CN107164342A - A kind of epoxide hydrolase in Kidney bean source and its application - Google Patents

A kind of epoxide hydrolase in Kidney bean source and its application Download PDF

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CN107164342A
CN107164342A CN201710474254.0A CN201710474254A CN107164342A CN 107164342 A CN107164342 A CN 107164342A CN 201710474254 A CN201710474254 A CN 201710474254A CN 107164342 A CN107164342 A CN 107164342A
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epoxide hydrolase
coli
pveh2
seq
amino acid
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邬敏辰
李闯
宗迅成
王瑞
李剑芳
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Jiangnan University
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/001—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by metabolizing one of the enantiomers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y303/00—Hydrolases acting on ether bonds (3.3)
    • C12Y303/02—Ether hydrolases (3.3.2)

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Abstract

The invention discloses a kind of epoxide hydrolase in Kidney bean source and its application, belong to biological technical field.The invention discloses a kind of epoxide hydrolase gene (pveh2) and corresponding amino acid sequence for deriving from legume Kidney bean (Phaseolus vulgaris)；Prepare restructuring epoxide hydrolase is expressed there is provided recombinant plasmid pET28a (+) pveh2 and genetically engineered E.coli BL21 (DE3) containing the gene/pET28a (+) pveh2, and using the bacterial strain；There is provided a kind of method for institute's producing enzyme progress living things catalysis of being fermented using the recombinant bacterial strain built in the present invention, the pure R type Styryl oxides (ee of mapping is prepared>99%), also, its ultimate yield reaches 49.3%, close to theoretical value 50%.

Description

A kind of epoxide hydrolase in Kidney bean source and its application
Technical field
The present invention relates to a kind of epoxide hydrolase in Kidney bean source and its application, belong to biological technical field,.
Background technology
The epoxides of chiral purity and its corresponding hydrolysate vicinal diamines are considered as that a class has many of high added value Function chiral building block, is widely used in the synthesis of chiral drug and fine chemicals.However, in some chiral epoxides In preparation process, the reaction result of the chemical method such as Sharpless asymmetric Epoxidations, Jacobsen epoxidations is often unable to reach Desired chiral purity, the enantiomer excess rate of product (referring to the single configuration epoxides remained and the glycol of generation) (Enantiomeric excess, ee) value generally only has the catalyst such as 30%~80%, and used heavy metal also to give ring Bring huge threat in border.In recent years, the Kinetic Resolution and enantiomer regression nature Hydrolyze method that biology enzyme is mediated because of it more Single-minded stereoselectivity and the visual field for gradually coming into people, epoxide hydrolase (Epoxide hydrolase, EH, EC 3.3.2.x it is exactly) the important enzyme of one type, the enzyme can make hydrone solid selection with the ring opening hydrolysis of catalysis epoxidation thing Property add on the oxygen-containing three-membered ring of epoxides, generate corresponding 1,2- glycol, be a kind of typical α/β folded form hydrolysis Enzyme.EH wide material sources, react without co-factor, substrate spectrum be wide, reaction condition is gentle, it is considered to be one kind have much potentiality to be exploited and The biocatalyst of researching value.
At present, people are found that more than 300 kinds of EH from animal, plant and microorganism, and fixed according to the subcellular fraction of enzyme The specificity of position and substrate is divided into seven subfamilies, wherein soluble epoxide enzyme be the more EH of current research it One.Bellucci etc. have studied the stereochemical properties for being present in microsome epoxide hydrolase in mammal body, utilize The alkyl-substituted Styryl oxide derivatives of some β of its catalyzing hydrolysis, as a result obtain corresponding R types glycol.Butterfly etc. is helped from space A kind of new epoxide hydrolase AuEH2 is successfully excavated out in U.S. aspergillus, finds it to R type Styryl oxides (Styrene Oxide, SO) height of the compatibility compared with S types, it is believed that the enzyme racemic (rac-) SO chiral resolution reaction in have it is very big Application potential.Than above-mentioned several sources, plant EH is then more prone to obtain and raw material is also relatively inexpensive.Currently including Soybean (Glycine max), pigeonpea (Cajanus cajan), Medicago truncatula (Medicago truncatula), Ben Shi cigarette Coding EH ORFs is determined in Plant Genomes such as careless (Nicotiana benthamiana), wherein studying most deep That enter is potato EH, and its crystal structure was just obtained by the parsing of X ray diffracting spectrum early in 2006.Recently, Xu Jian The two kinds of EH (MbEHA, MbEHB) found with seminar from mung bean (Vigna radiata) are with to nitro Styryl oxide The enantioselectivity of complementation is shown in the case of for substrate, the concern of related scholar is caused.
