CN103820417A - Esterolytic enzyme, coding gene, carrier, engineering bacterium and application of coding gene - Google Patents
Esterolytic enzyme, coding gene, carrier, engineering bacterium and application of coding gene Download PDFInfo
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- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
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- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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Abstract
The invention provides esterolytic enzyme from bacillus megaterium, a coding gene of the esterolytic enzyme, a carrier containing the coding gene, an engineering bacterium and application of the coding gene. The amino acid sequence of the esterolytic enzyme is shown as SEQ ID NO. 2; the nucleotide sequence of the encoding gene is shown as SEQ ID NO.1. The recombined esterolytic enzyme provided by the invention can be used for synthesizing a stereoselective catalytic chiral compound and has high catalytic activity and stereoselectivity.
Description
(1) technical field
The present invention relates to one and there is the stereoselective ester hydrolase of S-isomer and encoding gene thereof, the carrier that contains this encoding gene, engineering bacteria and application thereof.
(2) background technology
Ester hydrolase includes lipase and esterase, and it is the biological catalyst that a class has important use in chirality is synthetic.These enzymes can be identified very wide substrate, and the biology of > 40% can not have ester hydrolase catalysis to complete to thing building-up reactions at present, and these reactions have regioselectivity and the stereoselectivity high of mild condition, reflection.Wait until application widely at aspects such as Enzymatic kinetic resolution racemate, amine and conversion prochirality alcohol.In addition, they also can be used in selective esterification, transesterification and polyreaction.
Due to the growth to chiral drug and intermediate demand, utilize biological process or enzyme process synthesizing chiral compound day by day to obtain people's attention and industrial applications.The key issue of biological process synthesizing chiral compound is to find the biological catalyst with height stereoselectivity catalysis.Because ester hydrolase tool has been widely used, screening new ester hydrolase is just becoming the study hotspot of enzyme engineering.Although it is very wide that ester hydrolase distributes at microbial world, their stereoselectivity catalysis is different.In same organism, often there is multiple ester hydrolase, due to the stereoselectivity property of there are differences between them, be utilized as optical purity (the Geun-Joong Kim that tends to reduce product when biomass cells or their thick enzyme preparation carry out enzyme process synthesizing chiral compound, Journal of Molecular Catalysis B:Enzymatic17,2002:29-38).Address this problem the generation that can reduce by the control measures of engineering side reaction, also can obtain single zymin by separation and purification and carry out catalyzed reaction, but often more complicated and high cost of these processes, then be difficult for being used in producing.If by engineered means, the gene of Direct Cloning and the needed enzyme of expression, just can easily reach the above object.
(3) summary of the invention
The object of the invention be to provide a kind of screen acquisition there is the stereoselective ester hydrolase of S-isomer, the carrier that contains this encoding gene, engineering bacteria and application thereof.
The technical solution used in the present invention is:
One has the stereoselective ester hydrolase of S-isomer, and its aminoacid sequence is as shown in SEQ ID NO.2.
The inventor is by a large amount of microorganism of screening, find that bacillus megaterium can produce one and have highly stereoselective ester hydrolase, ester hydrolase of the present invention is from bacillus megaterium (Bacillus megaterium) WZ009, this bacterial strain deposit number is CCTCC NO:M2010171, in CN102168036A, discloses.The one that contriver obtains bacillus megaterium by pcr amplification has the stereoselective ester hydrolase gene of S-isomer BMEST, measure its nucleotide sequence, as shown in SEQ ID No.1 in sequence table, wherein encoding sequence (CDS) from the 1st base of DNA to the 1401st stop, ATG is transcription initiation codon, and TAA is Transcription Termination password; And obtain corresponding aminoacid sequence, as shown in SEQ ID No.2 in list.
Due to the singularity of aminoacid sequence; any fragment that contains the peptide protein of aminoacid sequence shown in SEQ ID NO.2 or its variant; as its examples of conservative variations, bioactive fragment or derivative; as long as the fragment of this peptide protein or peptide protein variant and aforementioned amino acid sequence homology, more than 90%, all belong to the row of protection domain of the present invention.Concrete described change can comprise amino acid whose disappearance, insertion or replacement in aminoacid sequence; Wherein, change for the conservative property of variant, the amino acid of replacing has the structure similar to original acid or chemical property, and as replaced Isoleucine with leucine, variant also can have non-conservation and change, as replaced glycine with tryptophane.
The invention still further relates to the encoding gene of described ester hydrolase.Concrete, described gene nucleotide series is as shown in SEQ ID NO.1.
