CN102994470A - Pronucleus expression of epoxide hydrolase gene (EH-B) and preparation of chiral epichlorohydrin - Google Patents
Pronucleus expression of epoxide hydrolase gene (EH-B) and preparation of chiral epichlorohydrin Download PDFInfo
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Abstract
本发明提出一种源于Aspergillus usamii E001的新型B类环氧化物水解酶基因成熟肽cDNA序列的克隆及原核表达方法，其核苷酸序列为SEQ ID NO：1，相应的氨基酸序列为SEQID NO：2，相应的基因命名为Aus EH-B。手性气相柱分析rEH对(R)-环氧氯丙烷具有良好的立体选择性，生产(S)-环氧氯丙烷对映体过量值达99％。为该环氧化物水解酶的产业化生产奠定基础，对EH酶动力学拆分法研究为生物催化技术产业化制备手性环氧氯丙烷提供基础。The present invention proposes a method for cloning and prokaryotic expression of a novel class B epoxide hydrolase gene mature peptide cDNA sequence derived from Aspergillus usamii E001, the nucleotide sequence of which is SEQ ID NO: 1, and the corresponding amino acid sequence is SEQ ID NO : 2, and the corresponding gene was named Aus EH-B. The chiral gas column analysis of rEH has good stereoselectivity to (R)-epichlorohydrin, and the enantiomeric excess value of (S)-epichlorohydrin is up to 99%. It lays the foundation for the industrial production of the epoxide hydrolase, and the research on the kinetic resolution method of EH enzyme provides the basis for the industrial preparation of chiral epichlorohydrin by biocatalytic technology.
Description
Technical field
The present invention relates to derive from clone and the prokaryotic expression of a kind of novel B type epoxide hydrolase (Aus EH-B) gene mature peptide cDNA sequence of Aspergillus usamii (Aspergillus usamii) E001 bacterial strain, utilize biochirality to split the cinnamic method of preparation chiral epoxy, belong to technical field of bioengineering.
Background technology
Chipal compounds is important chiral ligand, is widely used in all kinds of asymmetric reactions, and particularly the application in the synthetic field of medicine is more extensive.Chiral epichlorohydrin is a kind of important organic synthesis key intermediate, in order to preparing multiple high-optical-purity pharmaceutical intermediate and natural product, such as the derivative of aryloxy propanol amine medicine, cyclopentanone, antifungal drug (S)-Ketoconazole, Zarator, VBT etc.At present, racemic epoxy chloropropane is cheap and easy to get, and chiral epichlorohydrin is expensive, and the latter's quotation is substantially at the former more than 6 times, and therefore preparing its chiral monomer by the splitting racemation epoxy chloropropane has very large industrial applications prospect.Product 3-chloro-1 is cut in its fractionation to pieces, and 2-the third two also is a kind of important chirality synthesis material.The method of splitting racemation epoxy chloropropane can be divided into chemical method and biological process two classes: chemical method uses expensive S len-Co catalyzer usually, can obtain ee and reach (S)-epoxy chloropropane yield of 99% and can reach 30%, its shortcoming is that cost height and environmental pollution are serious.
Epoxide hydrolase (Epoxide hydrolase, EC, 3.3.2.-) claim again epoxide hydratase or epoxy hydratase, and it is corresponding 1 to be that the stereoselective addition epoxide of a class catalysis water molecules is hydrolyzed to, the hydrolase of 2-glycol.In the method for chemical method and Biological preparation chiral purity epoxide, the Enzymatic kinetic resolution method is because its high enantioselectivity is become numerous investigators' attention always.This enzyme extensively is present in plant, insect, Mammals and the microbe.Epoxide hydrolase is a kind of enzyme that does not rely on cofactor, its wide material sources, and the substrate spectrum width is wide, and enantio-selectivity is high, has very high industrial applications prospect, its research has been become the focus of current research.
