CN108165538A - The Kidney bean epoxide hydrolase mutant that a kind of catalytic activity improves - Google Patents
The Kidney bean epoxide hydrolase mutant that a kind of catalytic activity improves Download PDFInfo
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- CN108165538A CN108165538A CN201810033882.XA CN201810033882A CN108165538A CN 108165538 A CN108165538 A CN 108165538A CN 201810033882 A CN201810033882 A CN 201810033882A CN 108165538 A CN108165538 A CN 108165538A
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- C12Y303/02—Ether hydrolases (3.3.2)
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Abstract
The invention discloses the Kidney bean epoxide hydrolase mutant that a kind of catalytic activity improves, and belong to genetic engineering and field of protein expression.The mutant of the present invention is on the basis of the amino acid shown in SEQ ID NO.2, by the valine mutation of the 106th into isoleucine.The mutant enzyme PvEH1 that shaking flask induction 10h of the present invention is obtainedV106ICatalytic activity is 234.75U/g, is 1.49 times of wild-type enzyme (157.15U/g).
Description
Technical field
The present invention relates to the Kidney bean epoxide hydrolase mutant that a kind of catalytic activity improves, and belong to genetic engineering and egg
White matter expresses field.
Background technology
Epoxide hydrolase (epoxide hydrolases, EHs, EC 3.3.2.-) be widely present in animal, plant,
In the natural environments such as microorganism, it can form phase by adding epoxides ring opening hydrolysis on a molecular water to epoxide ring
The glycol answered, participation of this process without co-factor and metal ion.Since epoxide hydrolase has in catalyzing hydrolysis
There are highly-solid selectively, reaction condition mild and the features such as environmental pollution is small, people increasingly pay attention to it in biology in recent years
The application value of catalyzing hydrolysis racemic epoxides.
Most of EHs can be divided into three classes according to the difference of catalytic mechanism：First, limonene -1,2- epoxides hydrolyzes
Enzyme family is represented as R.erythropLis EH (EC 3.3.2.8)；Second is that leukotriene A4Hydrolase；Third, α/β-folding epoxy
Compound hydrolase family.Most study is α/β-folding epoxides hydrolase family at present.It is fast with structure biology
Speed development, the structure of epoxide hydrolase to be parsed there are many oneself, such as：Radioactive soil bacillus epoxide hydrolase
Agrobacterium radiobacter AD1 EH (ArEH), aspergillus niger epoxide hydrolase Aspergillus niger
EH (AnEH), people epoxide hydrolase Homo sapiens sEH (HssEH), mouse epoxide hydrolase Mus msculus
She (MmsEH), mycobacterium tuberculosis epoxide hydrolase Mycobacterium tuberculosis EHA (MtEHA), with
And from potato epoxide hydrolase Solanum tuberosum EH (StEH) etc..
The catalyst mechanism of α/β-folding epoxides hydrolase family is generally divided into two steps：1) two tyrosine residues pass through
Oxygen atom on ammonia keyed engagement epoxy substrate ethylene oxide, then the asparagicacid residue nucleophillic attack in epoxide hydrolase
One of carbon atom on epoxide ring forms dihydric alcohol -ester-covalently bound dimer of enzyme；2) water of a molecule due to by
Activation to Asp/Glu-His residues generates an alkyl, so as to attack the covalent dimer formed before, releases ortho position two
Alcohol product, nucleopilic reagent must live again.
Although in recent years epoxide hydrolase due to without co-factor or metal ion when it is reacted participation, reaction item
The advantages that part is mild, environmentally friendly is in widespread attention on optically pure substrate and product is prepared, but in laboratory and
In plant-scale application, epoxide hydrolase limits its further development because of the defects of its enzyme activity is relatively low.Protein
The shortcomings that advantage of engineering is that above series of can be overcome to a certain extent.
Invention content
The invention solves first problem be to provide the epoxide hydrolase (PvEH1) that a kind of catalytic activity improves
Mutant.
The mutant is：
(a) protein of the amino acid sequence as shown in SEQ ID NO.1；
(b) hybridize under strict conditions with (a) amino acid sequence limited and encode with epoxide hydrolase activity
Amino acid sequence.
In one embodiment of the invention, the nucleotide sequence for encoding the gene of the mutant is SEQ ID
Sequence shown in NO.3.
The present invention also provides the methods for obtaining the mutant, are in the amino acid sequence base as shown in SEQ ID NO.2
On plinth, by the valine mutation of the 106th into isoleucine (V106I).
