CN105349503A - Carbonyl reductase AcCR and encoding gene and application thereof - Google Patents
Carbonyl reductase AcCR and encoding gene and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of gene engineering and particularly relates to carbonyl reductase AcCR and an encoding gene and application thereof. The amino acid sequence of the carbonyl reductase AcCR is shown as SEQ ID NO.1. The nucleotide sequence of the gene for encoding the carbonyl reductase AcCR is shown as SEQ ID NO.2. The carbonyl reductase AcCR comes from bacillus aceticus XZY003 and can catalyze 13 carbonyl compounds to generate corresponding chiral alcohol of single enantiomers. The carbonyl reductase AcCR and glucose dehydrogenase GDH are co-expressed in a prokaryotic expression system or a eukaryotic expression system, the biological reaction process of biological catalytic conversion is conducted through the carbonyl reductase, in-situ regeneration of coenzyme is achieved, production cost is greatly reduced, the optical purity of an obtained product is high, the reaction process is efficient, and conditions are moderate.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of carbonyl reductase AcCR and encoding gene thereof and application.
Background technology
Optically pure chiral alcohol and derivative thereof are the important building blocks of synthesis of chiral medicine, functional materials and Chiral pesticide.According to estimates, the tens billion of dollar of sales volume E Gaoda of global chiral alcohol building block.Due to these chiral alcohol building blocks chiral drug synthesis in importance and huge commercial value, the exploitation of its synthetic technology becomes the commanding elevation of global Ge great drugmaker development strategy already.At present, the synthesis of chiral alcohol building block mainly contains chemistry and biological two kinds of methods.There is severe reaction conditions (High Temperature High Pressure) in traditional chemical process, the problems such as the not high and environmental pollution of expensive catalyst (precious metal), optical purity of products, therefore, urgently research and develop relatively green approach to make up the deficiency of traditional chemical routes, biocatalysis is a kind of excellent selection.Biocatalysis has high stereoselectivity, gentle reaction conditions, advantages of environment protection because of it, the particularly develop rapidly of the technology of enzyme molecular modification in recent years (information biology, genetically engineered, protein engineering), has become in process of sustainable development the important method expanding traditional chemical synthesis.The method that biocatalysis obtains chiral alcohol is divided into again the fractionation of chiral alcohol racemic modification and direct-reduction prochiral ketone to generate the chiral alcohol of enantiomer-pure.Biocatalysis generates catalyzer mainly oxydo-reductase and the various biomass cells of chiral alcohol.The microorganism cells of energy catalysis resolution of racemates alcohol and asymmetric reduction prochiral ketone is of a great variety, comprises bacterium, actinomycetes, mould, yeast etc.But most of microbe cell catalysis prochiral ketone asymmetric reduction process follows Prelog rule, and the configuration of its product can be predicted by Prelog rule.Therefore, the enzyme or the microorganism cells that need to follow trans-Prelog rule especially carry out the chiral alcohol that catalytic asymmetric reduction prochiral ketone generates corresponding single configuration.
In the research in earlier stage of this seminar, from " Chinese Kefir grains " bacterium grain, successful separation and purification obtains a strain Novel acetate bacillus Acetobactersp.CCTCCM209061, and this bacterial strain can follow a series of carbonyl compound asymmetric reduction of trans-Prelog rule catalysis.Obviously, this new strains has obvious advantage following in the asymmetric synthesis of trans-Prelog rule catalysis chiral alcohol, shows huge application potential.But this bacterial strain still exists some problems, e.g., enzyme is lived not high, and enzyme system is complicated, and there is side reaction, culture medium cost is higher etc.In addition, the yield of enzyme of wild type strain is often lower, thus limits the concentration of substrate of its catalyzed reaction, the raising of initial velocity of reaction and productive rate etc.The microorganism of following the reduction of trans-Prelog rule catalysis prochiral ketone is little already, and in these microorganisms, have quite a few microorganism not high for the reducing activity of prochiral ketone, and substrate spectrum is narrow, productive rate is low or selectivity is not high.The microorganism strains with high reactivity and good selectivity is rare especially.Therefore, the carbonyl reductase (alcoholdehydrogenase) played a crucial role in this bacterial strain then has important Study and appliance and is worth.