Although the epoxide hydrolase of hitherto reported is respectively provided with the activity of certain asymmetric hydrolysis epoxides, still Only more than 40 kind EH are directed to one or more epoxides and show high enantioselectivity (i.e. mapping selection rate E values at present> 30), as far as we know, in addition to the potato EH that foreign scholar studies, the EH of plant origin is there is no at present efficiently to be torn open Rac-SO is divided to prepare high chiral pure R-SO, therefore, being excavated by molecular biology method can express with high selectivity Its gene constructed expression system of EH, and be applied to the research work such as the preparation of optical voidness epoxides there is Important Economic And social benefit.
The content of the invention
First purpose of the present invention is to provide a kind of epoxide hydrolase PvEH2, such as shown in (a) or (b)：
(a) amino acid sequence is as shown in SEQ ID NO.2；
(b) amino acid sequence in (a) passes through substitution, lacks or adds one or several amino acid and with epoxidation Thing hydrolysing activity as protein derived from (a).
Second object of the present invention is to provide a kind of gene for encoding the epoxide hydrolase PvEH2, gene life Entitled pveh2, the nucleotide sequence of the gene is as shown in SEQ ID NO.1, sequence 951bp, encodes 316 amino Acid.
Third object of the present invention is to provide a kind of recombination bacillus coli for being used to produce R- Styryl oxides, is with big Enterobacteria is host, the epoxide hydrolase shown in expression SEQ ID NO.2.
In one embodiment of the invention, the recombination bacillus coli with pET-28a (+) be carrier.
In one embodiment of the invention, the recombination bacillus coli with E.coli BL21, E.coli JM109, E.coli DH5 α or E.coli TOP10 are host.
In one embodiment of the invention, the recombination bacillus coli is built as follows：(1) expand Sequence shown in SEQ ID NO.1, restriction enzyme Nde I and Xho I restriction enzyme site are introduced at gene two ends；(2) use State two kinds of restriction endonucleases and handle sequence and expression vector pET-28a (+) shown in SEQ ID NO.1 respectively, and by above-mentioned enzymolysis, digestion Product is stayed overnight in 17 DEG C of connections, obtains recombinant expression plasmid pET-28a (+)-pveh2；(3) by recombinant plasmid transformed to large intestine bar In bacterium BL21 (DE3) competent cell, you can obtain recombinant bacterial strain E.coli BL21 (DE3)/pET28a (+)-pveh2.
Fourth object of the present invention is to provide a kind of bioconversion method of R- Styryl oxides, and methods described is application Epoxide hydrolase shown in SEQ ID NO.2, catalysis racemation epoxy vinylbenzene production chiral purity (ee>99%) R- rings Oxygen vinylbenzene.
In one embodiment of the invention, methods described be will restructuring epoxide hydrolase PvEH2 according to 0.1~ The ratio of 0.5U/mg substrates be dissolved in concentration and be during pH is 7.0~8.0 cushioning liquid be incubated at 20~35 DEG C after 10min plus Enter racemation epoxy vinylbenzene, and carry out Kinetic Resolution reaction under the same conditions.
In one embodiment of the invention, methods described be will restructuring epoxide hydrolase PvEH2 according to The ratio of 0.017U/mg substrates is dissolved in the NaH that the pH that concentration is 100mmol/l is 7.0~7.22PO4-Na2HPO4Buffering is molten In liquid system, it is incubated at 20~35 DEG C after 10min and adds racemation epoxy vinylbenzene, and enter action edge under the same conditions Learn resolution reaction.
In one embodiment of the invention, cell of the epoxide hydrolase with enzyme liquid, with bioactivity, The form application of solid polypeptide formulation.
In one embodiment of the invention, to express SEQ ID NO, the restructuring of epoxide hydrolase shown in 2 is big Enterobacteria carries out bioconversion.
In one embodiment of the invention, methods described is according to 1% after described recombination bacillus coli is activated (v/v) inoculum concentration is forwarded to the LB culture mediums containing 0.1mg/ml kanamycin sulfates, and in the OD of fermentation system600Value reaches Final concentration of 0.05~1.0mmol/l IPTG is added during to 0.5~1.0,6~7h, centrifugation, collection are then cultivated at 25 DEG C Thalline, thalline is used for the bioconversion of R- Styryl oxides.
