CN100354422C - Ester hydrolase and its gene and recombinant enzyme - Google Patents
Ester hydrolase and its gene and recombinant enzyme Download PDFInfo
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- CN100354422C CN100354422C CNB2005100231721A CN200510023172A CN100354422C CN 100354422 C CN100354422 C CN 100354422C CN B2005100231721 A CNB2005100231721 A CN B2005100231721A CN 200510023172 A CN200510023172 A CN 200510023172A CN 100354422 C CN100354422 C CN 100354422C
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
The present invention discloses a gene PPEST from pseudomonas putida and for coding ester hydrolase, which provides a gene containing a PPEST sequence, a corresponding amino acid sequence, an expression vector pPPEST-PET containing PPEST, a gene engineering strain for expressing the ester hydrolase, namely E. coli cell BL21(DE3)/pPPEST-PET containing the vector, and a method for recombining the ester hydrolase. The recombinant ester hydrolase of the present invention can be used for the synthesis of a chiral compound (S type) with stereoselective catalysis.
Description
Technical field
The invention belongs to bioengineering field, relate to a kind of ester hydrolase and gene thereof and recombinase, comprise this ester hydrolase expression carrier and prepare the method and the engineering strain thereof of its recombinase, and the application of this ester hydrolase in stereoselectivity catalysis chipal compounds is synthetic.
Background technology
Ester hydrolase includes lipase and esterase, and it is a class has important use in chirality is a synthetic biological catalyst.These enzymes can be discerned very wide substrate, and biology at present＞40% can not finished by ester hydrolase catalysis the thing building-up reactions, and these reactions have the regioselectivity and the solid of mild condition, reaction and select characteristics such as height.Be widely used at aspects such as enzyme kinetics resolution of racemic ester, amine and conversion prochirality alcohol.In addition, they also can be used in selective esterification, transesterificationization and the polyreaction.
Owing to, utilize biological process or enzyme process synthesizing chiral compound to obtain people's attention and industrial applications day by day to chiral drug and intermediate growth of requirement.The key issue of biological process synthesizing chiral compound is to seek the biological catalyst with height stereoselectivity catalysis.Because ester hydrolase has purposes widely, screens the research focus that new ester hydrolase is just becoming enzyme engineering.Though it is very wide that ester water enzyme distributes at microbial world, their stereoselectivity catalytic capability is different.In same organism, often there is multiple ester hydrolase, because the stereoselectivity property of there are differences between them, tend to reduce optical purity (the Geun-Joong Kim of product when utilizing microorganism cells or their thick enzyme preparation to carry out the enzyme process synthesizing chiral compound, Journal of Molecular Catalysis B:Enzymatic 17,2002:29-38).Address this problem the generation (PerBerglund that can reduce side reaction by the control measures of engineering, Biomolecular Engineering, 18,2001:13-22), also can obtain single zymin and carry out catalyzed reaction by separation and purification, but these processes are often complicated and high cost, are difficult for aborning being used.If by engineered means, directly clone and express the gene of needed enzyme, just can easily reach the above object.
Summary of the invention
The inventor has been and has solved above-mentioned problem, by screening a large amount of microorganisms, finds that pseudomonasputida can produce a kind of highly stereoselective ester hydrolase that has, and can be used for the synthetic of catalysis chipal compounds; For the output that improves this ester hydrolase with eliminate that isozyme is to the negative impact of enzymic catalytic reaction in the wild type strain, the present invention has cloned this gene and this gene has been expressed in intestinal bacteria.Therefore, the technical problem to be solved in the present invention, being provides a kind of ester hydrolase and gene and recombinase, comprise this ester hydrolase expression carrier and prepare the method and the engineering strain thereof of its recombinase, and the application of this ester hydrolase in stereoselectivity catalysis chipal compounds is synthetic.