Microorganism cells catalysis prochiral ketone is reduced into the oxydo-reductase in the microorganism cells played a major role in single enantiomer alcohol process, carbonyl reductase (alcoholdehydrogenase) is wherein one of important stereoselectivity catalyzer, the substrate conversion of latent chirality can be become have the important medicine intermediate of chirality, in the reaction of asymmetric synthesis, therefore play a part to lift lumping weight light.For active whole-cell catalytic reaction, because there is many emulative oxydo-reductase in active cells, substrate conversion is likely become unwanted product by these enzymes, reduces the selectivity of substrate.Make its current space-time yield that there is reaction low, the problem that side reaction stereoselectivity that is more and that react in some cases is not high.
Summary of the invention
In order to overcome the deficiencies in the prior art and shortcoming, primary and foremost purpose of the present invention is to provide a kind of carbonyl reductase AcCR.
Another object of the present invention is to provide the gene of above-mentioned carbonyl reductase AcCR of encoding.
Another object of the present invention is the application providing above-mentioned carbonyl reductase AcCR.
4th object of the present invention is the recombinant bacterium providing a kind of above-mentioned carbonyl reductase AcCR and Hexose phosphate dehydrogenase GDH coexpression, and this bacterial strain can realize the solubility expression of carbonyl reductase AcCR.
Object of the present invention is achieved through the following technical solutions:
A kind of carbonyl reductase AcCR, its aminoacid sequence is as follows:
MARVAGKVAIVSGAANGIGKAAAQLLAKEGAKVVIGDLKEEDGQKAVAEIKAAGGEAAFVKLNVTDEAAWKAAIEQTLKLYGRLDIAVNNAGIAYSGSVESTSLEDWRRVQSINLDGVFLGTQVAIEAMKKSGGGSIVNLSSIEGLIGDPMLAAYNASKGGVRLFTKSAALHCAKSGYKIRVNSVHPGYIWTPMVAGLTKEDAAARQKLVDLHPIGHLGEPNDIAYGILYLASDESKFVTGIELVMDGRSTAQ；
Encode the gene of above-mentioned carbonyl reductase AcCR, its nucleotide sequence is as follows:
ATGGCACGTGTAGCAGGCAAGGTTGCCATTGTTTCTGGGGCCGCTAATGGCATTGGCAAGGCAGCCGCACAGCTTTTGGCCAAGGAAGGCGCAAAAGTTGTTATTGGTGATTTAAAAGAAGAAGATGGGCAGAAAGCTGTTGCAGAAATTAAGGCAGCAGGTGGTGAAGCCGCATTTGTCAAACTGAATGTAACAGATGAGGCTGCATGGAAAGCCGCTATTGAGCAAACGCTTAAGCTTTATGGGCGGCTGGATATTGCAGTGAACAATGCAGGCATTGCGTATTCTGGCAGTGTAGAAAGCACATCTCTGGAAGATTGGCGGCGCGTTCAGTCTATCAATCTGGATGGCGTGTTTTTGGGCACACAGGTGGCTATTGAGGCCATGAAGAAGTCGGGCGGTGGATCCATTGTCAATCTGTCTTCCATTGAAGGACTGATTGGGGACCCAATGTTGGCCGCCTATAACGCCAGTAAAGGTGGGGTAAGGCTGTTTACAAAATCTGCGGCCCTACATTGCGCCAAATCTGGATACAAAATTCGGGTAAACTCAGTGCATCCCGGCTATATCTGGACACCTATGGTGGCCGGTTTAACAAAGGAAGATGCTGCTGCACGCCAAAAGCTGGTGGATCTGCACCCCATTGGCCACTTGGGTGAGCCCAACGATATTGCTTACGGTATTTTGTATCTTGCCTCTGATGAATCCAAGTTTGTTACAGGGATCGAACTGGTCATGGATGGGAGGTCAACAGCACAATGA；
Described carbonyl reductase AcCR is preparing the application in chiral alcohol and derivative thereof;
Containing the recombinant vectors of carbonyl reductase AcCR gene, be to be connected with carrier by the nucleotide sequence of the gene by GST label, the above-mentioned carbonyl reductase AcCR that encodes to obtain;
Expressing a recombination engineering bacteria of carbonyl reductase AcCR, is be transformed in E. coli expression strains by the above-mentioned recombinant vectors containing carbonyl reductase AcCR gene to obtain;
Described E. coli expression strains is preferably BL21 (DE3) pLysS;
A recombinant vectors containing carbonyl reductase AcCR gene and Hexose phosphate dehydrogenase GDH gene to be connected with carrier by the nucleotide sequence of the gene by GST label, the above-mentioned carbonyl reductase AcCR that encodes and the nucleotide sequence of the gene of encodes glucose desaturase GDH to obtain;
Described carrier is preferably pETDuet-1;