In one embodiment of the invention, methods described is will to contain restructuring epoxides water by induced expression Thalline or the clasmatosis of enzyme are solved, the pure enzyme of supernatant or supernatant after purified is dissolved in the pH that concentration is 100mmol/l For 7.2 NaH2PO4-Na2HPO4In (100mM, pH 7.2) buffer solution system, then it is incubated after 10min and adds at 25 DEG C Racemation epoxy vinylbenzene, carries out Kinetic Resolution reaction.
The present invention also provides the recombination bacillus colis of the production R- Styryl oxides in food, biology, chemical field Using.
Beneficial effects of the present invention：(1) present invention discover that a kind of epoxide hydrolase gene pveh2, its nucleotides sequence Row are as shown in SEQ ID No.1, the gene code a kind of new epoxide hydrolase PvEH2, its amino acid sequence such as SEQ Shown in ID No.2；(2) present invention finds the recombinase of epoxide hydrolase gene expression as shown in SEQ ID No.1 New opplication；Compared with the epoxide hydrolase in other sources, the selectivity of catalysis is stronger；(3) restructuring that the present invention is built is big Enterobacteria carries out bioconversion as catalyst, can be using racemation epoxy vinylbenzene as substrate, through enantioselective hydrolysis Produce chiral purity (ee>99%) R- Styryl oxides, yield is improved extremely by originally reported the 45.6% of potato EH 49.3%.
Brief description of the drawings
Fig. 1 is recombinant expression carrier pET-28a (+)-pveh2 of present invention physical map.
Fig. 2 is 12%SDS-PAGE collection of illustrative plates, wherein, M is molecular weight of albumen Marker；1 is E.coli BL21 (DE3)；2 For without the E.coli BL21 (DE3) of induction/pET28a (+)-pveh2；3 be to be induced through 0.2mmol/l IPTG E.coli BL21(DE3)//pET28a(+)-pveh2。
Fig. 3 is hydrolysis process of the restructuring epoxide hydrolase to racemation epoxy vinylbenzene；Wherein ,-▼-be Substrate SO enantiomeric excess rate ees；- ▲-is conversion ratio c；- ●-it is S-SO；—■—R-SO.
Embodiment
The present invention is further described with reference to specific embodiment：
The concentration of each configuration Styryl oxide utilizes chiral gas phase capillary chromatograph (CYCLOSIL-B, Agilent company) Detected, the calculating of assay method, conversion ratio and ee values is referring to disclosed in 2016《New Kidney bean epoxide hydrolase Heterogenous expression and mapping regression nature catalysis characteristics》.
The measure of epoxide hydrolase：The thalline that taking 250 μ l PvEH2 crude enzyme liquids or pure enzyme liquid or expression has PvEH2 hangs Supernatant liquid is added in 225 μ l sodium phosphate buffers and mixed, and is placed at 25 DEG C and preheats 10min, adds 25 μ l rac-SO (female Liquid concentration is 200mmol/l, is dissolved in methanol), the initial concentration for making substrate SO in catalyst system and catalyzing is 10mmol/l, during reaction 200 μ l are taken to be extracted into 1ml ethyl acetate (n-hexyl alcohol containing 1mmol/l is as internal standard compound) from system per 10min, upper strata is organic Detected using gas chromatography.Enzyme activity is defined as the epoxy benzene second that epoxide hydrolase per minute is catalyzed 1.0 μm of ol Enzyme amount required for alkane hydrolyzes or generated 1.0 μm of ol benzoglycols is 1U.
The clone of the epoxide hydrolase gene of embodiment 1 and the structure of expression plasmid
The total serum IgE (using TRIZOL kits, Invitrogen companies) of Kidney bean is extracted, is carried out with reference to kit specification Operation.RT-PCR kit PCR PrimeScriptTM RT reagent Kit of the transcriptive process,reversed with reference to Takara companies With gDNAEraser (Perfect Real Time) specification, the cDNA then obtained using reverse transcription is template, upstream and downstream Primer sequence is respectively SEQ ID NO:3 and SEQ ID NO.4 (introduce Nde I and Xho I digestion with restriction enzyme in primer Site, primer is synthesized by Shanghai Sheng Gong Co., Ltds), pveh2 full-length genes, its nucleotides are expanded using conventional PCR method Sequence such as SEQ ID NO.1, PCR reaction conditions are：94 DEG C of pre-degenerations 2min, 94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C are prolonged 60s is stretched, is circulated 30 times, then extend 10min at 72 DEG C.PCR primer 1.0% agarose nucleic acid gel electrophoresis detection and purifying Purpose fragment is reclaimed with DNA glue reclaims kit (Shanghai life work) afterwards, recovery product and expression vector pET-28a (+) are through identical Restriction enzyme processing after, use T4DNA ligase connects both overnight at 17 DEG C, obtains recombinant expression carrier pET- 28a (+)-pveh2, and be conducted into E.coli BL21 (DE3) competent cell, it is coated on containing 0.1mg/ml sulphur On the LB flat boards of sour kanamycins, 37 DEG C of culture 16h, picking positive colony is transferred in the LB containing identical kanamycins content Shaken cultivation in fluid nutrient medium, extracts plasmid and double digestion identification, pveh2 sequences such as sequence chart is carried out with Nde I and Xho I Shown in SEQ ID No.1, the amino acid sequence derived is as shown in SEQ ID No.2.
