CN104630242A - Carbonyl reductase gene, codase, vector, engineering bacterium and application thereof - Google Patents
Carbonyl reductase gene, codase, vector, engineering bacterium and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant carbonyl reductase gene derived from Burkholderia gladioli ZJB-12126 and a codase thereof, a recombinant vector containing the gene, a recombinant gene engineering bacterium converted from the recombinant vector and application in catalyzing asymmetric reduction of prochiral carbonyl compound. 2-benzoylaminomethyl-3-one butyrate, tert-butyl (R)-6-cyano-5-hydroxy-3-carbonyl hexanoate and ethyl 4,4,4-trifluoro acetoacetate used as substrates are subjected to biological catalytic reaction to prepare high-optical-purity (2S,3R)-2-benzoylaminomethyl-3-hydroxy-butyrate, tert-butyl 6-cyano-(3R,5R)-dihydroxy hexanoate and ethyl (S)-4,4,4-trifluoro-3-hydroxy-butanoate. The recombinant Escherichia coli can be used as a biocatalyst to perform the biological catalytic reaction, thereby providing an alternative new enzyme source for biological catalytic synthesis of the drug chiral intermediate.
Description
(1) technical field
The invention belongs to technical field of bioengineering, be specifically related to that one derives from the carbonyl reductase gene of gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB-12126, codase, the recombinant vectors containing this gene, this recombinant vectors transform the recombination engineering bacteria that obtains and application in catalytic asymmetric reduction prochiral carbonyl compounds.
(2) background technology
Carbonyl reductase (Carbonyl reducatase, E.C.1.1.1.x) be that a class can the enzyme of bidirectional reversible redox reaction between catalysis alcohols and aldehydes/ketone, and need coenzyme NAD (H) (Reduced nicotinamide-adenine dinucleotide) or NADP (H) (Triphosphopyridine nucleotide, reduced) as hydrogen carrier.NADH and NADPH participates in its reduction reaction as electron donor, NAD and NADP then participates in its oxidizing reaction as electron acceptor(EA).Short-chain dehydrogenase superfamily (Short-chaindehydrogenase/reductase is mostly belonged at present according to the carbonyl reductase of bibliographical information, SDR), medium chain alcohol hydrogen enzyme superfamily (Medium-chaindehydrogenase/reductase, MDR), aldehyde ketone reductase enzyme superfamily (Aldo-keto reductase, AKR).Although three has similar catalysis, evolution and structure differ greatly.Carbonyl reductase is the very ancient family of a class, is distributed widely in occurring in nature, in all kinds of animal, microorganism and plant, because microbe species is various, distribution wide, is the main source of carbonyl reductase.
In recent years, along with developing rapidly of genomics, proteomics and information biology, increasing rapidly of the gene data announced in genome and geneseq database, from a large amount of data resources, find novel carbonyl reductase gene, excavate its biological information contained, for the exploitation service of new and effective biological catalyst becomes a reality.Gene excavating technology is a novel enzyme triage techniques, is applied widely at present.Utilize 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, a large amount of carbonyl reductases has been excavated in the bacterial strains such as Candida magnoliae CmCR and Acetobacter sp.AcCR.The wherein gene of part carbonyl reductase successful expression in different hosts (Escherichia coli, Pichia pastoris, Arxula adeninivorans etc.), obtain and produce enzyme activity and the higher genetic engineering bacterium of selectivity, and be successfully applied to the asymmetric reduction reaction of catalysis of carbonyl compounds.However, many carbonyl reductases have substrate specificity, greatly limit its range of application.In addition, the catalytic efficiency of many enzymes is lower, also limit its industrial applications.Screening has the Novel carbonyl reductase of wider substrate spectrum, and studying it can the carbonyl compound of efficient highly selective catalysis, not only can widening its range of application, promote its application potential, also laying the foundation for realizing suitability for industrialized production.
Chiral alcohol is widely used in the synthesis of chiral drug and other chiral fine chemicals.The synthetic method of current chiral alcohol comprises physical partition method, Split Method and method of asymmetrically reducing etc.Wherein, utilize the asymmetric reduction synthesis of chiral alcohol of prochiral carbonyl compounds, its theoretical yield reaches 100%, is a kind of main method of producing chiral alcohol at present.Chemistry method of asymmetrically reducing mainly utilizes chiral metal complex as the asymmetric reduction of catalyzer for carbonyl, although this chemical process part is used for industrial production, but this reaction conditions is harsher, chiral metal complex synthesis is complicated and expensive, the residual of heavy metal is had to cause product separation difficulty in product, environmental pollution is comparatively large, and therefore its application receives certain restriction.Biocatalysis method of asymmetrically reducing not only has chemistry, the regio-and stereo-selectivity of height, and reaction conditions is gentle, avoid the heavy-metal residual in product, environmentally friendly, compensate for the deficiency of chemical process, therefore, the application of carbonyl asymmetric reduction reaction in chiral alcohol synthesis of biocatalysis more and more comes into one's own in recent years.
(2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester is the chiral building block of synthesis carbapenem and penems medicine intermediate 4-AA (4-AA).Current acquisition (2S, the main path of 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester intermediate generates (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester with chiral catalyst (R)-BINAP-Ru title complex asymmetry catalysis 2-benzoyl aminomethyl-3-ketone butyric ester.But due to this reaction conditions need the price comparison of high-pressure hydrogenation, ruthenium complex expensive and with product be separated comparatively difficulty etc., be unfavorable for its industrial applications.Current biological catalysis synthesis (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester report is less.Saccharomycopsis malanga NBRC 171096 catalysis generates (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester, and there is higher enantioselectivity (enantiomeric excess value ee>96.2%), but catalytic efficiency low (productive rate only 4%).US Patent No. 20130034895 have studied the carbonyl reduction Enzyme catalyzed synthesis (2S deriving from Lactobacillus kefir, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester, there is higher enantioselectivity (ee is 60 ~ 99%), but in this reaction process, coenzyme recycle is realized as the second substrate using Virahol, because Virahol and substrate exist competitive inhibition, cause maximum conversion rate to be affected, and the generation of by product acetone have certain detrimental effect to enzyme.
6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester is the chiral intermediate of HMG-CoA enzyme inhibitors Stains medicine, can be obtained by the method for chemosynthesis and biocatalysis synthesis.Need to use inflammable and explosive n-Butyl Lithium, borine in chemical method synthesis, and need to carry out under <-65 DEG C of cold condition, energy consumption is large, add 6-cyano group-(3R again, 5R)-dihydroxyl hecanoic acid t-butyl ester diastereomeric induction is insufficient, and the optical purity of product is difficult to reach requirement.In recent years, utilize enzyme process to replace chemical method to improve reaction conditions, reduce reaction cost, the selectivity improving product becomes the focus of research.The bacterial strain that highly-solid selectively catalysis (the R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester screened at present generates 6-cyano group-(3R, 5R)-dihydroxyl 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 house journal US7879585B2 has cloned a kind of ketoreductase and has transformed from Saccharomyces cerevisiae, catalytic production 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester transformation efficiency is 99.7%, de>99%.