The described preparation method containing the recombinant vectors of carbonyl reductase AcCR gene and Hexose phosphate dehydrogenase GDH gene, comprises following steps:
(1) with bacillus aceticus strain X ZY003 (Acetobactersp.XZY003, preserving number: CCTCCM209061) genomic dna be template, according to the nucleotide sequence design primer of the gene of the above-mentioned carbonyl reductase AcCR of coding, pcr amplification obtains carbonyl reductase AcCR gene (accr); Amplified production is connected with vector pGEX-2T, obtains recombinant vectors pGEX-accr; GST label in order to obtain contributing to increasing solubility expression of protein by carbonyl reductase AcCR gene clone to the object on pGEX-2T carrier;
(2) with recombinant vectors pGEX-accr for template, design primer, pcr amplification obtains the carbonyl reductase AcCR gene (gaccr) with GST label, and is connected on co-expression carrier pETDuet-1, obtains recombinant expression vector pETDuet-gaccr;
(3) with the genomic dna of Bacillus subtilis 168 for template, design primer, pcr amplification obtains Hexose phosphate dehydrogenase GDH gene (gdh), and be connected on the obtained recombinant plasmid vector pETDuet-gaccr of step (2), obtain the recombinant vectors (pETDUet-gaccr-gdh) containing carbonyl reductase AcCR gene and Hexose phosphate dehydrogenase GDH gene;
Primer described in step (1) is as follows, and wherein restriction enzyme site is respectively AvaI and EcoRI (underscore):
Primer 1:5'-TCC
cCCGGGaATGGCACGTGTAGCAGGCAAGGTT-3';
Primer 2: 5'-CCG
gAATTCcTCATTGTGCTGTTGACCTCCCATCCAT-3';
Primer described in step (2) is as follows, and wherein restriction enzyme site is respectively PstI and SalI (underscore):
Primer 3:5'-TGCA
cTGCAGaTGTCCCCTATACTAGGTTATTGGG-3';
Primer 4:5'-GC
gTCGACtCATTGTGCTGTTGACCTCCCATCCAT-3';
Primer described in step (3) is as follows, and wherein restriction enzyme site is respectively BglII and XhoI (underscore):
Primer 5:5'-GA
aGATCTcATGTATCCGGATTTAAAAGGAAAAGTCGTCG-3';
Primer 6:5'-CCG
cTCGAGtTAACCGCGGCCTGCCTGGAAT-3';
A recombination engineering bacteria for carbonyl reductase AcCR and Hexose phosphate dehydrogenase GDH coexpression is transformed in prokaryotic expression system or eukaryotic expression system by the above-mentioned recombinant vectors containing carbonyl reductase AcCR gene and Hexose phosphate dehydrogenase GDH gene to obtain;
Described prokaryotic expression system is preferably E. coli expression strains BL21 (DE3) pLysS;
The recombination engineering bacteria of above-mentioned carbonyl reductase AcCR and Hexose phosphate dehydrogenase GDH coexpression prepares application in chiral alcohol and derivative thereof in catalytic asymmetric reduction chiral carbonyl compounds of diving;
A kind of microbial cell catalyst, comprises the recombination engineering bacteria of above-mentioned carbonyl reductase AcCR and Hexose phosphate dehydrogenase GDH coexpression;
The preparation method of described microbial cell catalyst, comprises following steps:
By the recombination recombinant of above-mentioned carbonyl reductase AcCR and Hexose phosphate dehydrogenase GDH coexpression to OD
600=1.0 ~ 1.2, then under the condition of 20 ~ 22 DEG C, adding final concentration is that after the IPTG of 0.4 ~ 0.5mmol/L carries out induction 15 ~ 18h, collected by centrifugation thalline, obtains microbial cell catalyst;
Described microbial cell catalyst may be used for the chiral alcohol that catalysis of carbonyl compound generates corresponding single enantiomer;
The present invention has following advantage and effect relative to prior art:
(1) the invention provides a kind of carbonyl reductase AcCR deriving from bacillus aceticus XZY003, this carbonyl reductase is membranin, can generate the chiral alcohol of corresponding single enantiomer by catalysis 13 kinds of carbonyl compound.