In one embodiment of the invention, with the recombination matter of the gene PvEH1 of carrying coding epoxide hydrolase
Grain is template, designs and synthesizes primer, and rite-directed mutagenesis is carried out by PCR, obtains carrying the recombination matter of the gene of encoding mutant body
Grain converts expressive host；Culture host is allowed to produce epoxide hydrolase mutant.
The present invention also provides a kind of genetic engineering bacterium for expressing the epoxide hydrolase mutant, the genetic engineerings
The construction method of bacterium includes the following steps：Using the recombinant plasmid of the gene PvEH1 of carrying coding epoxide hydrolase as template,
Primer is designed and synthesized, rite-directed mutagenesis is carried out by PCR, obtains carrying the recombinant plasmid of the gene of encoding mutant body, translation table
Up to host.
In one embodiment of the invention, the recombination of the gene PvEH1 for carrying coding epoxide hydrolase
Plasmid is pET28a (+)-pveh1, and expressive host is E.coli BL21 (DE3).
The present invention also provides a kind of application epoxide hydrolase mutant hydrolysis o-methyl-phenyl glycidol ethers
Method, be using the genetic engineering bacterium of the mutant or the expression mutant as catalyst, in phosphate buffer
(Na2HPO4-NaH2PO4, pH 7.0) in catalysis racemic o-methyl-phenyl glycidol ether (concentration of substrate 10mM).
The mutant naming method of the present invention：
Mutant is represented using " amino acid that Original amino acid position is replaced ".Such as V106I, the ammonia of position 106 is represented
Base acid is substituted for isoleucine Ile by the valine Val of parent's epoxide hydrolase, and the number of position corresponds to parent's epoxy
The corresponding site of the amino acid sequence of compound hydrolase.
Beneficial effects of the present invention：The mutant of the present invention is on the basis of the amino acid shown in SEQ ID NO.2, is incited somebody to action
The valine mutation of the 106th is into isoleucine.The mutant enzyme PvEH1 that shaking flask induction 10h of the present invention is obtainedV106ICatalytic activity
It it is 1.49 times of wild-type enzyme (157.15U/g) for 234.75U/g.
Specific embodiment
In order to be more clearly understood that the technology contents of the present invention, spy lifts following embodiment and is described in detail, and purpose is only
It is to be best understood from the protection domain that present disclosure is not intended to limit the present invention.
Embodiment 1：It is mutated the structure of enzyme gene and its expression plasmid
First, the acquisition of plasmid pET-28a (+)-pveh1
The culture medium of recombination E.coli BL21 (DE3)/pET-28a (+)-pveh1, which forms, is：Peptone 1%, yeast extract
0.5%, NaCl 1%.
It is in 37 DEG C, 215rpm shaken cultivations 12h in 5mL test tubes that recombinant bacterium is inoculated in culture medium liquid amount.Culture knot
Thalline is centrifuged 1min under 12,000rpm and collects cell, utilizes plasmid extraction kit Pure Plasmid by Shu Hou
Mini Kit (health is century bio tech ltd) extraction plasmid pET-28a (+)-pveh1；Wherein plasmid pET-28a
The amino acid sequence of epoxide hydrolase PvEH1 in (+)-pveh1 (nucleotide sequence such as SEQ as shown in SEQ ID NO.2
Shown in ID NO.4).
2nd, the structure of recombination bacillus coli E.coli BL21 (DE3)/pET-28a (+)-pveh1 (V106I)
It is as follows to design and synthesize specific rite-directed mutagenesis primer：
With V106I-F, V106I-R (SEQ ID NO.5 and SEQ ID NO.6) be upstream and downstream primer, with pET28a (+)-
Pveh1 is template, utilizes QuickChangeTMKit carries out PCR, and PCR conditions are as follows：95 DEG C of pre-degeneration, 4min；Denaturation 98
DEG C, 10s；55 DEG C of annealing, 15s；72 DEG C of extension, 8min；It is recycled 30 times from be denaturalized to extending, last fully 72 DEG C of extension,
10min.System is 25 μ L, using the plasmid of wild type pveh1 as template.
After the completion of PCR, 0.5 μ L Dpn I and 1 μ 10 × T of L Buffer are added in, 25 DEG C are digested overnight.Conversion is with reference to raw work
Super competent cell reagent preparation box operation instructions.By the mutant enzyme expression vector of acquisition by the limited public affairs of the farsighted enlightening biology in Shanghai
Department's sequencing confirms base sequence.
