CN105018443B - A kind of epoxide hydrolase mutant and preparation method thereof - Google Patents
A kind of epoxide hydrolase mutant and preparation method thereof Download PDFInfo
- Publication number
- CN105018443B CN105018443B CN201510460244.2A CN201510460244A CN105018443B CN 105018443 B CN105018443 B CN 105018443B CN 201510460244 A CN201510460244 A CN 201510460244A CN 105018443 B CN105018443 B CN 105018443B
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- epoxide hydrolase
- replaces
- mutant
- arginine
- amino acid
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Classifications
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- C—CHEMISTRY; METALLURGY
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Abstract
The present invention discloses a kind of epoxide hydrolase mutant and preparation method thereof, and epoxide hydrolase mutant is the 8th of the amino acid sequence as shown in SEQ ID NO.1, the 26th, the 83rd, the 90th and the 122nd amino acid to be carried out simple point mutation or multipoint mutation obtains.Preparation method：By in the gene cloning to plasmid pET28a (+) for encoding epoxide hydrolase, construction recombination plasmid pET28a (+) EH；Using rolling ring PCR amplification plasmid pET28a (+) EH, the open loop recombinant vector of the gene order containing coding epoxide hydrolase mutant is obtained；By the PCR reaction solution transformed competence colibacillus bacillus coli DH 5 alpha containing the gene for encoding correct mutant, the correct replicon of picking, and extract correct mutant plasmid；Convert e. coli bl21 (DE3), induced expression.Compared with wild type epoxide hydrolase, epoxide hydrolase of the present invention has high temperature stability and pH value accommodation, can greatly improve the service life of epoxide hydrolase in industrialized production.
Description
Technical field
The invention belongs to technical field of enzyme engineering, and in particular to a kind of epoxide hydrolase mutation that thermal stability improves
Body and preparation method thereof.
Background technology
Epoxide hydrolase (epoxide hydrolase, EH, EC 3.3.2.10) be catalyzing hydrolysis epoxides or
Its salt generates the general name of corresponding vicinal diamines or the class of enzymes of its salt, and catalysis reaction is without any coenzyme, prothetic group or metal ion
It participates in.Epoxide hydrolase is distributed widely in mammal, plant and microorganism.In recent years, three-dimensional single-minded due to having
Property it is good, enzymatic reaction is fast, product optical purity and yield are high, and isolates and purifies the advantages that product is simpler, microbe-derived
Epoxide hydrolase have received widespread attention and concrete application.Miura in 1977 et al. is for the first time successfully by epoxides water
Solve the industrialized production (US Patent4010072) that enzyme is applied to L (+)-tartaric acid.
L (+)-tartaric acid, also known as (2R, 3R) -2,3- dihydroxy -1, 4- succinic acid are a kind of naturally occurring organic acids,
It may be used as acid in the food industry, tartaric acid list (double) acid anhydride ester is important food additives；In pharmaceuticals industry, it
It is the resolving agent of most common chiral drug and intermediate；As resist agent, color fixing agent etc. in dyeing industry；It is also used as gold
Belong to ion masking agent, for being electroplated, the industries such as process hides and mirror processed.
Past, the main method of production L (+)-tartaric acid are to extract and process the by-product winestone of grape wine, can also be with
Glucose is that primary raw material is prepared by fermentation, conversion and extraction.Currently, using maleic anhydride as raw material, using containing epoxy
The microorganism conversion of compound hydrolase is the main mode of production.Catalysis Principles is as follows：First by maleic anhydride and hydrogen peroxide
Cis-form epoxy succinic acid is made in reaction, and cis-form epoxy succinic acid or its salt hydrolysis are by recycling microbial epoxidation object hydrolase
L (+)-tartaric acid or salt.
The microorganism that can produce the epoxide hydrolase prepared for L (+)-tartaric acid reported at present has rhizobium
Category, pseudomonas, Nocardia, corynebacterium, Rhod, achromobacter, acetobacter, agrobacterium
Category, alcaligenes and acinetobacter.The most commonly used is the Rhods with greater catalytic efficiency in industrial production.2007
Liu et al. reports Rhodococcus sp Rhodococcus opacus ML-0004 epoxide hydrolase genes for the first time, and by it in original
Nucleus (E.coli) is expressed.(Appl Microbiol Biotechnol 2007；74:99-106) however the enzyme is warm
Stability is very poor, and to very temperature sensitive, preservation 30min can then lose 60% vigor at 45 DEG C, after preserving 30min at 50 DEG C
Complete deactivation.The weak thermal stability limit service life of biocatalyst, limits its catalytic efficiency, also increases industrial life
Cost in production.Therefore, the thermal stability for improving this enzyme is most important currently with biological catalysis production L (+)-tartaric acid
One of problem.
Currently, by genetic engineering means engineered protein primary structure, enhancing protein three-dimensional structure rigidly improves
The means of heat stability of protein have become mainstream.According to protein structure and emic degree of understanding, protein
Transformation means are broadly divided into " orthogenesis ", " design and rational " and " half design and rational ".Since the eighties in last century, " orientation
Evolve " it has successfully been applied on protein " engineered ex vivo ".Since " orthogenesis " is not required to it is to be understood that specific protein
The structure of matter and its corresponding function, it is established that can simulate natural evolution to purpose egg after rational evolution Filtering system
White matter is transformed and screens, therefore a large amount of protein that structural rigidity is low and structural information is few result in transformation
(FEBS Lett.276(2009)1750–1761).Although significant effect, " orthogenesis " has reforming direction blindness, work
The shortcomings that work amount is big and Filtering system is difficult to set up.
In order to make transformation more with purpose and reduce workload, researcher proposes the concept of " design and rational ".
" design and rational " refers to appliance computer simulation under the premise of known target protein structural information and its function mechanism
The influence in terms of structure and function that operation prediction can generate after being transformed to protein, and the side for passing through rite-directed mutagenesis
Formula takes prediction result in experiment (Appl.Microbiol.Biotechnol.99 (2014) 1205-1216) to." rationality is set
The characteristics of meter " is using with a high credibility by the obtained protein structures of X-ray diffraction crystallization of protein body, accurately
Predict that protein substitutes.Although " design and rational " presents efficient and small workload spy really in terms of predicting protein transformation
Point.But the process often extremely complex hardships, and success rate of early-stage preparations protein crystal and parsing protein three-dimensional structure
It cannot ensure.Do not have to utilize " design and rational " engineered protein Quality Research to be easy in the case of ready-made protein structures
The structural research to target protein has been evolved into, main direction of studying is deviated from.