The epoxide hydrolase PvEH2 of embodiment 2 preparation
Genetically engineered E.coli BL21 (DE3) in embodiment 1/pET28a (+)-pveh2 is inoculated in the training of LB liquid Support in base (kanamycin sulfate containing 0.1mg/ml), 37 DEG C of 10~16h of culture (optimization, be 12h), then again with 1% (v/v) Inoculum concentration transfer in the LB culture mediums containing identical component continue expand culture, as cell concentration OD600Value reaches 0.8~ 1.0 (optimization, IPTG is added when being 0.8) to its final concentration of 0.2mmol/l, 4~9h of Fiber differentiation (optimizations at 25 DEG C , it is 7h) 8000r/min centrifugations 5min at 4 DEG C afterwards, collects bacterial sediment, can be sent out per 100ml culture mediums according to above-mentioned steps Ferment obtains 0.5~0.8g wet thallus.The SDS-PAGE analysis results of somatic cells are as shown in Fig. 2 apparent point of destination protein Son is about 37.9kDa..
Applications of the epoxide hydrolase PvEH2 of embodiment 3 in chiral epoxy vinylbenzene is prepared
By the wet thallus obtained in embodiment 2 through centrifugation according to every g correspondence 10ml NaH2PO4-Na2HPO4(100mM,pH 7.2) ratio of buffer solution is suspended again, bacterium cell suspension directly as biological enzyme agent, using racemation epoxy vinylbenzene as Substrate is converted, and prepares R type Styryl oxides.Concrete operations are：2.5ml bacterium cell is added in 10ml Ep plastic tubes to hang Liquid (the dense 50mg/ml of bacterium, vigor is 0.04U/ml), 2.25ml buffer solutions, 0.25ml concentration are 200mmol/l racemation epoxy Vinylbenzene (in the final concentration of 10mmol/l of catalyst system and catalyzing), converts 120min under conditions of being vibrated at 25 DEG C, is eventually adding phase The ethyl acetate of same volume is extracted and terminating reaction.
PvEH2 is as shown in Figure 3 for the reaction process of Styryl oxide under these conditions.After testing, when reacting 40min eesMore than 70%, conversion ratio reaches 42%；Conversion ratio is up to 45% when reacting 80min；Ee is obtained when reacting complete>99% R Type Styryl oxide, yield reaches 49.3% (theoretical yield of chiral resolution is 50%).
Reference examples 1
PvEH2 enzymes are prepared as described in Example 2, difference is, as fermentation system OD600When value reaches 1.5, at 30 DEG C During lower addition 0.5mmol/l IPTG Fiber differentiation 6h, the restructuring PvEH2 produced is basic with the inclusion bodies without the enzyme activity In the presence of.
Reference examples 2
As described in Example 3 using PvEH2 is recombinated to racemation epoxy vinylbenzene progress bioconversion, difference is, Conversion process controls temperature to be 35 DEG C, is influenceed by the stronger autolysis of substrate, causes when reacting 70~75min, S-SO hydrolysis is complete, obtains 97.6%ee R-SO, its ultimate yield is only 32.4%.In addition, restructuring PvEH2 is in 40 DEG C of conditions After lower holding 1h, its remaining vigor is only the 8.3% of protoenzyme.
Reference examples 3
The effect that the EH enzyme hydrolysis in other sources prepares R-SO is as shown in table 1.
