weight).Reaction reacts 4h under the conditions of 30 DEG C, 700rpm, every 1h timing samplings, sample is terminated with HCl (6.0M) to react, and dilutes 4 times, 0.22 μm through centrifugation (12000rpm, 2min), ultra-pure water Analysis is detected using the Chiral HPLC Method in embodiment 1 after membrane filtration.As a result such as following table：

Table 2

Catalytic result shows that most of racemic fragrance 2- hydroxy acids (1a-1m) pass through the reaction of 2h, are efficiently converted For corresponding fragrant (R) -2- hydroxy acids, yield is more than 98%, e.e. values and is more than 99%.Even if racemic fragrance 2- hydroxy acids (1f- 1h, 1i-1k) there are ortho position, meta or para positions to replace on phenyl ring, and the stereoeffect of generation does not influence whole catalysis effect but Rate.For racemic fragrance 2- hydroxy acids (1n-1q), due to having a small amount of (S) -2- hydroxy acids unconverted complete after reaction or having few Intermediate ketone acid accumulation is measured, causes yield relatively relatively low, but also reach 80% or more.And for racemic fragrance 2- hydroxy acids (1r-1t), the three enzymes coexpression system do not have catalytic activity substantially.Generally speaking, due to excavate arrived it is a kind of efficient Ketone acid reductase LlKAR, and it is chiral fragrance 2- hydroxy acids to find that it can effectively be catalyzed aromatic series 2- keto acid transformations, by its with The three enzyme coexpression redox of HADH and GDH coupling structures single bacterium double-mass model cascades system, outer disappears to most of to realize The efficient deracemization of fragrance 2- hydroxy acids is revolved, there is important application value in actual production.

Embodiment 7：Recombinant bacterium E.coli (HADH-LlKAR-GDH) is catalyzed 200mM o-chloromandelic acid deracemizations

Reaction carries out in the transformation system of 20mL, and system includes KH₂PO₄-K₂HPO₄It is buffer solution (100mM, pH7.0), outer Racemization o-chloromandelic acid (final concentration 200mM), glucose (final concentration 200mM) co-express weight by three enzymes that embodiment 4 obtains Group bacterium (dosage is calculated as 20g/L with dry cell weight).Reaction reacts 20h under the conditions of 30 DEG C, 700rpm, every 2h timing samplings, Sample is terminated with HCI (6.0M) to react, using the HPLC in embodiment 1 after centrifugation (12000rpm, 2min), dilution, mistake film Method is detected analysis.PH is automatically controlled 7.0 with the NaOH of 3.0M in reaction process, is contained after the reaction was complete (R)- The conversion fluid of o-chloromandelic acid.Reaction process is catalyzed as shown in fig. 6, the racemic o-chloromandelic acid of 200mM is complete in 16h It is converted into optical voidness (R)-o-chloromandelic acid, yield and e.e. values are all higher than 99%, and rate of non-occupied time is up to 55.9g/ (Ld).

Under similarity condition, the recombinant bacterium E.coli (HADH-LeKAR-GDH) obtained in 4 method of embodiment is catalyst, knot Fruit is：E.coli (HADH-LeKAR-GDH) is when urging 200mM racemic o-chloromandelic acids to go racemization, because intermediate ketone acid is a large amount of Accumulation, reaction is until 22h, and racemic o-chloromandelic acid cannot still be fully converted to (R)-o-chloromandelic acid, as a result such as Fig. 5 institutes Show.

Embodiment 8：Recombinant bacterium E.coli (HADH-LlKAR-GDH) is catalyzed 300mM o-chloromandelic acid deracemizations

Reaction carries out in the transformation system of 20mL, and system includes KH₂PO₄-K₂HPO₄It is buffer solution (100mM, pH7.0), outer Racemization o-chloromandelic acid (100mM), glucose (100mM), three enzymes that are obtained by embodiment 4 co-express recombinant bacterium (dosage with Dry cell weight is calculated as 20g/L).After reaction starts under the conditions of 30 DEG C, 700rpm, racemic neighbour's chlorine almond is added every 4h Sour (100mM) and glucose (100mM), are added 2 times altogether.Reaction terminates anti-every 1h timing samplings, sample HCI (6.0M) It answers, analysis is detected using the HPLC methods in embodiment 1 after centrifugation (12000rpm, 2min), dilution, mistake film.Reaction PH is automatically controlled 7.0 with the NaOH of 3.0M in the process, and the conversion fluid of (R)-o-chloromandelic acid is contained after the reaction was complete.It urges Change reaction process as shown in fig. 7, the racemic o-chloromandelic acid of final 300mM is completely converted in 16h as optical voidness (R)- O-chloromandelic acid, yield and e.e. values are all higher than 99%, and rate of non-occupied time is up to 83.8g/ (Ld).(R)-o-chloromandelic acid is anti- The important chiral building block of thrombus medicine (clopidogrel) synthesis, therefore catalysis reaction has great application value.