Therefore researcher has also been proposed a concept for being combined both " orthogenesis " and " design and rational " " half reason
Property design ".This concept is related to a series of protein engineering means, is on the one hand advised in the case where allowing relatively large prediction error
Fixed " orthogenesis " is mutated direction to reduce screening scale, and the protein structure on the other hand evaded in " design and rational " is parsed
Journey greatly improves the efficiency of protein engineering transformation." half design and rational " includes mainly multiple alignment (MSAs), homologous to build
Mould, virtual mutation and fixed point saturation mutation (Curr.Opin.Biotechnol.21 (2010) 734-743.).
Currently, the protein knot of the epoxide hydrolase from Rhodococcus sp Rhodococcus opacus ML-0004
Structure information and its function mechanism are still unintelligible.There is the epoxide hydrolase compared with heat-flash stability to be mutated in order to obtain
Body, while improving working efficiency as far as possible, the method that the present invention is combined using " orthogenesis " with " half design and rational " is to it
Carry out primary structure transformation.
Invention content
For the not high technical problem of the thermal stability of epoxide hydrolase, the present invention provides a kind of epoxides water
Solve enzyme mutant and preparation method thereof.
A kind of epoxide hydrolase mutant, the epoxide hydrolase mutant are by such as SEQ ID NO.1 institutes
Show that the 8th, the 26th, the 83rd, the 90th and the 122nd amino acid of amino acid sequence carries out simple point mutation or multiple spot is prominent
Become and obtains.
Further, the epoxide hydrolase mutant is one of following：
(1) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine (D8K)；
(2) the 26th phenylalanine of the amino acid sequence as shown in SEQ ID NO.1 replaces with valine or tryptophan
(sour F26V/F26W)；
(3) the 83rd isoleucine of the amino acid sequence as shown in SEQ ID NO.1 replaces with arginine (I83R)；
(4) the 90th serine of the amino acid sequence as shown in SEQ ID NO.1 replaces with arginine (S90R)；(5)
122 glutamine of the amino acid sequence as shown in SEQ ID NO.1 replace with arginine (Q122R)；
(6) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 26th
The phenylalanine of position replaces with valine or tryptophan (D8K＆F26V/D8K＆F26W)；
(7) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 83rd
The isoleucine of position replaces with arginine (D8K＆I83R)；
(8) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 90th
The serine of position replaces with arginine (D8K＆S90R)；
(9) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 122nd
The glutamine of position replaces with arginine (D8K＆Q122R)；(10) the 26th of the amino acid sequence as shown in SEQ ID NO.1
Phenylalanine replaces with valine or tryptophan, while the 83rd isoleucine replaces with arginine (F26V＆I83R/F26W＆
I83R)；
(11) the 26th phenylalanine of the amino acid sequence as shown in SEQ ID NO.1 replaces with valine or color ammonia
Acid, while the 90th serine replaces with arginine (F26V＆S90R/F26W＆S90R)；
(12) the 26th phenylalanine of the amino acid sequence as shown in SEQ ID NO.1 replaces with valine or color ammonia
Acid, while the 122nd glutamine replaces with arginine (F26V＆Q122R/F26W＆Q122R)；
(13) the 83rd isoleucine of the amino acid sequence as shown in SEQ ID NO.1 replaces with arginine, while
90 serines replace with arginine (I83R＆S90R)；
(14) the 83rd isoleucine of the amino acid sequence as shown in SEQ ID NO.1 replaces with arginine, while
122 glutamine replace with arginine (I83R＆Q122R)；
(15) the 90th serines replace with arginine, while the 122nd glutamine replaces with arginine
(S90R&Q122R)；
(16) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 26th
The phenylalanine of position replaces with valine or tryptophan, while the 83rd isoleucine replaces with arginine (D8K＆F26V＆
I83R/D8K&F26W&I83R)；
(17) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 26th
The phenylalanine of position replaces with valine or tryptophan, while the 90th serine replaces with arginine (D8K＆F26V＆
S90R/D8K&F26W&S90R)；
(18) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 26th
The phenylalanine of position replaces with valine or tryptophan, while the 122nd glutamine replaces with arginine (D8K＆F26V＆
Q122R/D8K&F26W&Q122R)；
(19) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 83rd
The isoleucine of position replaces with arginine, while the 90th serine replaces with arginine (sour D8K＆I83R＆S90R)；
(20) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 83rd
The isoleucine of position replaces with arginine, while the 122nd glutamine replaces with arginine (D8K＆I83R＆Q122R)；
(21) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 90th
The serine of position replaces with arginine, while the 122nd glutamine replaces with arginine (D8K＆S90R＆Q122R)；
(22) the 26th phenylalanine of the amino acid sequence as shown in SEQ ID NO.1 replaces with valine or color ammonia
Acid, while the 83rd isoleucine replaces with arginine, while the 90th serine replaces with arginine (S90R＆F26V＆
I83R/S90R&F26W&I83R)；
(23) the 26th phenylalanine of the amino acid sequence as shown in SEQ ID NO.1 replaces with valine or color ammonia
Acid, while the 83rd isoleucine replaces with arginine, while the 122nd glutamine replaces with arginine (Q122R＆
F26V&I83R/Q122R&F26W&I83R)；
(24) the 26th phenylalanine of the amino acid sequence as shown in SEQ ID NO.1 replaces with valine or color ammonia
Acid, while the 90th serine replaces with arginine, while the 122nd glutamine replaces with arginine (S90R＆
F26V&Q122R/S90R&F26W&Q122R)；
(25) the 83rd isoleucine of the amino acid sequence as shown in SEQ ID NO.1 replaces with arginine, while
90 serines replace with arginine, while the 122nd glutamine replaces with arginine (S90R＆I83R＆Q122R)；
(26) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 26th
The phenylalanine of position replaces with valine or tryptophan, while the 83rd isoleucine replaces with arginine, while the 90th
Serine replace with arginine (D8K＆S90R＆F26V＆I83R/D8K＆S90R＆F26W＆I83R)；
(27) the 26th phenylalanine of the amino acid sequence as shown in SEQ ID NO.1 replaces with valine or color ammonia
Acid, while the 83rd isoleucine replaces with arginine, while the 90th serine replaces with arginine, while the 122nd
The glutamine of position replaces with arginine (Q122R＆S90R＆F26V＆I83R/Q122R＆S90R＆F26W＆I83R)；
(28) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 83rd
The isoleucine of position replaces with arginine, while the 90th serine replaces with arginine, while the 122nd glutamine
Replace with arginine (D8K＆S90R＆I83R＆Q122R)；
(29) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 26th
The phenylalanine of position replaces with valine or tryptophan, while the 90th serine replaces with arginine, while the 122nd
Glutamine replaces with arginine (D8K＆S90R＆F26V＆Q122R/D8K＆S90R＆F26W＆Q122R)；
(30) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 26th
The phenylalanine of position replaces with valine or tryptophan, while the 83rd isoleucine replaces with arginine, while the 122nd
Glutamine replace with arginine (D8K＆I83R＆F26V＆Q122R/D8K＆I83R＆F26W＆Q122R)；
(31) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 26th
The phenylalanine of position replaces with valine or tryptophan, while the 83rd isoleucine replaces with arginine, while the 90th
Serine replace with arginine (D8K＆I83R＆F26V＆S90R/D8K＆I83R＆F26W＆S90R)；
(32) the 8th aspartic acid of the amino acid sequence as shown in SEQ ID NO.1 replaces with lysine, while the 26th
The phenylalanine of position replaces with valine or tryptophan, while the 83rd isoleucine replaces with arginine, while the 90th
Serine replace with arginine, while the 122nd glutamine replaces with arginine (D8K＆I83R＆F26V＆Q122R＆
S90R/D8K&I83R&F26W&Q122R&S90R)。
The present invention also provides a kind of gene of the coding epoxide hydrolase mutant, the bases of one of which mutant
The base sequence of cause such as SEQ ID NO.16.
