CN108517319B - Epoxide hydrolase mutant and its application in mapping normalizing hydrolysis epoxides - Google Patents
Epoxide hydrolase mutant and its application in mapping normalizing hydrolysis epoxides Download PDFInfo
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Abstract
The present invention relates to epoxide hydrolase mutant and its applications in mapping normalizing hydrolysis epoxides.Present invention discloses a kind of epoxide hydrolase mutant, the mutant has very high catalytic activity and stereoselectivity.Present invention further teaches the applications of the epoxide hydrolase mutant.Utilize the mapping normalizing hydrolysis of epoxide hydrolase mutant catalytic racemization styrene oxide and its derivative of the invention, with the advantages such as mapping regression nature degree is high, reaction condition is mild, environmental-friendly, optical purity of products is high, mutational site obtained has universality in other epoxide hydrolase, has boundless application prospect.
Description
Technical field
The invention belongs to chemical synthesis and enzyme engineering field, more particularly it relates to epoxide hydrolase mutant and
Its application in hydrolysis epoxides.
Background technique
It is the important intermediate and chiral building block of many pharmaceutical synthesis by the vicinal diols that epoxide-derivative obtains, has
Extensive synthesis application value.Such as with p-nitrophenyl ethylene oxide be set out substrate synthesis p-nitrophenyl ethyl glycol be
The important chiral intermediate of β-blocker class drug Nifenalol and DCI are synthesized, β-blocker class drug loses with the anti-rhythm of the heart
Normal and anginal effect is mainly used for treating cardiovascular disease, has important application value.And the drug effect of this kind of drug with
The optical purity of product is closely related, and optical purity is higher, then its drug effect is just more obvious.
It is the substrate that sets out with styrene oxide and its derivative, is to prepare high optical voidness using chemistry or bioanalysis hydrolysis
Spend the important method of chiral aryl vicinal diols.In chemical method, usually in metallic catalyst and corresponding chiral ligand with cooperation
Under, catalysis epoxidation object selective hydrolysis generates corresponding vicinal diols with optical activation, this method reaction condition compared with
It for harshness and needs heavy metal as catalyst, be easy to cause environmental pollution, and be difficult to break through traditional power fractionation
50% yield limitation.
Epoxide hydrolase (Epoxide hydrolases, abbreviation EHs, EC 3.3.2.3) can be in the presence of hydrone
The hydrolysis of catalysis epoxidation object generates corresponding chiral ortho glycol.As green catalyst, epoxide hydrolase has coming for enzyme
Source is extensive, stereoselectivity is good, the advantages that not needing metal ion and coenzyme in reaction process, has been widely used in racemic
The enantioselective hydrolysis of epoxides is split, and prepares the epoxides and vicinal diols of high-optical-purity.Nevertheless, big portion
The dynamics Hydrolysis Resolution for dividing epoxide hydrolase that can only be catalyzed racemic epoxides, theoretical maximum yield is only 50%.
The mapping regression nature hydrolysis for having a handful of epoxide hydrolase that can be catalyzed the epoxides with specific structure is anti-
It answers.This fermentoid has complementary regioselectivity for the substrate of opposite enantiomer configuration, so that a kind of in catalytic reaction process
Spatial configuration is retained after the substrate hydrolysis of configuration, and spatial configuration inverts after the substrate hydrolysis of another configuration, most
Two kinds of enantiomters of whole racemic substrate are all hydrolyzed, obtain the hydrolysate of same configuration, this unique substrate
Hydrolysis method is known as the hydrolysis of mapping regression nature.The complete conversion that substrate may be implemented by the hydrolysis of mapping regression nature, breaks through easily
The 50% yield limitation that traditional power is split, significantly improves the yield of product, reduces cost.But in reported epoxy
In hydrolase, only seldom several enzymes have this catalysis characteristics.From plant potato epoxide hydrolase (StEH) and
Mung bean epoxide hydrolase (VrEHs) can be hydrolyzed with styrene catalyzed oxide and its derivative mapping regression nature generates high optics
The product glycol of purity.2004, Furstoss seminar utilized chlorobenzene between the epoxide hydrolase mapping regression nature hydrolysis of potato
Ethylene oxide, it can be achieved that substrate complete conversion, product (R)-m-chloro benzoglycols ee value be 91% (Tetrahedron:
Asymmetry,2004,15,2801-2805).Epoxide hydrolase VrEH2 from mung bean being capable of the hydrolysis pair of mapping regression nature
Nitrostyrolene oxide obtains (R)-p-nitrophenyl ethylene glycol, and the final ee value of product is 82.4% (Organic
Letters,2006,8,1737-1740).So far, the reported ring with mapping regression nature epoxides hydrolysis property
The stereoselectivity of oxygen hydrolysis enzyme is all imperfect, and the optical purity of hydrolysate vicinal diols is not ideal enough, and medical hand is not achieved
Property 98% or more ee value of building block requirement, and enzyme activity it is poor, catalyst amount is big.
In conclusion having reported that a few epoxide hydrolase can be with the mapping regression nature of catalysed partial epoxides at present
Complete hydrolysis generates corresponding optical activity vicinal diols, but that there is catalytic activity is lower for these known epoxide hydrolase,
The problems such as stereoselectivity is not high, and the optical purity of hydrolysate is undesirable.Therefore, it is necessary to develop, catalysis reaction efficiency is high, selects
The good enzyme catalyst of selecting property, to reduce catalyst amount, satisfaction is inexpensive, process is easy to operate, the segregative industrialization of product
Demand.
Summary of the invention
The purpose of the present invention is to provide mung bean epoxide hydrolase mutant and its applications in hydrolysis epoxides.
In the first aspect of the present invention, epoxide hydrolase mutant, epoxy of the mutant relative to wild type are provided
Hydrolase, the amino acid in space structure near substrate binding pocket mutate.
In a preferred embodiment, the substrate binding pocket nearby includes: the close position of catalytic triads.
In another preferred example, the amino acid near the substrate binding pocket include: the 33rd, 105,151,176,
196,263 amino acids.
In another preferred example, epoxide hydrolase of the mutant relative to wild type, substrate knot in space structure
One or more amino acid mutations nearby occur for the bag that heals up；For example, the mutation of 1~10,1~5,1~3 amino acid occurs.
In another preferred example, the amino acid sequence of the mutant corresponds to SEQ ID No.2, and the 263rd sports
Asparagine, valine or glutamine.
In another preferred example, the amino acid sequence of the mutant corresponds to SEQ ID No.2, further includes by random
It is mutated one or more mutation selected from the group below: the 176th mutation are as follows: asparagine；151st mutation are as follows: glutamine or
Leucine；105th mutation are as follows: leucine；33rd mutation are as follows: asparagine or leucine；196th mutation are as follows: the third ammonia
Acid；3rd mutation are as follows: glutamic acid；4th mutation are as follows: isoleucine；20th mutation are as follows: alanine；31st mutation are as follows:
Cysteine；46th mutation are as follows: isoleucine；56th mutation are as follows: valine；96th mutation are as follows: glycine；121st
Position mutation are as follows: glycine；136th mutation are as follows: valine；144th mutation are as follows: phenylalanine；181st mutation are as follows: figured silk fabrics
Propylhomoserin；270th mutation are as follows: arginine；281st mutation are as follows: glutamic acid；296th mutation are as follows: cysteine；Or the 306th
Position mutation are as follows: alanine.
In another aspect of this invention, the polynucleotides of separation are provided, the nucleic acid is the coding epoxy hydrolysis
Enzyme mutant.
In another aspect of this invention, a kind of carrier is provided, it contains the polynucleotides.
In another aspect of this invention, a kind of genetically engineered host cell is provided, it contains the carrier or base
Because of the polynucleotides described in being integrated in group.
In another aspect of this invention, a kind of method producing the epoxide hydrolase mutant is provided, comprising steps of
(1) the culture host cell obtains culture；With
(2) the epoxide hydrolase mutant is separated from culture.
In another aspect of this invention, the purposes of the epoxide hydrolase mutant is provided, for being used as catalyst, is urged
Change mapping regression nature and hydrolyzes epoxides.
In another aspect of this invention, the method for a kind of catalytic activity improving epoxide hydrolase and selectivity, institute are provided
The method of stating includes: to be mutated the amino acid in the space structure of the epoxide hydrolase of wild type near substrate binding pocket；
Preferably, the substrate binding pocket nearby includes: the close position of catalytic triads.
