CN104630242B - A kind of carbonyl reduction enzyme gene, codase, carrier, engineering bacteria and its application - Google Patents

Abstract

The invention discloses the recombination engineering bacteria that a kind of restructuring carbonyl reduction enzyme gene from gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB 12126 and its codase, the recombinant vector containing the gene, the recombinant vector convert to obtain, and its application in catalytic asymmetric reduction prochiral carbonyl compounds；Respectively with the ketone butyrate of 2 benzoyl aminomethyl 3, (R) the carbonyl hecanoic acid t-butyl ester of 6 cyano group, 5 hydroxyl 3 and 4, 4, 4 trifluoroacetic ethyl acetoacetates are substrate, carry out (the 2S that biocatalytic reaction prepares high-optical-purity, 3R) the butyric ester of 2 benzoyl aminomethyl 3, 6 cyano group (3R, 5R) dihydroxy hecanoic acid t-butyl ester and (S) 4, 4, the 3-hydroxyethyl butyrate of 4 trifluoro 3, using recombination bacillus coli biocatalytic reaction can be carried out as biocatalyst, alternative new enzyme source is provided for the living things catalysis synthesis of medicine chiral intermediate.

Description

A kind of carbonyl reduction enzyme gene, codase, carrier, engineering bacteria and its application

(1) technical field

The invention belongs to technical field of bioengineering, and in particular to one kind derives from gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB-12126 carbonyl reduction enzyme gene, codase, the recombinant vector containing the gene, The recombinant vector converts obtained recombination engineering bacteria and answering in catalytic asymmetric reduction prochiral carbonyl compounds With.

(2) background technology

Carbonyl reductase (Carbonyl reducatase, E.C.1.1.1.x) be one kind can be catalyzed alcohols and aldehydes/ketone it Between bidirectional reversible redox reaction enzyme, and need coenzyme NAD (H) (NADH) or NADP (H) (nicotinamide-adenine dinucleotide phosphate) is used as hydrogen carrier.NADH and NADPH participates in its reduction instead as electron donor Should, NAD and NADP then participate in its oxidation reaction as electron acceptor.Carbonyl reductase according to the literature belongs to mostly at present Short-chain dehydrogenase superfamily (Short-chain dehydrogenase/reductase, SDR), middle chain alcohol hydrogen enzyme superfamily (Medium-chain dehydrogenase/reductase, MDR), aldehyde ketone reductase superfamily (Aldo-keto Reductase, AKR).Although three has similar catalysis, evolving and differed greatly in structure.Carbonyl reductase It is a kind of very ancient family, is distributed widely in nature, in all kinds of animals, microorganism and plant, due to microorganism Species is various, distribution is wide, is the main source of carbonyl reductase.

In recent years, developing rapidly with genomics, proteomics and bioinformatics, genome and gene order The gene data announced in database increases rapidly, found from substantial amounts of data resource new carbonyl reduction enzyme gene, Its biological information contained is excavated, has become reality for the exploitation service of new and effective biocatalyst.Gene excavating skill Art is a novel enzyme triage techniques, is widely applied at present.Using gene excavating from Pichia finlandica PfODH、Clostridium ljungdahlii BDH1、Candida glabrata CgKR1、Serratia quinivorans SQ_SDR、Polygonum minus PmADH、Arabidopsis thaliana AtADH、 Oenococcus oeni Adh3, Chryseobacterium sp.ChKRED20, Candida magnoliae CmCR and Substantial amounts of carbonyl reductase has been excavated in the bacterial strains such as Acetobacter sp.AcCR.The gene of which part carbonyl reductase is The success table in different hosts (Escherichia coli, Pichia pastoris, Arxula adeninivorans etc.) Reach, obtain producing enzyme vigor and selective higher genetic engineering bacterium, and be successfully applied to the asymmetry of catalysis of carbonyl class compound Reduction reaction.Nevertheless, many carbonyl reductases have substrate specificity, its application greatly limit.It is in addition, many The catalytic efficiency of enzyme is relatively low, also limit its industrial applications.Novel carbonyl reductase of the screening with wider substrate spectrum, research It can efficiently high selectivity catalysis carbonyls, can not only widen its application, lift its application potential, also for Realize that industrialized production lays the foundation.

Chiral alcohol is widely used in the synthesis of chiral drug and other chiral fine chemicals.The synthesis side of chiral alcohol at present Method includes physical partition method, Split Method and method of asymmetrically reducing etc..Wherein, using prochiral carbonyl compounds asymmetry also Former synthesis of chiral alcohol, its theoretical yield are a kind of current main methods for producing chiral alcohol up to 100%.Chemical asymmetric reduction Method is mainly utilize chiral metal complex as asymmetric reduction of the catalyst for carbonyl, although chemical method part For industrial production, but the reaction condition is harsher, chiral metal complex synthesis is complicated and expensive, in product The residual for having heavy metal causes product separation difficulty, and environmental pollution is larger, therefore its application receives certain limitation.Biology is urged Changing method of asymmetrically reducing not only has chemistry, the regio- and stereo-selectivity of height, and reaction condition is gentle, avoids product In heavy-metal residual, it is environmentally friendly, compensate for the deficiency of chemical method, therefore, the carbonyl of living things catalysis is not right in recent years Application of the reduction reaction in chiral alcohol synthesis is claimed increasingly to be taken seriously.

(2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate ester is synthesis carbapenem and penems medicine intermediate The chiral building block of 4-AA (4-AA).Currently acquired (2S, 3R) -2- benzoyl aminomethyls -3- hydroxyl fourths The main path of acid esters intermediate be with chiral catalyst (R)-BINAP-Ru complex asymmetry catalysis 2- benzoyl aminomethyls- 3- ketone butyrate generation (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate ester.But because the reaction condition needs high pressure to add Hydrogen, the price of ruthenium complex are unfavorable for its industrial applications costly and with the separation more difficulty of product etc..Mesh Preceding biological catalysis synthesis (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate ester report is less.Saccharomycopsis Catalysis generation (2S, the 3R) -2- of malanga NBRC 171096 benzoyl aminomethyls -3-hydroxybutyrate ester, and there is higher pair Reflect selectivity (enantiomeric excess value ee>96.2%), but catalytic efficiency is low (yield only 4%).United States Patent (USP) US20130034895 have studied carbonyl reduction Enzyme catalyzed synthesis (2S, 3R) -2- benzene from Lactobacillus kefir Formyl aminomethyl -3-hydroxybutyrate ester, there is higher enantioselectivity (ee is 60~99%), but in the course of reaction, Realize that coenzyme recycles using isopropanol as the second substrate, because isopropanol and substrate have competitive inhibition, cause Maximum conversion rate is affected, and the generation of accessory substance acetone has certain detrimental effect to enzyme.

6- cyano group-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester is the hand of HMG-CoA enzyme inhibitor Stains medicines Property intermediate, the method that chemical synthesis and living things catalysis synthesize can be passed through and obtained.Needed to use in chemical method synthesis inflammable Explosive n-BuLi, borine, and need to exist<Carried out under -65 DEG C of cryogenic conditions, energy consumption is big, then add 6- cyano group-(3R, 5R) - The induction of dihydroxy hecanoic acid t-butyl ester diastereomeric is insufficient, and the optical purity of product is difficult to reach requirement.In recent years, enzyme process generation is utilized Improve reaction condition for chemical method, reduce reaction cost, improving the selectivity of product turns into the focus of research.Screen at present Highly-solid selectively catalysis (R) -6- cyano group -5- hydroxyl -3- carbonyls hecanoic acid t-butyl esters generation 6- cyano group-(3R, 5R)-dihydroxy The bacterial strain of hecanoic acid t-butyl ester has：Saccharomyces cerevisiae、Pichia angusta、Pichia haplophila、 Beauveria bassiana、Pichia pastoris、Pichia membranefaciens、Candida humicola、 Kluyveromyces drosophilarum, Rhodotorula glutinis and Pichia caribbic etc..Codexis is public Department patent US7879585B2 has cloned a kind of ketoreductase and transformed from Saccharomyces cerevisiae, Catalytic production 6- cyano group-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester conversion ratio is 99.7%, de>99%.