The present invention also provides a kind of recombinant vectors or recombinant cell carrying the gene.
The present invention also provides a kind of methods obtaining the epoxide hydrolase mutant, include the following steps：(1) will
In the gene cloning to plasmid pET28a (+) for encoding epoxide hydrolase, construction recombination plasmid pET28a (+)-EH；(2) it sets
Complementary primer of the meter containing the codon base for representing mutating acid is obtained using rolling ring PCR amplification plasmid pET28a (+)-EH
To the open loop recombinant vector of the gene order containing coding epoxide hydrolase mutant；(3) volume will be contained obtained by step (2)
The PCR reaction solution of the gene of the correct mutant of code passes through DpnI endonuclease digestions, removes unmutated protoplasm grain, reaction solution is direct
Transformed competence colibacillus bacillus coli DH 5 alpha, the correct replicon of picking, and extract correct mutant plasmid；(4) by correct mutant plasmid
Convert e. coli bl21 (DE3), induced expression.
The gene of coding epoxide hydrolase is from Rhodococcus sp Rhodococcus opacus ML-0004 in step (1)
It obtains.It still further comprises and epoxide hydrolase is purified using " the nickel column " of HisTrap FF crude 1ml.
The induced expression process is：The recombinant bacterium that step (4) is obtained is trained in the LB of the kanamycins containing 50 μ g/L
It supports 37 DEG C of Liquid Cultures in base to stay overnight, the 37 DEG C of cultures of the rear LB fermentation broths for accessing the kanamycins containing 50 μ g/L are extremely
OD600=0.6 is cooled to 28 DEG C of cultures, and the derivant IPTG that ultimate density 0.1mM is added induces 8-12h, harvests interior expression ring
The thalline of oxide hydrolase.
About the research of epoxide hydrolase according to the present invention also in the elementary stage, this is only encoded at present
The protein sequence (GenBank accession number ABF01020) of protein, corresponding nucleic acid sequence (GenBank accession number
DQ471957) and the Catalysis Principles of this albumen is predicted to be reported.Therefore the present invention utilizes " half design and rational " using main
Research Thinking this epoxide hydrolase is transformed.L (+)-winestone generated due to the catalysis of this epoxide hydrolase
Acid and its salt can be detected with " ammonium metavanadate development process ", therefore the present invention is devised for this epoxide hydrolase
" high flux screening method ", the means for introducing " orthogenesis " have carried out a wheel screening to this epoxide hydrolase.This epoxidation
Object hydrolase and belong to halogenated acids dehalogenation hydrolase superfamily (haloacid dehalogenase-like hydrolases
(HAD) superfamily) four enzymes with high thermal stability with conserved sequence similitude, therefore using MSAs to this
The protein sequence and this four protein sequences of the involved wild type epoxide hydrolase of invention are compared, and are predicted and are obtained
To the advantageous mutation of thermal stability." homologous modeling " is utilized to establish wild type epoxide hydrolase according to the present invention
Protein model uses " virtual mutation " prediction nucleic acid alternative, obtains to the advantageous mutation of thermal stability later.Utilize this
" the high flux screening method " established in invention is prominent to having obtained to the advantageous catastrophe point of heat stability of protein be saturated
Become, further improves the thermal stability of the epoxide hydrolase.
By to the rite-directed mutagenesis and combinatorial mutagenesis to selecting site, present invention gained epoxide hydrolase mutant
Thermal stability be remarkably reinforced, single-point mutants D8K, F26V/F26W, I83R, S90R, Q122R, two point combinatorial mutagenesis D8K＆
F26V、D8K&F26W、D8K&I83R、D8K&S90R、F26V&I83R、F26W&I83R、F26V&S90R、F26W&S90R、F26V&
Q122R, F26W＆Q122R, I83R＆S90R, I83R＆Q122R, S90R＆Q122R, 3 combinatorial mutagenesis D8K＆F26V＆I83R,
D8K&F26W&I83R、D8K&F26V&S90R、D8K&F26W&S90R、D8K&F26V&Q122R、D8K&F26W&Q122R、D8K&
I83R&S90R、D8K&I83R&Q122R、D8K&S90R&Q122R、S90R&F26V&I83R、S90R&F26W&I83R、Q122R&
F26V&I83R、Q122R&F26W&I83R、S90R&F26V&Q122R、S90R&F26W&Q122R、S90R&I83R&Q122R、
D8K＆S90R＆F26V＆I83RD8K＆S90R＆F26W＆I83R, 4 combinatorial mutagenesis Q122R＆S90R＆F26V＆I83R, Q122R＆
S90R&F26W&I83R、D8K&S90R&I83R&Q122R、D8K&S90R&F26V&Q122R、D8K&S90R&F26W&Q122R、
D8K&I83R&F26V&Q122R、D8K&I83R&F26W&Q122R、D8K&I83R&F26V&S90R、D8K&I83R&F26W&
The thermal stability of S90R and 5 combinatorial mutagenesis D8K＆I83R＆F26V＆Q122R＆S90, D8K＆I83R＆F26W＆Q122R＆S90R
It is significantly increased, and mutation combination D8K＆I83R＆F26W＆Q122R＆S90R also significantly increases the adaptability of pH value.