Befloxatone (Befloxatone) chemical name 5 (R)-(methoxyl methyl)-3-[4-[4,4, fluorine-based-3 (the R)-hydroxybutoxy of 4-tri-] phenyl] oxazolidone-2, it is a kind of strong selectivity reversibility monoamine oxidase A (MAO-A) inhibitor, interact with the Specific amino acid of MAO-A flavin adenine dinucleotide (FAD) cofactor and avtive spot, be used for Cure of depression clinically.(R)-4,4,4-trifluoro 3-hydroxy ethyl butyrates are chiral intermediates of synthesis befloxatone.Zhang etc. first reported in bacterial strain Bacillus pumilus Phe-C3 containing NADPH dependent form carbonyl reductase and glucose-6-phosphate dehydrogenase (G6PD) (G-6-PDH), energy catalysis 4,4,4-trifluoroacetic ethyl acetoacetate generates (R)-4,4,4-trifluoro 3-hydroxy ethyl butyrate, when concentration of substrate is 60mM, productive rate reaches 67%, ee and reaches 95%.The people such as He screen a strain Saccharomyces uvarum SW-58, and utilize water-organic solvent two-phase system catalysis 4,4,4-trifluoroacetic ethyl acetoacetate generates (R)-4,4,4-trifluoro 3-hydroxy ethyl butyrate, when concentration of substrate is 0.2mM, after 4h, transformation efficiency reaches 85%, ee and reaches 85.2%.The people such as Kara utilize this reaction of commercial enzyme ADH evo-1.1.200 catalysis, and concentration of substrate is transformation efficiency 39.2% after 5mM, 24h, and selectivity is significantly improved (ee>99.9%).In addition, (S)-4,4,4-trifluoro 3-hydroxy ethyl butyrate be also a kind of important chiral intermediate, its biological catalysis asymmetric synthesis has no report.
As can be seen here, the biocatalysis technique studying these medicine chiral intermediates is significant, can not only provide new route for the synthesis of these compounds, can provide new enzyme source again for it.Up to now, the application of carbonyl reductase in these medicine chiral intermediate asymmetric reductions in Burkholderia gladioli bacterial strain is had not yet to see.
(3) summary of the invention
The object of the invention is to provide that one derives from gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB-12126 carbonyl reductase, gene, the recombinant vectors containing this gene, this recombinant vectors transform the recombination engineering bacteria obtained, and at preparation (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester, (S)-4-chloro-3-hydroxyl ethyl butyrate, 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester and 4, application in the chirality pharmaceutical intermediate compounds such as 4,4-trifluoroacetic ethyl acetoacetate.
The technical solution used in the present invention is:
The invention provides the carbonyl reductase gene that one derives from gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB-12126, the nucleotides sequence of described gene is classified as shown in SEQ ID NO:1, and described carbonyl reductase gene obtains by the following method:
Utilize round pcr, be the carbonyl reductase gene order that template clone is about 0.8kb with the total genomic dna derived from gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB-12126 bacterial strain under the effect of primer 1 (5 '-ATGGCAGACGTCAACAGCCTGTTC-3 '), primer 2 (5 '-TCAGACCGTGCTGGTGAGGCC-3 '), called after adh5 (nucleotides sequence is classified as shown in SEQ ID NO:1).This fragment is connected on pGEM-T carrier, obtains cloning vector pGEM-T-adh5, vector intestinal bacteria are obtained the recombination bacillus coli containing carrier pGEM-T-adh5.To recombinant plasmid order-checking, and utilize software to analyze sequencing result, this sequence contains the open reading frame of a long 774bp.
The present invention express 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 for template, obtains the carbonyl reductase gene for expressing by pcr amplification.
Any the nucleotide sequence that the replacement of one or more Nucleotide, insertion or disappearance process obtain is carried out to nucleotide sequence shown in SEQ ID NO:1, as long as itself and this Nucleotide has the homology of more than 90%, all belong to protection scope of the present invention.
The invention provides a kind of restructuring carbonyl reductase of being encoded by described carbonyl reductase gene adh 5, called after BgADH5, the aminoacid sequence of described restructuring carbonyl reductase is for shown in SEQ ID NO:2.
The polypeptide fragment that any process of inserting amino acid in aminoacid sequence shown in SEQ ID NO:2, lack or replacing obtains or its mutant; as long as aminoacid sequence has more than 95% homology shown in itself and SEQ ID NO:2, all belong to protection scope of the present invention.
The present invention relates to the recombinant vectors containing described carbonyl reductase gene.
The present invention relates to and utilize described recombinant vectors to transform the recombination engineering bacteria obtained, be specially: carbonyl reductase gene is connected with expression vector pET28a, construct the heterogenous expression recombinant plasmid pET28a-adh5 containing carbonyl reductase gene.Recombinant expression pET28a-adh5 is converted in E. coli BL21 (DE3), obtains recombination bacillus coli BL21 (the DE3)/pET28a-adh5 containing recombinant plasmid pET28a-adh5.
The invention still further relates to the application of carbonyl reductase gene in preparation restructuring carbonyl reductase, be specially: build the recombinant vectors containing described carbonyl reductase gene, described recombinant vectors is converted in Host Strains (preferred E. coli BL21 (DE3)), the recombination engineering bacteria obtained carries out inducing culture, nutrient solution is separated the somatic cells obtained containing restructuring carbonyl reductase, the carbonyl reductase crude enzyme liquid obtained after broken carries out purifying, obtains the pure enzyme of carbonyl reductase.
The invention still further relates to described restructuring carbonyl reductase and prepare application in chiral alcohol medicine chiral intermediate at asymmetric reduction prochiral carbonyl compounds, describedly to be applied as: the wet thallus obtained through fermentation culture with the engineering bacteria containing restructuring carbonyl reductase gene is for catalyzer, be in the damping fluid of 6 ~ 10 in pH value, add substrate, cosubstrate and NAD (P)
+, 20 ~ 40 DEG C, reaction under 50 ~ 250rpm condition (preferably 30 DEG C, 150r/min under react 12h), after reacting completely, obtain the mixed solution containing chiral alcohol medicine chiral intermediate; After reaction terminates, mixed solution is extracted with ethyl acetate twice, merges organic layer and with anhydrous magnesium sulfate drying, filter, Rotary Evaporators removing ethyl acetate, obtains medicine chiral intermediate after drying; Described substrate is 2-benzoyl aminomethyl-3-ketone butyric ester, (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester and 4,4, one in 4-trifluoroacetic ethyl acetoacetate, when described substrate is 2-benzoyl aminomethyl-3-ketone butyric ester, 2-benzoyl aminomethyl-3-ketone butyric ester adds with the form of 400mmol/L dimethyl sulphoxide solution; Described cosubstrate is glucose, ammonium formiate, Virahol or dehydrated alcohol, preferred glucose, when described cosubstrate is glucose, adds Hexose phosphate dehydrogenase and forms cosubstrate system, when described cosubstrate is ammonium formiate, adds hydrogenlyase and form cosubstrate system; The consumption of described catalyzer counts 20 ~ 200g/L damping fluid (preferred 50g/L) with wet thallus weight, the starting point concentration of described substrate is 10 ~ 100mmol/L damping fluid (preferred 20mmol/L), the consumption of described cosubstrate is 10 ~ 200g/L damping fluid (preferred 50g/L), described NAD (P)
+consumption be 0.01 ~ 5mmol/L damping fluid (preferred 2mmol/L), described glucose takes off the wet thallus weighing scale that desaturase or formate dehydrogenase enzyme dosage obtain through fermentation culture to take off desaturase or hydrogenlyase thalline containing glucose, is 20 ~ 200g/L damping fluid (preferred 50g/L).Described substrate adds (i.e. substrate dimethyl sulfoxide (DMSO) be mixed with 400mmol/L solution) with the form of 400mmol/L DMSO solution.