Sequence table

<110>Zhejiang Polytechnical University

<120>Ketone acid reductase, gene, engineering bacteria and the application in synthesis of chiral fragrance 2- hydroxy acids

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Thr Lys Leu Leu Val Leu Ser Asn Gly Leu Gly Asn Ile Glu Val Met

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Ala Lys His Val Ser Arg His Gln Ile Leu Ala Gly Val Thr Leu Trp

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Thr Ser Ser Leu Ile Lys Pro Gly Glu Ile His Val Thr Gly Ser Gly

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Ser Ile Lys Leu Gln Ala Ile Gly Asp Ala Asp Val Gln Ser Ile Ala

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Asp Ala Leu Asn Gln Ala Gly Leu Asn Ala Glu Ile Thr Pro Asp Val

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Met Thr Ala Ile Trp His Lys Ala Gly Ile Asn Ala Val Leu Asn Pro

180 185 190

Leu Ser Val Leu Leu Asn Ala Asn Ile Ala Glu Phe Gly Thr Ala Gly

195 200 205

Asn Ala Met Asp Leu Ala Leu Asn Ile Leu Asp Glu Met Lys Gln Val

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Met Lys Ile Ala Ile Ala Gly Phe Gly Ala Leu Gly Ala Arg Leu Gly

1 5 10 15

Ile Met Leu Gln Ala Ala Gly His Asp Val Thr Gly Ile Asp Gly Trp

20 25 30

Pro Ala His Ile Ala Ala Ile Asn Thr Lys Gly Leu Thr Val Val His

35 40 45

Asp Asp Gln Asp Pro Lys Val Tyr Tyr Leu Pro Val Met Thr Pro Gln

50 55 60

Glu Val Thr Gly Thr Phe Asp Leu Ile Ile Leu Leu Thr Lys Thr Pro

65 70 75 80

Gln Leu Asp Arg Met Leu Thr Asp Ile Ala Pro Ile Ile Thr Asp Gln

85 90 95

Thr Gln Leu Leu Ile Leu Ser Asn Gly Leu Gly Asn Ile Glu Val Met

100 105 110

Ala Lys His Val Ala Lys Ser Gln Ile Val Ala Gly Val Thr Leu Trp

115 120 125

Thr Ser Ser Leu Val Lys Pro Gly Glu Ile His Thr Thr Gly Ser Gly

130 135 140

Ser Ile Lys Leu Gln Ala Leu Ala Gly Gly Asp Ala Gln Pro Ile Val

145 150 155 160

Glu Ala Leu Asn Glu Ala Gly Leu Asn Ala Glu Leu Val Pro Asp Val

165 170 175

Met Thr Ala Ile Trp His Lys Ala Gly Ile Asn Ala Val Leu Asn Pro

180 185 190

Leu Ser Val Leu Leu Asp Ala Asn Ile Ala Glu Phe Gly Thr Ala Gly

195 200 205

Asn Ala Met Asp Met Ala Leu Asn Ile Leu Asp Glu Met Lys Gln Val

210 215 220

Gly Ala Ser Gln Gly Ile Lys Val Asp Val Ala Gly Ile Ile Ala Asp

225 230 235 240

Leu Ser Arg Leu Leu Lys Pro Glu Asn Ala Gly Asn His Tyr Pro Ser

245 250 255

Met Tyr Gln Asp Ile Gln Asn Gly Lys Arg Thr Glu Ile Asp Phe Leu

260 265 270

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275 280 285

Phe Asn Ala Leu Val Thr Arg Leu Ile His Ala Lys Glu Asp Ile Val

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Arg Thr Lys Leu Ala Lys Glu Lys Glu Asn Phe Glu Ile

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Ala Glu His Ile Ala Ala Ile Asn Thr Lys Gly Leu Thr Val Thr Glu