In another aspect of this invention, the purposes of the epoxide hydrolase mutant is provided, for as hydrolysis epoxy
The catalyst of compound；Preferably, described is hydrolyzed to the hydrolysis of mapping regression nature；Preferably, the epoxides is racemic
Epoxides.
In another preferred example, the epoxides has formula (I) structure:
Wherein, R represents 1~5 group (such as R1~R5), and the group is selected from: hydrogen, hydroxyl, cyano, nitro, substitution or
Non-substituted C1-C4 alkyl, substituted or non-substituted C2-C4 alkenyl, substituted or non-substituted C2-C4 alkynyl group, halogen, or
Two neighboring group is connected with each other and collectively forms ring structure with female ring；Preferably, the ring be hexatomic ring, more preferably for
Phenyl ring；Preferably, the group is located at the o-, m- or p- position position of parent nucleus.
In another preferred example, the epoxides is nitrostyrolene oxide (pNSO) or aoxidizes to cyano styrene
Object, to obtain beta-adrenergic blockers prodrug (R)-p-nitrophenyl ethylene glycol or R)-to cyano benzoglycols product.
In another preferred example, R is the H, NO of contraposition2, CN, halogen, CH3, CF3, Ph group (includes: p-H, p-NO2, p-
CN, p-F, p-Cl, p-Br, p-CH3, p-CF3, p-Ph)；
R is the halogen of meta position, CN, NO2It (include: m-Cl, m-Br, m-F, m-CN, m-NO2)；
R is the halogen at ortho position, NO2It (include: o-Cl, o-Br, o-F, o-NO2)；
R is and is stored in halogen (including the m, p-2F of meta position and contraposition；M, p-2Cl)；Or
R is and is stored in halogen (including the o, p-2F of ortho position and contraposition；O, p-2Cl).
In another aspect of this invention, a kind of method for hydrolyzing epoxides is provided, which comprises with epoxides
It is reacted for substrate using the epoxide hydrolase mutant as catalyst, obtains the product through hydrolyzing.Preferably, described
Epoxides have formula (I) structure.
In another preferred example, the concentration of the epoxide is 1-1000mmol/L；Preferably 10~
500mmol/L, the dosage of the epoxide hydrolase are 2~200U/mmol epoxide.
In another preferred example, the host cell is added in reaction system, participates in reaction；Preferably, described
Host cell be added in the form of lyophilized cells.
In another aspect of this invention, a kind of composition (including viable cell culture) is provided, is contained in the composition
The epoxide hydrolase mutant, and acceptable carrier (including culture medium) pharmaceutically or on commercial synthesis；Or contain
The host cell；Preferably, the host cell is lyophilized cells.
In another aspect of this invention, a kind of kit is provided, wherein containing the epoxide hydrolase mutant；Or contain
There is the composition.
Other aspects of the invention are apparent to those skilled in the art due to this disclosure
's.
Specific embodiment
The present inventor passes through in-depth study, finds some acid residues sites and its catalytic activity of epoxide hydrolase
Related to stereoselectivity conspicuousness, the site is more located in its space structure near substrate binding pocket, neighbouring to urge
Change triplet, obtains a collection of epoxide hydrolase mutation by carrying out the transformation of half design and rational to these sites on this basis
Body.Compared with wild-type enzyme, the mutant has very high catalytic activity and stereoselectivity.With existing optical activity
The synthetic method of benzoglycols and its derivative is compared, and using the styrene catalyzed oxide of epoxide hydrolase mutant and its is spread out
The mapping regression nature hydrolysis of biology, has the significant advantages such as mapping regression nature degree is good, optical purity of products is high, and involved
Some mutational sites on catalysis have universality, the mutation of other epoxide hydrolase can be promoted the use of well
It evolves in transformation, there is boundless application prospect.
As used herein, unless otherwise stated, " the epoxide hydrolase mutant ", " saltant type epoxide hydrolase ",
" saltant type VrEH2 ", " VrEH2 mutant " are used interchangeably, and are referred to corresponding to wild type epoxide hydrolase (such as SEQ ID
No.2), the polypeptide constituted after mutation.
If desired indicate wild type epoxide hydrolase, will be denoted as " wild type epoxide hydrolase ", " VrEH2 " or
" wild polypeptide (albumen) ", amino acid sequence are SEQ ID No.2, nucleotide sequence such as SEQ ID No.1, Huo Zheye
It can be the degenerate sequence of the sequence.Preferably, the wild type epoxide hydrolase derives from mung bean.
As used herein, " isolated epoxide hydrolase " refer to epoxide hydrolase mutant substantially free of naturally with its phase
Other albumen, lipid, carbohydrate or the other materials closed.Those skilled in the art can be purified with the purified technology of protein of standard
Epoxide hydrolase mutant.Substantially pure albumen can generate single master tape in non-reducing polyacrylamide gel.
As used herein, " substrate binding pocket " refers in the space structure of epoxide hydrolase interacts with substrate
The position of (in conjunction with).
As used herein, " catalytic triads ", which refer to, carries out the three of catalytic action in the active site center complex of hydrolase
A amino acid residue, the catalytic triads of mung bean epoxide hydrolase of the present invention include the 101st aspartic acid, and the 262nd
The aspartic acid and the 297th histidine of position." the catalytic triads close position " refer in amino acid sequence or
Close to the position of catalytic triads sequence on space structure, for example, 262 in catalytic triads are aspartic acid, then the
263 are its neighbouring positions.
Albumen of the invention can be recombinant protein, native protein, synthetic proteins, preferably recombinant protein.Egg of the invention
It is white to can be native purified product or chemically synthesized product, or use recombinant technique from protokaryon or eucaryon host (example
Such as, bacterium, yeast, higher plant, insect and mammalian cell) in generate.
The invention also includes segment, the derivative and analogue of the epoxide hydrolase mutant.As used herein, term
" segment ", " derivative " and " analog ", which refers to, is kept substantially the identical biology of native annulus oxygen hydrolysis enzyme mutant of the invention
Learn function or active albumen.Protein fragments of the invention, derivative or the like can be (i) have it is one or more conservative or
Non-conservative amino acid residue (preferably conservative amino acid) substituted albumen, and such substituted amino acid residue
Can be may not be by genetic code encoding, or (ii) has substituent group in one or more amino acid residues
Albumen, or (iii) additional amino acid sequence are fused to this protein sequence and the albumen (such as leader sequence or secretion sequence that are formed
Or for purifying the sequence of this albumen or proprotein sequence or fusion protein).According to the definition of this paper these segments, derivative
It is belonged to scope known to those skilled in the art with analog.However, the epoxide hydrolase mutant and its segment,
In the amino acid sequence of derivative and analogue, correspond to wild type epoxide hydrolase, it is recited above to there is the present invention certainly
Any group of mutation, namely amino acid mutation occurs near substrate binding pocket in the space structure of epoxide hydrolase.
In the present invention, term " epoxide hydrolase mutant " further includes (but being not limited to): several (usually 1-20
A, more preferably 1-10, also more preferably such as 1-8,1-5,1-3 or 1-2) amino acid missing, insertion and/or substitution,
And C-terminal and/or N-terminal addition or lack it is one or several (usually within 20, within preferably 10, more
Goodly it is within 5) amino acid.For example, in the art, when being substituted with similar nature or similar amino acid, usually
The function of protein will not be changed.For another example, adding one or several amino acid in C-terminal and/or N-terminal will not generally also change
The function of kink of preserved egg white matter.The term further includes the active fragment and reactive derivative of epoxide hydrolase mutant.But at these
In variant form, correspond to wild type epoxide hydrolase, there is the present invention any group of mutation recited above certainly, namely in ring
Amino acid mutation nearby occurs for substrate binding pocket in the space structure of oxygen hydrolysis enzyme.
The present invention also provides the polynucleotides for encoding epoxide hydrolase mutant of the present invention or its conservative variation's albumen
Sequence.
Polynucleotides of the invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people
The DNA of work synthesis.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.
The polynucleotides for encoding the maturation protein of the mutant include: a coded sequence for encoding mature albumen；It is mature
The coded sequence of albumen and various additional coding sequences；The coded sequence (and optional additional coding sequence) of maturation protein and
Non-coding sequence.
" polynucleotides of coding albumen " can be the polynucleotides including encoding this albumen, be also possible to further include adding
The polynucleotides of coding and/or non-coding sequence.