Further, the wet thallus that the engineering bacteria containing restructuring carbonyl reductase gene of the present invention obtains through fermentation culture is prepared as follows: the engineering bacteria containing restructuring carbonyl reductase gene is seeded to the LB liquid nutrient medium containing final concentration 50 μ g/mL kalamycin resistance, 37 DEG C, 12h is cultivated under 200rpm, be seeded to fresh in the LB liquid nutrient medium of final concentration 50 μ g/mL kalamycin resistance again with volumetric concentration 1% inoculum size, in 37 DEG C, under 150rpm, be cultured to thalline OD
600reach 0.6 ~ 0.8, add the IPTG that final concentration is 0.1mM, at 28 DEG C after inducing culture 12h, 4 DEG C, the centrifugal 5min of 5000rpm, abandoning supernatant, collects wet thallus.
Coenzyme wet thallus of the present invention is prepared as follows: (will preferably derive from the GDH of Exiguobacterium sibiricum 255-15 containing Hexose phosphate dehydrogenase (GDH), GenBank:ACB59697.1) recombinant bacterium BL21 (DE3)/pET28b-gdh) or (preferably derive from the FDH of Candida boidinii containing hydrogenlyase (FDH), GenBank:AF004096) recombinant bacterium BL21 (DE3)/pET28b-fdh is seeded to the LB liquid nutrient medium containing final concentration 50 μ g/mL kalamycin resistance, 37 DEG C, 12h is cultivated under 200rpm, be seeded to fresh in the LB liquid nutrient medium of final concentration 50 μ g/mL kalamycin resistance again with 1% inoculum size (v/v), in 37 DEG C, thalline OD is cultured under 150rpm
600reach 0.6 ~ 0.8, add the IPTG that final concentration is 0.1mM, at 28 DEG C after inducing culture 12h, 4 DEG C, the centrifugal 5min of 5000rpm, abandoning supernatant, collecting precipitation, namely obtains respective wet thalline.
Further, preferred described restructuring carbonyl reductase is preparing the application in medicine chiral intermediate: the wet thallus obtained through fermentation culture with the engineering bacteria containing restructuring carbonyl reductase gene is for catalyzer, be in the damping fluid of 6 ~ 10 in pH value, add substrate, glucose, Hexose phosphate dehydrogenase and NAD (P)
+, 30 DEG C, react under 150rpm condition, after reacting completely, obtain the mixed solution of drug containing chiral intermediate (i.e. chiral alcohol); Described substrate is 2-benzoyl aminomethyl-3-ketone butyric ester, (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester and 4,4, one in 4-trifluoroacetic ethyl acetoacetate, when described substrate is 2-benzoyl aminomethyl-3-ketone butyric ester, described 2-benzoyl aminomethyl-3-ketone butyric ester adds with the form of 400mmol/L dimethyl sulphoxide solution; The consumption of described catalyzer counts 50g/L damping fluid with the weight of wet thallus, the starting point concentration of described substrate is 20mmol/L damping fluid, the consumption of described glucose is 50g/L damping fluid, the consumption of described Hexose phosphate dehydrogenase is with the wet thallus weighing scale obtained through fermentation culture containing the thalline of Hexose phosphate dehydrogenase, for 50g/L damping fluid, described NAD (P)
+consumption be 2mmol/L damping fluid.
Further, carbonyl reductase BgADH5 containing described carbonyl reductase gene of the present invention generates (2S as biological catalyst at conversion of substrate 2-benzoyl aminomethyl-3-ketone butyric ester (formula I), application in 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester (formula II), the reaction formula related to as shown in Figure 6, in formula I, R is the alkyl of C1-C6, particularly, the reaction system of above-mentioned application is: with phosphate buffered saline buffer (100mM, pH 7.0) be reaction medium, the wet thallus obtained through fermentation culture with the engineering bacteria containing restructuring carbonyl reductase gene is for catalyzer, add Hexose phosphate dehydrogenase, 2-benzoyl aminomethyl-3-ketone butyric ester, NADP
+and glucose, 30 DEG C, rotating speed 150r/min Water Under bath shaking table reaction 12h, after reaction terminates, reaction solution equal-volume extraction into ethyl acetate twice, merge organic layer and with anhydrous magnesium sulfate drying, filter, Rotary Evaporators removing ethyl acetate, obtain enriched material, drying, is (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester, described substrate adds with the form of 400mmol/L DMSO solution, the final concentration 20mmol/L damping fluid of substrate, NADP
+consumption is 2mmol/L damping fluid, and glucose consumption is 50g/L damping fluid, and the consumption of described Hexose phosphate dehydrogenase is with the wet thallus weighing scale obtained through fermentation culture containing the thalline of Hexose phosphate dehydrogenase, and be 50g/L damping fluid, catalyst levels is 50g/L damping fluid.
Further, when described substrate is (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, its reaction formula as shown in Figure 7, particularly, the reaction system of above-mentioned application is: the wet thallus obtained through fermentation culture with the engineering bacteria containing restructuring carbonyl reductase gene for catalyzer, with potassium phosphate buffer (100mM, pH 7.0) be reaction medium, add Hexose phosphate dehydrogenase, glucose, NADP
+substrate (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester (formula III), 30 DEG C, react 12h under rotating speed 150r/min condition, after reaction terminates, add equal-volume extraction into ethyl acetate twice, merge organic layer and use anhydrous magnesium sulfate drying, filter, Rotary Evaporators removing ethyl acetate, enriched material drying is 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester (formula IV); Described restructuring carbonyl reductase wet thallus consumption is 50g/L damping fluid, glucose dehydro enzyme dosage counts 50g/L damping fluid with the wet thallus consumption obtained through fermentation culture containing the thalline of Hexose phosphate dehydrogenase, and glucose consumption is 50g/L damping fluid, NADP
+consumption is 2mmol/L damping fluid, concentration of substrate 20mmol/L damping fluid.
Further, described substrate is 4,4, during 4-trifluoroacetic ethyl acetoacetate, its reaction formula as shown in Figure 8, particularly, the reaction system of above-mentioned application is: the wet thallus obtained through fermentation culture with the engineering bacteria containing restructuring carbonyl reductase gene is for catalyzer, with potassium phosphate buffer (100mM, pH 7.0) for reaction medium, add Hexose phosphate dehydrogenase, glucose, NADP
+, substrate 4,4,4-trifluoroacetic ethyl acetoacetate (formula V), 30 DEG C, under rotating speed 150r/min condition, react 12h, after reaction terminates, add equal-volume extraction into ethyl acetate twice, merge organic layer and use anhydrous magnesium sulfate drying, filter, Rotary Evaporators removing ethyl acetate, enriched material drying is (S)-4,4,4-trifluoro 3-hydroxy ethyl butyrate (formula VI); Described restructuring carbonyl reductase wet thallus consumption 50g/L damping fluid, glucose dehydro enzyme dosage count 50g/L damping fluid, glucose consumption for 50g/L damping fluid, NADP with the thalline containing Hexose phosphate dehydrogenase through the wet thallus weight that fermentation culture obtains
+consumption is 2mmol/L damping fluid, concentration of substrate 20mmol/L damping fluid.
Catalyzer of the present invention also comprises other forms such as the pure enzyme of carbonyl reductase BgADH5, crude enzyme liquid or thick enzyme powder.When adopting carbonyl reductase recombination engineering bacteria to make catalyzer, in conjunction with a kind of coenzyme recycle system, two enzyme Double bottom thing coenzyme recycle system such as glucose/Hexose phosphate dehydrogenase (GDH), formic acid/hydrogenlyase (FDH) and single enzyme Double bottom such as ethanol, Virahol thing coenzyme recycle system need be comprised.Usually adding cosubstrate replaces NAD (P) H to react, and conventional cosubstrate is: glucose 10 ~ 200g/L, ethanol or Virahol 2 ~ 30% (v/v accounts for the quality percent by volume of total transformation system); Preferably cell concentration is 20 ~ 200g/L.