Epoxide hydrolase is microbe-derived extensively, but the epoxide hydrolase with good chiral separation effect is less, Botes etc. are with 1, the 2-octylene oxide is that substrate filters out the bacterial strain that 25 yeast that do not belong to together have the epoxide hydrolase activity from 187 bacterial strains, Yeates etc. filter out 45 yeast that do not belong to together and have the hydrolysis substrate activity from 409 bacterial strains take the nitro epoxy styrene as substrate, but only have the epoxide hydrolase selectivity of 3 Basidiomycotina bacterial strain yeast higher, i.e. trichosporon (Trichosporon), red winter spore belongs to (Rhodosporidium) and Rhodotorula (Rhodotorula).Furstoss and Archelas etc. screen the asymmetric hydrolysis that a strain Aspergillus niger LCP 521 carries out epoxy compounds from numerous bacterial strains, synthesized the two hydroxyl Geraniols of 6,7-.Present research for epoxide hydrolase, mainly concentrate on the optimization of original strain culture condition is lived with increase yield of enzyme or raising enzyme, also some investigator adopts genetic engineering technique and protein engineering to improve the stereoselectivity of epoxide hydrolase.
The present invention derives from Aspergillus usamii (Aspergillus usamii) E001 bacterial strain and has the epoxide hydrolase enzymic activity, and a kind of novel B type epoxide hydrolase (Aus EH-B) gene mature peptide cDNA clone and the prokaryotic expression of sequence are provided.Genetically engineered epoxide hydrolase purity is high, do not contain other proteolytic enzyme, and stereoselectivity is stronger; Escherichia expression system has efficient intracellular expression recombinant protein, with low cost, high productivity, the advantage such as simple to operate; And because general substrate is organic solvent, the enzyme molecule is subjected to the concentration of substrate restraining effect, and intracellular expression Host Strains coating forms natural protective barrier, more is conducive to carry out under high concentration of substrate biocatalysis, adapts to industrialization production requirements; The present invention utilizes the restructuring epoxide hydrolase to prepare the cinnamic research of chiral epoxy and has no report.
Summary of the invention
The clone and the prokaryotic expression that the purpose of this invention is to provide the novel B class epoxide hydrolase gene mature peptide cDNA sequence of a kind of Aspergillus of coming from usamii E001, utilize the restructuring epoxide hydrolase to prepare the cinnamic method of chiral epoxy, for the industrialization production of this epoxide hydrolase lays the foundation.According to the Subcellular Localization of enzyme and the specificity of substrate EH is divided into soluble epoxide hydrolase (sEH), microsome epoxide hydrolase (mEH), neotonin epoxide hydrolase (JhEH), the slack enzyme of cholesterol epoxide water (ChEH), hydroxyl epoxy olefin(e) acid lytic enzyme (hepoxilinhydrolase), leukotriene A 4 hydrolase (LAH) and seven subfamilies of limonene lytic enzyme (LEH).Bioinformatic analysis shows that a kind of novel epoxy compound lytic enzyme that derives from Aspergillus usamii E001 bacterial strain belongs to microsome epoxide hydrolase (mEH) class, belongs to a class novel epoxy compound lytic enzyme among the aspergillus EH.Owing to only being 56% with studying more A.Niger LCP521EH homology, with this enzyme called after Aus EH-B, its corresponding unnamed gene is Aus EH-B.Aus EH-B has higher catalytic activity and chiral selectivity, in the chiral epoxy compound is produced larger suitability for industrialized production and application potential and economic worth is arranged.
A.usamii E001 bacterial strain is by Southern Yangtze University screening and preservation, in the Vol.31 at " food and fermentation industries " magazine in 2005, and No.4, Pg.50 is open, and the inventor promises to undertake that this bacterial strain provided to the public in 20 years.
Technical scheme of the present invention: a kind of novel category-B epoxide hydrolase (Aus EH-B) that is derived from A.usamii E001, its gene (Aus EH-B) mature peptide cDNA nucleotides sequence is classified SEQ ID NO:1 as.
Described Aus EH-B aminoacid sequence of being derived by mature peptide cDNA sequence is SEQ ID NO:2.
The activity determination method of described restructuring Aus EH-B:
In the 1.5mLEP pipe, respectively add the suitable recombinase bacterium liquid of 0.85mL potassium phosphate buffer (pH7.0) dilution, at 30 ℃ of lower pre-temperature 5min, then respectively add 150 μ L200mM epoxy chloropropane and clock immediately behind the reaction 20min.Get the 1mL conversion fluid and add an amount of anhydrous sodium sulphate, after the vibration centrifugal (12000r/min, 5min), get supernatant liquor and carry out the chirality gas phase analysis.Enzyme unit definition alive: under 30 ℃, the required enzyme amount of per minute catalysis 1 μ moL racemation epoxy chloropropane is defined as an enzyme activity unit (U), and the epoxide hydrolase enzyme activity represents (U/mg) with every milligram of contained enzyme activity unit of dry mycelium.