Epoxide hydrolase mutant nucleotide full length sequence or its segment of the invention can usually use PCR amplification method, again
Group method or artificial synthesized method obtain.For PCR amplification method, can disclosed related nucleotide sequence according to the present invention, especially
It is that open reading frame sequence carrys out design primer, and with the commercially available library cDNA or presses conventional method well known by persons skilled in the art
The prepared library cDNA expands as template and obtains related sequence.When sequence is longer, it is often necessary to carry out twice or repeatedly
Then the segment that each time amplifies is stitched together by PCR amplification by proper order again.
Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will
It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.
In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, logical
After first synthesizing multiple small fragments, it is then attached the very long segment of available sequence again.At present, it is already possible to passing through completely
Synthesis is learned to obtain encoding the DNA sequence dna of albumen of the present invention (or its segment, or derivatives thereof).Then the DNA sequence dna can be drawn
Enter in various existing DNA moleculars (or such as carrier) as known in the art and cell.In addition, can will also be dashed forward by chemical synthesis
Change is introduced into protein sequence of the present invention.
The present invention also relates to the carriers comprising polynucleotides of the invention, and with carrier or epoxide hydrolase of the invention
The genetically engineered host cell of mutant code sequence, and generate through recombinant technique the side of albumen of the present invention
Method.
By the recombinant dna technology of routine (Science, 1984；224:1431), using polynucleotide of the invention
Sequence come express or produce recombination epoxide hydrolase mutant.In general there are following steps:
(1) polynucleotides (or variant) of coding collar oxygen hydrolysis enzyme mutant of the invention, or with contain the multicore
The recombinant expression carrier of thuja acid converts or suitable host cell of transduceing；
(2) host cell that is cultivated in suitable culture medium；
(3) be separated from culture medium or cell, protein purification.
In the present invention, epoxide hydrolase mutant polynucleotide sequence be can be plugged into recombinant expression carrier.Term " recombination
Expression vector " refers to bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus
Or other carriers.As long as any plasmid and carrier can be used in short, can replicate and stablize in host.Expression vector
One important feature is to usually contain replication orgin, promoter, marker gene and translation control element.
Method well-known to those having ordinary skill in the art can be used to construct the DNA sequence dna of mutant code containing epoxide hydrolase and conjunction
Suitable transcription/translation control signal expression vector.These methods include recombinant DNA technology in vi, DNA synthetic technology, in vivo
Recombinant technique etc..The DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to synthesize.
Expression vector further includes the ribosome bind site and transcription terminator of translation initiation.Expression vector preferably include one or
Multiple selected markers, to provide the phenotypic character for selecting the host cell of conversion, such as eukaryotic culture
Dihyrofolate reductase, neomycin resistance and green fluorescent protein (GFP), or kanamycins or ammonia benzyl for Escherichia coli
Penicillin resistance.
Carrier comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence, can be used for converting suitable
When host cell, allow it to expression protein.
Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high
Equal eukaryocytes, such as plant cell.Representative example has: Escherichia coli, streptomyces, Agrobacterium；Fungal cell's such as yeast；It plants
Object cell etc..In a specific embodiment of the present invention, using Escherichia coli as host cell, it is used for high efficient expression target protein.
When polynucleotides of the invention are expressed in higher eucaryotic cells, if will when being inserted into enhancer sequence in the carrier
Transcription can be made to be enhanced.Enhancer is the cis-acting factors of DNA, generally about has 10 to 300 base-pairs, acts on and open
Mover is to enhance the transcription of gene.
Persons skilled in the art are aware that how to select carrier, promoter, enhancer and host cell appropriate.
In a specific embodiment of the present invention, using E.coli as host strain, with LB culture medium culture, which contains egg
White peptone 10g/L, yeast extract 10g/L, NaCl 5g/L, pH 7.0.Condition of culture conventionally carries out, and temperature is shaken at 37 DEG C
Bed revolving speed is 180rpm.Can certainly commonly it be known according to the difference of the factors such as host cell species and cultural method by this field
Knowledge makes appropriate choice, as long as enabling transformant to grow and efficiently generating epoxide hydrolase mutant of the present invention i.e.
It can.When expressing epoxide hydrolase VrEH2 mutant using recombinant expression transformants, large intestine preferable following methods: will be recombinated
Bacillus (preferably E.coli BL21 (DE3)) is seeded in the LB culture medium containing kanamycins according to 1% inoculum concentration, 37 DEG C of trainings
After supporting 2h or so, the optical density OD of culture solution is measured600Value is added final concentration of when it reaches (preferably 0.6) 0.5-0.7
The isopropyl-beta D-thio galactopyranoside (IPTG) of 0.1-1.0mmol/L (preferably 0.2mmol/L) induces, inducing temperature
For 16-25 DEG C (preferably 25 DEG C), induction time 12-24h (preferably 12h).In the embodiment of the present invention, recombination epoxy water is realized
Solve the high efficient expression of enzyme.
Epoxide hydrolase mutant of the invention can be formulated in pharmaceutically or on commercial synthesis in acceptable carrier,
It obtains and is adapted for catalysis reaction or the composition suitable for storage.It can also be placed in kit, in order to use or sell
It sells.
In a specific embodiment of the invention, the acquisition of epoxide hydrolase mutant and storage method can be such that by
The fermentation liquid (including shaking flask culture and ferment tank) of recombination bacillus coli, in supercentrifuge with 12,000rpm centrifugation
20min collects recombinant Bacillus coli cells and takes after being cleaned twice with 0.85%~0.9% physiological saline after collection
Thallus is placed in -80 DEG C of cryogenic freezings and saves out.The thallus of acquisition can use Tris-HCl buffer (100mM) weight of pH 6.5
Outstanding, concentration control carries out ultrasonication (ultrasound under conditions of 0.05g/mL~0.1g/mL (preferably 0.1g/mL) is in ice-water bath
Broken instrument sets power as 400W, and work 3s, interval 7s, recycles 99 times altogether).Broken liquid is in 4 DEG C of refrigerated centrifuges
14000rpm is centrifuged 45min, and obtained supernatant is the crude enzyme liquid for containing soluble epoxide hydrolase mutant, obtains thick enzyme
Liquid can carry out vitality test and protein purification.In addition collected crude enzyme liquid is placed at -80 DEG C after freezing 12h, using true
For 24 hours, freeze-drying enzyme powder can be obtained in the low temperature drying of vacuum freecing-dry machine.The freeze-drying enzyme powder obtained is stored in 4 DEG C of refrigerators, in case
The use of sequential hydrolysis reaction.
The vigor of epoxide hydrolase mutant catalysis p-nitrophenyl ethylene oxide hydrolysis of the present invention is available such as lower section
Method measurement: being measured using reversed-phase HPLC (C18 column), and live body system includes 50 μ L substrates (20mM is dissolved in DMSO), 50 μ L
The enzyme solution of suitable concentration and 400 μ L kaliumphosphate buffers (100mM, pH 6.5).In 2mL EP pipe, 30 DEG C, 1000rpm oscillation
After reacting 10min on device, 100 μ L of sampling, which are added in 400 μ L methanol, terminates reaction, 12,000rpm centrifugation 2min, 0.22 μm of film mistake
With the relative amount of C18 column analysis substrate and product after filter, mobile phase is methanol: water=75:25,30 DEG C of column temperature, flow velocity
0.8mL/min, Detection wavelength 270nm.Since response of the HPLC detector for same substrate and product is consistent, that is, react
When one mol substrate generate a product of moles, when being detected with same instrument, the variation of substrate and peak areas
It is consistent, it is possible to which that reaction conversion ratio c is calculated according to substrate peak area As and peak areas Ap:
The ratio that enzyme can be calculated according to the concentration of conversion ratio, reaction time and enzyme is living, other epoxide substrates can
To carry out the measurement of Rate activity referring to this universal method.
Epoxide hydrolase mutant of the invention can be applied to hydrolysis epoxides, be especially applied to mapping regression nature
The racemic epoxides of catalyzing hydrolysis, the more particularly styrene oxide and its derivative of hydrolysis of racemic, prepare optics
Homochiral vicinal diols, beta-adrenergic blockers intermediate, further more particularly catalysis p-nitrophenyl ethylene oxide and
Cyano styrene oxide mapping regression nature is hydrolyzed to prepare optical voidness beta-adrenergic blockers intermediate p-nitrophenyl
Ethylene glycol and application to cyano-phenyl ethylene glycol.