Gladiolus bulkholderia cepasea (Burkholderiagladioli) ZJB-12126 of carbonyl reductase gene of the present invention can be provided, deposit number is CCTCC M 2012379, be preserved in China typical culture collection center, preservation address is Wuhan, China Wuhan University, preservation date is on September 25th, 2012, discloses in previous patent application (CN103045504A).
Beneficial effect of the present invention is mainly reflected in: the invention provides the carbonyl reductase gene that one derives from gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB-12126, this carbonyl reductase gene can be connected the recombinant expression pET28a-adh5 building and obtain containing this gene with expression vector, be converted in e. coli bl21 (DE3) again, obtain recombination bacillus coli, these intestinal bacteria contain restructuring carbonyl reductase, recombination bacillus coli can be utilized to carry out biocatalytic reaction as biological catalyst, for the biocatalysis synthesis of medicine chiral intermediate provides alternative new enzyme source.
Restructuring carbonyl reductase BgADH5 is as biological catalyst, with 2-benzoyl aminomethyl-3-ketone butyric ester for substrate preparation (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester, product ee>99%, 12h substrate conversion efficiency is 51.4%, compared with B.gladioli ZJB-12126 wild mushroom in CN103045504A (ee 81%), enantioselectivity is improved.With (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester be substrate prepare 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester time, product de is 98%, 12h substrate conversion efficiency is 87.4%.As compared to the Saccharomyces cerevisiae wild-type ketoreductase YDL (reaction times 24h, transformation efficiency 85%) in patent US7879585B2, transformation efficiency improves.When being substrate with 4,4,4-trifluoroacetic ethyl acetoacetate, also can carry out conversion reaction preparation corresponding (S)-4,4,4-trifluoro 3-hydroxy ethyl butyrate, ee>99%, 12h substrate conversion efficiency is 88.4%.Although having been reported of biological method of asymmetrically reducing synthesis (R)-4,4,4-trifluoro 3-hydroxy ethyl butyrate, (S)-4,4,4-the biological process asymmetric synthesis of trifluoro 3-hydroxy ethyl butyrate do not have report.
(4) accompanying drawing explanation
Fig. 1 is cloning vector pGEM-T-adh5 physical map;
Fig. 2 is pET28a-adh5 recombinant plasmid physical map;
Fig. 3 is carbonyl reductase gene PCR amplification low melting-point agarose gel (argrose) electrophorogram; Wherein, swimming lane 1 is DL2000DNA Marker; Swimming lane 2 to increase the carbonyl reductase gene fragment obtained for utilizing primer 1 and primer 2; Swimming lane 3,4 to increase the carbonyl reductase gene fragment obtained for utilizing primer 3 and primer 4.
The enzyme of the positive recombinant plasmid pET28a-adh5 of Fig. 4 cuts structure iron; Wherein, swimming lane 1 is DL 10000DNA Marker fragment; Swimming lane 2 is carbonyl reductase gene; Swimming lane 3 is pET28a-adh5/Nco I sample; Swimming lane 4 is pET28a-adh5/Xho I sample; Swimming lane 5 is pET28a-adh5/Nco I and Xho I sample;
Fig. 5 is the SDS-PAGE figure after carbonyl reduction enzyme purification: swimming lane 1 is protein molecular weight Marker, and swimming lane 2,3 is the carbonyl reductase BgADH5 after purifying.
Fig. 6 is (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate Lipase absobed equation.
Fig. 7 is 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester synthesis equation.
Fig. 8 be (S)-4,4,4-trifluoro 3-hydroxy ethyl butyrate synthesis equation.
(5) embodiment
Below in conjunction with 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 sequencing information, excavate wherein a large amount of carbonyl reductases, one of them has catalysis 2-benzoyl aminomethyl-3-ketone butyric ester, (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester and 4, 4, 4-trifluoroacetic ethyl acetoacetate generates (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester, 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester and (S)-4, 4, the enzyme of 4-trifluoro 3-hydroxy ethyl butyrate function is the carbonyl reductase BgADH5 that the present invention relates to.
Utilize the FastDNA of MPBio company
spin test kit extracts the total genomic dna of gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB12126 thalline, with this genomic dna for template, under the effect of primer 1 (5 '-ATGGCAGACGTCAACAGCCTGTTC-3 '), primer 2 (5 '-TCAGACCGTGCTGGTGAGGCC-3 '), carry out pcr amplification.PCR reaction system (cumulative volume 50 μ L): 10 × Pfu DNA Polymerase Buffer 5 μ L, 10mM dNTP mixture (each 2.5mM of dATP, dCTP, dGTP and dTTP) 1 μ L, concentration is cloning primer 1, each 1 μ L of primer 2 of 50 μMs, genomic dna 1 μ L, Pfu DNA Polymerase 1 μ L, seedless sour water 40 μ L.
Adopt the PCR instrument of Biorad, PCR reaction conditions: denaturation 95 DEG C of 5min, 95 DEG C of sex change 30s, 60 DEG C of annealing 45s, 72 DEG C extend 1min, totally 30 circulations, and last 72 DEG C extend 10min.
PCR reaction solution detects with 0.9% agarose gel electrophoresis and cuts glue and reclaims this fragment of purifying, utilizes Taq DNA polymerase to hold to fragment 5 ' and introduces base A.Under the effect of T4DNA ligase enzyme, this fragment is connected with pGEM-T carrier, obtain cloning recombinant plasmids pGEM-T-adh5, see Fig. 1.By this recombinant plasmid transformed in e. coli jm109, coating containing final concentration is that the LB of 50 μ g/ml sodium ampicillin resistances is dull and stereotyped, random picking positive colony order-checking, utilize software analysis sequencing result, result shows: the nucleotide sequence length increased through primer 1 and primer 2 is 774bp (its nucleotide sequence is as shown in SEQ ID NO:1), the open reading frame (aminoacid sequence is SED ID NO.2) that this sequence encoding one is complete.
Embodiment 2: the structure of recombination bacillus coli BL21 (DE3)/pET28a-adh5
According to embodiment 1 analytical results design primer 3 (5 '-
cCATGGcAGACGTCAACAGCCTGTTC-3 '), primer 4 (5 '-
cTCGAGgAC
CGTGCTGGTGAGGCC-3 '), and in primer 3 and primer 4, introduce Nco I and XhoI restriction enzyme site (underscore mark) respectively.Under the initiation of primer 3 and primer 4, high-fidelity PfuDNA polysaccharase is utilized to increase, obtain the long carbonyl reductase gene order (its nucleotide sequence is as shown in SEQ ID NO:1) for 774bp, Nco I and Xho I restriction enzyme (TaKaRa) is utilized to process amplified fragments after order-checking, and utilize T4DNA ligase enzyme (TaKaRa) by this fragment with connecting with the commercialization carrier pET28a (Invitrogen) of identical restriction enzyme ferment treatment, construction of expression vector pET28a-adh5.The expression vector pET28a-adh5 of structure is converted in e. coli bl21 (DE3) (Invitrogen), the LB coated containing final concentration 50 μ g/mL kalamycin resistance is dull and stereotyped, 8-16h is cultivated at 37 DEG C, random picked clones, extracting plasmid carries out enzyme and cuts qualification, qualification result as shown in Figure 4, there is single band in line at 3 swimming lanes and 4 swimming lanes in positive recombinant plasmid pET28a-adh5 single endonuclease digestion as can be seen from Figure 4, after double digestion there are two bands in 5 swimming lanes, and a band is consistent with goal gene clip size.This result illustration purpose gene has been cloned into Nco I and the Xho I site of pET28a, namely obtains recombination bacillus coli E.coli BL21 (DE3)/pET28a-adh5.