35 40 45

Asp Asp Gly Ser Ser Lys Lys Tyr Phe Ile Pro Val Met Thr Ser Lys

50 55 60

Glu Val Thr Gly Glu Phe Asp Leu Val Ile Leu Leu Thr Lys Thr Pro

65 70 75 80

Gln Leu Asp Arg Met Leu Thr Asp Ile Gln Pro Leu Ile Thr Lys Gln

85 90 95

Thr Gln Leu Leu Val Leu Ser Asn Gly Leu Gly Asn Val Glu Val Met

100 105 110

Ala Lys His Val Ser Ser Gln Gln Ile Ile Ala Gly Val Thr Leu Trp

115 120 125

Thr Ser Asp Leu Val Gln Pro Gly Glu Ile His Val Thr Gly Thr Gly

130 135 140

Ser Ile Lys Leu Gln Ala Ile Asp His Ala Asp Ile Thr Ala Val Val

145 150 155 160

Thr Ala Leu Asn Glu Ala Gly Leu Asn Ala Glu Val Ser Asp Asn Val

165 170 175

Val Glu Ala Ile Trp His Lys Ala Gly Ile Asn Ser Val Leu Asn Pro

180 185 190

Leu Thr Val Leu Leu Asp Ala Asn Ile Ala Glu Phe Gly Thr Ala Gly

195 200 205

Asn Gly Met Asp Leu Ala Leu Asn Ile Leu Asp Glu Ile Lys Gln Val

210 215 220

Gly Asp Val Ala Gly Val Asn Val Asp Val Asn Ser Ile Leu Ser Asp

225 230 235 240

Leu Ser Asn Leu Leu Lys Pro Glu Asn Ala Gly Asn His Tyr Pro Ser

245 250 255

Met Tyr Gln Asp Ile Gln Ala Gly Lys Arg Thr Glu Ile Asp Phe Leu

260 265 270

Asn Gly Tyr Phe Ala Lys Leu Gly Arg Glu Asn His Ile Ala Thr Pro

275 280 285

Phe Asn Ala Leu Val Thr Arg Leu Ile His Ala Lys Glu Asp Ile Glu

290 295 300

Arg Val Lys Leu Ala Lys Gln Gln Glu Thr Phe Glu Ile

305 310 315

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<213>Leuconostoc mesenteroides (Leuconostoc mesenteroides)

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atgaaaatcg ctatcgctgg tttcggtgct ctgggtgctc gtgttggtgt tatgctgcag 60

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atcaaagctg gtaaacacac cgaaatcgac ttcctgaacg gttacttcgc taaactgggt 840

tctgaacacg acgttgctat gccgttcaac gctctggtta cccgtctgat ccacgctaaa 900

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<210> 8

<211> 317

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<213>Leuconostoc mesenteroides (Leuconostoc mesenteroides)

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Met Lys Ile Ala Ile Ala Gly Phe Gly Ala Leu Gly Ala Arg Val Gly

1 5 10 15

Val Met Leu Gln Gln Ala Gly His Glu Val Thr Gly Ile Asp Gly Trp

20 25 30

Ala Ala His Ile Ala Ala Ile Ser Thr Asp Gly Leu Thr Val His Gln

35 40 45

Asp Asp Gly Ala Thr Lys Lys Tyr Tyr Ile Pro Val Met Thr Ala Lys

50 55 60

Glu Ile Asp Gly Lys Phe Asp Leu Ile Ile Leu Leu Thr Lys Thr Pro

65 70 75 80

Gln Leu Asp Met Met Leu Thr Asp Ile Lys His Ile Ile Thr Lys Asn

85 90 95

Thr Lys Leu Leu Val Leu Ser Asn Gly Leu Gly Asn Ile Glu Val Met

100 105 110

Glu Lys His Val Asn Arg Asn Gln Ile Leu Ala Gly Val Thr Leu Trp

115 120 125

Thr Ser Glu Leu Ile Asn Pro Gly Glu Ile Arg Val Thr Gly Thr Gly

130 135 140

Ser Ile Lys Leu Gln Ala Ile Gly Glu Ala Asn Ala Lys Pro Ile Val

145 150 155 160

Ser Ala Leu Asn Lys Ala Gly Leu Asn Val Thr Leu Ser Gln Asn Val

165 170 175

Ile Glu Ala Ile Trp His Lys Ala Gly Ile Asn Ser Val Leu Asn Pro

180 185 190

Leu Thr Val Leu Leu Asp Ala Asn Ile Ala Glu Phe Gly Met Ala Gly

195 200 205

Asn Gly Met Asp Leu Ser Leu Asn Ile Leu Asp Glu Ile Lys Lys Ile

210 215 220

Gly Glu Leu Glu Gly Ile Asn Val Asp Val Asn Ala Ile Met Lys Asp

225 230 235 240

Leu Ala Leu Leu Ile Arg Pro Glu Asn Ala Gly Asn His Tyr Pro Ser

245 250 255

Met Tyr Gln Asp Ile Lys Ala Gly Lys His Thr Glu Ile Asp Phe Leu

260 265 270

Asn Gly Tyr Phe Ala Lys Leu Gly Ser Glu His Asp Val Ala Met Pro

275 280 285

Phe Asn Ala Leu Val Thr Arg Leu Ile His Ala Lys Glu Asp Ile Glu

290 295 300

Arg Thr Lys Leu Ala Lys Lys Gln Glu Thr Phe Glu Ile

305 310 315

<210> 9

<211> 918

<212> DNA

<213>Klebsiella oxytoca (Klebsiella oxytoca)