The clone of described Aus EH-B mature peptide cDNA sequence and the method for expression are as follows:
(1) then the A.usamii E001 gene cDNA sequence from having cloned carries out opening code-reading frame analysis and signal peptide prediction to this sequence, and according to a pair of Auele Specific Primer of consequence devised of prediction, primer sequence is as follows:
AuEHB-F:5 '-
GAATTCATGGCACTCGCTTACAGCAA-3 ' contains EcoR I restriction enzyme site;
AuEHB-R: 5 '-
GCGGCCGCTCATTTTCTACCAGCCCATAC-3 ' contains Not I restriction enzyme site;
Extract total RNA of A.usamii E001, carry out RT-PCR according to the specification sheets among RNA PCR Kit (AMV) Ver.3.0 of TaKaRa production: article one chain that carries out the synthetic cDNA of reverse transcription take Oligo dT-Adaptor Primer as primer; Carry out take M13Primer M4 and AuEHB-F as primer first round PCR (94 ℃, 2min; 94 ℃, 30s, 51 ℃, 30s, 72 ℃, 80s, 30 circulations; 72 ℃, 10min); Take the PCR product of the first round carry out as template, AuEHB-F and AuEHB-R as primer second take turns PCR (94 ℃, 2min; 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 70s, 30 circulations; 72 ℃, 10min); With two-wheeled PCR product with 1% agarose gel electrophoresis analysis, 250bp DNA LadderMarker does contrast, be connected with purpose band rubber tapping recovery and with pUCm-T, transform JM109 and obtain recombinant plasmid pUCm-T-AusEH-B, after enzyme is cut evaluation, serve Hai Shenggong and carry out sequencing, obtain Aus EH-B mature peptide cDNA sequence.
(2) pUCm-T-AusEH-B that sequencing result is correct and pET-28-a-c (+) plasmid all carry out double digestion with EcoR I and Not I, the enzyme that rubber tapping is reclaimed is cut product and is connected under the effect of T4DNA ligase enzyme, obtain recombinant plasmid pET-28a-AusEH-B, and recombinant plasmid is carried out sequencing.
(3) structure, expression, product purification and the determination of activity of E.coli Rosetta (DE3)/EH-B: with pET-28-a-AusEH-B Transformed E .coli Rosetta (DE3), containing paraxin and the Kna resistant panel is screened transformant.Recombinant plasmid EcoR I and Not I enzyme are cut checking.Choosing positive transformant contains in the LB substratum of Kna/ chlorampenicol resistant in 2mL, 37 ℃ of shaking culture are spent the night, inoculum size with 1% is transferred in the 30mL same medium, 37 ℃ of shaking culture are to logarithmic growth mid-term (OD600 is 0.6～0.8), add people IPTG to final concentration be 0.5mmol/L, induce and spend the night.Simultaneously not induce engineering bacteria and to contain the negative contrast of bacterial strain of empty plasmid.After inducing end, collect thalline and lyophilize, it is active to utilize Chiral gas chromatography to measure epoxide hydrolase.
Beneficial effect of the present invention: the purpose of this invention is to provide clone and the prokaryotic expression of the novel B class epoxide hydrolase gene mature peptide cDNA sequence of a kind of Aspergillus of coming from usamii E001, utilize the restructuring epoxide hydrolase to prepare the cinnamic method of chiral epoxy.Bioinformatic analysis shows that this epoxide hydrolase belongs to the category-B of this fermentoid, so called after Aus EH-B, its corresponding unnamed gene is Aus EH-B.Produce the successful structure of epoxide hydrolase recombinant bacterium, through gas phase as a result detection display higher enzymatic properties and enantio-selectivity.Therefore, in the chiral separation dynamics research of epoxy chloropropane, play an important role.The present invention has established theoretical basis for the industrialization production of this enzyme, and larger suitability for industrialized production and application potential and economic worth are arranged, and has also established theoretical basis for the research of other bacterial strain novel epoxy compound lytic enzymes.
Description of drawings
Fig. 1: the structure synoptic diagram of recombinant plasmid pET-28a-AusEH-B
Fig. 2: the SDS-PAGE electrophorogram of restructuring E.coli Rosetta (DE3)/Aus EH-B
Embodiment
Below in conjunction with specific embodiment, further set forth working method of the present invention.But these embodiment only are used for describing the present invention in detail, limit the scope of the invention and be not used in.