Embodiment 3: restructuring carbonyl reductase (BgADH5) wet thallus
Recombination bacillus coli E.coliBL21 (the DE3)/pET28a-adh5 thalline containing recombinant expression pET28a-adh5 embodiment 2 obtained is seeded to the LB liquid nutrient medium containing final concentration 50 μ g/mL kalamycin resistance, 37 DEG C, 12h is cultivated under 200rpm, be seeded to fresh in the LB liquid nutrient medium of final concentration 50 μ g/ml kalamycin resistance again with 1% inoculum size (v/v), in 37 DEG C, under 150rpm, be cultured to thalline OD
600reach 0.6 ~ 0.8, add the IPTG that final concentration is 0.1mM, at 28 DEG C after inducing culture 12h, 4 DEG C, the centrifugal 5min of 5000rpm, abandoning supernatant, collecting precipitation, namely obtains recombination bacillus coli BL21 (the DE3)/pET28a-adh5 wet thallus containing recombinant expression.This thalline can directly as biological catalyst or for protein purification.
Embodiment 4: the separation and purification of carbonyl reductase (BgADH5)
By the thalline (i.e. recombination bacillus coli BL21 (DE3)/pET28a-adh5 wet thallus) of acquisition in embodiment 3 with binding buffer liquid (50mM, pH 8.0 sodium phosphate buffer, containing 300mM NaCl, 10mM imidazoles) resuspended after, through ultrasonication, the centrifugal 40min of 12000rpm, supernatant is with after the Ni affinity chromatography resin that above-mentioned binding buffer liquid is equilibrated is hatched, use dcq buffer liquid (50mM again, pH 8.0 sodium phosphate buffer, containing 300mM NaCl, 20mM imidazoles) rinse extremely substantially without foreign protein, subsequently with elution buffer (50mM, pH 8.0 sodium phosphate buffer, containing 300mM NaCl, 250mM imidazoles) wash-out collect target protein, target protein is merged and with dialysis buffer liquid (50mM after electroresis appraisal purity, pH 8.0 sodium phosphate buffer) dialyse 48h, getting trapped fluid adopts Coomassie Brilliant Blue mensuration protein content to be 2mg/mL, enzyme liquid being diluted to final concentration is 0.5mg/mL packing, frozen in-80 DEG C (carbonyl reductase BgADH5 protein electrophoresis figure is shown in accompanying drawing 5), obtain the pure enzyme of carbonyl reductase BgADH5.
Embodiment 5: the determination of activity of restructuring carbonyl reductase BgADH5
The pure enzyme of carbonyl cyclase BgADH5 obtained with the separation and purification of embodiment 4 method is for catalytic substrate 2-benzoyl aminomethyl-3-ketone methyl-butyrate.
Catalyst system composition and catalytic condition as follows: 10mL phosphate buffered saline buffer (100mM, pH 7.0) in add the pure enzyme of carbonyl reductase BgADH5 (final concentration is 0.1mg/mL), 2-benzoyl aminomethyl-3-ketone methyl-butyrate (final concentration 20mmol/L, 2-benzoyl aminomethyl-3-ketone methyl-butyrate adds with the form of 400mmol/L DMSO solution), NAD (P) H (final concentration 5mmol/L damping fluid) forms reaction system.30 DEG C, react 5min sampling detection enzyme under rotating speed 150r/min condition and live.Under similarity condition, the trapped fluid obtained through dialysing with e. coli bl21 (DE3) and e. coli bl21 (DE3)/pET28a bacterial cell disruption supernatant in contrast.
Mei Huo unit (U) is defined as: 30 DEG C, under pH 7.0 condition, the enzyme amount needed for 1min internal consumption 1 μm of ol NAD (P) H is defined as 1U.The consumption of NAD (P) H adopts microplate reader to measure under 340nm.Enzyme according to the consumption calculating restructuring carbonyl reductase BgADH5 of NAD in system (P) H is lived.Measurement result is in table 1.
Table 1 is recombinated the enzyme activity determination of carbonyl reductase BgADH5
| Bacterial strain/plasmid | Enzyme lives (U/mg) |
| E. coli bl21 (DE3) | 0 |
| E. coli bl21 (DE3)/pET28a | 0 |
| E. coli bl21 (DE3)/pET28a-adh5 | 2.6 |
Embodiment 6: the mensuration of restructuring carbonyl reductase BgADH5 coenzyme Preference
Catalyst system composition and catalytic condition as follows: 10mL phosphate buffered saline buffer (100mM, pH 7.0) in add embodiment 4 prepare the pure enzyme of restructuring carbonyl reductase BgADH5 (final concentration is 0.1mg/mL), 2-benzoyl aminomethyl-3-ketone methyl-butyrate (final concentration 20mmol/L, 2-benzoyl aminomethyl-3-ketone methyl-butyrate adds with the form of 400mmol/L DMSO solution), NADH or NADPH (2mmol/L damping fluid) forms reaction system.30 DEG C, after reacting 5min under rotating speed 150r/min condition, sampling detects enzyme and lives (method is with embodiment 5).Under similarity condition, not add the reaction solution of coenzyme in contrast.Measurement result is in table 2.
Table 2 is recombinated carbonyl reductase BgADH5 coenzyme Preference
| Coenzyme type | Enzyme lives (U/mg) |
| Contrast | 0 |
| NADH | 0.06 |
| NADPH | 2.7 |
Result shows, when this carbonyl reductase take NADPH as coenzyme, enzyme activity is far away higher than using NADH as the enzyme activity of coenzyme, and therefore this can infer that this enzyme is a kind of NADPH cofactor-dependent carbonyl reductase.
Embodiment 7: restructuring carbonyl reductase BgADH5 Cofactor Regeneration Systems
Using recombination bacillus coli BL21 (the DE3)/pET28a-adh5 wet thallus containing recombinant expression of embodiment 3 method acquisition as biological catalyst, with 2-benzoyl aminomethyl-3-ketone methyl-butyrate for substrate.
(1) selection of carbonyl reductase BgADH5 Cofactor Regeneration Systems
Hexose phosphate dehydrogenase (GDH) (is derived from Exiguobacterium sibiricum 255-15, GenBank:ACB59697.1) recombinant bacterium BL21 (DE3)/pET28b-gdh) thalline (glycerine pipe) and hydrogenlyase (FDH) (derive from Candida boidinii, GenBank:AF004096) recombinant bacterium BL21 (DE3)/pET28b-fdh) thalline (glycerine pipe) is seeded to LB liquid nutrient medium containing final concentration 50 μ g/mL kalamycin resistance respectively, 37 DEG C, 12h is cultivated under 200rpm, be seeded to fresh in the LB liquid nutrient medium of final concentration 50 μ g/mL kalamycin resistance again with 1% inoculum size (v/v), in 37 DEG C, thalline OD is cultured under 150rpm
600reach 0.6 ~ 0.8, add the IPTG that final concentration is 0.1mM, at 28 DEG C after inducing culture 12h, 4 DEG C, the centrifugal 5min of 5000rpm, abandoning supernatant, collecting precipitation, namely obtains Hexose phosphate dehydrogenase wet thallus or hydrogenlyase wet thallus.This thalline can directly apply to Cofactor Regeneration Systems.