In a preferred embodiment of the present invention, the optional self-drifting of the epoxide (I):
Wherein, R represents 1~5 group (such as R1~R5), and the group is selected from: hydrogen, hydroxyl, cyano, nitro, substitution or
Non-substituted C1-C4 alkyl, substituted or non-substituted C2-C4 alkenyl, substituted or non-substituted C2-C4 alkynyl group, halogen, or
Two neighboring group is connected with each other and collectively forms ring structure with female ring；Preferably, the ring be hexatomic ring, more preferably for
Phenyl ring；Preferably, the group is located at the o-, m- or p- position position of parent nucleus.
Term " alkyl " refer to linear chain or branched chain saturation, containing 1-4 carbon atom (preferably 1-3 or 1-2 carbon atom)
Aliphatic hydrocarbon group.For example, alkyl includes but is not limited to methyl, ethyl, n-propyl, isopropyl, normal-butyl, isobutyl group, uncle
Butyl.
Term " alkenyl " includes containing at least one carbon-carbon double bond and 2-4 carbon atom (preferably 2-3 carbon atom)
Straight chain and branched hydrocarbyl.
Term " alkynyl group " includes containing at least one triple carbon-carbon bonds and 2-4 carbon atom (preferably 2-3 carbon atom)
Straight chain and branched hydrocarbyl.
Term " halogen " refers to F, Cl, Br or I.
The concentration of the epoxide is 10~500mmol/L, and the dosage of the epoxide hydrolase is 10~100U/
Mmol epoxide.Buffer needed for mapping regression nature hydrolytic process is this field conventional phosphoric acid salt buffer, such as phosphorus
Sour potassium buffer, concentration are preferably 100mmol/L.The mapping regression nature hydrolysis can be under oscillation or stirring condition
It carries out.The temperature of the mapping regression nature hydrolysis is 25~35 DEG C, preferably 30 DEG C.The mapping regression nature hydrolysis is anti-
The time answered is subject to the substrate completely reacted time, and preferred reaction time is lower than for 24 hours.
In the present invention, ee value can be calculated by analyzing the content of two kinds of enantiomters of reaction product, can be borrowed
HPLC is helped to be analyzed, illustrative liquid phase chromatogram condition are as follows: chiral column Chiralcel OD-H (0.46cm × 25cm) is used,
Mobile phase is n-hexane: isopropanol=85:15, flow velocity 1mL/min；Chromatogram column temperature is 30 DEG C；Detection wavelength is 270/
220nm。
Work as fully reacting, when conversion ratio is greater than 99%, stops reaction, select organic solvent to be extracted, organic solvent can
Ethyl acetate, butyl acetate, methylene chloride, methyl tertiary butyl ether(MTBE) (ethyl acetate) are selected, is selected isometric organic molten
Agent repeats extraction three times, combining extraction liquid, is cleaned with saturated salt solution and the dry 10h or more of anhydrous sodium sulfate, mistake is added afterwards twice
Anhydrous sodium sulfate is filtered out, the extract liquor containing product is then removed into organic solvent using Rotary Evaporators to get optical voidness
The crude extract of chiral product.Crude extract chromatograph using petroleum ether and ethyl acetate as mobile phase pure by silica gel column chromatography
The pure and mild optically pure product of elevated chemical can be obtained in change.
Compared with prior art, progress effect of the invention is: providing new epoxide hydrolase mutant, efficiently urges
Change hydrolysis epoxide, such as substrate p-nitrophenyl ethylene oxide can be directed to, to cyano styrene oxide and its structure
The mapping regression nature of analog hydrolyzes, and prepares optically pure (R) -4- nitro -1- styrene glycol, (R) -4- cyano -1- phenyl
Ethylene glycol and its analogue.
It is catalyzed using epoxide hydrolase mutant of the invention, without adding co-factor (coenzyme), reaction cost is low.It is described
Epoxide hydrolase mutant, can catalytic level be up to the hydrolysis of Hydrophobic Styrene's oxide of 500mM or derivatives thereof,
Realize 99% or more conversion ratio, the optical purity of product is up to 99%.
The hydrolysis of hydrophobicity epoxides relative to the catalysis of other epoxide hydrolase, epoxide hydrolase of the invention
Mutant has catalytic activity height, concentration of substrate high, and substrate can convert (high conversion rate is in 99%) completely, obtain single configuration
Converted product, the advantages such as optical purity height of reaction product.Epoxide hydrolase of the present invention is easily obtained, and reaction condition is mild,
Environmental-friendly, reaction process is simple and efficient, easily operated, and product is easy to extract, therefore has good prospects for commercial application.
Each reaction described above and testing conditions, can be advanced optimized, and can be used and pass through reality according to common sense in the field
It tests and is verified.Following instance illustrates exemplified embodiment of the present invention, to help skilled in the art to understand this Shen
Other targets, feature, advantage and various aspects please.Although it should be appreciated that showing the preferred embodiment of the application, below
Description and specific embodiment are merely to illustrating and providing, without being construed as limiting to the scope of the present invention.The scope of the present invention root
It is determined according to the appended claims.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Described in " Molecular Cloning:A Laboratory guide " (Science Press, 2002, the third edition) that part such as J. Pehanorm Brooker etc. is write
Condition, or according to the normal condition proposed by manufacturer.
Material source used in the following example is as follows:
Expression plasmid pET28a: it is purchased from Novagen company；
E.coli BL21 (DE3) competent cell: it is purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.；
Ago-Gel DNA QIAquick Gel Extraction Kit and plasmid extraction kit: the limited public affairs of Beijing Tiangeng biochemical technology are purchased from
Department；
Restriction enzyme BamH I, Xho I and Dpn I: being the commercial product of NEB company.
T4DNA ligase and PrimeSTAR HS: for Takara Products.
Plasmid pET28a-VrEH2 is voluntarily constructed, preservation by inventor unit one belongs to.
Embodiment 1, epoxide hydrolase VrEH2 mutation construction
Plasmid pET28a-VrEH2 is constructed as follows: using the mRNA sequence separated in mung bean flour as template, with following primers F 1
It is expanded with R1, obtains the amplified fragments comprising the base sequence as shown in sequence table SEQ ID No.1, it is inserted into expression
It is obtained in the BamH I/Xho I site of plasmid pET28a.
F1:5 '-CGCGGATCCATGGAAGAAATAGAACA-3 ' (SEQ ID No.3)；
R1:5 '-CCGTAAAACTTCTTGATAAAATCGTA-3 ' (SEQ ID No.4)；
The present inventor utilizes X-ray diffraction method, and parsing obtains the protein structures of epoxide hydrolase VrEH2,
In structure basis, the binding pattern of epoxide substrate is simulated using molecular docking, passes through POVME software prediction substrate
Binding pocket.Then, around by analysis substrate active pocketAmino acid in range has locked influence VrEH2 mapping and has returned
The candidate amino acid of one stereoselectivity, the amino acid including the sites such as the 33rd, 105,151,176,196,263.Using combination
The strategy of active site saturation mutation (Combinatorial active-site saturation test, CAST) is to selected
The candidate amino acid mutational site selected carries out semi-saturation mutation, constructs mutant library, therefrom screens the mutation of highly-solid selectively
Body.On this basis, further mutation transformation, building mutation are carried out using more excellent mutant of the strategy of random mutation to acquisition
Body library, the excellent mutant that therefrom screening discovery significantly improves vigor.Guarantor bacterium is carried out with 20% glycerol, is placed in -80 DEG C of guarantors
It deposits.