(2) present invention is directed at the chiral alcohol Problems existing that current microorganism cells catalysis of carbonyl compound generates corresponding single enantiomer, adopt and derive from the carbonyl reductase AcCR of bacillus aceticus XZY003 and the mode of Hexose phosphate dehydrogenase GDH coexpression, prepare microbial cell catalyst, both the bioprocesses utilizing carbonyl reductase to carry out Biocatalytic Conversion had been achieved, achieve again the in-situ regeneration of coenzyme, the method adopts whole-cell catalytic, there is the efficient regenerating coenzyme recycle system, thus improve the catalytic efficiency of the genetic engineering bacterium of restructuring, do not need to add expensive coenzyme, significantly reduce production cost, and the optical purity obtaining product is high, reaction process is efficient, and mild condition.
Accompanying drawing explanation
Fig. 1 is the building process figure of recombinant co-expression carrier pETDuet-gaccr-gdh.
Fig. 2 is the procedure chart of recombination bacillus coli BL21 (DE3) pLysS (pETDuet-gaccr-gdh) catalysis of carbonyl compound asymmetric reduction reaction.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Bacillus aceticus strain X ZY003 (Acetobactersp.XZY003, preserving number: CCTCCM209061) is at Chinese patent " bacillus aceticus and apply the method that it produces enantiomer-pure (R)-Organosilicon Alcohol in Organic " (application number: open 200910146261.3);
The substratum cultivating this bacterial strain is glucose 8.3g/L, fructose 2.5g/L, soy peptone 83.9g/L, initial pH5.7.Specifically see reference document (Optimizationofcultureconditionstoproducehighyieldsofacti veAcetobacterspCCTCCM209061cellsforanti-Prelogreductiono fprochiralketones)
The acquisition of embodiment 1 carbonyl reductase AcCR gene accr
Bacillus aceticus XZY003 is 30 DEG C in temperature, and rotating speed is that under the condition of 100rpm, 24h is cultivated in concussion; Then centrifugal (4 DEG C of thalline, 9000rpm) 5min, collect thalline, with the brine 3 times that mass percent is 0.85%, to remove residual substratum, then the genomic dna that test kit extracts bacillus aceticus XZY003 is extracted, the method that concrete grammar provides see test kit by the bacterial genomes of Shanghai Jierui Biology Engineering Co., Ltd.With the genomic dna of the bacillus aceticus XZY003 extracted for template, design primer 1 (5'-TCC
cCCGGGand primer 2 (5'-CCG AATGGCACGTGTAGCAGGCAAGGTT-3')
gAATTCcTCATTGTGCTGTTGACCTCCCATCCAT-3') be amplification upstream and downstream primer, wherein restriction enzyme site is respectively AvaI and EcoRI (underscore); Pcr amplification obtains object fragment carbonyl reductase AcCR gene accr.Wherein, the enzyme reagent kit that pcr amplification is used is the KOD enzyme that Japan is spun; PCR reaction system (25 μ L) is: KODFXbuffer12.5 μ L; 2mMdNTP5 μ L; Primer 10.75 μ L; Primer 2 0.75 μ L; Template DNA 1 μ L; ddH
2o5 μ L; PCR reaction conditions is: 94 DEG C of 2min; 98 DEG C of 15s, 60 DEG C of 30s, 68 DEG C of 40s, 30 circulations; 68 DEG C of 7min; Pcr amplification product is carried out reclaim (adopting the glue of Shanghai Jierui Biology Engineering Co., Ltd to reclaim test kit) and sequencing analysis, obtaining a segment length is that the sequence of 762bp is shown in SEQIDNO.2, for carbonyl reductase AcCR gene accr, this sequence encoding 253 amino acid, are shown in SEQIDNO.1.
