CN105018443A - Epoxide hydrolase mutant and preparation method thereof - Google Patents
Epoxide hydrolase mutant and preparation method thereof Download PDFInfo
- Publication number
- CN105018443A CN105018443A CN201510460244.2A CN201510460244A CN105018443A CN 105018443 A CN105018443 A CN 105018443A CN 201510460244 A CN201510460244 A CN 201510460244A CN 105018443 A CN105018443 A CN 105018443A
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- arginine
- seq
- aminoacid sequence
- epoxide hydrolase
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y303/00—Hydrolases acting on ether bonds (3.3)
- C12Y303/02—Ether hydrolases (3.3.2)
- C12Y303/0201—Soluble epoxide hydrolase (3.3.2.10)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
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- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an epoxide hydrolase mutant and a preparation method thereof. The epoxide hydrolase mutant is prepared by carrying out single-point mutation or multipoint mutation on 8th, 25th, 83rd, 90th and 122nd amino acids of amino acid sequence disclosed as SEQ ID NO.1. The preparation method comprises the following steps: cloning a gene for coding the epoxide hydrolase into a plasmid pET28a(a+) to construct a recombinant plasmid pET28a(a+)-EH; amplifying the plasmid pET28a(a+)-EH by using rolling ring PCR (polymerase chain reaction) to obtain an open-ring recombinant vector containing the gene sequence of the coded epoxide hydrolase mutant; transforming competence Escherichia coli DH5alpha by using the PCR reaction solution containing the gene for coding the correct mutant, selecting the correct replicon, and extracting the correct mutant plasmid; and transforming Escherichia coli BL21(DE3), and inducing the expression. Compared with the wild type epoxide hydrolase, the epoxide hydrolase disclosed by the invention has high temperature stability and wide pH value application range; and the service life of the epoxide hydrolase in industrial production is greatly prolonged.
Description
Technical field
The invention belongs to technical field of enzyme engineering, be specifically related to epoxide hydrolase mutant of a kind of thermostability raising and preparation method thereof.
Background technology
Epoxide hydrolase (epoxide hydrolase, EH, EC 3.3.2.10) be the general name that catalytic hydrolysis epoxide or its salt generate the class of enzymes of corresponding vicinal diamines or its salt, catalyzed reaction is without the need to the participation of any coenzyme, prothetic group or metal ion.Epoxide hydrolase is distributed widely in Mammals, plant and microorganism.In recent years, owing to having, stereospecificity is good, enzymatic reaction is fast, product optical purity and yield high, and the advantage such as separation and purification product is simpler, microbe-derived epoxide hydrolase receives extensive concern and embody rule.Epoxide hydrolase is successfully applied to L (+)-tartaric suitability for industrialized production (US Patent4010072) by the people such as Miura in 1977 first.
L (+)-tartrate, has another name called (2R, 3R)-2,3-dihydroxyl-1, 4-succinic acid, is a kind of naturally occurring organic acid, can be used as acidic flavoring agent in food service industry, and tartrate list (two) acid anhydride ester is important foodstuff additive; In pharmaceutical industries, it is the most frequently used chiral drug and the resolving agent of intermediate; As reserving agent, laking agent etc. in dyeing industry; Can also as metal ion masking agent, for industries such as plating, process hides and mirrors processed.
In the past, (+)-tartaric main method extracts and processes by product winestone vinous to produce L, also can be that main raw material is by fermentation, conversion and extraction preparation with glucose.At present, take MALEIC ANHYDRIDE MIN 99.5 as raw material, utilize the microbial transformation containing epoxide hydrolase to be the main mode of production.Catalysis Principles is as follows: first MALEIC ANHYDRIDE MIN 99.5 and hydroperoxidation are obtained cis-form epoxy succinic acid, and cis-form epoxy succinic acid or its salt hydrolysis are L (+)-tartrate or salt by recycling microbial epoxidation thing lytic enzyme.
The current microorganism that can produce the epoxide hydrolase prepared for L (+)-tartrate reported has rhizobium, Rhodopseudomonas, Nocardia, corynebacterium, Rhod, achromobacter, acetobacter, Agrobacterium, Alkaligenes and acinetobacter.What commonly use in industrial production is the Rhod with greater catalytic efficiency.The reported first such as Liu in 2007 rhodococcus Rhodococcus opacus ML-0004 epoxide hydrolase gene, and it is expressed at prokaryotic cell prokaryocyte (E.coli).(Appl Microbiol Biotechnol 2007; 74:99-106) but this enzyme heat stability extreme difference, to very temperature sensitive, at 45 DEG C, preserve 30min then can lose 60% vigor, complete deactivation after preserving 30min at 50 DEG C.Weak thermostability limits the work-ing life of biological catalyst, limits its catalytic efficiency, too increases the cost in industrial production.Therefore, the thermostability improving this enzyme utilizes biological catalysis to produce one of L (+)-tartrate sixty-four dollar question at present.