Transformation system: potassium phosphate buffer (100mM, pH 7.0) 10mL, carbonyl reductase BgADH5 wet thallus amount 0.5g (dry cell weight is 0.048g), substrate 2-benzoyl aminomethyl-3-ketone methyl-butyrate 20mmol/L damping fluid (2-benzoyl aminomethyl-3-ketone methyl-butyrate adds with the form of 400mmol/L DMSO solution), NADP
+0.5m mol/L damping fluid, cosubstrate (cosubstrate is one of following: the Virahol of the glucose of 50g/L damping fluid, the ammonium formiate of 50g/L damping fluid, 30% (v/v) damping fluid or the dehydrated alcohol of 30% (v/v) damping fluid), (desaturase is one of following to desaturase: Hexose phosphate dehydrogenase (GDH) or hydrogenlyase (FDH), desaturase wet thallus consumption is 0.5g), 30 DEG C, rotating speed 150r/min shaking table reaction 12h.
Reaction terminates rear equal-volume extraction into ethyl acetate twice, and sample treatment is identical with embodiment 9.Measure transformation efficiency and the ee of product.Under similarity condition, not add the reaction solution of coenzyme circulating system in contrast.The results are shown in Table 3.
The different coenzyme recycle system of table 3 is on the impact of catalyzed reaction
Result shows that productive rate can reach 27.9%, and its selectivity is all higher than other Cofactor Regeneration Systems when selecting glucose/GDH as Cofactor Regeneration Systems.
(2) optimization of Hexose phosphate dehydrogenase biomass in carbonyl reductase BgADH5 regenerating coenzyme circulating system
Transformation system: potassium phosphate buffer (100mM, pH 7.0) 10mL, (dry cell weight is 0.048g to carbonyl reductase BgADH5 wet thallus amount 0.5g, prepared by embodiment 3 method), 2-benzoyl aminomethyl-3-ketone butyric ester 0.05g (final concentration 20mmol/L, 2-benzoyl aminomethyl-3-ketone butyric ester adds with the form of 400mmol/LDMSO), NADP
+0.5mmol/L damping fluid, glucose 50g/L damping fluid, Hexose phosphate dehydrogenase wet thallus amount prepared by step (1) is respectively 0.1,0.25,0.5,0.75,1.0g (dry cell weight is respectively 0.009,0.023,0.048,0.076,0.01g), 30 DEG C, rotating speed 150r/min shaking table reaction 12h.Reaction terminates rear equal-volume extraction into ethyl acetate twice, and sample treatment is identical with embodiment 9.Measure transformation efficiency and the ee of product.Under similarity condition, not add the reaction solution of Hexose phosphate dehydrogenase in contrast.Optimum result is in table 4.
The optimization of GDH biomass in table 4BgADH5 regenerating coenzyme circulating system
| GDH (wet thallus g) | Transformation efficiency (%) |
| 0 | 39 |
| 0.1 | 43 |
| 0.25 | 49 |
| 0.5 | 51 |
| 0.75 | 51 |
| 1.0 | 51 |
Result shows when BgADH5 biomass and Hexose phosphate dehydrogenase biomass weight ratio are 1:1, and regenerating coenzyme circulation can meet the demand of BgADH5 to NADPH substantially.Above catalytic reaction products ee 99%.
(3) optimization of glucose concn in carbonyl reductase BgADH5 regenerating coenzyme circulating system.
Transformation system: potassium phosphate buffer (100mM, pH 7.0) 10mL, (dry cell weight is 0.048g to carbonyl reductase BgADH5 wet thallus amount 0.5g, prepared by embodiment 3), 2-benzoyl aminomethyl-3-ketone butyric ester 0.05g (final concentration 20mmol/L damping fluid, 2-benzoyl aminomethyl-3-ketone butyric ester adds with 400mmol/LDMSO solution form), NADP
+0.5mmol/L damping fluid, glucose concn is respectively 10g/L damping fluid, 50g/L damping fluid, 100g/L damping fluid, 150g/L damping fluid and 200g/L damping fluid.Hexose phosphate dehydrogenase wet thallus amount 0.5g (dry cell weight is 0.05g) prepared by step (1), reacts 12h under rotating speed 150r/min by 30 DEG C.Reaction terminates rear equal-volume extraction into ethyl acetate twice, and sample treatment is identical with embodiment 8.Measure transformation efficiency and the ee of product.Under similarity condition, not add the reaction solution of glucose in contrast.Optimum result is in table 5.
The optimization of glucose concn in table 5 carbonyl reductase BgADH5 regenerating coenzyme circulating system
| Glucose (g/L) | Transformation efficiency (%) |
| 0 | 32 |
| 10 | 45 |
| 50 | 51 |
| 100 | 51 |
| 150 | 51 |
| 200 | 51 |
Result shows that, when glucose concn is 50g/L, regenerating coenzyme circulating system can meet the demand of carbonyl reductase BgADH5 to NADPH substantially.Above catalytic reaction products ee 99%.
(4) NADP in carbonyl reductase BgADH5 regenerating coenzyme circulating system
+the optimization of concentration.
Transformation system: potassium phosphate buffer (100mM, pH 7.0) 10mL, (dry cell weight is 0.048g to carbonyl reductase BgADH5 wet thallus amount 0.5g, prepared by embodiment 3), 2-benzoyl aminomethyl-3-ketone butyric ester 0.05g (final concentration 20mmol/L damping fluid, 2-benzoyl aminomethyl-3-ketone butyric ester adds with the form of 400mmol/LDMSO solution), NADP
+be respectively 0.01,0.02,0.05,0.1,0.5,1.0 and 2.0mmol/L damping fluid.Glucose concn is 50g/L damping fluid.Hexose phosphate dehydrogenase wet thallus amount 0.5g (dry cell weight is 0.05g) prepared by step (1), reacts 12h under rotating speed 150r/min by 30 DEG C.Reaction terminates rear equal-volume extraction into ethyl acetate twice, and sample treatment is identical with embodiment 8.Measure transformation efficiency and the ee of product.Under similarity condition, not add NADP
+reaction solution in contrast.Optimum result is in table 6.
The optimization of NADP+ concentration in table 6 carbonyl reductase BgADH5 regenerating coenzyme circulating system
| NADP+(mM) | Transformation efficiency (%) |
| 0 | 30 |
| 0.01 | 38 |
| 0.02 | 40 |
| 0.05 | 46 |
| 0.1 | 51 |
| 0.5 | 51 |
| 1.0 | 51 |
| 2.0 | 51 |
Result shows that the transformation efficiency of carbonyl reductase BgADH5 catalytic substrate is with NADP
+concentration increases and increases, and works as NADP
+when concentration is greater than 0.1mM, transformation efficiency tends towards stability.Above catalytic reaction products ee 99%.
Embodiment 8: the application of the full cell of restructuring carbonyl reductase BgADH5 in preparation 4-AA intermediate (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester
(1) using recombination bacillus coli BL21 (the DE3)/pET28a-adh5 wet thallus containing recombinant expression of embodiment 3 method acquisition as biological catalyst, with 2-benzoyl aminomethyl-3-ketone butyric ester for substrate, carry out biocatalytic reaction preparation (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate methyl esters.