The acquisition of embodiment 2 glucose dehydrogenase gene gdh
The gene source of Hexose phosphate dehydrogenase is in Bacillus subtilis 168 (purchased from Guangdong Culture Collection), the method that culture medium prescription and culture condition provide by DSMZ, the extraction of Bacillus subtilis genes group DNA is extracted test kit by the bacterial genomes of Shanghai Jierui Biology Engineering Co., Ltd and is extracted.With the genomic dna of Bacillus subtilis 168 for template, design primer 5 (5'-GA
aGATCTand primer 6 (5'-CCG CATGTATCCGGATTTAAAAGGAAAAGTCGTCG-3')
cTCGAGtTAACCGCGGCCTGCCTGGAAT-3') be respectively upstream and downstream primer, wherein, restriction enzyme site is respectively BglII, XhoI (underscore); Pcr amplification obtains object fragment Hexose phosphate dehydrogenase GDH gene gdh.PCR reaction system (25 μ L) is: KODFXbuffer12.5 μ L; 2mMdNTP5 μ L; Primer 50.75 μ L; Primer 60.75 μ L; Template DNA 1 μ L; ddH
2o5 μ L; PCR reaction conditions is: 94 DEG C of 2min; 98 DEG C of 15s, 60 DEG C of 30s, 68 DEG C of 40s, 30 circulations; 68 DEG C of 7min; Carried out by the amplified production of PCR reclaiming (adopting the glue of Shanghai Jierui Biology Engineering Co., Ltd to reclaim test kit), carrying out sequencing analysis, obtain the sequence that a segment length is 786bp, is Hexose phosphate dehydrogenase GDH gene gdh.
The structure of embodiment 3 recombinant vectors pGEX-accr
(1) carbonyl reductase AcCR gene accr fragment (recovery product) obtained in embodiment 1 and plasmid pGEX-2T (purchased from Guangzhou Qi Yun Bioisystech Co., Ltd) is carried out double digestion respectively; Wherein, gene fragment enzyme is cut system system (30 μ L) and is: ddH
2o15 μ L; Buffer3 μ L; Gene fragment 10 μ L; The each 1 μ L of AvaI+EcoRI; Plasmid pGEX-2T enzyme cuts system (20 μ L): ddH
2o6 ~ 14 μ L; Buffer2 μ L; Carrier 2 ~ 10 μ L; The each 1 μ L of AvaI+EcoRI; Enzyme tangent condition is: 37 DEG C of enzymes cut 30min;
(2) product after being cut by step (1) enzyme reclaims respectively, and recovery method and step reclaim test kit see the PCR primer of Shanghai Jierui Biology Engineering Co., Ltd.Connected by recovery product, linked system (20 μ L) is: carrier 5 μ L; Gene fragment 10 μ L; 10 × T4buffer2 μ L; T4DNAligase1 μ L; ddH
2o2 μ L; Condition of contact is: 22 DEG C connect 30min;
(3) connection product conversion bacillus coli DH 5 alpha step (2) obtained, step of converting is as follows: 10 μ L are connected product and mixes with 100 μ L bacillus coli DH 5 alpha competent cells, ice bath 30min, 42 DEG C of heat shock 90s, ice bath 2min, add 890 μ LLB substratum, 37 DEG C, 200rpm, after 1h cultivated by shaking table, get 100 μ L nutrient solutions, coating ammonia benzyl resistant panel (containing 100 μ g/mL sodium ampicillins), 37 DEG C, incubated overnight, picking positive transformant carries out order-checking detection validation, preserve positive transformant, then extracting plasmid, obtain recombinant vectors pGEX-accr.