Compare original epoxide hydrolase, ratios of the mutant D8K＆I83R＆F26W＆Q122R＆S90R after Ni column purifications at 37 DEG C
Vigor (U/mg) is reduced to 75.1, K by 76.5m(mmol/L) 29.1, K are increased to by 25.3cat×103(min-1) become from 5.82
6.20.It is 55-60 DEG C that optimal reactive temperature has been increased to by 35-40 DEG C, and the half-life period at 50 DEG C has 8.5min to be increased to
293.2.1min.As it can be seen that heat will greatly improved in saltant type epoxide hydrolase D8K＆I83R＆F26W＆Q122R＆S90R
The catalysis efficiency of wild type epoxide hydrolase is not significantly impacted on the basis of stability.
Description of the drawings
Fig. 1 is that epoxide hydrolase EH and four come from halogenated acids dehalogenation hydrolase superfamily (haloacid
Dehalogenase-like hydrolases (HAD) superfamily) enzyme multiple alignment's result.
Fig. 2 is epoxide hydrolase EH homology model configuration schematic diagrames.
Fig. 3 is the SDS-PAGE electrophoresis of epoxide hydrolase EH and 5 kinds of mutant.W：Wild type epoxides hydrolyzes
Enzyme, 1：Saltant type Q122R.2：Saltant type F26W.3：Saltant type I83R.4：Saltant type D8K.5：Saltant type S90R.
Fig. 4 be wild type epoxide hydrolase and saltant type Q122R＆F26W＆I83R＆D8K＆S90R at different temperatures
Specific enzyme activity (U/mg).
Fig. 5 is that wild type oxide hydrolase and saltant type Q122R＆F26W＆I83R＆D8K＆S90R locate at different temperatures
Remnant enzyme activity (%) after reason.
Fig. 6 be wild type epoxide hydrolase and saltant type Q122R＆F26W＆I83R＆D8K＆S90R at various ph values
The relative activity (%) of the enzyme of reaction.
Fig. 7 is wild type oxide hydrolase and saltant type Q122R＆F26W＆I83R＆D8K＆S90R in different pH condition
Lower treated remnant enzyme activity (%).
Specific implementation mode
The detection method of parameter is corresponded in following embodiment：
Enzyme activity determination method
By the sodium hydrogen cis-epoxysuccinate substrate (pH 8.0) of 0.9mL 1mol/L after 37 DEG C keep the temperature 5min, it is added
0.1mol enzyme solutions simultaneously react 60min at 37 DEG C, measure the content of reaction solution mesotartaric acid.Under the above-described reaction conditions, per minute
It generates 1 μm of ol tartaric acid and is defined as an enzyme-activity unit, indicated with U.Under these conditions, the enzyme activity list contained by every milligram of albumen
Digit is defined as Rate activity, is indicated with U/mg.
The detection method of tartaric acid content
It takes the ammonium metavanadate of 2.5mL 1% in the volumetric flask of 25mL, after adding suitable above-mentioned reaction solution, then adds 1mL
The sulfuric acid of 1mol/L is settled to 25mL with distilled water, the light absorption value at 530nm is surveyed after mixing, and according to the standard curve of formulation
Calculate tartaric acid concentration.
The assay method of albumen concentration
Taking 5mL coomassie brilliant blue stainings liquid, (0.1g Coomassie brilliant blues are dissolved in 95% ethyl alcohol of 50mL, and 100mL 85% is added
Phosphoric acid adds water to be settled to 1L), appropriate protein liquid is added, the light absorption value at 595nm is surveyed after mixing, and bent according to specified standard
Line computation albumen concentration.
KmAnd KcatThe assay method of value
Equivalent enzyme solution is respectively placed in final concentration of 100mmol/L, 70mmol/L, 45mmol/L, 35mmol/L,
In the sodium hydrogen cis-epoxysuccinate (pH 7.5) of 25mmol/L, 20mmol/L, 15mmol/L, 10mmol/L, reacted at 37 DEG C
20min measures the content of reaction solution mesotartaric acid, the bis- counting backward techniques of Lineweaver-Burk is used in combination, calculate the K of enzymemAnd Kcat,
Unit is respectively mmol/L and × 103min-1。
The assay method of enantiomeric excess value
The engineering bacteria cell containing epoxide hydrolase gene of isopropylthio-β-D-galactoside of learning from else's experience induction,
It is dissolved in two sodium solution of 100mL 1mol/L cis-form epoxy succinic acids, for 24 hours, CaCl is added in 30 DEG C of oscillating reactions2Aqueous solution, mistake
Filter and water washing and precipitating, then refine, concentrate, crystallize and dry through sulfuric acid solution, cation and anion exchange column, respectively obtain L (+)-wine
Stone acid.By the sterling L (+) of acquisition-tartaric acid dissolving, its specific rotatory power is measured using polarimeter.After testing, all wild
The optical purity of type epoxide hydrolase products obtained therefrom L (+)-tartaric acid reaches 99% or more.
The experimental methods of molecular biology of actual conditions is not specified in following embodiment, according to normal condition, reference《Point
Sub- cloning experimentation guide》(New York：Cold Spring Harbor Laboratory Press, 2001) condition described in
It carries out.
Embodiment 1：The structure of recombinant bacterium
According to Rhodococcus sp Ml-0004 (Rhodococcus opacus ML- disclosed in U.S.'s GenBank databases
0004) nucleic acid sequence (accession number DQ471957) of epoxide hydrolase carries out codon optimization, it is made to be more suitable in engineering
It is replicated and is expressed in bacterium E.coli.By gene order (the gene order such as SEQ ID NO.2 institutes after optimization after optimization
Show) send Services Co., Ltd of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd to make recombinant plasmid pUC57-EH, epoxide hydrolase gene
(EH) between restriction endonuclease sites Bam H I and Hind III.