Ester hydrolase gene of the present invention can derive from pseudomonasputida (Pseudomonas putida), as P.putida ATCC 17522, and P.putida NCIB 11772 bacterial strains such as grade.The technical solution used in the present invention is by making up the DNA library of pseudomonasputida, screening obtains pseudomonasputida ester hydrolase gene PPEST from the library, measured its nucleotide sequence, shown in SEQ ID No.1 in the sequence table, wherein, its encoding sequence (CDS) stops from the 5th base to the of DNA 1150, and GTG is the transcription initiation codon, and TGA is the Transcription Termination password; And obtain amino acid sequence corresponding, shown in SEQ ID No.2 in the sequence table.Certainly, as is known to the person skilled in the art, ester hydrolase gene of the present invention can also be proteinic other nucleotide sequence that coding is made up of the aminoacid sequence shown in the SEQ ID No.2 in the sequence table; And ester hydrolase of the present invention is only for being the protein with composition of the aminoacid sequence shown in the SEQ IDNo.2 in the sequence table, can also be through replacement, disappearance or the interpolation of one or several amino-acid residue and to have same enzyme active by sequence 2 deutero-protein with the aminoacid sequence in the sequence 2, such as adding one or several amino acid, merge, do not influence the situations such as difference on the modified forms of sequence as amino acid with vector encoded at C-terminal and/or N-terminal.
Another object of the present invention provides the expression vector that comprises above-mentioned ester hydrolase gene nucleotide series.It is with ordinary method the nucleotide sequence of ester hydrolase gene of the present invention to be connected in to make up on the various carriers to form, and this carrier can be commercially available plasmid, clay, phage or virus vector etc.
Preferably will be connected with cloning vector pUC18-T, form cloning vector pPPEST-T with the PPEST gene product that pcr amplification arrives.PPPEST-T and expression vector pET22b (+) use digestion with restriction enzyme respectively, form the complementary sticky end, connect through the T4 dna ligase, form PPEST expression carrier (plasmid) pPPEST-PET.
A further object of the present invention provides a kind of engineering strain, it is that the expression vector that will comprise ester hydrolase gene nucleotide series of the present invention is transformed into the engineering strain that obtains among the competence intestinal bacteria E.coli BL21 (DE3), as plasmid pPPEST-PET being transformed intestinal bacteria E.coli BL21 (the DE3)/pPPEST-PET that wherein obtains containing pPPEST-PET.
Another purpose of the present invention provides a kind of method for preparing the ester hydrolase of recombinating, and it comprises with the invention described above expression vector transformed host cell, cultivates transformant, obtains the ester hydrolase of reorganization.
Wherein, this host cell can be bacterium or insect etc.Preferably intestinal bacteria, obtaining corresponding transformant is above-mentioned engineering strain: intestinal bacteria E.coli BL21 (DE3)/pPPEST-PET.
This bacterial strain can be induced at the IPTG of routine down (at OD
600=0.5-0.6 adds during the left and right sides, to its concentration be 0.1-1mmol/L all can) efficiently express ester hydrolase albumen, be reorganization ester hydrolase of the present invention, be used to prepare optically pure chipal compounds (S type).
Description of drawings
Fig. 1 is the pcr amplification electrophoretogram of PPEST of the present invention, and wherein, 1 is the pcr amplification product of PPEST; 2 is DNA Marker (100bp DNA ladder, New England Biolab company); 3 is DNA Marker (λ-HindIII digest, Takala company).
Fig. 2 analyzes collection of illustrative plates for HindIII and the NdeI double digestion of plasmid pPPEST-T of the present invention, wherein,
1 is DNA Marker (λ-HindIII digest, Takala company); 2 is pPPEST-T/HindIII+IndeI.
Fig. 3 analyzes collection of illustrative plates for HindIII and the NdeI double digestion of expression plasmid pPPEST-PET of the present invention, and wherein, 1 is DNA Marker (λ-HindIII digest, Takala company); 2 is pPPEST-PET/HindIII+IndeI.
Fig. 4 is the PAGE electrophorogram of engineering strain E.coli BL21 of the present invention (DE3)/pPPEST-PET expression product, and wherein 1 is lower molecular weight standard protein (Shanghai Bo Ya Bioisystech Co., Ltd);
2 is the product before engineering strain E.coli BL21 (DE3)/pPPEST-PET induces; 3 products of inducing 3h through IPTG for engineering strain E.coli BL21 (DE3)/pPPEST-PET.
Fig. 5 is the recombinate C of reaction product of ester hydrolase hydrolysis flurbiprofen axetil of the present invention
18HPLC analyzes collection of illustrative plates, and wherein, 1 is flurbiprofen; 2 is flurbiprofen axetil.