The clone of embodiment 1 A.usamii E001EH-B mature peptide cDNA sequence
Extract total RNA of A.usamii E001, carry out RT-PCR according to the specification sheets among RNA PCR Kit (AMV) Ver.3.0 of TaKaRa production: article one chain that carries out the synthetic cDNA of reverse transcription take Oligo dT-Adaptor Primer as primer; Carry out take M13Primer M4 and AuEHB-F as primer first round PCR (94 ℃, 2min; 94 ℃, 30s, 51 ℃, 30s, 72 ℃, 80s, 30 circulations; 72 ℃, 10min); Take the PCR product of the first round carry out as template, AuEHB-F and AuEHB-R as primer second take turns PCR (94 ℃, 2min; 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 70s, 30 circulations; 72 ℃, 10min); With two-wheeled PCR product with 1% agarose gel electrophoresis analysis, 250bp DNA LadderMarker does contrast, be connected with purpose band rubber tapping recovery and with pUCm-T, transform JM109 and obtain recombinant plasmid pUCm-T-AusEH-B, after enzyme is cut evaluation, serve Hai Shenggong and carry out sequencing, obtain Aus EH-B mature peptide cDNA sequence.
The pUCm-T-AusEH-B that sequencing result is correct and pET-28-a-c (+) plasmid all carry out double digestion with EcoR I and Not I, the enzyme that rubber tapping is reclaimed is cut product and is connected under the effect of T4DNA ligase enzyme, obtain recombinant plasmid pET-28a-AusEH-B, and recombinant plasmid is carried out sequencing.
Structure, expression, product purification and the determination of activity of E.coli Rosetta (DE3)/EH-B: with pET-28-a-AusEH-B Transformed E .coli Rosetta (DE3), containing paraxin and the Kna resistant panel is screened transformant.Recombinant plasmid EcoR I and NotI enzyme are cut checking.Choosing positive transformant contains in the LB substratum of Kna/ chlorampenicol resistant in 2mL, 37 ℃ of shaking culture are spent the night, inoculum size with 1% is transferred in the 30mL same medium, 37 ℃ of shaking culture are to logarithmic growth mid-term (OD600 is 0.6～0.8), add people IPTG to final concentration be 0.5mmol/L, induce and spend the night.Simultaneously not induce engineering bacteria and to contain the negative contrast of bacterial strain of empty plasmid.After inducing end, collect thalline and lyophilize.Molecular weight through SDS-PAGE electrophoresis showed restructuring Aus EH-B is 43kDa; Measure the epoxide hydrolase activity and reach 750U/mg, chirality gas phase post is analyzed rEH (R)-epoxy chloropropane is had good stereoselectivity, produces (S)-epoxy chloropropane enantiomeric excess value and reaches 99%, and productive rate is 19.8%.
Claims (2)
1. novel B class epoxide hydrolase that derives from Aspergillus usamii E001, its cDNA sequence and aminoacid sequence are respectively SEQ IDNO:1 and SEQ IDNO:2.