Befloxatone (Befloxatone) chemical name 5 (R)-(methoxyl methyl) -3- [4- [(R)-hydroxyls of 4,4,4- tri- fluorine-based -3 Base butoxy] phenyl] oxazolidone -2 are a kind of strong selectivity invertibity monoamine oxidase A (MAO-A) inhibitor, with MAO-A The Specific amino acid of flavin adenine dinucleotide (FAD) (FAD) co-factor and avtive spot interacts, and is clinically used for treating suppression Strongly fragrant disease.(R) -4,4,4- trifluoro 3-hydroxy ethyl butyrates are the chiral intermediates for synthesizing Befloxatone.Zhang etc. is reported first Contain NADPH dependent forms carbonyl reductase and glucose-6-phosphate dehydrogenase (G6PD) in bacterial strain Bacillus pumilus Phe-C3 (G-6-PDH) 4,4,4- trifluoroacetic ethyl acetoacetates generation (R) -4,4,4- trifluoro 3-hydroxy ethyl butyrates, substrate can, be catalyzed When concentration is 60mM, yield is up to 67%, ee up to 95%.He et al. screens one plant of Saccharomyces uvarum SW-58, And utilize water-organic solvent two-phase system catalysis 4,4,4- trifluoroacetic ethyl acetoacetate generation three fluoro- 3- hydroxyls of (R) -4,4,4- Ethyl butyrate, when concentration of substrate is 0.2mM, conversion ratio is up to 85%, ee up to 85.2% after 4h.Kara et al. utilizes commercial enzyme ADH Evo-1.1.200 is catalyzed the reaction, and conversion ratio 39.2% after concentration of substrate 5mM, 24h, be selectively significantly improved (ee >99.9%).In addition, (S) -4,4,4- trifluoro 3-hydroxy ethyl butyrates are also a kind of important chiral intermediate, and its biology is urged Change method asymmetric syntheses has no report.

As can be seen here, it is significant to study the living things catalysis technique of these medicine chiral intermediates, can not only be this The synthesis of a little compounds provides new route, and and can provides new enzyme source for it.So far, Burkholderia is had not yet to see Application of the carbonyl reductase in these medicine chiral intermediate asymmetric reductions in gladioli bacterial strains.

(3) content of the invention

It is an object of the present invention to provide one kind to derive from gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB-12126 carbonyl reductases, gene, the recombinant vector containing the gene, the recombinant vector convert obtained recombination work Journey bacterium, and preparing (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate ester, (S) -4- chloro-3-hydroxyls ethyl butyrate, 6- Answering in cyano group-chirality pharmaceutical intermediate compound such as (3R, 5R)-dihydroxy hecanoic acid t-butyl ester and 4,4,4- trifluoroacetic ethyl acetoacetates With.

The technical solution adopted by the present invention is：

The present invention provides one kind and derives from gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB- 12126 carbonyl reduction enzyme gene, the nucleotides sequence of the gene are classified as SEQ ID NO：Shown in 1, the carbonyl reductase base Because obtaining by the following method：

Using round pcr, primer 1 (5 '-ATGGCAGACGTCAACAGCCTGTTC-3 '), primer 2 (5 '- TCAGACCGTGCTGGTGAGGCC-3 ') in the presence of with from gladiolus bulkholderia cepasea (Burkholderia Gladioli) total genomic dna in ZJB-12126 bacterial strains is the carbonyl reductase gene order that template clone is about 0.8kb, Being named as adh5, (nucleotides sequence is classified as SEQ ID NO：Shown in 1).The fragment is connected on pGEM-T carriers, clone is obtained and carries Body pGEM-T-adh5, carrier conversion Escherichia coli are obtained to the recombination bacillus coli of the pGEM-T-adh5 containing carrier.To recombinating matter Grain sequencing, and sequencing result is analyzed using software, the sequence contains long 774bp ORFs.

Present invention expression primer 3 (5 '-CCATGGCAGACGTCAACAGCCTGTTC-3 ') and primer 4 (5 '-CTCGAGGACCGTGCTGGTGAGGCC-3 '), restriction enzyme site is respectively Nco I and Xho I (underscore), with cloning vector PGEM-T-adh5 is template, expands to have obtained the carbonyl reduction enzyme gene for expressing by PCR.

It is any to SEQ ID NO:Nucleotide sequence shown in 1 carries out substitution, insertion or the missing of one or more nucleotides The nucleotide sequence obtained is handled, as long as it has more than 90% homology with the nucleotides, belongs to the protection of the present invention Scope.

The present invention provides a kind of restructuring carbonyl reductase encoded by the carbonyl reductase gene adh 5, is named as BgADH5, the amino acid sequence of the restructuring carbonyl reductase is SEQ ID NO:Shown in 2.

It is any to SEQ ID NO:The processing that amino acid is inserted, lacked or replaced in amino acid sequence shown in 2 obtains Polypeptide fragment or its mutant, as long as itself and SEQ ID NO:Amino acid sequence shown in 2 has more than 95% homology, belongs to In protection scope of the present invention.

The present invention relates to the recombinant vector containing the carbonyl reduction enzyme gene.

The recombination engineering bacteria that the recombinant vector converts to obtain is the present invention relates to the use of, is specially：By carbonyl reduction Enzyme gene constructs the heterogenous expression recombinant plasmid pET28a- containing carbonyl reduction enzyme gene with expression vector pET28a connections adh5.Recombinant expression pET28a-adh5 is converted into E. coli BL21 (DE3), obtained containing restructuring matter Grain pET28a-adh5 recombination bacillus coli BL21 (DE3)/pET28a-adh5.

The invention further relates to application of the carbonyl reduction enzyme gene in Prepare restructuring carbonyl reductase, is specially：Structure contains There is the recombinant vector of the carbonyl reductase gene, the recombinant vector is converted to Host Strains (preferably E. coli BL21 (DE3)) in, the recombination engineering bacteria of acquisition carries out Fiber differentiation, and nutrient solution is isolated to contain restructuring carbonyl reduction The somatic cells of enzyme, the carbonyl reductase crude enzyme liquid obtained after broken are purified, and obtain the pure enzyme of carbonyl reductase.