(4) recombinant vectors step (3) obtained transforms in expressive host BL21 (DE3) pLysS (purchased from Guangzhou Qi Yun Bioisystech Co., Ltd), obtain bacterial strain BL21 (DE3) pLysS (pGEX-accr), then this bacterial strain of liquid culture OD
600=1.2, under the condition of 20 DEG C, adding final concentration is that the IPTG of 0.4mmol/L carries out induction 15 ~ 18h, then thalline is collected, carry out SDS-PAGE and determine restructuring AcCR successful expression, measure enzyme and live as 304.9U/g-dw, obtain the carbonyl reductase AcCR of solubility expression.
The structure of embodiment 4 recombination bacillus coli BL21 (DE3) pLysS (pETDuet-gaccr-gdh)
With the recombinant plasmid pGEX-accr obtained in embodiment 3 for template, design primer 3 (5'-TGCA
cTGCAGand primer 4 (5'-GC ATGTCCCCTATACTAGGTTATTGGG-3')
gTCGACtCATTGTGCTGTTGACCTCCCATCCAT-3'), carry out pcr amplification, obtain the carbonyl reductase AcCR gene gaccr with GST label gene, wherein, PCR reaction system and condition are see embodiment 1, above-mentioned pcr amplification product and co-expression plasmid PETDuet-1 (purchased from Shanghai JaRa Bioisystech Co., Ltd) are carried out double digestion respectively, enzyme used be Fermentas cut enzyme FastDigest soon
fastDigest
enzyme used cuts system and condition see embodiment 3, reclaimed by digestion products, reclaim product and connect, linked system and condition are see embodiment 3, product conversion bacillus coli DH 5 alpha will be connected, step of converting is as follows: 10 μ L are connected product and mixes with 100 μ L bacillus coli DH 5 alpha competent cells, ice bath 30min, 42 DEG C of heat shock 90s, ice bath 2min, add 890 μ LLB substratum, 37 DEG C, 200rpm, after 1h cultivated by shaking table, get 100 μ L nutrient solutions, coating ammonia benzyl resistant panel (containing 100 μ g/mL sodium ampicillins), 37 DEG C, incubated overnight, picking positive transformant carries out sequence verification, preserve positive transformant, then extracting plasmid, obtain recombinant expression vector pETDuet-gaccr, in order to building co-expression carrier pETDuet-gaccr-gdh (Fig. 1),
The Hexose phosphate dehydrogenase GDH gene gdh fragment with BglII, XhoI restriction enzyme site embodiment 2 obtained and recombinant expression vector pETDuet-gaccr carry out double digestion respectively, restriction endonuclease used be Fermentas cut enzyme FastDigest soon
fastDigest
enzyme used cuts system and condition see embodiment 3; Reclaimed by digestion products, reclaim product and connect, linked system and condition are see embodiment 3; To connect product conversion bacillus coli DH 5 alpha, step of converting is described above; Extract the recombinant vectors pETDuet-gaccr-gdh that order-checking is correct, transformed in expressive host BL21 (DE3) pLysS (purchased from Guangzhou Qi Yun Bioisystech Co., Ltd), the bacterial strain obtained is recombination bacillus coli BL21 (DE3) pLysS (pETDuet-gaccr-gdh) that will build.