Catalyst system composition and catalytic condition as follows: 10mL phosphate buffered saline buffer (100mM, pH 7.0) in add 0.5g carbonyl reductase BgADH5 wet thallus (dry cell weight is 0.049g), (dry cell weight is 0.05g to 0.5g Hexose phosphate dehydrogenase wet thallus, prepared by embodiment 7 method), 2-benzoyl aminomethyl-3-ketone methyl-butyrate 0.05g (final concentration 20mmol/L damping fluid, 2-benzoyl aminomethyl-3-ketone methyl-butyrate adds with the form of 400mmol/L DMSO solution), NADP
+2mmol/L damping fluid, glucose 50g/L damping fluid forms reaction system.30 DEG C, rotating speed 150r/min Water Under bath shaking table reaction 12h, reaction terminates rear equal-volume extraction into ethyl acetate twice, merge organic layer and with anhydrous magnesium sulfate drying, filter, Rotary Evaporators removing ethyl acetate, enriched material HPLC moving phase is dissolved, and HPLC detects transformation efficiency and ee.Under similarity condition, using the reaction solution added without thalline as blank, replace above-mentioned recombination bacillus coli BL21 (DE3)/pET28a-adh5 as negative control using e. coli bl21 (DE3)/pET28a wet thallus.After 12h, the transformation efficiency of substrate is 51.4%, ee is 99%, and compared with original strain gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB-12126 (ee is 81%), selectivity is significantly improved.Blank and negative control all show substrate non-activity.
The ee of (2S, 3R)-2-benzoyl aminomethyl-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) Liquid Detection of 2-benzoyl aminomethyl-3-ketone methyl-butyrate and (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester
High performance liquid chromatograph device: Shimadzu LC-20AD system-SPD-20A UV-detector.
Detection transformation efficiency is chromatographic column is Hypersil ODS2C18 (4.6mm × 250mm, 2.5 μm), moving phase: water: acetonitrile=75:25, flow velocity 1mL/min, column temperature 40 DEG C, determined wavelength: 254nm.The retention time of 2-benzoyl aminomethyl-3-ketone butyric ester is 10.5min.The retention time of 2-benzoyl aminomethyl-3-hydroxybutyrate ester two pairs of enantiomers is respectively 6.0min and 6.9min.
During detection ee, chiral chromatographic column is Chiralpak AY-H (250 × 4.6mm, 5 μm), moving phase: normal hexane: ethanol=76:24, flow velocity 1.0mL/min.(2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester, (2R, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester, (2R, 3S) retention time of-2-benzoyl aminomethyl-3-hydroxybutyrate ester and (2S, 3S)-2-benzoyl aminomethyl-3-hydroxybutyrate ester respectively: 5.8,6.7,7.3 and 10.4min.The retention time of substrate 2-benzoyl aminomethyl-3-ketone butyric ester two kinds of configurations is respectively at 9.8min and 11.0min.
Embodiment 9: the full cell of restructuring carbonyl reductase BgADH5 is preparing the application in atorvastatin pharmaceutical intermediate 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester
(1) recombination bacillus coli BL21 (the DE3)/pET28a-adh5 wet thallus containing recombinant expression obtained in embodiment 3 is as biological catalyst, with (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester for substrate, carry out bioconversion reaction and prepare 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester.
Catalyst system composition and catalytic condition as follows: add in 10mL potassium phosphate buffer (pH 7.0) 0.5g recombinate carbonyl reductase BgADH5 wet thallus (dry cell weight is 0.049g) and 0.5g Hexose phosphate dehydrogenase wet thallus (dry cell weight is 0.05g, prepared by embodiment 7 method), (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester 0.0458g (final concentration is 20mmol/L damping fluid), NADP
+2mmol/L damping fluid, glucose 50g/L damping fluid.30 DEG C, under rotating speed 150r/min condition, react 12h.Add equal-volume extraction into ethyl acetate twice after reaction terminates, merge organic layer and with anhydrous magnesium sulfate drying, filter, Rotary Evaporators removing ethyl acetate, enriched material moving phase is dissolved, liquid chromatographic detection productive rate and de.Under similarity condition, using the reaction solution of not mycetome as blank, replace above-mentioned recombination bacillus coli BL21 (DE3)/pET28a-adh5 wet thallus as negative control using e. coli bl21 (DE3)/pET28a wet thallus.The transformation efficiency of substrate is 87.4%, de 98%.As compared to the Saccharomyces cerevisiae wild-type ketoreductase YDL (reaction times 24h, transformation efficiency 85%) in patent US7879585B2, transformation efficiency improves.Blank and negative control all show substrate without effect.
(2) liquid phase detection method of (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester and 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester
Liquid chromatography instrument: Shimadzu LC-20AD system-SPD-20A UV-detector.
When detection transformation efficiency and ee, chromatographic column is Hypersil ODS2C18 (4.6mm × 250mm, 2.5 μm), moving phase: acetonitrile: water=1:3, flow velocity 1mL/min, determined wavelength 220nm.(R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, 6-cyano group-(3R, the retention time of 5R)-dihydroxyl hecanoic acid t-butyl ester and 6-cyano group-(3R, 5S)-dihydroxyl hecanoic acid t-butyl ester is respectively: 11.4,9.4 and 9.8min.
Embodiment 10: the application of the full cell of restructuring carbonyl reductase BgADH5 in preparation (S)-4,4,4-trifluoro 3-hydroxy ethyl butyrate
(1) recombination bacillus coli BL21 (the DE3)/pET28a-adh5 wet thallus containing recombinant expression obtained in embodiment 3 is as biological catalyst, with 4,4,4-trifluoroacetic ethyl acetoacetate is substrate, carry out bioconversion reaction preparation (S)-4,4,4-trifluoro 3-hydroxy ethyl butyrate.
Catalyst system composition and catalytic condition as follows: add in 10mL potassium phosphate buffer (pH 7.0) 0.5g recombinate carbonyl reductase BgADH5 wet thallus (dry cell weight is 0.049g) and 0.5g Hexose phosphate dehydrogenase wet thallus (dry cell weight is 0.05g, prepared by embodiment 7 method), 4,4,4-trifluoroacetic ethyl acetoacetate 0.037g (final concentration is 20mmol/L damping fluid), NADP
+2mmol/L damping fluid, glucose 50g/L damping fluid.30 DEG C, under rotating speed 150r/min condition, react 12h.Add equal-volume extraction into ethyl acetate twice after reaction terminates, merge organic layer and with anhydrous magnesium sulfate drying, filter, Rotary Evaporators removing ethyl acetate, enriched material moving phase is dissolved, gas chromatographic detection productive rate and ee.Under similarity condition, using the reaction solution of not mycetome as blank, replace above-mentioned recombination bacillus coli BL21 (DE3)/pET28a-adh5 wet thallus as negative control using e. coli bl21 (DE3)/pET28a wet thallus.The transformation efficiency of substrate is 88.4%, ee99%.Blank and negative control all show substrate without effect.Although having been reported of biological method of asymmetrically reducing synthesis (R)-4,4,4-trifluoro 3-hydroxy ethyl butyrate, (S)-4,4,4-the biological process asymmetric synthesis of trifluoro 3-hydroxy ethyl butyrate do not have report.
(2) 4,4,4-trifluoroacetic ethyl acetoacetates and (S)-4,4,4-vapor detection method of trifluoro 3-hydroxy ethyl butyrate
Gas-chromatography instrument: Shimadzu GC-14C system-fid detector.