Then double digestion is carried out respectively to pUC57-EH and pET28a (+) with Bam H I and Hind III, QIAquick is used in combination
GEL Extraction Kit kits carry out glue recycling small fragment and large fragment respectively.DNA fragmentation and with plasmid fragments in T4
In linked system, 16 DEG C of connections overnight, target gene are connected on pET28a (+), construction recombination plasmid pET28a (+)-EH.
Among recombinant plasmid is transferred to competent cell of the clone with bacterial strain E.coli DH5 α by thermal shock conversion method, into
Row amplification.Single bacterium colony E.coli DH5 α-pET28a (+)-EH on picking LB tablets, is incubated overnight in LB culture mediums, uses alkali
Cracking process extracts plasmid, and then recombinant plasmid is transferred to E.coli BL21 (DE3) competence using thermal shock conversion method.
Embodiment 2：The expression and purity of wild type and saltant type epoxide hydrolase
It is inoculated with wild type gene engineering bacteria and mutated genes engineering bacteria (being prepared by 1 method of embodiment) respectively to containing 50 μ
In the 50mL LB culture mediums of g/mL kanamycins (1% peptone, 0.5% yeast extract, 1% sodium chloride, pH7.0), 37 DEG C
Shaken cultivation, as cell concentration (OD600) when reaching 0.6-0.8, it is added 0.1mM isopropylthio-β-D-galactosides, 28 DEG C
Shaken cultivation 12h.
4 DEG C, 5000rpm centrifugation 10min collect thalline, normal saline flushing three times after, add in the ratio of every gram of thalline 5mL
Enter lysis buffer (50mmol/L Tris-HCl, 0.5mol/L NaCl, pH 7.5), the broken born of the same parents of ice precooling ultrasound, 4 DEG C,
12000rpm centrifuges 5min, and supernatant, loading to Ni-NTA affinity columns is taken (it is limited to be purchased from Shanghai life work biotechnology service
Company), with wash miscellaneous buffer solution (50mmol/L Tris-HCl, 0.5mol/L NaCl, 20mmol/L imidazoles, pH 7.5) wash away it is miscellaneous
Matter composition is eluted with elution buffer (50mmol/L Tris-HCl, 0.5mol/L NaCl, 200mmol/L imidazoles, pH 7.5)
Destination protein fully dialyses the enzyme solution of collection in 4 DEG C, 10mmol/L Tris-HCl (pH 7.5) buffer solution, polyethylene glycol
After concentration, with 10mmol/L Tris-HCl (pH 7.5) buffer solution containing 50% glycerine, in -20 DEG C of preservations, and pass through 12%
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel
Electropheresis, SDS-PAGE) detection purification effect.
Protein band after purification is single, and for purity up to 99% or more, molecular weight is about 28kDa, epoxide hydrolase
The SDS-PAGE electrophoresis of EH and 5 kinds of mutant is as shown in Figure 3.W：Wild type epoxide hydrolase, 1：Saltant type Q122R.
2：Saltant type F26W.3：Saltant type I83R.4：Saltant type D8K.5：Saltant type S90R.
Embodiment 3：The directional evolution mutant of Rhodococcus sp ML-0004 epoxide hydrolase genes
Extraction is connected with the recombinant plasmid pET- of the wild type epoxide hydrolase gene EH after rare codon optimization
28a (+) (structure of embodiment 1), introduces copy error in a manner of divalent manganesetion is added into polymerase chain reaction PCR.
The PCR reaction systems of orthogenesis are：The homemade 5mM MnCl of 1.0ul2, 0.5ul Taq polymerases (Takara
Bio Inc., Shiga, Japan), the buffer (10 ×), 14.0ul 25mM MgCl that 10.0ul suppliers provide2,4.0ul
Primer (SEQ ID NO.4), 0.5ul template DNAs after primer (SEQ ID NO.3) before dNTP Mixture, 1.0ul, 1.0ul
(50ng/ul) and 18.0ul steam water.
Its PCR program is：98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 60s (30 cycle).
Purified pcr product is handled 3 hours using restriction endonuclease BamH I and Hind III at 37 DEG C.Instead
It is 1ul BamH I, 1ul Hind III, 2ul 10 × M Buffer and 16ul aqua sterilisas to answer system.After " double digestion " processing
PCR product purified using nucleic acid electrophoresis, be tapped and recovered smaller fragment containing mutation sites.
Mutant fragments after purification are connected to pET-28a (+) empty plasmid obtained after same method " double digestion ", are carried out
Enzyme connects：The enzyme disjunctor system of 20ul includes the mixed of 1ul T4 ligases, 10 × T4 of 2ul ligases and 17ul Insert Fragments and carrier
Liquid is closed, the wherein ratio of Insert Fragment and carrier is 5:1.Connection reaction preserves overnight at 16 DEG C.
Connection product is all imported with thermal transition in e. coli bl21 (DE3) competent cell prepared in advance：
By in e. coli bl21 (DE3) competent cells for just having thawed of 20ul connection products addition 100ul, ice bath 30min, 42 DEG C
Then accurate thermal shock 45s, ice bath 10min down are added the LB culture mediums of 250ul sterilizings, are cultivated in 37 DEG C, the shaking table of 225rpm
1 hour.The cell after preculture is taken, EP pipes are centrifuged into 30min at 3,000 rpm, clear liquid is removed with liquid-transfering gun, it then will precipitation
Cell gently pick up, be completely transferred on the LB agar plates containing 50 μ g/mL kanamycins using rubbing method, trained at 37 DEG C
Support 10-12h.
After building mutant library, random picking single bacterium colony carries out heat stability of protein detection experiment from mutation library.It chooses
Doubtful mutant single bacterium colony is taken, " the 96 hole cell training of shallow bore hole of the 300ulLB culture mediums containing 50ug/ml kanamycins is added to
Culture 6 hours in foster ware "." 96 hole Tissue Culture Dish of shallow bore hole " is named as " motherboard ", is stored in -80 DEG C of refrigerator-freezers as connecing down
Come the seed cultivated and induced.50ul bacterium solutions are extracted from motherboard, are added to containing 50ug/ml kanamycins and the cis- rings of 0.1M
It is cultivated in the 600ul LB culture mediums of oxydisuccinic acid sodium." 96 hole Tissue Culture Dish of shallow bore hole " is named as " daughter board ".It will be sub
Plate is cultivated 2.5 hours in 37 DEG C, the shaking table of 225rpm, and 50ul 1.2mM IPTG, which are added, in each hole on plate is induced.