The invention further relates to the restructuring carbonyl reductase to prepare chiral alcohol in asymmetric reduction prochiral carbonyl compounds Application in medicine chiral intermediate, the application are：Obtained with the fermented culture of the engineering bacteria of the carbonyl reduction enzyme gene containing restructuring The wet thallus obtained is catalyst, in the buffer solution that pH value is 6~10, adds substrate, cosubstrate and NAD (P)⁺, 20~ 40 DEG C, reaction under the conditions of 50~250rpm (preferably 30 DEG C, 150r/min under react 12h), after reaction completely, acquisition contains chiral alcohol The mixed liquor of medicine chiral intermediate；After reaction terminates, mixed liquor is extracted with ethyl acetate twice, merges organic layer and uses nothing Water magnesium sulfate is dried, and filtering, Rotary Evaporators remove ethyl acetate, and medicine chiral intermediate is obtained after drying；The substrate is 2- benzoyl aminomethyl -3- ketone butyrate, (R) -6- cyano group -5- hydroxyls -3- carbonyls hecanoic acid t-butyl ester and 4,4,4- trifluoroacetyls One kind in ethyl acetate, when the substrate is 2- benzoyl aminomethyl -3- ketone butyrates, 2- benzoyl aminomethyl -3- ketone Butyrate is added in the form of 400mmol/L dimethyl sulphoxide solutions；The cosubstrate is glucose, ammonium formate, isopropanol Or absolute ethyl alcohol, preferred glucose, when the cosubstrate is glucose, addition glucose dehydrogenase forms cosubstrate body System, when the cosubstrate is ammonium formate, addition hydrogenlyase forms cosubstrate system；The dosage of the catalyst with Wet thallus weight is calculated as 20~200g/L buffer solutions (preferably 50g/L), and the initial concentration of the substrate delays for 10~100mmol/L Fliud flushing (preferably 20mmol/L), the dosage of the cosubstrate are 10~200g/L buffer solutions (preferably 50g/L), the NAD (P )⁺Dosage be 0.01~5mmol/L buffer solutions (preferably 2mmol/L), the glucose takes off dehydrogenase or formate dehydrogenase enzyme dosage In terms of the wet thallus weight that dehydrogenase or the fermented culture acquisition of hydrogenlyase thalline are taken off containing glucose, delay for 20~200g/L Fliud flushing (preferably 50g/L).The substrate adds that (i.e. substrate dimethyl sulfoxide (DMSO) is configured in the form of 400mmol/L DMSO solutions 400mmol/L solution).

Further, the wet thallus that the fermented culture of the engineering bacteria of the carbonyl reduction enzyme gene of the present invention containing restructuring obtains is pressed It is prepared by following method：Engineering bacteria containing restructuring carbonyl reduction enzyme gene is seeded to containing the μ g/mL kanamycins of final concentration 50 The LB fluid nutrient mediums of resistance, 12h are cultivated under 200rpm, then fresh contain is seeded to the inoculum concentration of volumetric concentration 1% by 37 DEG C In the LB fluid nutrient mediums of the μ g/mL kalamycin resistances of final concentration 50, in 37 DEG C, cultivated under 150rpm to thalline OD₆₀₀Up to 0.6 ~0.8, add final concentration of 0.1mM IPTG, at 28 DEG C after Fiber differentiation 12h, 4 DEG C, 5000rpm centrifugation 5min, discard Clear liquid, collect wet thallus.

Coenzyme wet thallus of the present invention is prepared as follows：(being preferably derived from for glucose dehydrogenase (GDH) will be contained Exiguobacterium sibiricum 255-15 GDH, GenBank:ACB59697.1) recombinant bacterium BL21 (DE3)/ PET28b-gdh) or (FDH) containing hydrogenlyase (is preferably derived from Candida boidinii FDH, GenBank: AF004096 recombinant bacterium BL21 (DE3)/pET28b-fdh) is seeded to the LB containing the μ g/mL kalamycin resistances of final concentration 50 Fluid nutrient medium, 37 DEG C, cultivate 12h under 200rpm, then be seeded to 1% inoculum concentration (v/v) and fresh contain the μ g/ of final concentration 50 In the LB fluid nutrient mediums of mL kalamycin resistances, in 37 DEG C, cultivated under 150rpm to thalline OD₆₀₀Up to 0.6~0.8, add eventually Concentration is 0.1mM IPTG, and at 28 DEG C after Fiber differentiation 12h, 4 DEG C, 5000rpm centrifugation 5min, abandoning supernatant, it is heavy to collect Form sediment, that is, obtain corresponding wet thallus.

Further, application of the preferably described restructuring carbonyl reductase in medicine chiral intermediate is prepared：With the carbonyl containing restructuring The wet thallus that the fermented culture of engineering bacteria of base reductase gene obtains is catalyst, in the buffer solution that pH value is 6~10, is added Enter substrate, glucose, glucose dehydrogenase and NAD (P)⁺, react under the conditions of 30 DEG C, 150rpm, after reaction completely, contained The mixed liquor of medicine chiral intermediate (i.e. chiral alcohol)；The substrate is 2- benzoyl aminomethyl -3- ketone butyrate, (R) -6- cyanogen Base -5- hydroxyls -3- carbonyls hecanoic acid t-butyl ester and 4, one kind in 4,4- trifluoroacetic ethyl acetoacetates, when the substrate is 2- benzene first During acyl aminomethyl -3- ketone butyrates, the 2- benzoyl aminomethyls -3- ketone butyrate is with 400mmol/L dimethyl sulphoxide solutions Form add；The dosage of the catalyst is calculated as 50g/L buffer solutions with the weight of wet thallus, and the initial concentration of the substrate is 20mmol/L buffer solutions, the dosage of the glucose is 50g/L buffer solutions, and the dosage of the glucose dehydrogenase is with containing glucose The wet thallus weight meter that the fermented culture of the thalline of dehydrogenase obtains, is 50g/L buffer solutions, the NAD (P)⁺Dosage be 2mmol/L buffer solutions.

Further, the carbonyl reductase BgADH5 of the present invention containing the carbonyl reduction enzyme gene is as biocatalyst In conversion of substrate 2- benzoyl aminomethyl -3- ketone butyrates (Formulas I) generation (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate Application in ester (Formula II), as shown in Figure 6, in Formulas I, R is C1-C6 alkyl, specifically, above-mentioned application to the reaction equation being related to Reaction system is：With phosphate buffer (100mM, pH 7.0) for reaction medium, with the work of the carbonyl reduction enzyme gene containing restructuring The wet thallus that the fermented culture of journey bacterium obtains is catalyst, adds glucose dehydrogenase, 2- benzoyl aminomethyl -3- ketone butyric acid Ester, NADP⁺And glucose, 30 DEG C, shaking bath reaction 12h under the conditions of rotating speed 150r/min, after reaction terminates, reaction solution with etc. Volume of ethylacetate is extracted twice, and is merged organic layer and is dried with anhydrous magnesium sulfate, filtered, Rotary Evaporators remove acetic acid second Ester, concentrate is obtained, dried, be (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate ester；The substrate is with 400mmol/ The form of L DMSO solutions adds, the final concentration 20mmol/L buffer solutions of substrate, NADP⁺Dosage is 2mmol/L buffer solutions, grape Sugared dosage is 50g/L buffer solutions, and the dosage of the glucose dehydrogenase is obtained with the fermented culture of the thalline containing glucose dehydrogenase The wet thallus weight meter obtained, is 50g/L buffer solutions, catalyst amount is 50g/L buffer solutions.

Further, when the substrate is (R) -6- cyano group -5- hydroxyl -3- carbonyl hecanoic acid t-butyl esters, its reaction equation is shown in Fig. 7 institutes Show, specifically, the reaction system of above-mentioned application is：Obtained with the fermented culture of the engineering bacteria of the carbonyl reduction enzyme gene containing restructuring Wet thallus is catalyst, with kaliumphosphate buffer (100mM, pH 7.0) for reaction medium, adds glucose dehydrogenase, grape Sugar, NADP⁺, substrate (R) -6- cyano group -5- hydroxyl -3- carbonyls hecanoic acid t-butyl esters (formula III), 30 DEG C, rotating speed 150r/min conditions Lower reaction 12h, reaction add isometric ethyl acetate after terminating and are extracted twice, and merge organic layer and are dried with anhydrous magnesium sulfate, Filtering, Rotary Evaporators remove ethyl acetate, and it is 6- cyano group-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester (formula that concentrate, which is dried, IV)；The restructuring carbonyl reductase wet thallus dosage is 50g/L buffer solutions, glucose dehydrogenase dosage with dehydrogenation containing glucose The wet thallus dosage that the fermented culture of the thalline of enzyme obtains is calculated as 50g/L buffer solutions, glucose dosage be 50g/L buffer solutions, NADP⁺Dosage is 2mmol/L buffer solutions, concentration of substrate 20mmol/L buffer solutions.