<400> 9

atgaaaatcg ctatcgctgg tgctggtgct atgggttgcc gtttcggtta catgctgctg 60

ggtgctggtc acgacgttac cctgatcgac ggttggcacg aacacgttaa cgctatctgc 120

tctaacggtc tgttcgttga aaccgaagtt tctcagcagt actacccgat cccggctatg 180

ctggctgacg aatctcaggg tgaattcgaa ctgatcatcc tgttcaccaa agctatgcag 240

ctggaccgta tgctgcagca catcaaaccg ctgctgccgg ctgctaaagt tgttatgatc 300

ctgtctaacg gtctgggtaa catcgaaacc ctggaaaaat acgttgaccg tcagaaaatc 360

tacgctggtg ttaccctgtg gtcttctgaa ctggaaggtc cgggtcacat catggctacc 420

ggtaccggta ccatcgaact gcagccggtt gcttctcagg acgctgctct ggaagaaaac 480

atcgttgctg ttctgaactc tgctggtctg aacgctgaaa tctctccgga cgttctgctg 540

tctatctgga aaaaagctgc tttcaactct gttatgaaca cctactgcgc tctgctggac 600

tgcaacgttg gtggtttcgg tcagctgccg ggtgctctgg acctggctca ggctgttgtt 660

gacgaattcg ttctggttgc tgcttctcag aacatcccgc tgtctggtga acgtgttatg 720

aacaccgtta aaaaagtttt cgacccgcgt gaatctggtc accactaccc gtctatgtac 780

caggacctgc agaaaggtcg tctgaccgaa atcgactacc tgaacggtgc tatcgctcgt 840

atcggtatgc agaacaacat cccggttccg gttaacaccc tgctgaccca gctgatccac 900

gctaaagaag ctcagtaa 918

<210> 10

<211> 305

<212> PRT

<213>Klebsiella oxytoca (Klebsiella oxytoca)

<400> 10

Met Lys Ile Ala Ile Ala Gly Ala Gly Ala Met Gly Cys Arg Phe Gly

1 5 10 15

Tyr Met Leu Leu Gly Ala Gly His Asp Val Thr Leu Ile Asp Gly Trp

20 25 30

His Glu His Val Asn Ala Ile Cys Ser Asn Gly Leu Phe Val Glu Thr

35 40 45

Glu Val Ser Gln Gln Tyr Tyr Pro Ile Pro Ala Met Leu Ala Asp Glu

50 55 60

Ser Gln Gly Glu Phe Glu Leu Ile Ile Leu Phe Thr Lys Ala Met Gln

65 70 75 80

Leu Asp Arg Met Leu Gln His Ile Lys Pro Leu Leu Pro Ala Ala Lys

85 90 95

Val Val Met Ile Leu Ser Asn Gly Leu Gly Asn Ile Glu Thr Leu Glu

100 105 110

Lys Tyr Val Asp Arg Gln Lys Ile Tyr Ala Gly Val Thr Leu Trp Ser

115 120 125

Ser Glu Leu Glu Gly Pro Gly His Ile Met Ala Thr Gly Thr Gly Thr

130 135 140

Ile Glu Leu Gln Pro Val Ala Ser Gln Asp Ala Ala Leu Glu Glu Asn

145 150 155 160

Ile Val Ala Val Leu Asn Ser Ala Gly Leu Asn Ala Glu Ile Ser Pro

165 170 175

Asp Val Leu Leu Ser Ile Trp Lys Lys Ala Ala Phe Asn Ser Val Met

180 185 190

Asn Thr Tyr Cys Ala Leu Leu Asp Cys Asn Val Gly Gly Phe Gly Gln

195 200 205

Leu Pro Gly Ala Leu Asp Leu Ala Gln Ala Val Val Asp Glu Phe Val

210 215 220

Leu Val Ala Ala Ser Gln Asn Ile Pro Leu Ser Gly Glu Arg Val Met

225 230 235 240

Asn Thr Val Lys Lys Val Phe Asp Pro Arg Glu Ser Gly His His Tyr

245 250 255

Pro Ser Met Tyr Gln Asp Leu Gln Lys Gly Arg Leu Thr Glu Ile Asp

260 265 270

Tyr Leu Asn Gly Ala Ile Ala Arg Ile Gly Met Gln Asn Asn Ile Pro

275 280 285

Val Pro Val Asn Thr Leu Leu Thr Gln Leu Ile His Ala Lys Glu Ala

290 295 300

Gln

305

<210> 11

<211> 918

<212> DNA

<213>Salmonella enteritidis (Salmonella enterica)