Subsequent daughter board is cultivated 10-16 hours in 28 DEG C, the shaking table of 225rpm.By the bacterium after culture and induction at 4000rpm from
Heart 5min.The 0.1M sodium acetate buffers of 125ul supernatants and 100ul pH4.8 are taken to be shown with pure 1% ammonium metavanadates of 25ul
Color.The absorbance of above-mentioned reaction solution is measured at 530nm, retaining color developing effect in mutant library, will close to the bacterium of wild type
It is extracted from daughter board, carries out thermal stability determination experiment.
Cell on selected daughter board 300ul 50mM phosphate buffers are resuspended, by re-suspension liquid in 50 DEG C of water-baths
Middle processing 15min is immediately placed in and is allowed to restore room temperature on ice.Contain to addition 300ul is restored into the re-suspension liquid of room temperature
The 50mM phosphate buffers of 0.2M sodium hydrogen cis-epoxysuccinates react 1 hour at 37 DEG C.Centrifuging and taking supernatant again, inclined vanadium
Sour ammonium color developing detection remnant enzyme activity.Saltant type by remnant enzyme activity high percentage in wild type screens, and extraction seed liquor is sent
It goes to Shanghai Sheng Gong Co., Ltds to be sequenced, and the seed liquor containing mutant enzyme is cultivated and induced, separation is pure
Its specific enzyme activity, thermal stability and kinetic parameter are measured after change.
It screens to obtain the genetic engineering bacterium of one plant of epoxide hydrolase containing high thermal stability by orthogenesis.Through
Gene sequencing learns, which carries mutant Q122R, the as on wild type epoxide hydrolase subject amino acid chain
122 are mutated by glutamine for arginine.
Preferably, Rate activity (U/mg) of the mutant enzyme -1 that mutant Q122R is obtained after Ni column purifications at 37 DEG C be
75.8, Km(mmol/L) it is 24.8, Kcat×103(min-1) it is 5.72.The optimal reactive temperature of mutant enzyme -1 is 35-40
DEG C, the half-life period at 50 DEG C is 31.6min.It can be seen that orthogenesis is introduced to be changed to arginine by 122 upper glutamine
Replacement have the effect of improve heat stability of protein, and without influence enzyme catalysis characteristics.
Embodiment 4：Rhodococcus sp ML-0004 epoxide hydrolase genes multiple alignment (MSAs)
Although being proved to effective, the mutant library of random screening required for orthogenesis experiment is excessively huge, the present invention
Multiple alignment is introduced to accelerate the speed that purpose epoxide hydrolase is transformed.In same superfamily, thermal stability
The same amino acid residue on conserved sequence shared between good protein may be to discriminate between the egg of they and thermal stability difference
Key between white matter.From Rhodococcus sp ML-0004 epoxide hydrolase EH with from belonging to halogenated acids dehalogenation water
Solve enzyme superfamily (haloacid dehalogenase-like hydrolases (HAD) superfamily) four have height
Enzyme (the GenBank accession number No.HAD_AGRTR of thermal stability:P60527.1,DhlS5I:P60527.1,L-DEX:
Q53464.1and PH0459:83753572) there is conserved sequence similitude (alignment is as shown in Fig. 1).Think at this
The shared conserved sequence of four homologous proteins can be to leading to high thermal stability, therefore intends looking for by way of multiple alignment
To the structural similarity of EH and this four thermophilic proteins, prediction may improve the amino acid substitution mode of heat stability of protein.
Phenylalanine in wild type epoxide hydrolase on the 26th site is sported figured silk fabrics by success prediction of the present invention
Propylhomoserin F26V can improve the thermal stability of epoxide hydrolase；And by the 83rd site in wild type type epoxide hydrolase
On tryptophan sport arginine S90R (I83R) thermal stability of epoxide hydrolase can be improved.
Embodiment 5：The homologous modeling of Rhodococcus sp ML-0004 epoxide hydrolases and virtual mutation
Due to up to the present there are no the protein three-dimensional structure about Rhodococcus sp ML-0004 epoxide hydrolases,
Therefore it needs to pass through the protein three-dimensional structure model that " homologous modeling " establishes Rhodococcus sp ML-0004 epoxide hydrolases, and
It is influenced caused by entire protein structure model using virtual sudden change means calculating single-point amino acid mutation to judge the mutation
Whether the structural rigidity of protein can be impacted, and then change the thermal stability of protein.
Using the homologous modeling module in software Discovery Studio3.0, in entire Protein Data Bank numbers
According to search pattern in library, highest halohydrin dehalogenase DehIVa (the GenBank accession number of structural similarity is obtained
No.Q51645), as template, epoxide hydrolase protein matter model is established, model is verified using software PROCHECK
Availability.It is established using halohydrin dehalogenase DehIVa as template by homologous modeling and model feasibility analysis, the present invention
Protein structure model (as shown in Figure 2).
Using Discovery Studio3.0 softwares, simulation is complete by amino acid residue all on all proteins model
Portion replaces with alanine, as a result, it has been found that, it is to be happened at Asp8 that maximum variation, which occurs, for the virtual front and back protein structure energy of mutation,
Asp 25,Phe26,Glu32,Gly34,Leu35,Asp 43,Glu48,Asp 52,Asp 60,Asp 63,Leu65,Phe89,
Ser90, Asp 91, Glu101, Gly106, on Ser112, Asp 152, and the sites Gly161.
In view of the structure of modification on protein surface loop is easier to change the structural stability of protein, therefore will
Virtual saturation mutation is carried out on the five amino acid residue being in the areas protein surface loop.This five amino acid residue is distinguished
It is Asp 8, Leu65, Ser90, Asp 91 and Gly161.Virtual saturation mutation is predicted the Aspartic acid mutations in the 8th site
For lysine D8K；It is the thermal stability S90R that arginine can improve protein by the mutant serine in 90 sites.It is prominent using fixed point
Become and above-mentioned prediction result is presented.