Embodiment 2：Mutant enzyme PvEH1V106IAcquisition
By E.coli BL21-pveh1V106ISingle bacterium colony is inoculated in LB culture mediums of the 2mL containing 100 μ g/mL kanamycins,
In 37 DEG C, 215r/min overnight incubations；The switching of 1mL culture solutions is taken to be cultivated in the identical culture mediums of 50mL to OD600For 0.6-
When 0.8, IPTG (final concentration 0.5mmol/L), 20 DEG C of induction 10h are added.Thalline is collected, is delayed per g wet thallus with 10mL sodium phosphates
Fliud flushing (Na2HPO4-NaH2PO4, 100mmol/L, pH 7.0) and suspending obtains bacteria suspension.
Embodiment 3：Mutant enzyme PvEH1V106IThe measure of catalytic activity
30 μ L bacteria suspensions and 920 μ L phosphate buffers are added in 2mL EP pipes, 25 DEG C of heat preservation 5min add 50 μ L
Racemic o-methyl-phenyl glycidol ether (rac-o-GMPE, final concentration 10mmol/L) is clocked immediately after reaction 10min, is negated
200 μ L of liquid is answered to add in 1mL ethyl acetate and terminate reaction.After micro porous filtration, sample analysis is using positive HPLC, OD-H column and ultraviolet
Detector.Mobile phase is n-hexane/isopropanol (80:20, v/v), flow velocity 0.8mL/min, Detection wavelength 220nm.Enzyme activity
Property unit definition：Under this determination condition, 1 epoxy is defined as with the enzyme amount needed for 1 μm of ol rac-o-GMPE of decomposition per minute
The active unit (IU) of compound hydrolase.Mutant enzyme PvEH1V106ICatalytic activity is 234.75U/g, is wild-type enzyme
1.49 times of (157.15U/g).
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention
Enclosing be subject to what claims were defined.
Sequence table
<110>Southern Yangtze University
<120>The Kidney bean epoxide hydrolase mutant that a kind of catalytic activity improves
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aaatttggca agacaggctt aactggtggc ctcaactact atagaaatct caacctcaat 720
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Claims (10)
1. a kind of Kidney bean epoxide hydrolase mutant, which is characterized in that the epoxide hydrolase mutant is：
(a) protein of the amino acid sequence as shown in SEQ ID NO.1；
(b) hybridize under strict conditions with (a) amino acid sequence limited and encode the ammonia with epoxide hydrolase activity
Base acid sequence.
2. a kind of gene for encoding epoxide hydrolase mutant described in claim 1, which is characterized in that the gene
Nucleotide sequence is the sequence shown in SEQ ID NO.3.
A kind of 3. method for obtaining epoxide hydrolase mutant described in claim 1, which is characterized in that be in such as SEQ
On the basis of amino acid sequence shown in ID NO.2, by the valine mutation of the 106th into isoleucine.
4. the carrier containing the gene of mutant described in coding claim 1.
5. express the genetic engineering bacterium of mutant described in claim 1.
6. genetic engineering bacterium according to claim 5, which is characterized in that the genetic engineering bacterium is with Escherichia coli, bud
What spore bacillus or saccharomycete were built for host.
7. nucleotide fragments of mutant described in claim 1, mutant described in coding claim 1, will containing coding right
It seeks the carrier of the gene of 1 mutant, express the application of the genetic engineering bacterium of mutant described in claim 1.
8. apply according to claim 7, which is characterized in that the application refer to medicine, pesticide, ferroelectric liquid crystals, fragrance,
Application in fine chemicals industry.
9. apply according to claim 7, which is characterized in that the application refers to the application in chiral living things catalysis.
10. apply according to claim 9, which is characterized in that the application is with mutant described in claim 1 or expression
The genetic engineering bacterium of mutant described in claim 1 is catalyst, and racemic o-methyl-phenyl is catalyzed in phosphate buffer
Glycidol ether.
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| CN109486789A (en) * | 2018-12-29 | 2019-03-19 | 江南大学 | A kind of Kidney bean epoxide hydrolase mutant that stereoselectivity improves |
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| CN106119220A (en) * | 2016-07-07 | 2016-11-16 | 江南大学 | A kind of Kidney bean Epoxide hydrolase mutant being catalyzed activity and the raising of mapping regression nature |
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| CN106119220A (en) * | 2016-07-07 | 2016-11-16 | 江南大学 | A kind of Kidney bean Epoxide hydrolase mutant being catalyzed activity and the raising of mapping regression nature |
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| CN109486789A (en) * | 2018-12-29 | 2019-03-19 | 江南大学 | A kind of Kidney bean epoxide hydrolase mutant that stereoselectivity improves |
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