<400> 11

atgaaaattg caatcgcagg tgcaggcgct atggggtgtc gttttggcta tatgctgctg 60

gaggccgggc acgacgtgac gcttatcgat agctggcagg agcatgtcga cgctattcgt 120

agcaaggggt tgtttgtcga aacggaaacg acgcagaagt attaccccat ccctgctatg 180

ttggctgatg aatcccaggg ggagtttgag ctggttattc tgtttaccaa agccatgcag 240

ttggatagca tgttacagcg tatcaagcca ttactgccag ccgcgaaagt cgtgatgatt 300

ctatctaacg gtctgggaaa tattgaaacg ctggagaaat atgtcgatcg gcataaaatc 360

tatgcgggtg tgacgttatg gtccagcgaa ctggaggggg ctgggcatat tatggccacc 420

ggtaccggaa cgattgaact gcagccgatt gccagccagg attcggctca agaggctaag 480

gtcattgcca cccttaatag cgctggattg aatgctgaaa taagccctga cgtattatta 540

tcgatctgga agaaagcagc ctttaatagc gtaatgaaca cctattgcgc gctactggat 600

tgtaatatcg gcggatttgg tcagcggcct ggtgctttag atttagcgca agccgtagtt 660

gatgagtttg tgttagttgc tgccagccag aatatttcgt tgactgagca aatggtgatg 720

aatacggtga agaaagtgtt cgatccgcgt gagagcggcc accactatcc ttctatgcat 780

caggatttac ataaaggccg actgactgaa atcgactatt taaatggtgc gattgcgcga 840

atcggcgttc agaacaatat tgccgtaccg gttaacacac tcctgacgca attgattcac 900

gctaaagaag cgcaataa 918

<210> 12

<211> 305

<212> PRT

<213>Salmonella enteritidis (Salmonella enterica)

<400> 12

Met Lys Ile Ala Ile Ala Gly Ala Gly Ala Met Gly Cys Arg Phe Gly

1 5 10 15

Tyr Met Leu Leu Glu Ala Gly His Asp Val Thr Leu Ile Asp Ser Trp

20 25 30

Gln Glu His Val Asp Ala Ile Arg Ser Lys Gly Leu Phe Val Glu Thr

35 40 45

Glu Thr Thr Gln Lys Tyr Tyr Pro Ile Pro Ala Met Leu Ala Asp Glu

50 55 60

Ser Gln Gly Glu Phe Glu Leu Val Ile Leu Phe Thr Lys Ala Met Gln

65 70 75 80

Leu Asp Ser Met Leu Gln Arg Ile Lys Pro Leu Leu Pro Ala Ala Lys

85 90 95

Val Val Met Ile Leu Ser Asn Gly Leu Gly Asn Ile Glu Thr Leu Glu

100 105 110

Lys Tyr Val Asp Arg His Lys Ile Tyr Ala Gly Val Thr Leu Trp Ser

115 120 125

Ser Glu Leu Glu Gly Ala Gly His Ile Met Ala Thr Gly Thr Gly Thr

130 135 140

Ile Glu Leu Gln Pro Ile Ala Ser Gln Asp Ser Ala Gln Glu Ala Lys

145 150 155 160

Val Ile Ala Thr Leu Asn Ser Ala Gly Leu Asn Ala Glu Ile Ser Pro

165 170 175

Asp Val Leu Leu Ser Ile Trp Lys Lys Ala Ala Phe Asn Ser Val Met

180 185 190

Asn Thr Tyr Cys Ala Leu Leu Asp Cys Asn Ile Gly Gly Phe Gly Gln

195 200 205

Arg Pro Gly Ala Leu Asp Leu Ala Gln Ala Val Val Asp Glu Phe Val

210 215 220

Leu Val Ala Ala Ser Gln Asn Ile Ser Leu Thr Glu Gln Met Val Met

225 230 235 240

Asn Thr Val Lys Lys Val Phe Asp Pro Arg Glu Ser Gly His His Tyr

245 250 255

Pro Ser Met His Gln Asp Leu His Lys Gly Arg Leu Thr Glu Ile Asp

260 265 270

Tyr Leu Asn Gly Ala Ile Ala Arg Ile Gly Val Gln Asn Asn Ile Ala

275 280 285

Val Pro Val Asn Thr Leu Leu Thr Gln Leu Ile His Ala Lys Glu Ala

290 295 300

Gln

305

<210> 13

<211> 1188

<212> DNA

<213>Pseudomonas aeruginosa (Pseudomonas aeruginosa)