Due to the singularity of nucleotide sequence, the variant of polynucleotide shown in any SEQ ID NO.1, as long as itself and this polynucleotide have 90% above homology, all belongs to the row of protection domain of the present invention.The variant of described polynucleotide refers to a kind of polynucleotide sequence that one or more Nucleotide changes that has.The variant of these polynucleotide can make raw displacement varient or the varient of non-life, comprises and replaces varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of polynucleotide, but can be from not changing in fact the function of peptide protein of its coding.
The invention still further relates to the recombinant vectors that contains described encoding gene, and utilize described recombinant vectors to transform the recombination engineering bacteria obtaining.
Described recombinant vectors is by ordinary method, the nucleotide sequence of ester hydrolase gene of the present invention to be connected on various carriers and to build and form, this carrier can be commercially available plasmid, clay, phage or virus vector etc., as pUC, and pBluescript (Stratagene), pLEX (Novagen, Inc., Madison, Wis.), PQE (Qiagen), pREP, pSE420 and pLEX (Invitrogen), but be not to only limit to these carriers.Preferably BMEST gene product pcr amplification being arrived is cloned with expression vector pEASY-E1 and is connected by TA, forms expression vector (plasmid) pEASY-E1-BMEST of BMEST gene.Expression vector pEASY-E1-BMEST transforms Trans1-T1 competent cell, amplification plasmid.
Described engineering bacteria is that the expression vector that comprises ester hydrolase gene nucleotide series of the present invention is transformed into the engineering strain obtaining in competence intestinal bacteria E.coli BL21 (DE3), wherein obtains intestinal bacteria E.coli BL21 (the DE3)/pEASY-E1-BMEST that contains pEASY-E1-BMEST as plasmid pEASY-E1-BMEST is transformed.
The invention still further relates to the application of described gene in preparation restructuring ester hydrolase.Concrete application comprises with the invention described above expression vector transformed host cell, cultivates transformant, obtains the ester hydrolase of restructuring.Wherein, this host cell can be protokaryon, eukaryotic microorganisms or insect etc.Preferably intestinal bacteria, obtaining corresponding transformant is above-mentioned engineering strain: intestinal bacteria E.coli BL21 (DE3)/pEASY-E1-BMEST.This bacterial strain can be under conventional IPTG induction (add during in left and right, OD600=0.5～0.6, to its concentration be 0.1～1mmol/L all can) high efficient expression ester hydrolase albumen, be restructuring ester hydrolase of the present invention.
The invention still further relates to described ester hydrolase and prepare the application in chipal compounds at stereoselectivity catalysis resolution of racemic substrate.Described racemic substrate is one of following: 4-chloro-3-hydroxybutanoic acid ester, 3-hydroxybutyrate ester, tetrahydrochysene furoic acid ester.Ester hydrolase of the present invention can be for the preparation of optically pure chipal compounds.
Beneficial effect of the present invention is mainly reflected in: ester hydrolase of the present invention can, for the preparation of optically pure chipal compounds, have higher catalysis activity and stereoselectivity.
(4) accompanying drawing explanation
Fig. 1 is the pcr amplification electrophoretogram of BMEST of the present invention, wherein, 1, DNA Marker(λ-Hind III digest, TakaRa company); 2, the pcr amplification product of BMEST.
Fig. 2 is plasmid pEASY-E1-BMEST PCR of the present invention checking electrophoretogram, wherein, 1, DNA Marker(λ-Hind III digest, TakaRa company); 2, recombinant plasmid pEASY-E1-BMEST PCR product;
Fig. 3 is the PAGE electrophorogram of engineering strain E.coli BL21 of the present invention (DE3)/pEASY-E1-BMEST expression product, wherein, and product before 1, engineering strain E.coli BL21 (DE3)/pEASY-E1-BMEST induction; 2, engineering strain E.coli BL21 (DE3)/pEASY-E1-BMEST induces after product through IPTG; 3, lower molecular weight standard protein (TakaRa company).
Fig. 4 is that expression plasmid pEASY-E1-BMEST builds schematic diagram.
(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:
Materials and methods in embodiment is:
" the molecular cloning experiment guide " that the molecule clone technology adopting is compiled referring to J. Pehanorm Brooker etc.
TransStartTM Taq DNA Polymerase is purchased from TransGen Biotech company.DNA marker, genome extract test kit, plasmid extraction kit, DNA recovery purification kit, agarose electrophoresis reagent all purchased from TaKaRa, the precious biotech firm in Dalian, and using method is with reference to catalogue.
Trans1-T1 competent cell, Beijing Quanshijin Biotechnology Co., Ltd.
Colon bacillus BL21 (DE3), TakaRa, the precious biotech firm in Dalian.
Ni-NTA Sefinose Kit, Bio Basic INC company.