Further, the substrate is 4, during 4,4- trifluoroacetic ethyl acetoacetate, its reaction equation as shown in Figure 8, specifically, on The reaction system for stating application is：The wet thallus obtained using the fermented culture of the engineering bacteria of the carbonyl reduction enzyme gene containing restructuring is catalysis Agent, with kaliumphosphate buffer (100mM, pH 7.0) for reaction medium, add glucose dehydrogenase, glucose, NADP⁺, substrate 4,4,4- trifluoroacetic ethyl acetoacetates (Formula V), react 12h under the conditions of rotating speed 150r/min by 30 DEG C, the bodies such as reaction adds after terminating Product ethyl acetate is extracted twice, and is merged organic layer and is dried with anhydrous magnesium sulfate, is filtered, and Rotary Evaporators remove ethyl acetate, It is (S) -4,4,4- trifluoro 3-hydroxy ethyl butyrates (Formula IV) that concentrate, which is dried,；The restructuring carbonyl reductase wet thallus is used Measure 50g/L buffer solutions, the wet thallus weight that glucose dehydrogenase dosage is obtained with the fermented culture of the thalline containing glucose dehydrogenase Amount is calculated as 50g/L buffer solutions, glucose dosage is 50g/L buffer solutions, NADP⁺Dosage is 2mmol/L buffer solutions, concentration of substrate 20mmol/L buffer solutions.

Catalyst of the present invention also includes other shapes such as the pure enzymes of carbonyl reductase BgADH5, crude enzyme liquid or thick enzyme powder State.When making catalyst using carbonyl reductase recombination engineering bacteria, a kind of coenzyme circulatory system need to be combined, including glucose/ Double enzyme Double bottom thing coenzyme circulatory systems such as glucose dehydrogenase (GDH), formic acid/hydrogenlyase (FDH) and ethanol, isopropanol Etc. single enzyme Double bottom thing coenzyme circulatory system.It is usually added into cosubstrate to be reacted instead of NAD (P) H, conventional cosubstrate For：10~200g/L of glucose, ethanol or isopropanol 2~30% (v/v, accounting for the quality percentage by volume of total transformation system)；Compared with Good cell concentration is 20~200g/L.

Gladiolus bulkholderia cepasea (the Burkholderia of carbonyl reduction enzyme gene of the present invention can be provided Gladioli) ZJB-12126, deposit number are CCTCC M 2012379, are preserved in China typical culture collection center, are protected Tibetan address is Wuhan, China Wuhan University, and preservation date is September in 2012 25, in previous patent application (CN103045504A) disclosed in.

The beneficial effects are mainly as follows：The invention provides one kind to derive from gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB-12126 carbonyl reduction enzyme gene, the carbonyl reduction enzyme gene can be with expression vectors Connection structure obtains the recombinant expression pET28a-adh5 containing the gene, then converts into e. coli bl21 (DE3), obtains Recombination bacillus coli is obtained, the Escherichia coli contain restructuring carbonyl reductase, can be used as living things catalysis by the use of recombination bacillus coli Agent carries out biocatalytic reaction, and alternative new enzyme source is provided for the living things catalysis synthesis of medicine chiral intermediate.

Carbonyl reductase BgADH5 is recombinated as biocatalyst, using 2- benzoyl aminomethyl -3- ketone butyrates as substrate Prepare (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate ester, product ee>99%, 12h substrate conversion efficiency are 51.4%, with B.gladioli ZJB-12126 wild mushrooms compare (ee 81%) in CN103045504A, and enantioselectivity is improved.With (R) -6- cyano group -5- hydroxyls -3- carbonyls hecanoic acid t-butyl ester is that substrate prepares 6- cyano group-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester When, product de is that 98%, 12h substrate conversion efficiencies are 87.4%.With the Saccharomyces in patent US7879585B2 Cerevisiae wild type ketoreductases YDL (reaction time 24h, conversion ratio 85%) is compared, and conversion ratio improves. When with 4,4,4- trifluoroacetic ethyl acetoacetates being substrate, conversion reaction can also be carried out and prepare corresponding (S) -4,4,4- tri- is fluoro- Ethyl 3-hydroxybutanoate, ee>99%, 12h substrate conversion efficiency are 88.4%.Although biological method of asymmetrically reducing synthesizes (R) -4,4, 4- trifluoro 3-hydroxy ethyl butyrates it has been reported that but (S) -4, the bioanalysises of 4,4- trifluoro 3-hydroxy ethyl butyrates it is asymmetric Synthesis does not have been reported that.

(4) illustrate

Fig. 1 is cloning vector pGEM-T-adh5 physical maps.

Fig. 2 is pET28a-adh5 recombinant plasmid physical maps.

Fig. 3 is that carbonyl reductase gene PCR expands low melting-point agarose gel (argrose) electrophoretogram；Wherein, swimming lane 1 For DL2000DNA Marker；Swimming lane 2 is to expand obtained carbonyl reductase genetic fragment using primer 1 and primer 2；Swimming lane 3, 4 be that obtained carbonyl reductase genetic fragment is expanded using primer 3 and primer 4.

Fig. 4 positive recombinant plasmids pET28a-adh5 digestion structure chart；Wherein, swimming lane 1 is DL 10000DNA Marker Fragment；Swimming lane 2 is carbonyl reduction enzyme gene；Swimming lane 3 is pET28a-adh5/Nco I samples；Swimming lane 4 is pET28a-adh5/ Xho I samples；Swimming lane 5 is pET28a-adh5/Nco I and Xho I samples.

Fig. 5 is the SDS-PAGE figures of carbonyl reductase after purification：Swimming lane 1 is protein molecular weight Marker, and swimming lane 2,3 is Carbonyl reductase BgADH5 after purification.

Fig. 6 is (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate Lipase absobed equation.

Fig. 7 is that 6- cyano group-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester synthesizes equation.

Fig. 8 is that (S) -4,4,4- trifluoro 3-hydroxy ethyl butyrates synthesize equation.

(5) embodiment

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：The amplification of carbonyl reductase gene adh 5

According to gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB-12126 genome sequencings Information, excavate wherein substantial amounts of carbonyl reductase, one of them have catalysis 2- benzoyl aminomethyl -3- ketone butyrate, (R) - 6- cyano group -5- hydroxyls -3- carbonyls hecanoic acid t-butyl ester and 4,4,4- trifluoroacetic ethyl acetoacetates generation (2S, 3R) -2- benzene carbon amides Three fluoro- 3- hydroxyls fourth of methyl -3-hydroxybutyrate ester, 6- cyano group-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester and (S) -4,4,4- The enzyme of acetoacetic ester function is carbonyl reductase BgADH5 of the present invention.

Utilize MPBio companiesSpin kits extract gladiolus bulkholderia cepasea The total genomic dna of (Burkholderia gladioli) ZJB12126 thalline, using the genomic DNA as template, in primer 1 Enter in the presence of (5 '-ATGGCAGACGTCAACAGCCTGTTC-3 '), primer 2 (5 '-TCAGACCGTGCTGGTGAGGCC-3 ') Performing PCR expands.PCR reaction systems (the μ L of cumulative volume 50)：10 × Pfu DNA Polymerase Buffer 5 μ L, 10mM dNTP Mixture (each 2.5mM of dATP, dCTP, dGTP and dTTP) 1 μ L, cloning primer 1, each 1 μ L of primer 2 that concentration is 50 μM, gene Group DNA1 μ L, Pfu DNA Polymerase 1 μ L, the seedless μ L of sour water 40.

Using Biorad PCR instrument, PCR reaction conditions：Pre-degeneration 95 DEG C of 5min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 30 circulations, last 72 DEG C of extensions 10min.