<400> 13

atgtctcaga acctgttcaa cgttgaagac taccgtaaac tggctcagaa acgtctgccg 60

aaaatggttt acgactacct ggaaggtggt gctgaagacg aatacggtgt taaacacaac 120

cgtgacgttt tccagcagtg gcgtttcaaa ccaaagaggt tagttgacgt atcgcgtcgt 180

tctctgcagg ctgaagttct gggtaaacgt cagtctatgc cgctgctgat cggtccgacc 240

ggtctgaacg gtgctctgtg gccgaaaggt gacctggctc tggctcaggc tgctaccaaa 300

gctggtatcc cgttcgttct gtctaccgct tctaacatgt ctatcgaaga cctggctcgt 360

cagtgcgacg gtgacctgtg gttccagctg tacgttatcc accgtgaaat cgctcagggt 420

atggttctga aagctctgca ctctggttac accaccctgg ttctgaccac cgacgttgct 480

gttaacggtt accgtgaacg tgacctgcac aaccgtttca aaatgccgat gtcttacacc 540

ccgaaagtta tgctggacgg ttgcctgcac ccgcgttggt ctctggacct ggttcgtcac 600

ggtatgccgc agctggctaa cttcgtttct tctcagacct cttctctgga aatgcaggct 660

gctctgatgt ctaggcagat ggacgctagc ttcaactggg aagcgctgcg ttggctgcgt 720

gacctgtggc cgcacaaact gctggttaaa ggtctgctgt ctgctgaaga cgctgaccac 780

tgcatcgctg aaggtgctga cggtgttatc ctgtctaacc acggtggtcg tcagctggac 840

tgcgctgttt ctccgatgga agttctggct cagtctgttg ctaaaaccgg taaaccggtt 900

ctgatcgact ctggtttccg tcgtggttct gacatcgtta aagctctggc tctgggtgct 960

gaagctgttc tgctgggtcg tgctaccctg tacggtctgg ctgctcgtgg tgaaaccggt 1020

gttgacgaag ttctgaccct gctgaaagct gacatcgacc gtaccctggc tcagatcggt 1080

tgcccggaca tcacctctct gtctccggac tacctgcagt ctgaaggtgt tacctctacc 1140

gctccggttg accacctgat cggtaaaggt acccacgctc tcgagtga 1188

<210> 14

<211> 395

<212> PRT

<213>Pseudomonas aeruginosa (Pseudomonas aeruginosa)

<400> 14

Met Ser Gln Asn Leu Phe Asn Val Glu Asp Tyr Arg Lys Leu Ala Gln

1 5 10 15

Lys Arg Leu Pro Lys Met Val Tyr Asp Tyr Leu Glu Gly Gly Ala Glu

20 25 30

Asp Glu Tyr Gly Val Lys His Asn Arg Asp Val Phe Gln Gln Trp Arg

35 40 45

Phe Lys Pro Lys Arg Leu Val Asp Val Ser Arg Arg Ser Leu Gln Ala

50 55 60

Glu Val Leu Gly Lys Arg Gln Ser Met Pro Leu Leu Ile Gly Pro Thr

65 70 75 80

Gly Leu Asn Gly Ala Leu Trp Pro Lys Gly Asp Leu Ala Leu Ala Gln

85 90 95

Ala Ala Thr Lys Ala Gly Ile Pro Phe Val Leu Ser Thr Ala Ser Asn

100 105 110

Met Ser Ile Glu Asp Leu Ala Arg Gln Cys Asp Gly Asp Leu Trp Phe

115 120 125

Gln Leu Tyr Val Ile His Arg Glu Ile Ala Gln Gly Met Val Leu Lys

130 135 140

Ala Leu His Ser Gly Tyr Thr Thr Leu Val Leu Thr Thr Asp Val Ala

145 150 155 160

Val Asn Gly Tyr Arg Glu Arg Asp Leu His Asn Arg Phe Lys Met Pro

165 170 175

Met Ser Tyr Thr Pro Lys Val Met Leu Asp Gly Cys Leu His Pro Arg

180 185 190

Trp Ser Leu Asp Leu Val Arg His Gly Met Pro Gln Leu Ala Asn Phe

195 200 205

Val Ser Ser Gln Thr Ser Ser Leu Glu Met Gln Ala Ala Leu Met Ser

210 215 220

Arg Gln Met Asp Ala Ser Phe Asn Trp Glu Ala Leu Arg Trp Leu Arg

225 230 235 240

Asp Leu Trp Pro His Lys Leu Leu Val Lys Gly Leu Leu Ser Ala Glu

245 250 255

Asp Ala Asp His Cys Ile Ala Glu Gly Ala Asp Gly Val Ile Leu Ser

260 265 270

Asn His Gly Gly Arg Gln Leu Asp Cys Ala Val Ser Pro Met Glu Val

275 280 285

Leu Ala Gln Ser Val Ala Lys Thr Gly Lys Pro Val Leu Ile Asp Ser

290 295 300

Gly Phe Arg Arg Gly Ser Asp Ile Val Lys Ala Leu Ala Leu Gly Ala

305 310 315 320

Glu Ala Val Leu Leu Gly Arg Ala Thr Leu Tyr Gly Leu Ala Ala Arg

325 330 335

Gly Glu Thr Gly Val Asp Glu Val Leu Thr Leu Leu Lys Ala Asp Ile

340 345 350

Asp Arg Thr Leu Ala Gln Ile Gly Cys Pro Asp Ile Thr Ser Leu Ser

355 360 365

Pro Asp Tyr Leu Gln Ser Glu Gly Val Thr Ser Thr Ala Pro Val Asp

370 375 380

His Leu Ile Gly Lys Gly Thr His Ala Leu Glu

385 390 395

<210> 15

<211> 786

<212> DNA

<213>Siberia microbacterium (Exiguobacterium sibiricum)