When detection transformation efficiency and ee, chiral chromatographic column is BGB-174 (30m × 0.25mm × 0.25 μM), injector temperature: 220 DEG C, detector temperature: 220 DEG C, column temperature: 120 DEG C.Carrier is helium, flow velocity: 1.5mL/min.4,4,4-trifluoroacetic ethyl acetoacetate, (R)-4,4,4-trifluoro 3-hydroxy ethyl butyrate and (S)-4,4,4-the retention time of trifluoro 3-hydroxy ethyl butyrate be respectively: 2.6,6.8 and 7.1min.
From above experimental result, the recombination bacillus coli containing carbonyl reductase gene that the present invention obtains has the ability of stronger carbonyl reduction, can directly to contain the somatic cells of enzyme for carrying out biocatalysis or conversion reaction in enzyme source.Carbonyl reductase BgADH5 (SED ID NO.2) is as conversion enzyme, 2-benzoyl aminomethyl-3-ketone butyric ester, (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester and 4 can be utilized, 4,4-trifluoroacetic ethyl acetoacetate is substrate, carry out bioconversion reaction and prepare high optically pure chiral alcohol medicine chiral intermediate (2S, 3R)-2-benzoyl aminomethyl-3-hydroxybutyrate ester, 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester and (S)-4,4,4-trifluoro 3-hydroxy ethyl butyrate.
Claims (10)
1. derive from a carbonyl reductase gene of gladiolus bulkholderia cepasea (Burkholderia gladioli) ZJB-12126, it is characterized in that the nucleotides sequence of described gene is classified as shown in SEQ ID NO:1.
2. the restructuring carbonyl reductase by carbonyl reductase genes encoding described in claim 1.
3. to recombinate as claimed in claim 2 carbonyl reductase, it is characterized in that the aminoacid sequence of described enzyme is for shown in SEQ ID NO:2.
4. one kind by the gene constructed recombinant vectors of carbonyl reductase described in claim 1.
5. one kind is transformed the recombination engineering bacteria obtained by recombinant vectors described in claim 4.
6. an application of being recombinated in carbonyl reductase in preparation by carbonyl reductase gene according to claim 1, it is characterized in that described being applied as: build the recombinant vectors containing described carbonyl reductase gene, described recombinant vectors is converted in Host Strains, the recombination engineering bacteria obtained carries out inducing culture, and nutrient solution is separated the somatic cells obtained containing restructuring carbonyl reductase.
7. to recombinate described in a claim 2 application of carbonyl reductase in asymmetric reduction prochiral carbonyl compounds.
8. apply as claimed in claim 7, be applied as described in it is characterized in that: the wet thallus obtained through fermentation culture with the engineering bacteria containing restructuring carbonyl reductase gene is for catalyzer, be in the damping fluid of 6 ~ 10 in pH value, add substrate, cosubstrate and NAD (P)
+, 20 ~ 40 DEG C, react under 50 ~ 250rpm condition, after reacting completely, obtain the mixed solution containing chiral alcohol; Described substrate is 2-benzoyl aminomethyl-3-ketone butyric ester, (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester and 4,4, one in 4-trifluoroacetic ethyl acetoacetate, when described substrate is 2-benzoyl aminomethyl-3-ketone butyric ester, 2-benzoyl aminomethyl-3-ketone butyric ester adds with the form of 400mmol/L dimethyl sulphoxide solution; Described cosubstrate is glucose, ammonium formiate, Virahol or dehydrated alcohol, when described cosubstrate is glucose, add Hexose phosphate dehydrogenase and form cosubstrate system, when described cosubstrate is ammonium formiate, adds ammonium formiate desaturase and form cosubstrate system; The consumption of described catalyzer counts 20 ~ 200g/L damping fluid with the weight of wet thallus, the starting point concentration of described substrate is 10 ~ 100mmol/L damping fluid, the consumption of described cosubstrate is 10 ~ 200g/L damping fluid, described glucose takes off the consumption of desaturase or ammonium formiate desaturase with the wet thallus weighing scale obtained through fermentation culture containing the thalline of Hexose phosphate dehydrogenase or ammonium formiate desaturase, be 20 ~ 200g/L damping fluid, described NAD (P)
+consumption be 0.01 ~ 5mmol/L damping fluid.
9. apply as claimed in claim 8, it is characterized in that described wet thallus is prepared as follows: the engineering bacteria containing restructuring carbonyl reductase gene is seeded to the LB liquid nutrient medium containing final concentration 50 μ g/ml kalamycin resistance, 37 DEG C, 12h is cultivated under 200rpm, be seeded to fresh in the LB liquid nutrient medium of final concentration 50 μ g/ml kalamycin resistance again with volumetric concentration 1% inoculum size, in 37 DEG C, under 150rpm, be cultured to thalline OD
600reach 0.6 ~ 0.8, add the IPTG that final concentration is 0.1mM, at 28 DEG C after inducing culture 12h, 4 DEG C, the centrifugal 5min of 5000rpm, abandoning supernatant, collects wet thallus.
10. apply as claimed in claim 8, it is characterized in that wet thallus that described application obtains through fermentation culture with the engineering bacteria containing restructuring carbonyl reductase gene is for catalyzer, be in the damping fluid of 6 ~ 10 in pH value, add substrate, glucose, Hexose phosphate dehydrogenase and NAD (P)
+, 30 DEG C, react under 150rpm condition, after reacting completely, obtain the mixed solution of drug containing chiral intermediate; Described substrate is 2-benzoyl aminomethyl-3-ketone butyric ester, (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester and 4,4, one in 4-trifluoroacetic ethyl acetoacetate, when described substrate is 2-benzoyl aminomethyl-3-ketone butyric ester, 2-benzoyl aminomethyl-3-ketone butyric ester adds with the form of 400mmol/L dimethyl sulphoxide solution; The consumption of described catalyzer counts 50g/L damping fluid with the weight of wet thallus, the starting point concentration of described substrate is 20mmol/L damping fluid, the consumption of described glucose is 50g/L damping fluid, described glucose takes off the consumption of desaturase with the wet thallus weighing scale obtained through fermentation culture containing the thalline of Hexose phosphate dehydrogenase, for 50g/L damping fluid, described NAD (P)
+consumption be 2mmol/L damping fluid.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105602913A (en) * | 2016-03-09 | 2016-05-25 | 浙江工业大学 | Restructuring carbonyl reductase mutant ReCR-Mut, encoding gene, engineering bacteria and application |
| CN105755095A (en) * | 2016-02-04 | 2016-07-13 | 浙江工业大学 | Method for synthesizing (R)-2-hydroxy acid by biological enzyme method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105755095A (en) * | 2016-02-04 | 2016-07-13 | 浙江工业大学 | Method for synthesizing (R)-2-hydroxy acid by biological enzyme method |
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| CN105602913A (en) * | 2016-03-09 | 2016-05-25 | 浙江工业大学 | Restructuring carbonyl reductase mutant ReCR-Mut, encoding gene, engineering bacteria and application |
| CN105602913B (en) * | 2016-03-09 | 2019-02-01 | 浙江工业大学 | Recombinate carbonyl reduction enzyme mutant ReCR-Mut, encoding gene, engineering bacteria and application |
| CN110643556A (en) * | 2019-08-23 | 2020-01-03 | 浙江工业大学 | Recombinant genetic engineering bacterium for co-expressing enal reductase and glucose dehydrogenase and application thereof |
| CN114634957A (en) * | 2020-12-15 | 2022-06-17 | 苏州引航生物科技有限公司 | Method for synthesizing 4AA intermediate through biocatalysis |
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