The inducing expression and its purifying of 2 epoxide hydrolase VrEH2 mutant of embodiment
The recombinant expression transformants strain of epoxide hydrolase VrEH2 mutant will be recombinantly expressed obtained in embodiment 1, connect
Kind is into the LB culture medium test tube containing 50 μ g/mL kanamycins, 37 DEG C, 180rpm shaking table shaken cultivation 12 hours, then presses 1%
(v/v) inoculum concentration is seeded in the 500mL triangular flask containing 100mL LB culture medium, is placed in 37 DEG C, the interior training of 180rpm shaking table
It supports, as the OD of culture solution600Reach 0.6 or so, IPTG to final concentration 0.2mmol/L is added and carries out inducing expression, 25 DEG C of inductions 12
After hour, culture solution is centrifuged 15min with 12,000rpm revolving speed, collects thallus, and with 0.85% brine thallus
Twice, resting cell is obtained.Gained wet cell 0.1g is weighed, 10mL Tris-HCl buffer (100mM, pH 6.5) weight is used
Outstanding, be ultrasonically treated as follows in ice-water bath: power 400W, work 3s, interval 7s, carries out 99 circulations, is crushed liquid at 4 DEG C
14000rpm is centrifuged 45 minutes, collects supernatant, and then carry out protein purification using nickel column.The following are protein purification buffers to match
Side: Buffer A:20mM Tris-HCl, pH 8.0,500mM NaCl, 20mM imidazoles, 375 μ L/L mercaptoethanols；Buffer
B:20mM Tris-HCl, pH 8.0,500mM NaCl, 500mM imidazoles, 375 μ L/L mercaptoethanols；Buffer C:25mM
Tris-HCl, pH 7.5,150mM NaCl, 1mM DTT.By the crude enzyme liquid of the epoxide hydrolase of broken acquisition using on peristaltic pump
Sample to use A liquid balance after nickel column on, after loading, with A liquid elution column in foreign protein, then with the B liquid of different gradients
Target protein is eluted, verifies whether then to collect the eluent containing target protein containing target protein using SDS-PAGE, benefit
It is concentrated by ultrafiltration with super filter tube (30kDa), then twice with the displacement of C liquid, to remove the imidazoles in protein solution.Albumen after purification
Activity and Stereoselective determination are carried out, is then dispensed, is saved backup after liquid nitrogen flash freezer in -80 DEG C.
3 epoxide hydrolase VrEH2 mutant activity of embodiment and Stereoselective determination
The activity of epoxide hydrolase VrEH2 mutant is measured using reversed-phase HPLC (C18 column), with p-nitrophenyl ethylene
Oxide is vitality test substrate, and live body system includes 50 μ L substrates (20mM is dissolved in DMSO), the enzyme solution of 50 μ L suitable concentrations
With 400 μ L kaliumphosphate buffers (100mM, pH 6.5).Reaction in 2mL EP pipe, 30 DEG C, under 1000rpm oscillating condition into
Row after reacting 10min, samples 100 μ L, and 400 μ L methanol are added and terminate reaction, 12,000rpm centrifugation 2min, centrifuged supernatant is used
0.22 μm of film filtering, then carries out HPLC analysis with C18 column, mobile phase is methanol: water=75:25, and 30 DEG C of column temperature, flow velocity
0.8mL/min, Detection wavelength 270/220nm.Result, reaction transformation time and zymoprotein concentration calculation enzyme are analyzed according to HPLC
Rate activity.
The stereoselectivity of epoxide hydrolase VrEH2 mutant is with the ee of enzymatic p-nitrophenyl ethylene oxide hydrolysate
Value is characterized, the ee value (ee of hydrolysatep) higher, the stereoselectivity of enzyme is better.
Table 1 provides the list of the epoxide hydrolase VrEH2 mutant of active particular sequence of the invention.In table
In 1, sequential labeling respectively refers to a series of subsequent sequences of table 1, and in active column, a plus sige "+" indicates mutant protein ratio
1.01-2 times is improved by the Rate activity for the protein that amino acid sequence shown in SEQ ID No.2 forms in sequence table；Two add
Number " ++ " indicates that the Rate activity of protein of the composition of amino acid sequence shown in mutant protein ratio SEQ ID No.2 improves
2.01-4 again.Three plus siges " +++ " indicate the protein of the composition of amino acid sequence shown in mutant protein ratio SEQ ID No.2
Rate activity improves 4.01-8 times.
The activity and selectivity of table 1, epoxide hydrolase VrEH2 mutant
Wherein, the Rate activity of maternal enzyme VrEH2 is 2.0U/mg, and the ee value of enzymatic hydrolysis product is 82.4%.It is preferred that being mutated
The Rate activity of body has 1-8 times of raising, and the ee value of enzymatic hydrolysis product is promoted to 98% or more.
The amino acid sequence difference of epoxide hydrolase VrEH2 mutant is as follows in upper table:
(1) the 3rd glutamic acid of amino acid sequence shown in SEQ ID No.2 replaces with glycine；263rd first sulphur
Propylhomoserin replaces with asparagine, and the 176th isoleucine replaces with asparagine,
(2) the 4th isoleucine of amino acid sequence shown in SEQ ID No.2 replaces with leucine, the 20th paddy
Propylhomoserin replaces with alanine；263rd methionine replaces with asparagine, and the 176th isoleucine replaces with asparagus fern
Amide,
(3) the 20th cysteine of amino acid sequence shown in SEQ ID No.2 replaces with alanine；263rd
Methionine replaces with asparagine, and the 176th isoleucine replaces with asparagine,
(4) the 31st histidine of amino acid sequence shown in SEQ ID No.2 replaces with cysteine, and the third of the 46th
Propylhomoserin replaces with isoleucine；263rd methionine replaces with asparagine, and the 176th isoleucine replaces with day
Winter amide,
(5) the 33rd phenylalanine of amino acid sequence shown in SEQ ID No.2 replaces with asparagine；263rd
Methionine replace with asparagine, the 176th isoleucine replaces with asparagine,
(6) the 56th alanine of amino acid sequence shown in SEQ ID No.2 replaces with valine, the 96th phenylpropyl alcohol
Propylhomoserin replaces with glycine；263rd methionine replaces with asparagine, and the 176th isoleucine replaces with asparagus fern
Amide,
(7) the 105th isoleucine of amino acid sequence shown in SEQ ID No.2 replaces with leucine, while the 56th
The alanine of position replaces with valine, and the 121st tyrosine sports glycine；263rd methionine replaces with asparagus fern
Amide, the 176th isoleucine replace with asparagine,
(8) the 136th arginine of amino acid sequence shown in SEQ ID No.2 replaces with valine, the 121st junket
Propylhomoserin replaces with glycine, and the 105th isoleucine mutation is leucine；263rd methionine replaces with asparagus fern acyl
Amine, the 176th isoleucine replace with asparagine,
(9) the 33rd phenylalanine of amino acid sequence shown in SEQ ID No.2 replaces with asparagine, and the 105th
Isoleucine mutation be leucine；263rd methionine replaces with asparagine, while the 176th isoleucine
Replace with asparagine.
(10) the 181st lysine of amino acid sequence shown in SEQ ID No.2 replaces with valine, and the third of the 46th
Propylhomoserin replaces with isoleucine；263rd methionine replaces with asparagine, and the 176th isoleucine replaces with day
Winter amide,
(11) the 270th lysine of amino acid sequence shown in SEQ ID No.2 replaces with arginine, the 151st
Isoleucine replaces with glutamine, and the 144th methionine sports phenylalanine；263rd methionine replaces with
Asparagine,
(12) the 281st glutamine of amino acid sequence shown in SEQ ID No.2 replaces with glutamic acid, while
151 isoleucines replace with leucine, while the 196th phenylalanine replaces with alanine；263rd first sulphur ammonia
Acid replaces with asparagine,
(13) the 296th alanine of amino acid sequence shown in SEQ ID No.2 replaces with cysteine, while the 46th
The alanine of position replaces with isoleucine, and the 105th isoleucine mutation is leucine, and the 121st tyrosine sports sweet
Propylhomoserin；263rd methionine replaces with asparagine, while the 176th isoleucine replaces with asparagine.
(14) the 306th glutamic acid of amino acid sequence shown in SEQ ID No.2 replaces with alanine, while the 46th
Alanine replace with isoleucine, the 105th isoleucine mutation is leucine, and the 121st tyrosine sports sweet ammonia
Acid；263rd methionine replaces with asparagine.
(15) the 263rd methionine of amino acid sequence shown in SEQ ID No.2 replaces with asparagine, while
176 isoleucines replace with asparagine, and the 33rd phenylalanine sports leucine, and the 151st isoleucine is prominent
Become glutamine.
(16) the 263rd methionine of amino acid sequence shown in SEQ ID No.2 replaces with valine, while
176 isoleucines replace with asparagine, and the 33rd phenylalanine sports leucine, and the 151st isoleucine is prominent
Become glutamine.