PCR reaction solutions purify the fragment with the detection of 0.9% agarose gel electrophoresis and gel extraction, are gathered using Taq DNA Synthase is held to fragment 5 ' and introduces base A.The fragment is attached with pGEM-T carriers under T4DNA connection enzyme effects, obtained Cloning recombinant plasmids pGEM-T-adh5, is shown in Fig. 1.By the recombinant plasmid transformed into e. coli jm109, it is coated on containing eventually Concentration is the LB flat boards of 50 μ g/ml ampicillin sodium resistances, random picking positive colony sequencing, is sequenced and tied using software analysis Fruit, the results showed that：The nucleotide sequence length expanded through primer 1 and primer 2 is 774bp (its nucleotide sequence such as SEQ ID NO：Shown in 1), the complete ORFs of the sequential coding one (amino acid sequence is SED ID NO.2).

Embodiment 2：Recombination bacillus coli BL21 (DE3)/pET28a-adh5 structure

According to the analysis result of embodiment 1 design primer 3 (5 '-CCATGGCAGACGTCAACAGCCTGTTC-3 '), primer 4 (5’-CTCGAGGAC

CGTGCTGGTGAGGCC-3 '), and Nco I and Xho I Restriction Enzymes are introduced in primer 3 and primer 4 respectively Enzyme site (underscore mark).Under the initiation of primer 3 and primer 4, expanded, obtained using high-fidelity Pfu archaeal dna polymerases Obtain a length of 774bp carbonyl reductase gene order (its nucleotide sequence such as SEQ ID NO：Shown in 1), Nco is utilized after sequencing I and Xho I restriction enzymes (TaKaRa) are handled amplified fragments, and should using T4DNA ligases (TaKaRa) Fragment is same to be attached with the commercial carrier pET28a (Invitrogen) of identical restriction enzyme ferment treatment, construction expression Carrier pET28a-adh5.The expression vector pET28a-adh5 of structure is converted to e. coli bl21 (DE3) (Invitrogen) in, the LB flat boards containing the μ g/mL kalamycin resistances of final concentration 50 is coated on, 8-16h is cultivated at 37 DEG C, with Machine picked clones, extracting plasmid carry out digestion identification, qualification result as shown in Figure 4, positive recombinant plasmid as can be seen from Figure 4 There is single band in line in 3 swimming lanes and 4 swimming lanes in pET28a-adh5 single endonuclease digestions, and 5 swimming lanes occur two after double digestion Band, a band are consistent with target gene clip size.The result illustration purpose gene be cloned into pET28a Nco I and Xho I sites, that is, obtain recombination bacillus coli E.coli BL21 (DE3)/pET28a-adh5.

Embodiment 3：Recombinate carbonyl reductase (BgADH5) wet thallus

The recombination bacillus coli E.coli BL21 containing recombinant expression pET28a-adh5 that embodiment 2 is obtained (DE3)/pET28a-adh5 thalline are seeded to the LB fluid nutrient mediums containing the μ g/mL kalamycin resistances of final concentration 50,37 DEG C, Cultivate 12h under 200rpm, then be seeded to 1% inoculum concentration (v/v) fresh containing the μ g/ml kalamycin resistances of final concentration 50 In LB fluid nutrient mediums, in 37 DEG C, cultivated under 150rpm to thalline OD₆₀₀Up to 0.6~0.8, add final concentration of 0.1mM's IPTG, at 28 DEG C after Fiber differentiation 12h, 4 DEG C, 5000rpm centrifugation 5min, abandoning supernatant, precipitation is collected, that is, obtain and contain table Up to recombination bacillus coli BL21 (DE3)/pET28a-adh5 wet thallus of recombinant plasmid.The thalline can be directly as living things catalysis Agent or for protein purification.

Embodiment 4：Carbonyl reductase (BgADH5's) isolates and purifies

By the thalline obtained in embodiment 3 (i.e. recombination bacillus coli BL21 (DE3)/pET28a-adh5 wet thallus) with knot Close after buffer solution (sodium phosphate buffer of 50mM, pH 8.0, NaCl containing 300mM, 10mM imidazoles) is resuspended, through ultrasonication, 12000rpm centrifuges 40min, supernatant with after the equilibrated Ni affinity chromatographys resin of above-mentioned combination buffer is incubated, then with flushing Buffer solution (sodium phosphate buffer of 50mM, pH 8.0, NaCl containing 300mM, 20mM imidazoles) is rinsed to substantially without foreign protein, then Eluted with elution buffer (sodium phosphate buffer of 50mM, pH 8.0, NaCl containing 300mM, 250mM imidazoles) and collect purpose egg In vain, after electroresis appraisal purity merge destination protein and with elution buffer (sodium phosphate buffer of 50mM, pH 8.0) dialyse 48h, Take trapped fluid to use Coomassie Brilliant Blue to determine protein content as 2mg/mL, enzyme liquid is diluted to final concentration of 0.5mg/mL points Dress, freeze in -80 DEG C (carbonyl reductase BgADH5 protein electrophoresis figures are shown in accompanying drawing 5), obtain the pure enzymes of carbonyl reductase BgADH5.

Embodiment 5：Recombinate carbonyl reductase BgADH5 determination of activity

The pure enzymes of carbonyl cyclase BgADH5 for isolating and purifying to obtain in the method for embodiment 4 are used to be catalyzed substrate 2- benzene carbon amides Methyl -3- ketone methyl butyrates.

Catalyst system and catalyzing forms and catalytic condition is as follows：Carbonyl is added in 10mL phosphate buffers (100mM, pH 7.0) also The pure enzymes of protoenzyme BgADH5 (final concentration of 0.1mg/mL), 2- benzoyl aminomethyl -3- ketone methyl butyrate (final concentration 20mmol/L, 2- benzoyl aminomethyl -3- ketone methyl butyrates are added in the form of 400mmol/L DMSO solutions), NAD (P) H (final concentrations 5mmol/L buffer solutions) form reaction system.30 DEG C, 5min sampling detection enzyme activity is reacted under the conditions of rotating speed 150r/min.Same batten Under part, obtained with e. coli bl21 (DE3) and e. coli bl21 (DE3)/pET28a bacterial cell disruptions supernatant through dialysis Trapped fluid is as control.

Enzyme-activity unit (U) is defined as：Under the conditions of 30 DEG C, pH 7.0, the enzyme in 1min needed for 1 μm of ol NAD (P) H of consumption Amount is defined as 1U.NAD (P) H consumption is determined using ELIASA under 340nm.According to NAD in system (P) H consumption gauge Calculate restructuring carbonyl reductase BgADH5 enzyme activity.Measurement result is shown in Table 1.

Table 1 recombinates carbonyl reductase BgADH5 enzyme activity determination

Embodiment 6：Recombinate the measure of carbonyl reductase BgADH5 coenzyme Preferences

Catalyst system and catalyzing forms and catalytic condition is as follows：Embodiment is added in 10mL phosphate buffers (100mM, pH 7.0) The 4 pure enzymes of restructuring carbonyl reductase BgADH5 (final concentration of 0.1mg/mL) prepared, 2- benzoyl aminomethyl -3- ketone methyl butyrates (final concentration 20mmol/L, 2- benzoyl aminomethyl -3- ketone methyl butyrate is added in the form of 400mmol/L DMSO solutions), NADH or NADPH (2mmol/L buffer solutions) forms reaction system.30 DEG C, taken after reacting 5min under the conditions of rotating speed 150r/min Sample detection enzyme activity (method is with embodiment 5).Under similarity condition, the reaction solution to be not added with coenzyme is used as control.Measurement result is shown in Table 2。

Table 2 recombinates carbonyl reductase BgADH5 coenzyme Preferences

Coenzyme type	Enzyme activity (U/mg)
		Control	0
NADH	0.06
		NADPH	2.7

As a result show, when the carbonyl reductase is using NADPH as coenzyme, enzyme activity is significantly larger than the enzyme using NADH as coenzyme Vigor, therefore this can speculate that the enzyme is a kind of NADPH cofactor-dependents carbonyl reductase.