<400> 15

atgtataatt ctctgaaagg caaagtcgcg attgttactg gtggtagcat gggcattggc 60

gaagcgatca tccgtcgcta tgcagaagaa ggcatgcgcg ttgttatcaa ctatcgtagc 120

catccggagg aagccaaaaa gatcgccgaa gatattaaac aggcaggtgg tgaagccctg 180

accgtccagg gtgacgtttc taaagaggaa gacatgatca acctggtgaa acagactgtt 240

gatcacttcg gtcagctgga cgtctttgtg aacaacgctg gcgttgagat gccttctccg 300

tcccacgaaa tgtccctgga agactggcag aaagtgatcg atgttaatct gacgggtgcg 360

ttcctgggcg ctcgtgaagc tctgaaatac ttcgttgaac ataacgtgaa aggcaacatt 420

atcaatatgt ctagcgtcca cgaaatcatc ccgtggccta ctttcgtaca ttacgctgct 480

tctaagggtg gcgttaaact gatgacccag actctggcta tggaatatgc accgaaaggt 540

atccgcatta acgctatcgg tccaggcgcg atcaacactc caattaatgc agaaaaattc 600

gaggatccga aacagcgtgc agacgtggaa agcatgatcc cgatgggcaa catcggcaag 660

ccagaggaga tttccgctgt cgcggcatgg ctggcttctg acgaagcgtc ttacgttacc 720

ggcatcaccc tgttcgcaga tggtggcatg accctgtacc cgagctttca ggctggccgt 780

ggttga 786

<210> 16

<211> 261

<212> PRT

<213>Siberia microbacterium (Exiguobacterium sibiricum)

<400> 16

Met Tyr Asn Ser Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Ser

1 5 10 15

Met Gly Ile Gly Glu Ala Ile Ile Arg Arg Tyr Ala Glu Glu Gly Met

20 25 30

Arg Val Val Ile Asn Tyr Arg Ser His Pro Glu Glu Ala Lys Lys Ile

35 40 45

Ala Glu Asp Ile Lys Gln Ala Gly Gly Glu Ala Leu Thr Val Gln Gly

50 55 60

Asp Val Ser Lys Glu Glu Asp Met Ile Asn Leu Val Lys Gln Thr Val

65 70 75 80

Asp His Phe Gly Gln Leu Asp Val Phe Val Asn Asn Ala Gly Val Glu

85 90 95

Met Pro Ser Pro Ser His Glu Met Ser Leu Glu Asp Trp Gln Lys Val

100 105 110

Ile Asp Val Asn Leu Thr Gly Ala Phe Leu Gly Ala Arg Glu Ala Leu

115 120 125

Lys Tyr Phe Val Glu His Asn Val Lys Gly Asn Ile Ile Asn Met Ser

130 135 140

Ser Val His Glu Ile Ile Pro Trp Pro Thr Phe Val His Tyr Ala Ala

145 150 155 160

Ser Lys Gly Gly Val Lys Leu Met Thr Gln Thr Leu Ala Met Glu Tyr

165 170 175

Ala Pro Lys Gly Ile Arg Ile Asn Ala Ile Gly Pro Gly Ala Ile Asn

180 185 190

Thr Pro Ile Asn Ala Glu Lys Phe Glu Asp Pro Lys Gln Arg Ala Asp

195 200 205

Val Glu Ser Met Ile Pro Met Gly Asn Ile Gly Lys Pro Glu Glu Ile

210 215 220

Ser Ala Val Ala Ala Trp Leu Ala Ser Asp Glu Ala Ser Tyr Val Thr

225 230 235 240

Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe

245 250 255

Gln Ala Gly Arg Gly

260

Claims

1. a kind of ketone acid reductase, it is characterised in that the ketone acid reduction enzyme amino acid sequence is SEQ ID NO.4, SEQ ID Shown in one of NO.8 or SEQ ID NO.10.

2. the encoding gene of ketone acid reductase described in a kind of claim 1, it is characterised in that the nucleotides sequence of the encoding gene It is classified as shown in one of SEQ ID NO.3, SEQ ID NO.7 or SEQ ID NO.9.