(17) the 263rd methionine of amino acid sequence shown in SEQ ID No.2 replaces with glutamine, while
176 isoleucines replace with asparagine, and the 33rd phenylalanine sports leucine, and the 151st isoleucine is prominent
Become glutamine.
The amino acid mutation site of above-mentioned each mutant is summarized as follows table 2.
The amino acid mutation site of table 2, each mutant
Pervasive Journal of Sex Research of the 4 hot spot amino acid sites of embodiment in epoxide hydrolase
In the mutant as designed by embodiment 1, the 33rd phenylalanine, the 151st isoleucine, the 176th
Isoleucine, the 196th phenylalanine, the 263rd methionine, the amino acid sites are located at around substrate binding pocketIn range, wherein 263 methionines are adjacent with 262 aspartic acids in catalytic triads, these amino acid are in function
The upper stereoselectivity with epoxide hydrolase is closely related, and in locations of structures, space in the epoxide hydrolase of separate sources
Upper these amino acid positioned at analogous location have conservative.
Investigate universality (table 3) of the amino acid mutation as described in Example 1 in epoxide hydrolase.Pass through multisequencing first
The amino acid positioned at analogous location determined in other epoxide hydrolase is compared, and is embodiment 3 by these amino acid mutations,
The stereoselectivity of amino acid in table 2, mutation epoxide hydrolase obtained has different degrees of raising, with corresponding mother
This epoxide hydrolase is compared, and for the epoxide substrate of different structure, after enzymatic complete hydrolysis, the ee value of the product of acquisition is aobvious
It writes and improves, the results are shown in Table 3.
The pervasive Journal of Sex Research of table 3, hot spot amino acid in epoxide hydrolase
Embodiment 5, VrEH2M15Mapping regression nature hydrolyzes p-nitrophenyl ethylene oxide
In the 100mL p-nitrophenyl of substrate containing 10mmol/L ethylene oxide, (formula (I) compound, wherein R is p-NO2)
In the kaliumphosphate buffer (100mmol/L, pH 6.5) of (1.65g/L), 136U is added and is recombinantly expressed as described in embodiment 2~3
Transformant (E.coli BL21/pET28a-VrEH2M15) lyophilized cells.It is reacted under the stirring of blender in 30 DEG C.
After reaction 3 hours, equivalent ethyl acetate is added and is extracted twice, combining extraction liquid is cleaned twice with saturated salt solution, is added anhydrous
Sodium sulphate is dried overnight.Measured with liquid chromatography: the substrate transformation rate is greater than 99%, and product ee value is 98.4% (R).
6 VrEH2 of embodimentM15Mapping regression nature is hydrolyzed to cyano styrene oxide
In 100mL substrate containing 10mmol/L to cyano styrene oxide (formula (I) compound, wherein R is p-CN)
In the kaliumphosphate buffer (100mmol/L, pH 6.5) of (1.63g/L), 175U is added and is recombinantly expressed as described in embodiment 2~3
Transformant (E.coli BL21/pET28a-VrEH2M15) lyophilized cells, reacted under the stirring of blender in 30 DEG C.
After reaction 4 hours, equivalent ethyl acetate is added and is extracted twice, combining extraction liquid is cleaned twice with saturated salt solution, is added anhydrous
Sodium sulphate is dried overnight.Measured with liquid chromatography: the substrate transformation rate is greater than 99%, and product ee value is 99.5% (R).
Embodiment 7~13, VrEH2M16Mutant mapping regression nature hydrolyzed styrene type oxide
In 100mL substrate containing 10mmol/L styrene oxide and its kaliumphosphate buffer (100mmol/ of derivative
L, pH 6.5) in, 200U recombinant expression transformants (E.coli BL21/pET28a- as described in embodiment 2~3 is added
VrEH2M16) lyophilized cells, reacted under the stirring of blender in 30 DEG C.Reaction process is detected using TCL, wait react
After the completion, three times with the extraction of isometric ethyl acetate, after combining extraction liquid is cleaned twice with saturated salt solution, add anhydrous sodium sulfate
Dry 12h, measures the ee value of conversion ratio and product.VrEH2M16Mapping regression nature hydrolyzed styrene oxide and its derivative
Conversion ratio and the analysis condition of product ee value are shown in Table 4, and reaction result is shown in Table 5.
The HPLC analysis condition of product in table 4, embodiment 7-13
The ee value of the conversion ratio and product that are reacted in table 5, embodiment 7-13
Embodiment 14~24, VrEH2M17Mutant mapping regression nature hydrolyzed styrene type oxide
In 100mL substrate containing 10mmol/L phenylethylene oxide and its kaliumphosphate buffer (100mmol/ of derivative
L, pH 6.5) in, 174U recombinant expression transformants (E.coli BL21/pET28a- as described in embodiment 2~3 is added
VrEH2M17) lyophilized cells, reacted under the stirring of blender in 30 DEG C.Reaction process is detected using thin layer chromatography,
To after the reaction was completed, three times with the extraction of isometric ethyl acetate, after combining extraction liquid is cleaned twice with saturated salt solution, add anhydrous
Sodium sulphate dries 12h, measures the ee value of conversion ratio and product.VrEH2M17Mapping regression nature hydrolyzed styrene oxide and its spread out
The conversion ratio of biology and the analysis condition of product ee value are shown in Table 6, and reaction result is shown in Table 7.
The HPLC analysis condition of product in table 6, embodiment 14-24
The ee value of the conversion ratio and product that are reacted in table 7, embodiment 14-24
Embodiment 25, VrEH2M15Mapping regression nature hydrolysis prepares (R)-p-nitrophenyl ethylene glycol
In the kaliumphosphate buffer of the 100mL p-nitrophenyl ethylene oxide of substrate containing 500mmol/L (82.5g/L)
In (100mmol/L, pH 6.5), 100U recombinant expression transformants (E.coli BL21/ as described in embodiment 2~3 is added
pET28a-VrEH2M15) lyophilized cells, reacted under the stirring of blender in 30 DEG C.After reaction 20 hours, substrate turns
Rate is greater than 99%, and equivalent ethyl acetate is added and is extracted twice, and combining extraction liquid is cleaned twice with saturated salt solution, is added anhydrous
Sodium sulphate is dried overnight.Then revolving removes ethyl acetate, and obtained crude product is separated using silica gel column chromatography, obtains product
(R)-p-nitrophenyl ethylene glycol 7.5g, separation yield are 81.9%, and product ee value is 98.4%.
Embodiment 26, VrEH2M15Mapping regression nature hydrolysis prepares (R)-to cyano benzoglycols
In 100mL substrate containing 500mmol/L to the kaliumphosphate buffer of cyano styrene oxide (81.5g/L)
In (100mmol/L, pH 6.5), 198U recombinant expression transformants (E.coli BL21/ as described in embodiment 2~3 is added
pET28a-VrEH2M15) lyophilized cells, reacted under the stirring of blender in 30 DEG C.After reaction 24 hours, substrate turns
Rate is greater than 99%, and equivalent ethyl acetate is added and is extracted twice, and combining extraction liquid is cleaned twice with saturated salt solution, is added anhydrous
Sodium sulphate is dried overnight.Then revolving removes ethyl acetate, and obtained crude product is separated using silica gel column chromatography, obtains product
(R)-and to cyano benzoglycols 7.7g, separating yield is 94.3%, and product ee value is 99.5%.
Embodiment 27, VrEH2M16Mapping regression nature hydrolysis prepares (R)-benzoglycols
In kaliumphosphate buffer (100mmol/L, the pH of the 100mL styrene oxide of substrate containing 500mmol/L (60g/L)
6.5) in, 200U recombinant expression transformants (E.coli BL21/pET28a-VrEH2 as described in embodiment 2~3 is addedM16) jelly
Stem cell is reacted under the stirring of blender in 30 DEG C.After reaction 24 hours, the substrate transformation rate is greater than 99%, be added etc.
Amount ethyl acetate is extracted twice, and combining extraction liquid is cleaned twice with saturated salt solution, and anhydrous sodium sulfate is added and is dried overnight.Then
Revolving removes ethyl acetate, and obtained crude product is separated using silica gel column chromatography, obtains product (R)-benzoglycols 5.2g, separates
Yield is 75%, and product ee value is 95.6%.