At present, by genetic engineering means engineered protein primary structure, the means that enhancing protein three-dimensional structure rigidity improves heat stability of protein become main flow.According to protein structure and emic degree of understanding, protein transformation means are mainly divided into " orthogenesis ", " design and rational " and " half design and rational ".From the eighties in last century, " orthogenesis " has successfully been applied on protein " engineered ex vivo ".Because " orthogenesis " does not need structure and its corresponding function of understanding specified protein, just simulating nature evolution can transform target protein matter and screen after setting up rational evolution Filtering system, therefore a large amount of protein that structure rigidity is low and structural information is few resulting in transformation (FEBS Lett.276 (2009) 1750 – 1761).Although Be very effective, " orthogenesis " have reforming direction blindly, the large and Filtering system of workload is difficult to the shortcoming set up.
In order to make transformation more have purpose and reduce workload, the concept that researchist proposes " design and rational "." design and rational " refers under the prerequisite of known target protein structural information with its function mechanism, the prediction of appliance computer simulation trial protein is transformed after the impact in structure and function that can produce, and will be predicted the outcome by the mode of rite-directed mutagenesis and take (Appl.Microbiol.Biotechnol.99 (2014) 1205 – 1216) in test to.The feature of " design and rational " utilizes the with a high credibility protein structures obtained by X-ray diffraction crystallization of protein body, and predicted protein matter substitutes exactly.Although " design and rational " presents efficient and that workload is little feature really in the transformation of predicted protein matter.But the process often very complicated hardships of early-stage preparations protein crystal and parsing protein three-dimensional structure, and success ratio can not get ensureing.Utilize " design and rational " engineered protein Quality Research to be easy to develop into the structural research to target protein when ready-made protein structures, deviate from main direction of studying.
Therefore researchist also been proposed a concept " half design and rational " that both combine by " orthogenesis " and " design and rational ".This concept relates to a series of protein engineering means, suddenly change direction to reduce screening scale allowing the situation further aspect of relatively large predicated error regulation " orthogenesis ", evade the protein structure resolving in " design and rational " on the other hand, greatly improve the efficiency of protein engineering transformation." half design and rational " mainly comprises multiple alignment (MSAs), homology modeling, virtual sudden change and fixed point saturation mutation (Curr.Opin.Biotechnol.21 (2010) 734 – 743.).
At present, protein structural information and its function mechanism of deriving from the epoxide hydrolase of rhodococcus Rhodococcus opacus ML-0004 are still unintelligible.In order to obtain having the epoxide hydrolase mutant compared with heat-flash stability, increase work efficiency as far as possible, the method that the present invention adopts " orthogenesis " to combine with " half design and rational " carries out primary structure transformation to it simultaneously.
Summary of the invention
For the technical problem that the thermostability of epoxide hydrolase is not high, the invention provides a kind of epoxide hydrolase mutant and preparation method thereof.
A kind of epoxide hydrolase mutant, described epoxide hydrolase mutant is by such as the 8th, the 26th, the 83rd, the 90th of aminoacid sequence shown in SEQID NO.1 and the amino acid of the 122nd carries out simple point mutation or multipoint mutation obtains.
Further, described epoxide hydrolase mutant is one of following:
(1) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin (D8K);
(2) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane (sour F26V/F26W);
(3) as shown in SEQ ID NO.1, the Isoleucine of the 83rd of aminoacid sequence replaces with arginine (I83R);
(4) as shown in SEQ ID NO.1, the Serine of the 90th of aminoacid sequence replaces with arginine (S90R); (5) as shown in SEQ ID NO.1, the glutamine of 122 of aminoacid sequence replaces with arginine (Q122R);
(6) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, and the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane (D8K & F26V/D8K & F26W) simultaneously;
(7) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, and the Isoleucine of the 83rd replaces with arginine (D8K & I83R) simultaneously;
(8) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, and the Serine of the 90th replaces with arginine (D8K & S90R) simultaneously;
(9) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, and the glutamine of the 122nd replaces with arginine (D8K & Q122R) simultaneously; (10) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane, and the Isoleucine of the 83rd replaces with arginine (F26V & I83R/F26W & I83R) simultaneously;
(11) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane, and the Serine of the 90th replaces with arginine (F26V & S90R/F26W & S90R) simultaneously;
(12) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane, and the glutamine of the 122nd replaces with arginine (F26V & Q122R/F26W & Q122R) simultaneously;
(13) as shown in SEQ ID NO.1, the Isoleucine of the 83rd of aminoacid sequence replaces with arginine, and the Serine of the 90th replaces with arginine (I83R & S90R) simultaneously;
(14) as shown in SEQ ID NO.1, the Isoleucine of the 83rd of aminoacid sequence replaces with arginine, and the glutamine of the 122nd replaces with arginine (I83R & Q122R) simultaneously;