Fig. 6 analyzes collection of illustrative plates for the recombinate chirality HPLC of reaction product of ester hydrolase hydrolysis flurbiprofen axetil of the present invention, and wherein, 1 is (S)-flurbiprofen.
Fig. 7 is the structure synoptic diagram of expression plasmid pPPEST-PET of the present invention.
Embodiment
Further specify the present invention with embodiment below.
Materials and methods in the following example is:
The molecule clone technology that is adopted is referring to " the molecular cloning experiment guide " of volumes such as the female Brooker of J. Sa.
Employed toolenzyme is all available from Takala company, and the concrete reaction conditions and the method for use are all with reference to catalogue.
Employed glue reclaims test kit and gives birth to worker company available from Shanghai, and using method is with reference to catalogue.
Following commercialization plasmid and e. coli strains are used for DNA library construction and gene clone.
PUC18 (Takala, Japan)
PUC18-T (Takala, Japan)
PET22b (+) (Novagen, the U.S.)
Escherichia coli DH5a (Takala, Japan)
E. coli bl21 (DE3) (Takala, Japan)
Embodiment 1: from pseudomonasputida clone PPEST gene
Digest the genomic dna that obtains from pseudomonasputida with Sau3AI, and on 0.7% sepharose, separate.The dna fragmentation that reclaims 2-8kb from gel also is connected with the pUC18 that handled, cuts with the BamHI enzyme with calf intestinal alkaline phosphatase (CIAP).To connect mixture and transform efficient competence escherichia coli DH5a (preparing the high state competent cell) with the method that Hanahan provided in " molecular cloning experiment guide ", coating contains the LB flat board of 50ug/ml Amp, in 37 ℃ of overnight incubation, obtain about 20000 clones altogether.
Bacterium colony on the LB flat board is transferred to contains 1-10mmol/L (as 5mmol/L) glycerine tri-n-butyl, on the new LB flat board of 0.1-1mmol/L (as 1mmol/L) IPTG, cultivate the bacterium colony that 48 hours, picking produce transparent circle for 37 ℃.Be inoculated in the LB substratum that contains 1-10 (as 5mmol/L) mmol/L flurbiprofen ethyl ester, 0.1-1mmol/L (as 1mmol/L) IPTG, 50-100ug/ml (as 60ug/ml) Amp, cultivate 24h for 37 ℃, analyze the optical purity of product flurbiprofen in the fermented liquid.Wherein there is a positive colony can produce the flurbiprofen of optical purity＞99%, this clone contains the plasmid of a 3kb size, this plasmid is carried out dna sequencing and restriction analysis, show that it has the gene PPEST of a coding ester hydrolase, its nucleotide sequence is shown in SEQ ID No.1, the size of the DNA of encoding mature peptide is 1142bp, and its corresponding amino acid sequence is shown in SEQ ID No.2.
Embodiment 2: cloned plasmids makes up
According to the PPEST gene order that embodiment 1 obtains, design forward primer 1 and reverse primer 2.Add the NdeI site at primer 1, primer 2 adds the HindIII site.
NdeI
HindIII
With the pseudomonasputida genomic dna is that template is carried out pcr amplification (utilizing the Takara test kit), and the PCR reaction mixture is by 2.5mmol/L dNTP, 20pmol primer 1 and primer 2, and 5 Taq of unit polysaccharases (Takara) and the damping fluid that provides are formed.Reaction conditions is as follows.Add the Taq polysaccharase to reaction mixture after the step 1.Step 2 to 4 repeats 29 times.
| Step | Temperature | Time |
| 1 | 97℃ | 5min | |
| 2 | 95 | 30sec | |
| 3 | 60℃ | 30sec | |
| 4 | 72℃ | 1min | |
| 5 | 72℃ | 10min |
Separate the PCR product with 0.7% sepharose, reclaim the dna fragmentation (as shown in Figure 1) of 1.1kb from gel, be connected with the pUC18-T carrier, the gained plasmid claims pPPEST-T, connects product transformed competence colibacillus escherichia coli DH5a.Plasmid pPPEST-T analyzes through HindIII and NdeI double digestion, proves and contains correct insertion fragment (as shown in Figure 2).