Embodiment 7：Recombinate carbonyl reductase BgADH5 Cofactor Regeneration Systems

Recombination bacillus coli BL21 (the DE3)/pET28a- containing recombinant expression obtained in the method for embodiment 3 Adh5 wet thallus is as biocatalyst, using 2- benzoyl aminomethyl -3- ketone methyl butyrates as substrate.

(1) selection of carbonyl reductase BgADH5 Cofactor Regeneration Systems

Glucose dehydrogenase (GDH) (is derived from into Exiguobacterium sibiricum 255-15, GenBank: ACB59697.1 recombinant bacterium BL21 (DE3)/pET28b-gdh)) thalline (glycerol tube) and hydrogenlyase (FDH) (derive from Candida boidinii, GenBank:AF004096 recombinant bacterium BL21 (DE3)/pET28b-fdh)) thalline (glycerol tube) point It is not seeded to the LB fluid nutrient mediums containing the μ g/mL kalamycin resistances of final concentration 50,37 DEG C, cultivates 12h under 200rpm, then with 1% inoculum concentration (v/v) is seeded in the fresh LB fluid nutrient mediums containing the μ g/mL kalamycin resistances of final concentration 50, in 37 DEG C, cultivated under 150rpm to thalline OD₆₀₀Up to 0.6~0.8, final concentration of 0.1mM IPTG is added, Fiber differentiation 12h at 28 DEG C Afterwards, 4 DEG C, 5000rpm centrifugation 5min, abandoning supernatant, collect precipitation, that is, obtain glucose dehydrogenase wet thallus or formate dehydrogenase Enzyme wet thallus.The thalline may be directly applied to Cofactor Regeneration Systems.

Transformation system：Kaliumphosphate buffer (100mM, pH 7.0) 10mL, carbonyl reductase BgADH5 wet thallus amounts 0.5g (dry cell weight 0.048g), substrate 2- benzoyl aminomethyl -3- ketone methyl butyrate 20mmol/L buffer solution (2- benzene carbon amide first Base -3- ketone methyl butyrate is added in the form of 400mmol/L DMSO solutions), NADP⁺0.5m mol/L buffer solutions, cosubstrate (cosubstrate is one of following：The glucose of 50g/L buffer solutions, the ammonium formate of 50g/L buffer solutions, 30% (v/v) buffer solution The absolute ethyl alcohol of isopropanol or 30% (v/v) buffer solution), (dehydrogenase is one of following to dehydrogenase：Glucose dehydrogenase (GDH) Or hydrogenlyase (FDH), dehydrogenase wet thallus dosage are 0.5g), 30 DEG C, rotating speed 150r/min shaking tables reaction 12h.

Reaction is extracted twice after terminating with isometric ethyl acetate, and sample treatment is same as Example 9.Determine product Conversion ratio and ee.Under similarity condition, the reaction solution to be not added with coenzyme circulating system is used as control.It the results are shown in Table 3.

Influence of the different coenzyme circulatory systems of table 3 to catalytic reaction

As a result show that, when selecting glucose/GDH as Cofactor Regeneration Systems, yield can be up to 27.9%, and it is selected Property is above other Cofactor Regeneration Systems.

(2) in carbonyl reductase BgADH5 regenerating coenzymes circulating system glucose dehydrogenase biomass optimization

Transformation system：Kaliumphosphate buffer (100mM, pH 7.0) 10mL, carbonyl reductase BgADH5 wet thallus amounts 0.5g (prepared by dry cell weight 0.048g, the method for embodiment 3), 2- benzoyl aminomethyl -3- ketone butyrate 0.05g (final concentrations 20mmol/L, 2- benzoyl aminomethyl -3- ketone butyrate are added in the form of 400mmol/L DMSO), NADP⁺0.5mmol/L Buffer solution, glucose 50g/L buffer solutions, glucose dehydrogenase wet thallus amount prepared by step (1) is respectively 0.1,0.25,0.5, 0.75th, 1.0g (dry cell weight is respectively 0.009,0.023,0.048,0.076,0.01g), 30 DEG C, rotating speed 150r/min shaking tables React 12h.Reaction is extracted twice after terminating with isometric ethyl acetate, and sample treatment is same as Example 9.Determine product Conversion ratio and ee.Under similarity condition, the reaction solution to be not added with glucose dehydrogenase is used as control.Optimum results are shown in Table 4.

The optimization of GDH biomasses in table 4BgADH5 regenerating coenzyme circulating systems

GDH (wet thallus g)	Conversion ratio (%)
		0	39
0.1	43
		0.25	49
0.5	51
		0.75	51
1.0	51

As a result show when BgADH5 biomasses and glucose dehydrogenase biomass weight ratio are 1:When 1, regenerating coenzyme circulation Demands of the BgADH5 to NADPH is disclosure satisfy that substantially.Above catalytic reaction products ee 99%.

(3) in carbonyl reductase BgADH5 regenerating coenzymes circulating system concentration of glucose optimization.

Transformation system：Kaliumphosphate buffer (100mM, pH 7.0) 10mL, carbonyl reductase BgADH5 wet thallus amounts 0.5g (prepared by dry cell weight 0.048g, embodiment 3), 2- benzoyl aminomethyl -3- ketone butyrate 0.05g (final concentration 20mmol/L Buffer solution, 2- benzoyl aminomethyl -3- ketone butyrates are added in the form of 400mmol/L DMSO solutions), NADP⁺0.5mmol/L Buffer solution, concentration of glucose be respectively 10g/L buffer solutions, 50g/L buffer solutions, 100g/L buffer solutions, 150g/L buffer solutions and 200g/L buffer solutions.Glucose dehydrogenase wet thallus amount 0.5g (dry cell weight 0.05g) prepared by step (1), 30 DEG C, rotating speed 12h is reacted under 150r/min.Reaction is extracted twice after terminating with isometric ethyl acetate, sample treatment and the phase of embodiment 8 Together.Determine the conversion ratio and ee of product.Under similarity condition, the reaction solution to be not added with glucose is used as control.Optimum results are shown in Table 5.

The optimization of concentration of glucose in the carbonyl reductase BgADH5 regenerating coenzyme circulating systems of table 5

Glucose (g/L)	Conversion ratio (%)
		0	32
10	45
		50	51
100	51
		150	51
200	51

As a result show that regenerating coenzyme circulating system substantially disclosure satisfy that carbonyl also when concentration of glucose is 50g/L Demands of the protoenzyme BgADH5 to NADPH.Above catalytic reaction products ee 99%.

(4) NADP in carbonyl reductase BgADH5 regenerating coenzymes circulating system⁺The optimization of concentration.

Transformation system：Kaliumphosphate buffer (100mM, pH 7.0) 10mL, carbonyl reductase BgADH5 wet thallus amounts 0.5g (prepared by dry cell weight 0.048g, embodiment 3), 2- benzoyl aminomethyl -3- ketone butyrate 0.05g (final concentration 20mmol/L Buffer solution, 2- benzoyl aminomethyl -3- ketone butyrates are added in the form of 400mmol/L DMSO solutions), NADP⁺Respectively 0.01st, 0.02,0.05,0.1,0.5,1.0 and 2.0mmol/L buffer solutions.Concentration of glucose is 50g/L buffer solutions.Step (1) is made Standby glucose dehydrogenase wet thallus amount 0.5g (dry cell weight 0.05g), 30 DEG C, 12h is reacted under rotating speed 150r/min.Reaction It is extracted twice after end with isometric ethyl acetate, sample treatment is same as Example 8.Determine product conversion ratio and ee.Under similarity condition, to be not added with NADP⁺Reaction solution as control.Optimum results are shown in Table 6.