Embodiment 1: from bacillus megaterium clone BMEST gene
According to DNA sequence dna (GenBank:CP001982.1) design degenerated primer 1 and the primer 2 of Bacillus megaterium DSM319 lipase.
Primer 1 sequence: ATGAATATCACAACGTTTGACG
Primer 2 sequence: TTATTTTTCTGTTGATATGCTTTTC
Carry out pcr amplification (utilizing TakaRa test kit) take bacillus megaterium (Bacillus megaterium) DSM319 genomic dna as template, PCR reaction system and reaction conditions are as shown in Table 1 and Table 2.Step 2 to 4 repetition 25 times.
Table 1:PCR amplification reaction system
Table 2:PCR amplification reaction condition
With 0.8% agarose gel electrophoresis detection pcr amplification product, product is single band, and size is about 1400bp(as shown in Figure 1).PCR product is carried out to purifying recovery, and concrete steps reclaim test kit specification sheets with reference to vast Tyke, Beijing genomic dna fast purifying, then use trace dna protein measurement instrument detectable level and purity.
Embodiment 2: the structure of expression vector and transformant
Object fragment and pEASY-E1 expression vector are carried out to A-T and be connected, linked system is as following table 3:
Table 3:
peASY-E1Ex
pression Vector linked system
Linked system is reacted 15min at 25 ℃, adds and connects product in 50
μin the Trans-T1 competent cell that L has just thawed, clone, concrete operations are with reference to pEASY-E1Expression Kit specification sheets.PCR method is identified the positive recombinant (as shown in Figure 2) of correction direction, is forwarded to LB substratum amplification vector.From Trans1-T1 thalline, extract recombinant plasmid pEASY-E1-BMEST with TaKaRa plasmid extraction kit, Transformed E .coli BL21 (DE3) competent cell, obtain positive colony and through PCR clone identification through Amp resistance culture base screening, contain correct recombinant plasmid (as shown in Figure 3), thereby obtain transformant one and express engineering strain E.coli BL21 (the DE3)/pEASY-E1-BMEST of ester hydrolase of the present invention.
Embodiment 3: the expression of ester hydrolase
Engineering strain E.coli BL21 (DE3)/pEASY-E1-BMEST is connected to 50mL, contains in the LB substratum of 80 μ g/mL penbritins, 37 ℃ of concussion overnight incubation.Get the fresh LB substratum that 500 μ L nutrient solutions are connected to 50mL, contain 80 μ g/mL penbritins, when being cultured to OD600 and being about 0.5-0.6, add IPTG to its concentration be 0.1mmol/L induction, 25 ℃, 200rpm continues to cultivate 10-12h overexpression target protein, centrifugal collection thalline.
Embodiment 4: the application of restructuring ester hydrolase
Get the recombination bacillus coli in embodiment 3, through ultrasonication, centrifugal removal thalline, supernatant liquor obtains partially purified restructuring ester hydrolase after HisTrapTMFF affinity column.This restructuring ester hydrolase of obtaining, be hydrolyzed the substrates such as racemic 4-chloro-3-hydroxyl ethyl butyrate, reaction solvent is that 50mM pH8.0Tris hydrochloride buffer, concentration of substrate are respectively 2%(volume ratio), enzyme dosage is 20mg/L damping fluid, 30 ℃ of water-bath temperature, after reaction 120min, reaction product is analyzed the optical purity of transformation efficiency and product through gas-chromatography (6890N and BGB-174 Chiral gas chromatography post that Agilent company produces), the results are shown in Table 4.
The bacillus megaterium WZ009(CCTCC NO:M2010171 of wild-type) be seeded to fermention medium, under the condition of 50mL liquid amount/250mL shaking flask, 30 ℃ of temperature, shaking speed 200rpm, cultivate 12 hours, obtain fermented liquid, centrifugal collection thalline, through ultrasonication, centrifugal removal thalline, supernatant liquor is crude enzyme liquid, in contrast, be hydrolyzed under the same conditions the substrates such as racemic 4-chloro-3-hydroxyl ethyl butyrate, the results are shown in Table 5; Fermention medium: glucose 7.2g/L, yeast extract paste 7g/L, CaCl
21.25g/L, solvent is water, initial pH6.5.
Gas-chromatography operational conditions is as follows: carrier gas is nitrogen.Injector temperature: 220 ℃.Air flow quantity 300mL/min, make-up gas flow 30mL/min.Splitting ratio 1:20, sample size 1 μ L.Column temperature rise program: 100 ℃ of maintenance 2min of initial temperature, 2.5 ℃/min is warming up to 150 ℃, keeps 2min, and 5 ℃/min is warming up to 210 ℃, keeps 4min.FID detects its temperature: 220 ℃.