Embodiment 3: the structure of expression vector and transformant
PPPEST-T plasmid and pET22b (+) are respectively behind NdeI and HindIII double digestion, separate enzyme with 0.7% sepharose and cut product, from gel, reclaim the dna fragmentation of 1.1kbp and 4.8kbp respectively, connect with the T4 dna ligase, the gained plasmid claims pPPEST-PET plasmid, connect product transformed competence colibacillus intestinal bacteria E.coli BL21 (DE3), through the resistance substratum screen positive colony and carry out HindIII and NdeI double digestion Analysis and Identification, proof contains correct insertion fragment (as shown in Figure 3), thereby obtains transformant---express engineering strain E.coliBL21 (the DE3)/pPPEST-PET of ester hydrolase of the present invention.
Embodiment 4: the expression of ester hydrolase
Picking contains the single bacterium colony of pPPEST-PET plasmid intestinal bacteria, is connected to 5ml, contains in the LB substratum of 50ug/ml kantlex, 37 ℃ of concussion overnight incubation.Get the fresh LB substratum that the 50ul nutrient solution is connected to 5ml, contains the 50ug/ml kantlex, be cultured to OD
600About=0.6, adding IPTG is that 1mmol/L induces to its concentration, continues to cultivate 3h.Centrifugal collection thalline, expression product detects the expression amount of ester hydrolase and enzyme activity (according to people such as C.Ruiz through PAGE electrophoresis and enzymic activity, Letters in AppliedMicrobiology 2003,37, the survey enzyme activating method that 354-359 adopts), as can be seen from Figure 4, with induce preceding comparison, have and predictive molecule target stripe of the same size at 4.0 kd places, the expression amount of target protein accounts for total protein weight more than 20%, and enzyme activity is the 50U/g dry mycelium.
Embodiment 5: the application of reorganization ester hydrolase
Get the recombination bacillus coli among the embodiment 4, through ultrasonic broken wall, centrifugal removal thalline, supernatant liquor is through 60% (NH
4)
2SO
4After post precipitation, throw out redissolve through obtaining partially purified reorganization ester hydrolase behind the phenyl-Sephrose fast flow post layer post.This reorganization ester hydrolase of obtaining, racemize flurbiprofen axetil and the Ketoprofen BP 93 ester of hydrolysis 10-100mmol/L (pH10.0,50mmol/L potassium phosphate buffer), 30 ℃ of temperature of reaction.Reaction product is through C
18(moving phase is methyl alcohol to HPLC: water=85: 15 (V/V), detect wavelength 254nm, flow velocity 0.8ml/min, 30 ℃ of detected temperatures) and chirality CHI-TBB HPLC post (moving phase is normal hexane: t-butyl methyl ether: Glacial acetic acid=70: 30: 0.1 (V/V), detect wavelength 254nm, flow velocity 1.0ml/min, 30 ℃ of detected temperatures) optical purity of analysis transformation efficiency and product (as shown in Figure 5 and Figure 6, wherein the retention time of (R)-flurbiprofen should be 12.153min, this HPLC collection of illustrative plates does not detect (R)-flurbiprofen), the result is as shown in the table.As can be known from the results, behind the different substrate process reorganization ester hydrolase catalytic hydrolysis, obtain the flurbiprofen and the Ketoprofen BP 93 of optical purity＞99% respectively.With the same reaction of thick ester hydrolase catalysis that the P.putida ATCC 17522 of wild-type is separated to, obtain flurbiprofen and the Ketoprofen BP 93 of optical purity 97-98%.Illustrate that utilization reorganization ester hydrolase reacts the side reaction of other isozyme that can eliminate wild bacterium generation, improves the optical purity of chipal compounds.