Embodiment 5 recombination bacillus coli BL21 (DE3) pLysS (pETDuet-gaccr-gdh) prepares application in optical activity alcohol in asymmetric reduction chiral carbonyl compounds of diving
(1) recombinant strains BL21 (DE3) pLysS (pETDuet-gaccr-gdh) embodiment 4 obtained, at 37 DEG C, is cultured to bacterial concentration OD under the condition of 200rpm
600=1.2, be then cooled to 20 DEG C, adding final concentration is after the IPTG of 0.4mmol/L carries out induction 15 ~ 18h, 4 DEG C, and the centrifugal 5min of 9000rpm collects thalline, obtains microbial cell catalyst (wet thallus);
(2) in the tool plug triangular flask of 10mL, 4mL citrate buffer (100mM is added respectively, pH5.5), be 50mM glucose and final concentration be 15mg/mL microbial cell catalyst containing final concentration in damping fluid, be placed in gas bath constant temperature oscillator (30 DEG C) and hatch 10min, add the carbonyl compound be dissolved in DMSO that final concentration is 5mM, open reaction, timing sampling, bottom product and product is extracted by 100 μ L (2 × 50 μ L) ethyl acetate (containing internal standard substance 5mM n-dodecane), with vortex mixed instrument oscillation extraction 5min, after centrifugal (12000rpm) 5min, get supernatant to detect for gas-chromatography (GC).The catalytic effect that recombinant bacterium BL21 (DE3) pLysS (pETDuet-gaccr-gdh) is applied to the latent chiral carbonyl compounds of asymmetry catalysis conversion sees appendix table 1 and Fig. 2.
The catalytic effect of table 1 recombination bacillus coli BL21 (DE3) pLysS (pETDuet-gaccr-gdh)
Comparative example
(1) with bacillus aceticus XZY003 genome for template, design primer 7 (5'-CATG
cCATGGaTATGGCACGTGTAGCAGGCAAGGTT-3') and
Primer 8:5'-CCG
cTCGAGtCATTGTGCTGTTGACCTCCCATCCAT-3', wherein restriction enzyme site is respectively NcoI and XhoI (underscore); Pcr amplification obtains object fragment carbonyl reductase AcCR gene accr; Pcr amplification product is reclaimed, and carries out double digestion (NcoI and XhoI) respectively with carrier pET22b+, enzyme cut after product reclaim and connect; Concrete grammar is see embodiment 1;
(2) with bacillus aceticus XZY003 genome for template, design primer 9 (5'-CATG
cCATGGand primer 10 (5'-CCG CGATGGCACGTGTAGCAGGCAAGGTT-3')
gAATTCgGTCATTGTGCTGTTGACCTCCCATCCAT-3'); Wherein restriction enzyme site is respectively NcoI and EcoRI; Pcr amplification product is reclaimed, and carries out double digestion (NcoI and EcoRI) respectively with carrier pET30b+, enzyme cut after product reclaim and connect; Concrete grammar is see embodiment 1;
(3) with bacillus aceticus XZY003 genome for template, design primer 11 (5'-TGCA
cTGCAGand primer 4 (5'-GC ATGGCACGTGTAGCAGGCAAGGTT-3')
gTCGACtCATTGTGCTGTTGACCTCCCATCCAT-3'), wherein restriction enzyme site is respectively PstI and SalI; Pcr amplification product is reclaimed, and carries out double digestion (PstI and SalI) respectively with carrier pETDuet-1, enzyme cut after product reclaim and connect; Concrete grammar is see embodiment 1;
(4) by step (1), and (3) connection product of obtaining transformation of E. coli DH5 α respectively (2), step of converting is as follows: 10 μ L are connected product and mixes with 100 μ L bacillus coli DH 5 alpha competent cells, ice bath 30min, 42 DEG C of heat shock 90s, ice bath 2min, add 890 μ LLB substratum, 37 DEG C, 200rpm, after 1h cultivated by shaking table, get 100 μ L nutrient solutions, resistant panel (containing 50 μ g/mL kantlex) received by coating ammonia benzyl resistant panel (containing 100 μ g/mL sodium ampicillins) or card, 37 DEG C, incubated overnight, picking positive transformant carries out order-checking detection validation, preserve positive transformant, then extracting plasmid, obtain recombinant vectors pET22b+-accr, pET30b+-accr and pETDuet-accr.