Embodiment 28, VrEH2M17Mapping regression nature hydrolysis prepares (R)-m-chloro Benzenediol
In the kaliumphosphate buffer of the 100mL m-chlorostyrene oxide of substrate containing 500mmol/L (77.3g/L)
In (100mmol/L, pH 6.5), 174U recombinant expression transformants as described in Example 2 (E.coli BL21/pET28a- is added
VrEH2M17) lyophilized cells, reacted under the stirring of blender in 30 DEG C.After reaction 24 hours, the substrate transformation rate is greater than
99%, equivalent ethyl acetate is added and is extracted twice, combining extraction liquid is cleaned twice with saturated salt solution, anhydrous sodium sulfate is added
It is dried overnight.Then revolving removes ethyl acetate, and obtained crude product is separated using silica gel column chromatography, obtains product (R)-m-chloro
Benzoglycols 7.3g, separation yield are 84.8%, and product ee value is 92.4% (R).
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
<110>East China University of Science
<120>epoxide hydrolase mutant and its application in hydrolysis epoxides
<130> 17A471
<160> 4
<170> SIPOSequenceListing 1.0
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<213>mung bean (Vigna radiata)
<400> 1
atggaaggcg tggaacacag aacggtggaa gtgaatggca taaaaatgca tgtagcagag 60
aaaggagagg gtcctgtcgt gttgttcctc catggcttcc ccgagctctg gtactcctgg 120
cgccaccaga ttctcgctct cagctcccga ggctaccgcg ctgttgcacc ggatttacgt 180
ggctacggtg atacagaggc accggtttca atcagcagct acacaggctt tcatatagtg 240
ggtgatctga ttgcgcttat agaccttctg ggtgtcgacc aagtcttcct tgtggctcat 300
gactggggtg ccatcattgg ttggtatctc tgcacgtttc accccgacag agtgaaggcc 360
tatgtctgcc tcagtgttcc tttgcttcac agagacccca acatcagaac cgtcgatgcc 420
atgcgtgcta tgtacggaga tgactactac atctgcagat ttcagaaacc aggcgaaatg 480
gaggctcaga tggctgaagt tggaactgag tatgtgttga aaaacattct gacaactcga 540
aaacctggtc ctccgatatt tcccaaggga gagtatggaa ccgggttcaa tccagatatg 600
ccgaattcct taccctcttg gctcacgcaa gatgatcttg cctattacgt atccaaatat 660
gagaaaacgg gcttcacagg acccttgaac tattacagaa atatgaactt aaattgggag 720
ctgacagcac catggagtgg aggaaaaatt caagtgccgg taaagttcat cacaggtgag 780
ttggacatgg tgtacacctc actgaacatg aaggagtata tccacggtgg aggtttcaaa 840
caagatgttc caaatttaga agaagtgatt gtgcagaaaa atgtagctca cttcaataat 900
caagaagcag ctgaggaaat caataatcat atttacgatt ttatcaagaa gttttaa 957
<210> 2
<211> 318
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Met Glu Gly Val Glu His Arg Thr Val Glu Val Asn Gly Ile Lys Met
1 5 10 15
His Val Ala Glu Lys Gly Glu Gly Pro Val Val Leu Phe Leu His Gly
20 25 30
Phe Pro Glu Leu Trp Tyr Ser Trp Arg His Gln Ile Leu Ala Leu Ser
35 40 45
Ser Arg Gly Tyr Arg Ala Val Ala Pro Asp Leu Arg Gly Tyr Gly Asp
50 55 60
Thr Glu Ala Pro Val Ser Ile Ser Ser Tyr Thr Gly Phe His Ile Val
65 70 75 80
Gly Asp Leu Ile Ala Leu Ile Asp Leu Leu Gly Val Asp Gln Val Phe
85 90 95
Leu Val Ala His Asp Trp Gly Ala Ile Ile Gly Trp Tyr Leu Cys Thr
100 105 110
Phe His Pro Asp Arg Val Lys Ala Tyr Val Cys Leu Ser Val Pro Leu
115 120 125
Leu His Arg Asp Pro Asn Ile Arg Thr Val Asp Ala Met Arg Ala Met
130 135 140
Tyr Gly Asp Asp Tyr Tyr Ile Cys Arg Phe Gln Lys Pro Gly Glu Met
145 150 155 160
Glu Ala Gln Met Ala Glu Val Gly Thr Glu Tyr Val Leu Lys Asn Ile
165 170 175
Leu Thr Thr Arg Lys Pro Gly Pro Pro Ile Phe Pro Lys Gly Glu Tyr
180 185 190
Gly Thr Gly Phe Asn Pro Asp Met Pro Asn Ser Leu Pro Ser Trp Leu
195 200 205
Thr Gln Asp Asp Leu Ala Tyr Tyr Val Ser Lys Tyr Glu Lys Thr Gly
210 215 220
Phe Thr Gly Pro Leu Asn Tyr Tyr Arg Asn Met Asn Leu Asn Trp Glu
225 230 235 240
Leu Thr Ala Pro Trp Ser Gly Gly Lys Ile Gln Val Pro Val Lys Phe
245 250 255
Ile Thr Gly Glu Leu Asp Met Val Tyr Thr Ser Leu Asn Met Lys Glu
260 265 270
Tyr Ile His Gly Gly Gly Phe Lys Gln Asp Val Pro Asn Leu Glu Glu
275 280 285
Val Ile Val Gln Lys Asn Val Ala His Phe Asn Asn Gln Glu Ala Ala
290 295 300
Glu Glu Ile Asn Asn His Ile Tyr Asp Phe Ile Lys Lys Phe
305 310 315
<210> 3
<211> 26
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cgcggatcca tggaagaaat agaaca 26
<210> 4
<211> 26
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<213>primer (Primer)
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ccgtaaaact tcttgataaa atcgta 26
Claims (29)
1. epoxide hydrolase mutant, which is characterized in that epoxide hydrolase of the mutant relative to wild type, space knot
Amino acid in structure near substrate binding pocket mutates；The amino acid sequence of the mutant corresponds to SEQ ID No.2,
263rd sports asparagine, valine or glutamine；
And further include by random mutation one or more mutation selected from the group below:
176th mutation are as follows: asparagine；
151st mutation are as follows: glutamine or leucine；
105th mutation are as follows: leucine；
33rd mutation are as follows: asparagine or leucine；
196th mutation are as follows: alanine；
3rd mutation are as follows: glutamic acid；
4th mutation are as follows: isoleucine；
20th mutation are as follows: alanine；
31st mutation are as follows: cysteine；
46th mutation are as follows: isoleucine；
56th mutation are as follows: valine；
96th mutation are as follows: glycine；
121st mutation are as follows: glycine；
136th mutation are as follows: valine；
144th mutation are as follows: phenylalanine；
181st mutation are as follows: valine；
270th mutation are as follows: arginine；
281st mutation are as follows: glutamic acid；
296th mutation are as follows: cysteine；Or
306th mutation are as follows: alanine.
2. epoxide hydrolase mutant as described in claim 1, which is characterized in that the substrate binding pocket nearby wraps
It includes: the close position of catalytic triads.