| Substrate | Product | Transformation efficiency (%) | Optical purity ee (%) |
| The flurbiprofen methyl esters | (S)-flurbiprofen | 42 | ＞99 |
| The flurbiprofen ethyl ester | 50 | ＞99 | |
| The Ketoprofen BP 93 methyl esters | (S)-Ketoprofen BP 93 | 46 | ＞99 |
| The Ketoprofen BP 93 ethyl ester | 50 | ＞99 |
Sequence table
＜110〉Shanghai Institute of Pharmaceutical Industry
＜120〉a kind of ester hydrolase and gene thereof and recombinase
<130>051016C
<160>2
<170>PatentIn version 3.1
<210>1
<211>1390
<212>DNA
＜213〉pseudomonasputida (Pseudomonas putida)
<220>
<221>CDS
<222>(5)..(1150)
<223>
<400>1
gcgg gtg cag gag gtg agt cct ctg gtg ctg aag ttc gaa gca gtg cgc 49
Val Gln Glu Val Ser Pro Leu Val Leu Lys Phe Glu Ala Val Arg
1 5 10 15
gaa acc ttc gcc gcg ctg ttc gat gat ccc cag gag cgt ggc gcc gcg 97
Glu Thr Phe Ala Ala Leu Phe Asp Asp Pro Gln Glu Arg Gly Ala Ala
20 25 30
ctg tgc atc cag gtt ggt ggc gaa acc gtg gtc gac ctg tgg gcc ggc 145
Leu Cys Ile Gln Val Gly Gly Glu Thr Val Val Asp Leu Trp Ala Gly
35 40 45
agt gcc gac aag gac ggc cag cag gcc tgg cac agc gat acc atc gcc 193
Ser Ala Asp Lys Asp Gly Gln Gln Ala Trp His Ser Asp Thr Ile Ala
50 55 60
aac ctg ttc tcc tgc acc aag acc ttc acc gct gtc acc gcc ctg caa 241
Asn Leu Phe Ser Cys Thr Lys Thr Phe Thr Ala Val Thr Ala Leu Gln
65 70 75
ctg gtc ggc gag ggc aag ctg acg ctc gat gcg ccg gtg gcc aac tac 289
Leu Val Gly Glu Gly Lys Leu Thr Leu Asp Ala Pro Val Ala Asn Tyr
80 85 90 95
tgg ccc gag ttc gcc cag gca ggc aaa cag gct atc acc ctg cgc cag 337
Trp Pro Glu Phe Ala Gln Ala Gly Lys Gln Ala Ile Thr Leu Arg Gln
100 105 110
ttg ctc agc cac cgt gcc ggc ttg ccg gcc atc cgt gag ctg ctg ccg 385
Leu Leu Ser His Arg Ala Gly Leu Pro Ala Ile Arg Glu Leu Leu Pro
115 120 125
gcc gaa gcg ctg tat gac tgg cag gcc atg gtc gat gcg ctg gct gcc 433
Ala Glu Ala Leu Tyr Asp Trp Gln Ala Met Val Asp Ala Leu Ala Ala
130 135 140
gaa acg ccc tgg tgg aca ccc ggc acc gag cac ggc tat gcc gcc att 481
Glu Thr Pro Trp Trp Thr Pro Gly Thr Glu His Gly Tyr Ala Ala Ile
145 150 155
acc tac ggc tgg ctg atc ggc gag ctg atc cgg cgt gcc gac ggc cgt 529
Thr Tyr Gly Trp Leu Ile Gly Glu Leu Ile Arg Arg Ala Asp Gly Arg
160 165 170 175
ggc ccc ggc gac tcg atc gtg gcc cgt acc gct cgg ccg ttg ggg ctg 577
Gly Pro Gly Asp Ser Ile Val Ala Arg Thr Ala Arg Pro Leu Gly Leu
180 185 190
gat ttt cat gtc ggc ctg gcg gac gat gag ttt tat cgc gtg gcg cac 625
Asp Phe His Val Gly Leu Ala Asp Asp Glu Phe Tyr Arg Val Ala His
195 200 205
atc gcc cgt ggc aag ggc aac gcc ggg gac gct gcg gcg cag cgc ctg 673
Ile Ala Arg Gly Lys Gly Asn Ala Gly Asp Ala Ala Ala Gln Arg Leu
210 215 220
ctg cag gtg acc atg cgc gag ccc gag gcc ctg tcg acc cgt gcc ttc 721
Leu Gln Val Thr Met Arg Glu Pro Glu Ala Leu Ser Thr Arg Ala Phe
225 230 235
acc aac ccg ccg gcg atc ctg acc agt acc aac aag ccg gag tgg cgg 769
Thr Asn Pro Pro Ala Ile Leu Thr Ser Thr Asn Lys Pro Glu Trp Arg
240 245 250 255
cgc atg cag cag cca gcg gcc aat ggc cat ggc aat gca cgc agc ctg 817