3. a kind of recombination engineering bacteria of the encoding gene structure of ketone acid reductase described in claim 2, it is characterised in that institute It is one of following to state recombination engineering bacteria：(1) ketone acid reduction enzyme coding gene is imported what host strain obtained；(2) by ketone acid It restores enzyme coding gene, 2- hydroxy acid dehydrogenations enzyme coding gene and glucose dehydrogenase encoding gene and imports what host strain obtained.

4. a kind of application of the ketone acid reductase described in claim 1 in catalytically synthesizing chiral fragrance 2- hydroxy acids.

5. application as claimed in claim 4, it is characterised in that the application process is：Enzyme coding gene is restored to contain ketone acid Ketone acid reductase supernatant of the wet thallus after ultrasonication that obtains of engineering bacterium fermentation culture and compiled containing glucose dehydrogenase Glucose dehydrogenase supernatant of the wet thallus that the engineering fermented and cultured of code gene obtains after ultrasonication is catalyst, with benzene Acetonic acid is substrate, using glucose as cosubstrate, with NAD⁺For coenzyme, with the KH of 100mM, pH7.0₂PO₄-K₂HPO₄Buffer solution For reaction medium, is reacted under the conditions of 35 DEG C, 700rpm, after the reaction was complete, contained the reaction solution of (R)-mandelic acid；The ketone Sour reductase supernatant dosage is calculated as 800U/mL buffer solutions with ketone acid reductase enzyme activity, and the glucose dehydrogenase supernatant is used Amount is calculated as 800U/mL buffer solutions with glucose dehydrogenase enzyme activity, and the glucose dosage is calculated as 200 with buffer solution volume~ 800mM, the substrate dosage are calculated as 100~400mM, NAD with buffer solution volume⁺Dosage is calculated as 0.5mM with buffer solution volume.

6. application as claimed in claim 4, it is characterised in that the application process is：To contain ketone acid reductase, 2- hydroxy acids The wet thallus that the fermented culture of the engineering bacteria of dehydrogenase and glucose dehydrogenase encoding gene obtains is catalyst, with racemic virtue Fragrant 2- hydroxy acids are substrate, and the substrate supplemented by glucose is constituted reaction system using the buffer solution of pH6.0~8.0 as reaction medium, 20~45 DEG C, after the reaction was complete under the conditions of 700rpm, obtain the conversion fluid containing fragrant (the R) -2- hydroxy acids of optical voidness.

7. application as claimed in claim 6, it is characterised in that the racemic fragrance 2- hydroxy acids are one of following：Mandelic acid, 2- fluorine mandelic acid, 4- fluorine mandelic acid, 2,4- difluoros mandelic acid, 3,5- difluoros mandelic acid, 2- chloro mandelic acids, 3- chloro mandelic acids, 4- Chloro mandelic acid, 2- bromines mandelic acid, 3- bromines mandelic acid, 4- bromines mandelic acid, 4- methyl-mandelic acids, 4- trifluoromethyls mandelic acid, 3- hydroxyls Base mandelic acid, 4- hydroxymandelic acids, 4- methoxv mandelic acids, 3 methoxy 4 hydroxymandelic acid, 3- hydroxy-4-methyl almonds Acid, 3- hydroxyl -4- trifluoromethyls mandelic acid, 3- methyl -4- methoxv mandelic acids.

8. application as claimed in claim 6, it is characterised in that in the reaction system, Final substrate concentrations 20-300mM, institute It is 10-300mM to state auxiliary concentration of substrate, and the catalyst amount is calculated as 4-20g/L with wet thallus dry weight.

9. application as claimed in claim 6, it is characterised in that the engineering bacteria be by ketone acid reductase, 2- hydroxy acid dehydrogenases and It is built-up that the encoding gene of glucose dehydrogenase imports host strain jointly；The encoding gene nucleotide of the 2- hydroxy acid dehydrogenases Sequence is shown in SEQ ID NO.13, and the encoding gene nucleotides sequence of the glucose dehydrogenase is classified as SEQ ID NO.15 institutes Show.

10. application as claimed in claim 6, it is characterised in that the catalyst is prepared as follows：Ketone acid reduction will be contained The engineering bacteria of enzyme, 2- hydroxy acid dehydrogenases and glucose dehydrogenase encoding gene is inoculated into containing 50 μ g/mL kanamycins and 50 μ g/mL In the LB liquid medium of streptomysin, 8~10h of shaken cultivation under the conditions of 37 DEG C, 150rpm obtains seed liquor；By seed liquor In the LB liquid medium for containing 50 μ g/mL kanamycins and 50 μ g/mL streptomysins by the inoculum concentration access of volumetric concentration 2%, in 37 DEG C, under the conditions of 150rpm shaken cultivation to OD₆₀₀Reach 0.4~0.8, IPTG to final concentration of 0.1mM be added, in 28 DEG C, Wet thallus is collected by centrifugation and with brine twice to get wet thallus in 10~12h of shaken cultivation under the conditions of 150rpm.