The optimization of NADP+ concentration in the carbonyl reductase BgADH5 regenerating coenzyme circulating systems of table 6

NADP+(mM)	Conversion ratio (%)
		0	30
0.01	38
		0.02	40
0.05	46
		0.1	51
0.5	51
		1.0	51
2.0	51

As a result show the conversion ratio of carbonyl reductase BgADH5 catalysis substrates with NADP⁺Concentration increases and increased, and works as NADP⁺ When concentration is more than 0.1mM, conversion ratio tends towards stability.Above catalytic reaction products ee 99%.

Embodiment 8：Recombinate the full cells of carbonyl reductase BgADH5 and prepare 4-AA intermediates (2S, 3R) -2- benzene carbon amides Application in methyl -3-hydroxybutyrate ester

(1) recombination bacillus coli BL21 (the DE3)/pET28a- containing recombinant expression obtained in the method for embodiment 3 Adh5 wet thallus, using 2- benzoyl aminomethyl -3- ketone butyrates as substrate, carries out biocatalytic reaction system as biocatalyst Standby (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters.

Catalyst system and catalyzing forms and catalytic condition is as follows：0.5g carbonyls are added in 10mL phosphate buffers (100mM, pH 7.0) Base reductase BgADH5 wet thallus (dry cell weight 0.049g), (dry cell weight is 0.5g glucose dehydrogenases wet thallus It is prepared by 0.05g, the method for embodiment 7), 2- benzoyl aminomethyl -3- ketone methyl butyrates 0.05g (final concentration 20mmol/L buffer solutions, 2- benzoyl aminomethyl -3- ketone methyl butyrates are added in the form of 400mmol/L DMSO solutions), NADP⁺2mmol/L is buffered Liquid, glucose 50g/L buffer solutions form reaction system.30 DEG C, shaking bath reaction 12h, reaction under the conditions of rotating speed 150r/min It is extracted twice after end with isometric ethyl acetate, merges organic layer and dried with anhydrous magnesium sulfate, filtered, Rotary Evaporators remove Ethyl acetate is removed, concentrate HPLC flows phased soln, HPLC detection conversion ratios and ee.Under similarity condition, to add without thalline The reaction solution entered replaces above-mentioned recombination bacillus coli as blank control with e. coli bl21 (DE3)/pET28a wet thallus BL21 (DE3)/pET28a-adh5 is as negative control.The conversion ratio of substrate is 51.4%, ee 99% after 12h, with original bacteria Strain gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB-12126 compares (ee 81%), selectivity To significantly improving.Blank control shows inactive to substrate with negative control.

The ee of (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters is calculated as follows：Ee%=[(A_(2S,3R)- A_(2R,3S))/(A_(2S,3R)+A_(2R,3S))] × 100, wherein A is peak area.

(2) 2- benzoyl aminomethyls -3- ketone methyl butyrate and (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate ester Liquid phase detects

High performance liquid chromatograph device：Shimadzu LC-20AD system-SPD-20A UV-detectors.

Detection conversion ratio is that chromatographic column is Hypersil ODS2C18 (4.6mm × 250mm, 2.5 μm), mobile phase：Water：Second Nitrile=75:25, flow velocity 1mL/min, 40 DEG C of column temperature, Detection wavelength：254nm.The reservation of 2- benzoyl aminomethyl -3- ketone butyrates Time is 10.5min.2- benzoyl aminomethyls -3-hydroxybutyrate ester two is respectively 6.0min to the retention time of enantiomter And 6.9min.

Chiral chromatographic column is Chiralpak AY-H (250 × 4.6mm, 5 μm) when detecting ee, mobile phase：N-hexane：Ethanol =76:24, flow velocity 1.0mL/min.(2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate ester, (2R, 3R) -2- benzene carbon amides Methyl -3-hydroxybutyrate ester, (2R, 3S) -2- benzoyl aminomethyls -3-hydroxybutyrate ester and (2S, 3S) -2- benzene carbon amide first The retention time of base -3-hydroxybutyrate ester is respectively：5.8th, 6.7,7.3 and 10.4min.Substrate 2- benzoyl aminomethyl -3- ketone The retention time of two kinds of configurations of butyrate is respectively in 9.8min and 11.0min.

Embodiment 9：Restructuring the full cells of carbonyl reductase BgADH5 prepare Atorvastatin pharmaceutical intermediate 6- cyano group- Application in (3R, 5R)-dihydroxy hecanoic acid t-butyl ester

(1) recombination bacillus coli BL21 (DE3)/pET28a- containing recombinant expression to be obtained in embodiment 3 Adh5 wet thallus, using (R) -6- cyano group -5- hydroxyl -3- carbonyls hecanoic acid t-butyl esters as substrate, carries out biology as biocatalyst Conversion reaction prepares 6- cyano group-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester.

Catalyst system and catalyzing forms and catalytic condition is as follows：0.5g restructuring carbonyls are added in 10mL kaliumphosphate buffers (pH 7.0) Reductase BgADH5 wet thallus (dry cell weight 0.049g) and 0.5g glucose dehydrogenases wet thallus (dry cell weight 0.05g, It is prepared by the method for embodiment 7), (final concentration of 20mmol/L delays (R) -6- cyano group -5- hydroxyl -3- carbonyl hecanoic acid t-butyl esters 0.0458g Fliud flushing), NADP⁺2mmol/L buffer solutions, glucose 50g/L buffer solutions.30 DEG C, 12h is reacted under the conditions of rotating speed 150r/min.Instead Isometric ethyl acetate is added after should terminating to be extracted twice, and is merged organic layer and is dried with anhydrous magnesium sulfate, is filtered, rotary evaporation Instrument removes ethyl acetate, concentrate flowing phased soln, liquid chromatographic detection yield and de.Under similarity condition, with without bacterium The reaction solution of body replaces above-mentioned recombination bacillus coli as blank control with e. coli bl21 (DE3)/pET28a wet thallus BL21 (DE3)/pET28a-adh5 wet thallus is as negative control.The conversion ratio of substrate is 87.4%, de 98%.With patent Saccharomyces cerevisiae wild type ketoreductase YDL (reaction time 24h, conversion ratio in US7879585B2 85%) compare, conversion ratio improves.Blank control is shown to substrate without effect with negative control.

(2) (R) -6- cyano group -5- hydroxyls -3- carbonyls hecanoic acid t-butyl ester and 6- cyano group-tertiary fourth of (3R, 5R)-dihydroxy caproic acid The liquid phase detection method of ester

Liquid chromatogram instrument：Shimadzu LC-20AD system-SPD-20A UV-detectors.

Chromatographic column is Hypersil ODS2C18 (4.6mm × 250mm, 2.5 μm) when detecting conversion ratio and ee, flowing Phase：Acetonitrile：Water=1:3, flow velocity 1mL/min, Detection wavelength 220nm.(R) -6- cyano group -5- hydroxyls -3- carbonyls hecanoic acid t-butyl ester, The retention time of 6- cyano group-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester and 6- cyano group-(3R, 5S)-dihydroxy hecanoic acid t-butyl ester Respectively：11.4th, 9.4 and 9.8min.

Embodiment 10：Recombinate the full cells of carbonyl reductase BgADH5 and prepare three fluoro- 3-hydroxybutyrate second of (S) -4,4,4- Application in ester

(1) recombination bacillus coli BL21 (DE3)/pET28a- containing recombinant expression to be obtained in embodiment 3 Adh5 wet thallus, with 4,4,4- trifluoroacetic ethyl acetoacetates for substrate, carries out bioconversion reaction and prepared as biocatalyst (S) -4,4,4- trifluoro 3-hydroxy ethyl butyrates.