The epoxide hydrolase of the separate sources of table 1 retains R-SO effect in rac-SO Kinetic Resolutions
Above-described is only present pre-ferred embodiments, and the scope of the present invention, above-described embodiment are not limited with this Can also further it be optimized.I.e. every letter made according to apllied claims of the invention and description Single, equivalent change and modification, are all the scope of the claims of the present patent application.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>A kind of epoxide hydrolase in Kidney bean source and its application
<160> 4
<170> PatentIn version 3.3
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atggaataca tagtacacag aacagtggaa gtcaatggca tcaaaatgca tgttgcagag 60
aaaggagagg gtcctgccgt cttgttcctc catggcttcc ctgaactatg gtacacctgg 120
cgccaccaga ttcttgatct cagctcccga ggatatcacg cggttgcacc agatctacga 180
ggctacggtg acacagaggc accagcttcc atgagcagct acagctgctt tgacatagtg 240
ggtgatctgg ttgcgcttat agaccttctg ggtgttgatc aagtcttcct tgtggctcat 300
gactggggtg ccatcatagg ttggtacctc tgcatgtttc gccccgacag agtcaaggcc 360
tatgtctgcc tcagtgtgcc tttctggccc agaaacccaa aggtgaagcc cgttgatgcc 420
atgcgggccc tatacggaga tgactactat atctgcagat tccaggaggc aggaaaggca 480
gaaggtgagt tagccaaaaa tagcactgaa gaggtattga aaaaacttct gacaaatcgc 540
acacctgggc caccaatctt gcaaaaagaa ggaatgggtt caaatctgaa cacttcaatg 600
ccccttcctt cttggctttc actccaagat ctcaagtact atgcttccaa atttgaaaag 660
acaggcttca ctggaggcct caactactac agaaatatca acttaaattg ggagctcaca 720
gcaccttgga ctggagcaca ggtcaaagtt ccagtgaagt tcattactgg tgatttggat 780
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35 40 45
Ser Arg Gly Tyr His Ala Val Ala Pro Asp Leu Arg Gly Tyr Gly Asp
50 55 60
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Gly Asp Leu Val Ala Leu Ile Asp Leu Leu Gly Val Asp Gln Val Phe
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Leu Val Ala His Asp Trp Gly Ala Ile Ile Gly Trp Tyr Leu Cys Met
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Phe Arg Pro Asp Arg Val Lys Ala Tyr Val Cys Leu Ser Val Pro Phe
115 120 125
Trp Pro Arg Asn Pro Lys Val Lys Pro Val Asp Ala Met Arg Ala Leu
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Tyr Gly Asp Asp Tyr Tyr Ile Cys Arg Phe Gln Glu Ala Gly Lys Ala
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Glu Gly Glu Leu Ala Lys Asn Ser Thr Glu Glu Val Leu Lys Lys Leu
165 170 175
Leu Thr Asn Arg Thr Pro Gly Pro Pro Ile Leu Gln Lys Glu Gly Met
180 185 190
Gly Ser Asn Leu Asn Thr Ser Met Pro Leu Pro Ser Trp Leu Ser Leu
195 200 205
Gln Asp Leu Lys Tyr Tyr Ala Ser Lys Phe Glu Lys Thr Gly Phe Thr
210 215 220
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Ala Pro Trp Thr Gly Ala Gln Val Lys Val Pro Val Lys Phe Ile Thr
245 250 255
Gly Asp Leu Asp Ser Val Tyr Thr Ser Leu Gly Met Lys Asn Tyr Ile
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Glu Ser Gly Ala Phe Lys Lys Asp Val Pro Asn Leu Glu Glu Val Ile
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Val Gln Glu Gly Val Ala His Phe Asn Asn Gln Glu Ala Ala Glu Asp
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Val Ser Asn His Ile Tyr Asp Phe Ile Asn Lys Phe
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Claims (10)

1. a kind of epoxide hydrolase, it is characterised in that amino acid sequence is such as shown in (a) or (b)：
(a) amino acid sequence is as shown in SEQ ID NO.2；
(b) amino acid sequence in (a) passes through substitution, lacks or adds one or several amino acid and with epoxides water Solution activity as protein derived from (a).
2. encode the gene of epoxide hydrolase described in claim 1, it is characterised in that nucleotide sequence such as SEQ ID Shown in NO.1.
3. a kind of recombination bacillus coli, it is characterised in that using Escherichia coli as host, the epoxy shown in expression SEQ ID NO.2 Compound hydrolase.
4. recombination bacillus coli according to claim 3, it is characterised in that with pET-28a (+) for carrier.
5. recombination bacillus coli according to claim 3, it is characterised in that with E.coli BL21, E.coli JM109, E.coli DH5 α or E.coli TOP10 are host.