Embodiment 6：Rhodococcus sp ML-0004 epoxide hydrolase gene rite-directed mutagenesis
In rite-directed mutagenesis experiment, by the subject amino acid sequence of wild type Rhodococcus sp ML-0004 epoxide hydrolases
Upper 26th phenylalanine residue (Phe, corresponding codon is TTC) rite-directed mutagenesis is that (Val, corresponding codon are valine
GTT), corresponding primer (SEQ ID NO.5, SEQ ID NO.6) is made；By the 83rd isoleucine (Ile, corresponding codon
For ATC) rite-directed mutagenesis is arginine (Arg, corresponding codon are CGT, CGC, CGA or CGG).CGT, CGC, CGA and CGG are same
Adopted codon, the present embodiment design primer (SEQ ID NO.7, SEQ ID NO.8) by taking CGT codons as an example；By the 8th day
Winter histidine residue (Asp, corresponding codon are GAC) sports lysine (Lys, corresponding codon are AAA or AAG).AAA and
AAG is synonym, they encode lysine, the present embodiment made by taking AAA pass phrase as an example primer (SEQ ID NO.9,
SEQ ID NO.10)；It is arginine (Arg, corresponding password by the 90th serine (Ser, codon TCT) rite-directed mutagenesis
Son is CGT, CGC, CGA or CGG).CGT, CGC, CGA and CGG are synonym, they encode arginine, the present embodiment
By taking CGT codons as an example, mutant primer (SEQ ID NO.11, SEQ ID NO.12) is designed.
The PCR reaction systems of rite-directed mutagenesis are：40 μ L, 10 × PCR buffer of distilled water, 5 μ L, 10mmol/L dNTPs
1 μ L, the 10mmol/L primers of 2 μ L, 1 μ L of carrier pET28a-EH-x, 1 μ L of Taq enzyme, the reaction system of totally 50 μ L.
Its PCR program is：94 DEG C of 50s, 66 DEG C of 30s, 72 DEG C of 6min, 30 cycles, last 72 DEG C of extensions 10min.Then
Above-mentioned PCR product is converted into bacillus coli DH 5 alpha competent cell, the picking on the LB agar plates containing 50 μ g/mL kanamycins
Bacterium colony carries out DNA sequencing, screening positive clone by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.It will correctly be mutated
The plasmid extraction where obtained epoxide hydrolase main body nucleotide sequence afterwards comes out, and is transformed into e. coli bl21
(DE3) in, induced expression is isolated and purified according to example 2, and measures its thermal stability.
It is 76.2, K that mutant F26V obtains the Rate activity (U/mg) at 37 DEG C after Ni column purificationsm(mmol/L) it is
24.4, Kcat×103(min-1) it is 5.64, the half-life period at 50 DEG C is 29.5min.Mutant I83R is after Ni column purifications
It is 75.3, K to obtain the Rate activity (U/mg) at 37 DEG Cm(mmol/L) it is 24.5, Kcat×103(min-1) it is 5.81,50
Half-life period at DEG C is 10.3min.It is 76.0 that mutant D8K obtains the Rate activity (U/mg) at 37 DEG C after Ni column purifications,
Km(mmol/L) it is 24.1, Kcat×103(min-1) it is 5.66, the half-life period at 50 DEG C is 25.3min.Mutant S90R
It is 75.3, K that the Rate activity (U/mg) at 37 DEG C is obtained after Ni column purificationsm(mmol/L) it is 24.4, Kcat×103(min-1) it is 5.77, the half-life period at 50 DEG C is 11.5min.
Embodiment 7：The fixed point saturation mutation of saltant type epoxide hydrolase
Design the gene order of degenerate primer fixed point saturation mutation 8,26,83,90 and 122 upper amino acids of gene expression.
PCR reaction systems are：40 μ L, 10 × PCR buffer of distilled water, 5 μ L, 10mmol/L dNTPs 1 μ L, the 10mmol/L of 2 μ L
Primer, 1 μ L of carrier pET28a-EH-x, 1 μ L of Taq enzyme, the reaction system of totally 50 μ L.
Its PCR program is：94 DEG C of 50s, 66 DEG C of 30s, 72 DEG C of 6min, 30 cycles, last 72 DEG C of extensions 10min.Success
Obtain one plant of engineering bacteria containing high thermal stability epoxide hydrolase.It finds to connect on the recombinant plasmid of its carrying by sequencing
There is new mutator, which replaces with tryptophan F26W by the codon of 26 coding valines.26 site saturation mutations
It is shown using primer sequence such as (SEQ ID NO.13, SEQ ID NO.14).
It is 77.1, K that mutant F26W obtains the Rate activity (U/mg) at 37 DEG C after Ni column purificationsm(mmol/L) it is
24.8, Kcat×103(min-1) it is 5.7, the half-life period at 50 DEG C is 37.2min.
Embodiment 8：Wild type epoxide hydrolase and saltant type epoxide hydrolase Q122R＆F26W＆I83R＆
The comparison of D8K＆S90R
Saltant type D8K, F26W, I83R, S90R and Q122R1 are combined together using rite-directed mutagenesis, obtained one plant of tool
There are the saltant type Q122R＆F26W＆I83R＆D8K＆S90R of heat-flash stability, saltant type epoxide hydrolase Q122R＆F26W＆
The amino acid sequence of I83R＆D8K＆S90R encodes the gene such as SEQ ID of the amino acid sequence as shown in SEQ ID NO.15
Shown in NO.16.
Its phenotype is measured：
(1) influence of the temperature to wild type and saltant type epoxide hydrolase activity and stability.
Purify obtained wild type epoxide hydrolase and saltant type epoxide hydrolase respectively at 25 DEG C, 30 DEG C,
35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C and 80 DEG C measure wild type according to above-mentioned detection method
The activity of epoxide hydrolase and saltant type epoxide hydrolase.As a result (referring to Fig. 4) is shown, wild type epoxides water
Solution enzyme has higher work in 25-60 DEG C (60% or more of highest enzyme activity), preferably 35-50 DEG C (90% or more of highest enzyme activity)
Power, highest Rate activity are 105U/mg.Saltant type epoxide hydrolase is at 25-70 DEG C (60% or more of highest enzyme activity), excellent
It is selected as 40-70 DEG C (80% or more of highest enzyme activity), more preferably 45-65 DEG C (90% or more of highest enzyme activity) has higher work
Power, highest Rate activity are 119U/mg.The optimum temperature of saltant type epoxide hydrolase is hydrolyzed higher than wild type epoxides
Enzyme, and the highest Rate activity of saltant type epoxide hydrolase is also above wild type epoxide hydrolase.