Arg Met Gln Gln Pro Ala Ala Asn Gly His Gly Asn Ala Arg Ser Leu
260 265 270
gcg ggc ttc tat gcc ggg ttg ctg gac ggc agc ctg ctc gaa tcc gaa 865
Ala Gly Phe Tyr Ala Gly Leu Leu Asp Gly Ser Leu Leu Glu Ser Glu
275 280 285
ctg ctc gac gaa ctg acc cgt gag cac agc ctc ggg cag gat cgc acc 913
Leu Leu Asp Glu Leu Thr Arg Glu His Ser Leu Gly Gln Asp Arg Thr
290 295 300
ctg ctc acc cag acc cgt ttt ggc ctg ggc tgc atg ctc gac cag ccc 961
Leu Leu Thr Gln Thr Arg Phe Gly Leu Gly Cys Met Leu Asp Gln Pro
305 310 315
gac gtg gcc aat gcc acc ttc ggc ctc ggg gcc cgc gcc ttc ggc cac 1009
Asp Val Ala Asn Ala Thr Phe Gly Leu Gly Ala Arg Ala Phe Gly His
320 325 330 335
cca ggt gcc ggt ggc tcg gtc ggt ttc gct gac ccg gaa cac gat gtg 1057
Pro Gly Ala Gly Gly Ser Val Gly Phe Ala Asp Pro Glu His Asp Val
340 345 350
gcc ttc ggt ttc gtg gtc aat acc ctc ggc cct tat gtg ctc atg gac 1105
Ala Phe Gly Phe Val Val Asn Thr Leu Gly Pro Tyr Val Leu Met Asp
355 360 365
ccg cga gcc cag aaa ctg gta cgc gtc ctt gcc agt tgc ctt tga 1150
Pro Arg Ala Gln Lys Leu Val Arg Val Leu Ala Ser Cys Leu
370 375 380
tcgggtaatg cgggtgttgg cccacccctt tgactatcct caatgccaac gcctgaaagg 1210
cgtaggcaaa attcctttct atttcatggt gtatcgctga tgtcttcaca caaaacctta 1270
gcactcgccc tgtgcctgac cgccgttacc ggctgcgcca gccactccca ggacggctcg 1330
aaggacagcg cctcgagctg gtggcccttc ggttcggaca aggttgccga gcaggaagtg 1390
<210>2
<211>381
<212>PRT
＜213〉pseudomonasputida (Pseudomonas putida)
<400>2
Val Gln Glu Val Ser Pro Leu Val Leu Lys Phe Glu Ala Val Arg Glu
1 5 10 15
Thr Phe Ala Ala Leu Phe Asp Asp Pro Gln Glu Arg Gly Ala Ala Leu
20 25 30
Cys Ile Gln Val Gly Gly Glu Thr Val Val Asp Leu Trp Ala Gly Ser
35 40 45
Ala Asp Lys Asp Gly Gln Gln Ala Trp His Ser Asp Thr Ile Ala Asn
50 55 60
Leu Phe Ser Cys Thr Lys Thr Phe Thr Ala Val Thr Ala Leu Gln Leu
65 70 75 80
Val Gly Glu Gly Lys Leu Thr Leu Asp Ala Pro Val Ala Asn Tyr Trp
85 90 95
Pro Glu Phe Ala Gln Ala Gly Lys Gln Ala Ile Thr Leu Arg Gln Leu
100 105 110
Leu Ser His Arg Ala Gly Leu Pro Ala Ile Arg Glu Leu Leu Pro Ala
115 120 125
Glu Ala Leu Tyr Asp Trp Gln Ala Met Val Asp Ala Leu Ala Ala Glu
130 135 140
Thr Pro Trp Trp Thr Pro Gly Thr Glu His Gly Tyr Ala Ala Ile Thr
145 150 155 160
Tyr Gly Trp Leu Ile Gly Glu Leu Ile Arg Arg Ala Asp Gly Arg Gly
165 170 175
Pro Gly Asp Ser Ile Val Ala Arg Thr Ala Arg Pro Leu Gly Leu Asp
180 185 190
Phe His Val Gly Leu Ala Asp Asp Glu Phe Tyr Arg Val Ala His Ile
195 200 205
Ala Arg Gly Lys Gly Asn Ala Gly Asp Ala Ala Ala Gln Arg Leu Leu
210 215 220
Gln Val Thr Met Arg Glu Pro Glu Ala Leu Ser Thr Arg Ala Phe Thr
225 230 235 240
Asn Pro Pro Ala Ile Leu Thr Ser Thr Asn Lys Pro Glu Trp Arg Arg
245 250 255
Met Gln Gln Pro Ala Ala Asn Gly His Gly Asn Ala Arg Ser Leu Ala
260 265 270
Gly Phe Tyr Ala Gly Leu Leu Asp Gly Ser Leu Leu Glu Ser Glu Leu
275 280 285
Leu Asp Glu Leu Thr Arg Glu His Ser Leu Gly Gln Asp Arg Thr Leu
290 295 300
Leu Thr Gln Thr Arg Phe Gly Leu Gly Cys Met Leu Asp Gln Pro Asp