(5) recombinant vectors step (4) obtained transforms in expressive host BL21 (DE3) pLysS (purchased from Guangzhou Qi Yun Bioisystech Co., Ltd), obtains bacterial strain BL21 (DE3) pLysS (pET22b+-accrr), BL21 (DE3) pLysS (pET30b+-accr) and BL21 (DE3) pLysS (pETDuet-accr).Then inducing culture is carried out with reference to embodiment 3 pairs of above-mentioned bacterial strains, collect thalline and use the resuspended thalline of damping fluid, carry out SDS-PAE, find that AcCR all has expression in above three kinds of recombinant bacteriums, but all do not detect that enzyme is lived, thalline ultrasonication in ice bath is clarified to solution, in collection, cleer and peaceful precipitation carries out SDS-PAGE respectively, find that the AcCR expressed is all in precipitation, the AcCR that above-mentioned three kinds of bacterial strain inducings are expressed does not have activated inclusion body, does not all realize the solubility expression of AcCR.
These results suggest that, the carbonyl reductase AcCR deriving from bacillus aceticus XZY003 provided by the invention belongs to a kind of membranin and is difficult to realize solubility expression, and additional GST label, has achieved solubility expression.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. a carbonyl reductase AcCR, is characterized in that: its aminoacid sequence is as shown in SEQIDNO.1.
2. the gene of coding carbonyl reductase AcCR according to claim 1, is characterized in that: its nucleotide sequence is as shown in SEQIDNO.2.
3. carbonyl reductase AcCR according to claim 1 is preparing the application in chiral alcohol and derivative thereof.
4. the recombinant vectors containing carbonyl reductase AcCR gene, is characterized in that: be obtain by being connected with carrier by the nucleotide sequence described in GST label, claim 2.
5. the recombinant vectors containing carbonyl reductase AcCR gene and Hexose phosphate dehydrogenase GDH gene, is characterized in that: be obtain by being connected with carrier by the nucleotide sequence of the nucleotide sequence described in GST label, claim 2 and the gene of encodes glucose desaturase GDH.
6. the recombinant vectors containing carbonyl reductase AcCR gene and Hexose phosphate dehydrogenase GDH gene according to claim 5, is characterized in that:
Described carrier is pETDuet-1.
7. the preparation method containing the recombinant vectors of carbonyl reductase AcCR gene and Hexose phosphate dehydrogenase GDH gene described in claim 5 or 6, comprises following steps:
(1) with the genomic dna of bacillus aceticus strain X ZY003 for template, nucleotide sequence according to claim 2 design primer, pcr amplification obtains carbonyl reductase AcCR gene; Amplified production is connected with vector pGEX-2T, obtains recombinant vectors pGEX-accr;
(2) with recombinant vectors pGEX-accr for template, design primer, pcr amplification obtains the carbonyl reductase AcCR gene with GST label, and is connected on co-expression carrier pETDuet-1, obtains recombinant expression vector pETDuet-gaccr;
(3) with the genomic dna of Bacillus subtilis 168 for template, design primer, pcr amplification obtains Hexose phosphate dehydrogenase GDH gene, and be connected on the obtained recombinant plasmid vector pETDuet-gaccr of step (2), obtain the recombinant vectors containing carbonyl reductase AcCR gene and Hexose phosphate dehydrogenase GDH gene.
8. a recombination engineering bacteria for carbonyl reductase AcCR and Hexose phosphate dehydrogenase GDH coexpression, is characterized in that: be transformed in prokaryotic expression system or eukaryotic expression system by the recombinant vectors containing carbonyl reductase AcCR gene and Hexose phosphate dehydrogenase GDH gene described in claim 5 or 6 to obtain.
9. the recombination engineering bacteria of carbonyl reductase AcCR according to claim 7 and Hexose phosphate dehydrogenase GDH coexpression, is characterized in that:
Described prokaryotic expression system is E. coli expression strains BL21 (DE3) pLysS.
10. the recombination engineering bacteria of the carbonyl reductase AcCR described in claim 8 or 9 and Hexose phosphate dehydrogenase GDH coexpression prepares application in chiral alcohol and derivative thereof in catalytic asymmetric reduction chiral carbonyl compounds of diving.
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