6. a kind of bioconversion method of R- Styryl oxides, it is characterised in that the epoxides shown in application SEQ ID NO.2 Hydrolase, catalysis racemation epoxy vinylbenzene production R- Styryl oxides.
7. method according to claim 6, it is characterised in that methods described be by the epoxide hydrolase according to It is during pH is 7.0~8.0 cushioning liquid, to be incubated at 20~35 DEG C that the ratio of 0.01~0.5U/mg substrates, which is dissolved in concentration, Racemation epoxy vinylbenzene is added after 10min, and carries out Kinetic Resolution reaction under the same conditions.
8. method according to claim 6, it is characterised in that the epoxide hydrolase is lived with enzyme liquid, with biology The cell of property or the form application of enzyme powder.
9. epoxide hydrolase described in claim 1 food, biology, chemical field application.
10. recombination bacillus coli described in claim 3 is in the application of fermentation arts.
CN201710474254.0A 2017-06-21 2017-06-21 A kind of epoxide hydrolase in Kidney bean source and its application Pending CN107164342A (en)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107937364A (en) * 2018-01-15 2018-04-20 江南大学 The Kidney bean epoxide hydrolase mutant that a kind of enantioselectivity improves
CN108374017A (en) * 2018-01-24 2018-08-07 中国科学院成都生物研究所 A kind of novel epoxidation of styrene enzyme and its function
CN108559737A (en) * 2018-01-15 2018-09-21 江南大学 A kind of Kidney bean epoxide hydrolase mutant that stereoselectivity improves
CN109355271A (en) * 2018-12-28 2019-02-19 江南大学 A kind of epoxide hydrolase and its application in ocean rhodotorula source
CN109486789A (en) * 2018-12-29 2019-03-19 江南大学 A kind of Kidney bean epoxide hydrolase mutant that stereoselectivity improves
CN109652354A (en) * 2018-12-28 2019-04-19 江南大学 A kind of recombination bacillus coli prepares (R)-to the method for chlorine Styryl oxide
CN109880876A (en) * 2018-12-28 2019-06-14 江南大学 A method of (R)-m-nitro ethylene glycol is prepared using Kidney bean epoxide hydrolase
CN114854714A (en) * 2022-05-27 2022-08-05 安徽工程大学 Kidney bean source epoxide hydrolase mutant, gene, vector, engineering bacterium, preparation method and application
CN115948487A (en) * 2022-07-18 2023-04-11 安徽工程大学 Method for preparing (R) -benzyl glycidyl ether by using epoxide enzyme fusion and application

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CN107937364A (en) * 2018-01-15 2018-04-20 江南大学 The Kidney bean epoxide hydrolase mutant that a kind of enantioselectivity improves
CN108559737A (en) * 2018-01-15 2018-09-21 江南大学 A kind of Kidney bean epoxide hydrolase mutant that stereoselectivity improves
CN108559737B (en) * 2018-01-15 2020-08-04 江南大学 Soybean epoxy hydrolase mutant with improved stereoselectivity
CN107937364B (en) * 2018-01-15 2020-03-24 江南大学 Kidney bean epoxide hydrolase mutant with improved enantioselectivity
CN108374017A (en) * 2018-01-24 2018-08-07 中国科学院成都生物研究所 A kind of novel epoxidation of styrene enzyme and its function
CN108374017B (en) * 2018-01-24 2021-05-25 中国科学院成都生物研究所 Novel styrene epoxidase and function thereof
CN109880876A (en) * 2018-12-28 2019-06-14 江南大学 A method of (R)-m-nitro ethylene glycol is prepared using Kidney bean epoxide hydrolase
CN109652354A (en) * 2018-12-28 2019-04-19 江南大学 A kind of recombination bacillus coli prepares (R)-to the method for chlorine Styryl oxide
CN109355271B (en) * 2018-12-28 2020-09-04 江南大学 Marine rhodotorula-derived epoxide hydrolase and application thereof
CN109355271A (en) * 2018-12-28 2019-02-19 江南大学 A kind of epoxide hydrolase and its application in ocean rhodotorula source
CN109486789A (en) * 2018-12-29 2019-03-19 江南大学 A kind of Kidney bean epoxide hydrolase mutant that stereoselectivity improves
CN114854714A (en) * 2022-05-27 2022-08-05 安徽工程大学 Kidney bean source epoxide hydrolase mutant, gene, vector, engineering bacterium, preparation method and application
CN115948487A (en) * 2022-07-18 2023-04-11 安徽工程大学 Method for preparing (R) -benzyl glycidyl ether by using epoxide enzyme fusion and application

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