Table 4: the restructuring ester hydrolase of purifying transforms productive rate and the e.e. value of different substrates
Table 5: wild strain crude enzyme liquid transforms productive rate and the e.e. value of different substrates
As can be known from the results, different substrates, after recombinase lytic enzyme catalytic hydrolysis, obtains respectively R-4-chloro-3-hydroxyl ethyl butyrate, R-3-3-hydroxyethyl butyrate and the R-tetrahydro furfuryl methyl of optical purity > 99%.The thick ester hydrolase catalytic effect that reaction result is separated to the B.megaterium WZ009 of wild-type is compared, and reaction is all greatly improved in the productive rate of R-configuration substrate and optical purity.Illustrate that utilization restructuring ester hydrolase reacts the side reaction of other isozyme that can eliminate wild mushroom generation, improves the optical purity of chipal compounds.
The above, be only preferred embodiment of the present invention, not technology contents of the present invention done to any pro forma restriction.Any simple modification, equivalent variations and modification that every foundation technical spirit of the present invention is done above embodiment, all fall within the scope of protection of the present invention.
Claims (8)
1. have the stereoselective ester hydrolase of S-isomer, its aminoacid sequence is as shown in SEQ ID NO.2.
2. the gene of ester hydrolase described in coding claim 1.
3. gene as claimed in claim 2, is characterized in that described gene nucleotide series is as shown in SEQ ID NO.1.
4. contain the recombinant vectors of gene described in claim 2 or 3.
5. one kind transforms the recombination engineering bacteria obtaining with recombinant vectors described in claim 4.
6. the application of gene in preparation restructuring ester hydrolase described in claim 2 or 3.
7. ester hydrolase claimed in claim 1 is prepared the application in chipal compounds at stereoselectivity catalysis resolution of racemic substrate.
8. application as claimed in claim 7, is characterized in that described racemic substrate is one of following: 4-chloro-3-hydroxybutanoic acid ester, 3-hydroxybutyrate ester, tetrahydrochysene furoic acid ester.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543191A (en) * | 2016-02-24 | 2016-05-04 | 中国科学院南海海洋研究所 | Esterase PHE21 and encoding gene and application thereof |
| CN106520896A (en) * | 2016-12-21 | 2017-03-22 | 江苏远大仙乐药业有限公司 | Method for preparing dexamethasone intermediate product through one-step microbial fermentation and transformation |
| CN110592049A (en) * | 2019-09-29 | 2019-12-20 | 北京工商大学 | Aspergillus niger ester hydrolase AnCu3, encoding gene and application thereof in DEHP hydrolysis |
| CN110628743A (en) * | 2019-08-20 | 2019-12-31 | 浙江工业大学 | Stereoselective esterase, coding gene, vector, engineering bacterium and application |
Citations (1)
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| CN102168036A (en) * | 2010-11-30 | 2011-08-31 | 浙江工业大学 | Preparation of R-4-chloro-3-hydroxybutyrate and S-3-hydroxy-butyrolactone by microbial transformation and strain |
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| CN102168036A (en) * | 2010-11-30 | 2011-08-31 | 浙江工业大学 | Preparation of R-4-chloro-3-hydroxybutyrate and S-3-hydroxy-butyrolactone by microbial transformation and strain |
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| EPPINGER,M ET AL.: "Genome Sequences of the Biotechnologically Important Bacillus megaterium Strains QM B1551 and DSM319", 《J. BACTERIOL.》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543191A (en) * | 2016-02-24 | 2016-05-04 | 中国科学院南海海洋研究所 | Esterase PHE21 and encoding gene and application thereof |
| CN105543191B (en) * | 2016-02-24 | 2019-06-21 | 中国科学院南海海洋研究所 | A kind of esterase PHE21 and its encoding gene and application |
| CN106520896A (en) * | 2016-12-21 | 2017-03-22 | 江苏远大仙乐药业有限公司 | Method for preparing dexamethasone intermediate product through one-step microbial fermentation and transformation |
| CN106520896B (en) * | 2016-12-21 | 2019-10-29 | 江苏远大仙乐药业有限公司 | A kind of method that the conversion of microorganism one-step fermentation prepares Dexamethasone Intermediate |
| CN110628743A (en) * | 2019-08-20 | 2019-12-31 | 浙江工业大学 | Stereoselective esterase, coding gene, vector, engineering bacterium and application |
| CN110592049A (en) * | 2019-09-29 | 2019-12-20 | 北京工商大学 | Aspergillus niger ester hydrolase AnCu3, encoding gene and application thereof in DEHP hydrolysis |
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