By the wild type epoxide hydrolase purified in embodiment 2 and saltant type epoxide hydrolase respectively at
After 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C are placed 30min, at 37 DEG C
Under conditions of, the activity of wild type and saltant type epoxide hydrolase is measured according to above-mentioned detection method, and according to control group
Calculate remaining vigor.Control group is tested：Wild type epoxide hydrolase and saltant type epoxide hydrolase -6 without
30min is kept the temperature, and directly surveys enzyme activity in 37 DEG C, and corresponding enzyme activity is defined as 100%.As a result (referring to Fig. 5) is shown, wild type epoxy
After compound hydrolase keeps the temperature 30min at 40 DEG C, remaining vigor is 90%, after 45 DEG C of heat preservation 30min, only remains 40% vigor.So
And after saltant type epoxide hydrolase -6 keeps the temperature 30min at 55 DEG C, remaining vigor is still 100%, after 60 DEG C of heat preservation 30min,
Also surplus 40% vigor is far above wild type epoxide hydrolase.This explanation, the temperature of saltant type epoxide hydrolase -6
Stability is significantly increased than wild type epoxide hydrolase；
(2) influence of the pH value to wild type and saltant type epoxide hydrolase activity and stability
Obtained wild type epoxide hydrolase and saltant type epoxide hydrolase -5 are purified respectively at different soda acids
Under degree, specially pH 2.0, pH 3.0, pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0, pH 10.0, pH
11.0, pH 12.0, pH 13.0 measure wild type epoxide hydrolase and saltant type epoxides according to above-mentioned detection method
The activity of hydrolase.The results show that wild type epoxide hydrolase vigor under conditions of pH value is less than 6 and is more than 10 is very low
(the 10% or less of highest enzyme activity)；There is higher vigor in pH 7.0-9.0, reaches 80% or more of highest enzyme activity, preferably pH
When 7.0-8.0, reach 90% or more of highest enzyme activity, optimum pH 8.Saltant type epoxide hydrolase -5 is low in pH value
In 5 and when higher than 12 by relatively low enzyme activity (between 0%-30%), catalysis activity has higher opposite enzyme between pH 6.0-11.0
(60% or more) living, preferably pH 6.0-10 (enzyme activity 80%), optimal pH 7.0-10.0.This illustrates that wild type epoxides hydrolyzes
The optimal reaction pH value of enzyme and saltant type epoxide hydrolase -5 is substantially similar, and saltant type epoxide hydrolase -5 is most
Suitable pH value range is wider, and pH value influences smaller to the catalytic effect of saltant type epoxides -5 (referring to Fig. 6).
Obtained wild type epoxide hydrolase and saltant type epoxide hydrolase are purified respectively at pH 2.0, pH
3.0、pH 4.0、pH 5.0、pH 6.0、pH 7.0、pH 8.0、pH 9.0、pH 10.0、pH 11.0、pH 12.0、pH 13.0
After placing 60min at room temperature, under conditions of pH 8.0, wild type epoxide hydrolase is measured according to above-mentioned detection method
With the activity of saltant type epoxide hydrolase -6, and remaining vigor is calculated according to control group.Control group is tested：Wild type ring
Oxide hydrolase and saltant type epoxide hydrolase -6 are placed without the pH gradient of 60min, directly survey enzyme in pH 8.0
Living, corresponding enzyme activity is defined as 100%.The results show that wild type epoxide hydrolase pH value be less than 6.0 and be more than 9.0
When place 1 hour after lose enzyme activity (remnant enzyme activity is below 10%) substantially, remnant enzyme activity is less than after the placement of pH 6.0 time 1h
40%, remnant enzyme activity is less than 70% after 9.0 times placement 1h of pH, and no appearance is bright after 1h is placed only at pH 7.0-8.0
Aobvious vigor declines.Saltant type epoxide hydrolase -5 between pH 5.0-10.0 enzyme activity kept stable (remnants 80% with
On), preferably pH 6.0-10 (preferably 90% or more).It is obvious that the tolerance of -5 couples of pH of saltant type epoxide hydrolase is remote
Far above wild type epoxide hydrolase (referring to Fig. 7).
(3) wild type epoxide hydrolase and saltant type epoxide hydrolase Q122R＆F26W＆I83R＆D8K＆S90R
Dynamics and stereospecificity
According to above-mentioned detection method, the wild type epoxide hydrolase and saltant type epoxides water that purifying obtains are measured
Solve the kinetic parameter (Km and Kcat) and enantiomeric excess value (stereospecificity of reflection enzyme) of enzyme.Wild type epoxides water
Solve enzyme and saltant type epoxide hydrolase -6 all characteristic features with Michaelis enzyme, Km values be respectively 25.3mmol/L and
29.1mmol/L, Kcat value are respectively 5.82 × 103min-1 and 6.2 × 103min-1, and enantiomeric excess value is respectively 99.5%
With 99.6%.
Measurement result show wild type epoxide hydrolase and saltant type epoxide hydrolase kinetic property and
Stereospecificity is almost the same.
Other mutant not illustrated in embodiment are prepared with reference to the method for 1~embodiment of embodiment 8.
Claims (3)
1. a kind of epoxide hydrolase mutant, which is characterized in that the epoxide hydrolase mutant is by such as SEQ
8th, the 26th, the 83rd, the 90th and the 122nd amino acid of amino acid sequence shown in ID NO.1 is carried out at the same time prominent
Become and obtains；
8th aspartic acid of amino acid sequence shown in SEQ ID NO.1 is replaced with into lysine, while the 26th phenylpropyl alcohol
Propylhomoserin replaces with valine or tryptophan, while the 83rd isoleucine replaces with arginine, while the 90th serine
Arginine is replaced with, while the 122nd glutamine replaces with arginine.
2. a kind of gene of coding epoxide hydrolase mutant as described in claim 1.
3. a kind of recombinant vector or recombinant cell carrying gene as claimed in claim 2.
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| CN101942471A (en) * | 2010-08-13 | 2011-01-12 | 杭州宝晶生物化工有限公司 | Epoxide hydrolase and preparation method thereof |
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| CN1279714A (en) * | 1997-10-24 | 2001-01-10 | 普瑞图斯股份有限公司 | Epoxide hydrolase |
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