Catalyst system and catalyzing forms and catalytic condition is as follows：0.5g restructuring carbonyls are added in 10mL kaliumphosphate buffers (pH 7.0) Reductase BgADH5 wet thallus (dry cell weight 0.049g) and 0.5g glucose dehydrogenases wet thallus (dry cell weight 0.05g, It is prepared by the method for embodiment 7), 4,4,4- trifluoroacetic ethyl acetoacetate 0.037g (final concentration of 20mmol/L buffer solutions), NADP⁺ 2mmol/L buffer solutions, glucose 50g/L buffer solutions.30 DEG C, 12h is reacted under the conditions of rotating speed 150r/min.Reaction adds after terminating Isometric ethyl acetate is extracted twice, and is merged organic layer and is dried with anhydrous magnesium sulfate, filtered, Rotary Evaporators remove acetic acid second Ester, concentrate flowing phased soln, gas chromatographic detection yield and ee.Under similarity condition, made with the reaction solution of not mycetome For blank control, with e. coli bl21 (DE3)/pET28a wet thallus replace above-mentioned recombination bacillus coli BL21 (DE3)/ PET28a-adh5 wet thallus is as negative control.The conversion ratio of substrate is 88.4%, ee99%.Blank control and negative control Show to substrate without effect.Although biological method of asymmetrically reducing synthesis (R) -4,4,4- trifluoro 3-hydroxy ethyl butyrates are Have been reported that, but (S) -4, the bioanalysis asymmetric syntheses of 4,4- trifluoro 3-hydroxy ethyl butyrates do not have been reported that.

(2) the vapor detection side of 4,4,4- trifluoroacetic ethyl acetoacetates and (S) -4,4,4- trifluoro 3-hydroxy ethyl butyrates Method

Gas-chromatography instrument：Shimadzu GC-14C systems-fid detector.

Detection conversion ratio and chiral chromatographic column is BGB-174 (30m × 0.25mm × 0.25 μM) during ee, injection port temperature Degree：220 DEG C, detector temperature：220 DEG C, column temperature：120℃.Carrier is helium, flow velocity：1.5mL/min.4,4,4- trifluoroacetyls The reservation of ethyl acetate, (R) -4,4,4- trifluoro 3-hydroxy ethyl butyrates and (S) -4,4,4- trifluoro 3-hydroxy ethyl butyrates Time is respectively：2.6th, 6.8 and 7.1min.

From above experimental result, the recombination bacillus coli containing carbonyl reduction enzyme gene that the present invention obtains have compared with The ability of strong carbonyl reduction, directly it can carry out living things catalysis or conversion reaction by enzyme source of the somatic cells containing enzyme.Carbonyl Reductase BgADH5 (SED ID NO.2) is used as conversion enzyme, can utilize 2- benzoyl aminomethyl -3- ketone butyrate, (R) - 6- cyano group -5- hydroxyls -3- carbonyls hecanoic acid t-butyl ester and 4,4,4- trifluoroacetic ethyl acetoacetates are substrate, carry out bioconversion reaction High optically pure chiral alcohol medicine chiral intermediate (2S, the 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate ester of preparation, 6- cyano group - (3R, 5R)-dihydroxy hecanoic acid t-butyl ester and (S) -4,4,4- trifluoro 3-hydroxy ethyl butyrates.

Claims (4)

1. one kind derives from gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB-12126 restructuring carbonyl Application of the base reductase in asymmetric reduction prochiral carbonyl compounds, it is characterised in that the ammonia of the restructuring carbonyl reductase Base acid sequence is SEQ ID NO：Shown in 2.

2. application as claimed in claim 1, it is characterised in that the application is：With the engineering of the carbonyl reduction enzyme gene containing restructuring Bacterium it is fermented culture obtain wet thallus be catalyst, in pH value be 6~10 buffer solution in, add substrate, cosubstrate with NAD(P)⁺, reacted under the conditions of 20~40 DEG C, 50~250rpm, after reaction completely, obtain the mixed liquor containing chiral alcohol；It is described Substrate is 2- benzoyl aminomethyl -3- ketone butyrate, (R) -6- cyano group -5- hydroxyls -3- carbonyls hecanoic acid t-butyl ester and 4,4,4- tri- One kind in acetyl fluoride ethyl acetate, when the substrate is 2- benzoyl aminomethyl -3- ketone butyrates, 2- benzene carbon amide first Base -3- ketone butyrate is added in the form of 400mmol/L dimethyl sulphoxide solutions；The cosubstrate be glucose, ammonium formate, Isopropanol or absolute ethyl alcohol, when the cosubstrate is glucose, addition glucose dehydrogenase forms cosubstrate system, when When the cosubstrate is ammonium formate, addition ammonium formate dehydrogenase forms cosubstrate system；The dosage of the catalyst is with wet The weight of thalline is calculated as 20~200g/L buffer solutions, and the initial concentration of the substrate is 10~100mmol/L buffer solutions, described auxiliary The dosage for helping substrate is 10~200g/L buffer solutions, and the glucose takes off the dosage of dehydrogenase or ammonium formate dehydrogenase with containing grape The wet thallus weight meter that the fermented culture of the thalline of glucocorticoid dehydrogenase or ammonium formate dehydrogenase obtains, is 20~200g/L buffer solutions, The NAD (P)⁺Dosage be 0.01~5mmol/L buffer solutions.

3. application as claimed in claim 2, it is characterised in that the wet thallus is prepared as follows：Restructuring carbonyl will be contained The engineering bacteria of reductase gene is seeded to the LB fluid nutrient mediums containing the μ g/ml kalamycin resistances of final concentration 50,37 DEG C, Cultivate 12h under 200rpm, then be seeded to and fresh resisted containing the μ g/ml kanamycins of final concentration 50 with the inoculum concentration of volumetric concentration 1% Property LB fluid nutrient mediums in, in 37 DEG C, culture is to thalline OD under 150rpm₆₀₀Up to 0.6~0.8, final concentration of 0.1mM is added IPTG, at 28 DEG C after Fiber differentiation 12h, 4 DEG C, 5000rpm centrifugation 5min, abandoning supernatant, collect wet thallus.

4. application as claimed in claim 2, it is characterised in that described application is with the engineering of the carbonyl reduction enzyme gene containing restructuring The wet thallus that the fermented culture of bacterium obtains is catalyst, in the buffer solution that pH value is 6~10, adds substrate, glucose, grape Glucocorticoid dehydrogenase and NAD (P)⁺, reacted under the conditions of 30 DEG C, 150rpm, after reaction completely, obtain the mixed of drug containing chiral intermediate Close liquid；The substrate be 2- benzoyl aminomethyl -3- ketone butyrate, (R) -6- cyano group -5- hydroxyls -3- carbonyls hecanoic acid t-butyl ester and One kind in 4,4,4- trifluoroacetic ethyl acetoacetates, when the substrate is 2- benzoyl aminomethyl -3- ketone butyrates, 2- benzene first Acyl aminomethyl -3- ketone butyrate is added in the form of 400mmol/L dimethyl sulphoxide solutions；The dosage of the catalyst is with wet bacterium The weight of body is calculated as 50g/L buffer solutions, and the initial concentration of the substrate is 20mmol/L buffer solutions, and the dosage of the glucose is 50g/L buffer solutions, the glucose take off the dosage of dehydrogenase with the wet of the fermented culture acquisition of the thalline containing glucose dehydrogenase Bacterium weight, is 50g/L buffer solutions, the NAD (P)⁺Dosage be 2mmol/L buffer solutions.