3. epoxide hydrolase mutant as described in claim 1, which is characterized in that the mutant includes:
(1) polypeptide as shown in SEQ ID No.2, the 3rd sports glutamic acid；263rd sports asparagine, and the 176th
Position sports asparagine,
(2) polypeptide as shown in SEQ ID No.2, the 4th sports isoleucine, and the 20th sports alanine；263rd
Asparagine is sported, the 176th sports asparagine,
(3) polypeptide as shown in SEQ ID No.2, the 20th sports alanine；263rd sports asparagine, and the 176th
Position sports asparagine,
(4) polypeptide as shown in SEQ ID No.2, the 31st sports cysteine, and the 46th sports isoleucine；The
263 sport asparagine, and the 176th sports asparagine,
(5) polypeptide as shown in SEQ ID No.2, the 33rd sports asparagine；263rd sports asparagine, together
When the 176th sport asparagine,
(6) polypeptide as shown in SEQ ID No.2, the 56th sports valine, and the 96th sports glycine；263rd
Asparagine is sported, the 176th sports asparagine,
(7) polypeptide as shown in SEQ ID No.2, the 105th sports leucine, and the 56th sports valine, and the 121st
Sport glycine；263rd sports asparagine, and the 176th sports asparagine,
(8) polypeptide as shown in SEQ ID No.2, the 136th sports valine, and the 121st sports glycine, and the 105th
Position sports leucine；263rd sports asparagine, and the 176th sports asparagine,
(9) polypeptide as shown in SEQ ID No.2, the 33rd sports asparagine, and the 105th sports leucine；263rd
Position sports asparagine, and the 176th sports asparagine,
(10) polypeptide as shown in SEQ ID No.2, the 181st sports valine, and the 46th sports isoleucine；The
263 sport asparagine, and the 176th sports asparagine,
(11) polypeptide as shown in SEQ ID No.2, the 270th sports arginine, and the 151st sports glutamine, the
144 sport phenylalanine；263rd sports asparagine,
(12) polypeptide as shown in SEQ ID No.2, the 281st sports glutamic acid, and the 151st sports leucine, and the 196th
Position sports alanine；263rd sports asparagine,
(13) polypeptide as shown in SEQ ID No.2, the 296th sports cysteine, and the 46th sports isoleucine, the
105 sport leucine, and the 121st sports glycine；263rd sports asparagine, and the 176th sports asparagus fern
Amide,
(14) polypeptide as shown in SEQ ID No.2, the 306th sports alanine, and the 46th sports isoleucine, the
105 sport leucine, and the 121st sports glycine；263rd sports asparagine,
(15) polypeptide as shown in SEQ ID No.2, the 263rd sports asparagine, and the 176th sports asparagine,
33rd sports leucine, and the 151st sports glutamine,
(16) polypeptide as shown in SEQ ID No.2, the 263rd sports valine, and the 176th sports asparagine, the
33 sport leucine, and the 151st sports glutamine, or
(17) polypeptide as shown in SEQ ID No.2, the 263rd sports glutamine, and the 176th sports asparagine,
33rd sports leucine, and the 151st sports glutamine.
4. isolated polynucleotides, which is characterized in that any epoxy of the polynucleotide encoding claim 1~3
Hydrolyze enzyme mutant.
5. a kind of carrier, which is characterized in that it contains polynucleotides as claimed in claim 4.
6. a kind of genetically engineered host cell, which is characterized in that it contains carrier described in claim 5 or genome
In be integrated with polynucleotides as claimed in claim 4.
7. a kind of method for producing any epoxide hydrolase mutant of claims 1 to 33, which is characterized in that including step
It is rapid:
(1) host cell as claimed in claim 6 is cultivated, culture is obtained；With
(2) any epoxide hydrolase mutant of claims 1 to 3 is separated from culture.
8. a kind of method of the catalytic activity for improving epoxide hydrolase and selectivity, which is characterized in that the described method includes: by wild
Amino acid in the space structure of the epoxide hydrolase of raw type near substrate binding pocket is mutated；The amino of the mutant
Acid sequence corresponds to SEQ ID No.2, and the 263rd sports asparagine, valine or glutamine；It and further include being selected from
One or more mutation of the following group:
176th mutation are as follows: asparagine；
151st mutation are as follows: glutamine or leucine；
105th mutation are as follows: leucine；
33rd mutation are as follows: asparagine or leucine；
196th mutation are as follows: alanine；
3rd mutation are as follows: glutamic acid；
4th mutation are as follows: isoleucine；
20th mutation are as follows: alanine；
31st mutation are as follows: cysteine；
46th mutation are as follows: isoleucine；
56th mutation are as follows: valine；
96th mutation are as follows: glycine；
121st mutation are as follows: glycine；
136th mutation are as follows: valine；
144th mutation are as follows: phenylalanine；
181st mutation are as follows: valine；
270th mutation are as follows: arginine；
281st mutation are as follows: glutamic acid；
296th mutation are as follows: cysteine；Or
306th mutation are as follows: alanine.
9. the purposes of any epoxide hydrolase mutant of claims 1 to 3, for being used as catalyst, catalytic water is unlinked
Oxide；Wherein, the epoxides has formula (I) structure:
Wherein, R represents 1~5 group, and the group is selected from: hydrogen, hydroxyl, cyano, nitro, substituted or non-substituted C1-C4 alkane
Base, substituted or non-substituted C2-C4 alkenyl, substituted or non-substituted C2-C4 alkynyl group, halogen or two neighboring group phase
It connects and collectively forms ring structure with female ring.
10. purposes as claimed in claim 9, which is characterized in that described is hydrolyzed to the hydrolysis of mapping regression nature.
11. purposes as claimed in claim 9, which is characterized in that the epoxides is racemic epoxides.
12. purposes as claimed in claim 9, which is characterized in that the ring is hexatomic ring.
13. purposes as claimed in claim 12, which is characterized in that the hexatomic ring is phenyl ring.
14. purposes as claimed in claim 12, which is characterized in that the group is located at the o-, m- or p- position position of parent nucleus.
15. purposes as claimed in claim 14, which is characterized in that R is the H, NO of contraposition2, CN, halogen, CH3, CF3, Ph base
Group；Or
R is the halogen of meta position, CN, NO2；Or
R is the halogen at ortho position, NO2；Or
R is and is stored in the halogen of meta position and contraposition；Or
R is and is stored in the halogen of ortho position and contraposition.
16. purposes as claimed in claim 15, which is characterized in that the epoxides includes compound selected from the group below:
Formula (I) compound, wherein R is p-H；
Formula (I) compound, wherein R is p-NO2；
Formula (I) compound, wherein R is p-CN；
Formula (I) compound, wherein R is p-F；
Formula (I) compound, wherein R is p-Cl；
Formula (I) compound, wherein R is p-Br；
Formula (I) compound, wherein R is p-CH3；
Formula (I) compound, wherein R is p-CF3；
Formula (I) compound, wherein R is p-Ph；
Formula (I) compound, wherein R is m-Cl；
Formula (I) compound, wherein R is m-Br；
Formula (I) compound, wherein R is m-CN；
Formula (I) compound, wherein R is m-NO2；
Formula (I) compound, wherein R is m-F；
Formula (I) compound, wherein R is o-Cl；
Formula (I) compound, wherein R is o-NO2；
Formula (I) compound, wherein R is m, p-2F；
Formula (I) compound, wherein R is m, p-2Cl；
Formula (I) compound, wherein R is o, p-2F；Or
Formula (I) compound, wherein R is o, p-2Cl.
17. a kind of method for hydrolyzing epoxides, which is characterized in that the described method includes: using epoxides as substrate, with right
It is required that 1~3 any epoxide hydrolase mutant is catalyst, is reacted, obtain the product through hydrolyzing；Wherein, institute
The epoxides stated has formula (I) structure:
Wherein, R represents 1~5 group, and the group is selected from: hydrogen, hydroxyl, cyano, nitro, substituted or non-substituted C1-C4 alkane
Base, substituted or non-substituted C2-C4 alkenyl, substituted or non-substituted C2-C4 alkynyl group, halogen or two neighboring group phase
It connects and collectively forms ring structure with female ring.
18. method as claimed in claim 17, which is characterized in that the ring is hexatomic ring.
19. method as claimed in claim 18, which is characterized in that the hexatomic ring is phenyl ring.
20. method as claimed in claim 17, which is characterized in that the group is located at the o-, m- or p- position position of parent nucleus.
21. method as claimed in claim 20, which is characterized in that R is the H, NO of contraposition2, CN, halogen, CH3, CF3, ph base
Group；Or
R is the halogen of meta position, CN, NO2；Or
R is the halogen at ortho position, NO2；Or
R is and is stored in the halogen of meta position and contraposition；Or
R is and is stored in the halogen of ortho position and contraposition.
22. method as claimed in claim 17, which is characterized in that the concentration of the epoxide is 1-1000mmol/L,
The dosage of the epoxide hydrolase is 2~200U/mmol epoxide.
23. method as claimed in claim 22, which is characterized in that the concentration of the epoxide is 10~500mmol/L.
24. method as claimed in claim 22, which is characterized in that the host cell of claim 6 is added to reaction system
In, participate in reaction.
25. method as claimed in claim 24, which is characterized in that the host cell is added in the form of lyophilized cells.
26. a kind of composition, which is characterized in that contain any epoxide hydrolase of claims 1 to 3 in the composition
Mutant, and acceptable carrier pharmaceutically or on commercial synthesis；Or contain host cell as claimed in claim 6.
27. composition as claimed in claim 26, which is characterized in that the host cell is lyophilized cells.
28. a kind of kit, which is characterized in that wherein containing any epoxide hydrolase mutant of claims 1 to 3.
29. a kind of kit, which is characterized in that wherein containing the composition described in claim 26.
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