305 310 315 320
Val Ala Asn Ala Thr Phe Gly Leu Gly Ala Arg Ala Phe Gly His Pro
325 330 335
Gly Ala Gly Gly Ser Val Gly Phe Ala Asp Pro Glu His Asp Val Ala
340 345 350
Phe Gly Phe Val Val Asn Thr Leu Gly Pro Tyr Val Leu Met Asp Pro
355 360 365
Arg Ala Gln Lys Leu Val Arg Val Leu Ala Ser Cys Leu
370 375 380
Claims (10)
1, a kind of ester hydrolase gene is one of following nucleotide sequences:
1) it has the base sequence shown in the SEQ ID No.1 in the sequence table;
2) protein of encoding and forming by the aminoacid sequence shown in the SEQ ID No.2 in the sequence table.
2, a kind of ester hydrolase is to have the protein that the aminoacid sequence shown in the SEQ ID No.2 is formed in the sequence table.
3, the expression vector that comprises the nucleotide sequence of the described ester hydrolase gene of claim 1.
4, expression vector according to claim 3, it is characterized in that it is made by following method: with the nucleotide sequence amplification of the described ester hydrolase gene of claim 1, be connected with plasmid pUC18-T, obtain cloning vector pPPEST-T, pPPEST-T and plasmid pET22b (+) are connected behind NdeI and HindIII double digestion respectively obtain expression plasmid pPPEST-PET again.
5, a kind of engineering strain that is used to express the described ester hydrolase of claim 2, it is that claim 3 or 4 described expression vectors are transformed into the engineering strain that obtains among the intestinal bacteria E.coli BL21.DE3.
6, a kind of method for preparing the ester hydrolase of recombinating, it comprises with claim 3 or 4 described expression vector transformed host cells, cultivates transformant, obtains the ester hydrolase of reorganization.
7, method according to claim 6 is characterized in that this host cell is intestinal bacteria.
8, method according to claim 7 is characterized in that this transformant is the described engineering strain of claim 5.
9, the reorganization ester hydrolase that makes of method according to claim 6.
10, the application of reorganization ester hydrolase according to claim 9 in stereoselectivity catalysis chipal compounds is synthetic.
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| CN114196653B (en) * | 2021-12-10 | 2023-06-20 | 安徽医科大学 | Application of recombinase ester Est1260 in reducing Jie Nibo gold ester |
Citations (1)
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|---|---|---|---|---|
| CN1354795A (en) * | 1998-11-10 | 2002-06-19 | 诺沃奇梅兹生物技术有限公司 | Polypeptides having lactonohydrolase activity and nucleic acids encoding same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1354795A (en) * | 1998-11-10 | 2002-06-19 | 诺沃奇梅兹生物技术有限公司 | Polypeptides having lactonohydrolase activity and nucleic acids encoding same |
Non-Patent Citations (1)
| Title |
|---|
| D-泛解酸内酯水解酶产生菌的筛选及产酶条件研究 汤一新.微生物学报，第42卷第1期 2002 * |
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