2.A.usamii the method for E001 novel epoxy compound lytic enzyme B gene order Cloning and Expression:
(1) then the A.usamii E001 gene cDNA sequence from having cloned carries out opening code-reading frame analysis and signal peptide prediction to this sequence, and according to a pair of Auele Specific Primer of consequence devised of prediction, primer sequence is as follows:
AuEHB-F:5 '-
GAATTCATGGCACTCGCTTACAGCAA-3 ' contains EcoR I restriction enzyme site;
AuEHB-R:5 '-
GCGGCCGCTCATTTTCTACCAGCCCATAC-3 ' contains Not I restriction enzyme site;
Extract total RNA of A.usamii E001, carry out RT-PCR according to the specification sheets among RNA PCR Kit (AMV) Ver.3.0 of TaKaRa production: article one chain that carries out the synthetic cDNA of reverse transcription take Oligo dT-Adaptor Primer as primer; Carry out take M13Primer M4 and AuEHB-F as primer first round PCR (94 ℃, 2min; 94 ℃, 30s, 51 ℃, 30s, 72 ℃, 80s, 30 circulations; 72 ℃, 10min); Take the PCR product of the first round carry out as template, AuEHB-F and AuEHB-R as primer second take turns PCR (94 ℃, 2min; 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 70s, 30 circulations; 72 ℃, 10min); With two-wheeled PCR product with 1% agarose gel electrophoresis analysis, 250bp DNA LadderMarker does contrast, be connected with purpose band rubber tapping recovery and with pUCm-T, transform JM109 and obtain recombinant plasmid pUCm-T-AusEH-B, after enzyme is cut evaluation, serve Hai Shenggong and carry out sequencing, obtain Aus EH-B mature peptide cDNA sequence;
(2) pUCm-T-AusEH-B that sequencing result is correct and pEH-28-a-c (+) plasmid all carry out double digestion with EcoR I and Not I, the enzyme that rubber tapping is reclaimed is cut product and is connected under the effect of T4DNA ligase enzyme, obtain recombinant plasmid pEH-28-a-AusEH-B, and recombinant plasmid is carried out sequencing;
(3) structure, expression, product purification and the determination of activity of E.coli Rosetta (DE3)/EH-B: with pEH-28-a-AusEH-B Transformed E .coli Rosetta (DE3), containing paraxin and the Kna resistant panel is screened transformant; Recombinant plasmid EcoR I and Not I enzyme are cut checking; Choosing positive transformant contains in the LB substratum of Kna/ chlorampenicol resistant in 2mL, 37 ℃ of shaking culture are spent the night, inoculum size with 1% is transferred in the 30mL same medium, 37 ℃ of shaking culture are to logarithmic growth mid-term (OD600 is 0.6～0.8), add people IPTG to final concentration be 0.5mmol/L, induce and spend the night; Simultaneously not induce engineering bacteria and to contain the negative contrast of bacterial strain of empty plasmid; After inducing end, collect thalline and lyophilize, it is active to measure the Chiral gas chromatography epoxide hydrolase.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450641A (en) * | 2014-09-17 | 2015-03-25 | 华东理工大学 | Epoxide hydrolase as well as encoding gene and application thereof |
| CN104531728A (en) * | 2014-12-10 | 2015-04-22 | 江南大学 | Cloning and heterologous expressing methods of epoxide hydrolase gene of aspergillus usaanii |
| CN104531630A (en) * | 2014-12-10 | 2015-04-22 | 江南大学 | Design and preparation method of epoxide hydrolase mutant |
| CN106244637A (en) * | 2016-10-31 | 2016-12-21 | 江南大学 | A kind of method that double-enzyme catalysis racemation epoxy vinylbenzene prepares (R) benzoglycols |
| CN109486789A (en) * | 2018-12-29 | 2019-03-19 | 江南大学 | A kind of Kidney bean epoxide hydrolase mutant that stereoselectivity improves |
| CN109609479A (en) * | 2018-12-29 | 2019-04-12 | 江南大学 | A mutant of Aspergillus usamia epoxide hydrolase with improved enantioselectivity |
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2012
- 2012-12-24 CN CN 201210562633 patent/CN102994470A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450641A (en) * | 2014-09-17 | 2015-03-25 | 华东理工大学 | Epoxide hydrolase as well as encoding gene and application thereof |
| CN104450641B (en) * | 2014-09-17 | 2018-03-06 | 华东理工大学 | A kind of epoxide hydrolase and its encoding gene and application |
| CN104531728A (en) * | 2014-12-10 | 2015-04-22 | 江南大学 | Cloning and heterologous expressing methods of epoxide hydrolase gene of aspergillus usaanii |
| CN104531630A (en) * | 2014-12-10 | 2015-04-22 | 江南大学 | Design and preparation method of epoxide hydrolase mutant |
| CN106244637A (en) * | 2016-10-31 | 2016-12-21 | 江南大学 | A kind of method that double-enzyme catalysis racemation epoxy vinylbenzene prepares (R) benzoglycols |
| CN106244637B (en) * | 2016-10-31 | 2019-05-17 | 江南大学 | A kind of method that double-enzyme catalysis racemation epoxy vinylbenzene prepares (R)-benzoglycols |
| CN109486789A (en) * | 2018-12-29 | 2019-03-19 | 江南大学 | A kind of Kidney bean epoxide hydrolase mutant that stereoselectivity improves |
| CN109609479A (en) * | 2018-12-29 | 2019-04-12 | 江南大学 | A mutant of Aspergillus usamia epoxide hydrolase with improved enantioselectivity |
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