The Serine of (15) the 90th replaces with arginine, and the glutamine of the 122nd replaces with arginine (S90R & Q122R) simultaneously;
(16) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, and the Isoleucine of the 83rd replaces with arginine (D8K & F26V & I83R/D8K & F26W & I83R) simultaneously;
(17) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, and the Serine of the 90th replaces with arginine (D8K & F26V & S90R/D8K & F26W & S90R) simultaneously;
(18) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, and the glutamine of the 122nd replaces with arginine (D8K & F26V & Q122R/D8K & F26W & Q122R) simultaneously;
(19) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the Isoleucine of the 83rd replaces with arginine simultaneously, and the Serine of the 90th replaces with arginine (sour D8K & I83R & S90R) simultaneously;
(20) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the Isoleucine of the 83rd replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine (D8K & I83R & Q122R) simultaneously;
(21) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the Serine of the 90th replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine (D8K & S90R & Q122R) simultaneously;
(22) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane, the Isoleucine of the 83rd replaces with arginine simultaneously, and the Serine of the 90th replaces with arginine (S90R & F26V & I83R/S90R & F26W & I83R) simultaneously;
(23) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane, the Isoleucine of the 83rd replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine (Q122R & F26V & I83R/Q122R & F26W & I83R) simultaneously;
(24) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane, the Serine of the 90th replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine (S90R & F26V & Q122R/S90R & F26W & Q122R) simultaneously;
(25) as shown in SEQ ID NO.1, the Isoleucine of the 83rd of aminoacid sequence replaces with arginine, the Serine of the 90th replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine (S90R & I83R & Q122R) simultaneously;
(26) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, the Isoleucine of the 83rd replaces with arginine simultaneously, and the Serine of the 90th replaces with arginine (D8K & S90R & F26V & I83R/D8K & S90R & F26W & I83R) simultaneously;
(27) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane, the Isoleucine of the 83rd replaces with arginine simultaneously, the Serine of the 90th replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine (Q122R & S90R & F26V & I83R/Q122R & S90R & F26W & I83R) simultaneously;
(28) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the Isoleucine of the 83rd replaces with arginine simultaneously, the Serine of the 90th replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine (D8K & S90R & I83R & Q122R) simultaneously;
(29) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, the Serine of the 90th replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine (D8K & S90R & F26V & Q122R/D8K & S90R & F26W & Q122R) simultaneously;
(30) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, the Isoleucine of the 83rd replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine (D8K & I83R & F26V & Q122R/D8K & I83R & F26W & Q122R) simultaneously;
(31) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, the Isoleucine of the 83rd replaces with arginine simultaneously, and the Serine of the 90th replaces with arginine (D8K & I83R & F26V & S90R/D8K & I83R & F26W & S90R) simultaneously;
(32) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, the Isoleucine of the 83rd replaces with arginine simultaneously, the Serine of the 90th replaces with arginine simultaneously, the glutamine of the 122nd replaces with arginine (D8K & I83R & F26V & Q122R & S90R/D8K & I83R & F26W & Q122R & S90R) simultaneously.
The present invention also provides a kind of gene of described epoxide hydrolase mutant of encoding, and wherein a kind of base sequence of gene of mutant is as SEQ ID NO.16.
The present invention also provides a kind of recombinant vectors or the reconstitution cell that carry described gene.
The present invention also provides a kind of method obtaining described epoxide hydrolase mutant, comprise the steps: (1) by the gene clone of coding epoxide hydrolase in plasmid pET28a (+), construction recombination plasmid pET28a (+)-EH; (2) design the complementary primer containing the codon base representing mutating acid, adopt and roll ring pcr amplification plasmid pET28a (+)-EH, obtain the open loop recombinant vectors of the gene order containing coding epoxide hydrolase mutant; (3) step (2) gained is contained the PCR reaction solution of the gene of correct mutant of encoding through DpnI endonuclease digestion, remove the protoplasm grain do not suddenlyd change, reaction solution direct transform competent E. coli DH5 α, the correct replicon of picking, and extract correct mutant plasmid; (4) by correct mutant plasmid transformation of E. coli BL21 (DE3), abduction delivering.
In step (1), the gene of coding epoxide hydrolase obtains from rhodococcus Rhodococcus opacusML-0004.Also comprise further and use " the nickel post " of HisTrap FF crude 1ml to carry out purifying to epoxide hydrolase.
Described abduction delivering process is: recombinant bacterium step (4) obtained 37 DEG C of liquid culture in the LB substratum of the kantlex containing 50 μ g/L are spent the night, the LB fermentation broth 37 DEG C that rear access contains the kantlex of 50 μ g/L is cultured to OD600=0.6, be cooled to 28 DEG C of cultivations, the inductor IPTG adding ultimate density 0.1mM induces 8-12h, expresses the thalline of epoxide hydrolase in results.
The research about epoxide hydrolase involved in the present invention is had also to be in the elementary stage, only have the protein sequence (GenBank accession number ABF01020) of this protein of coding at present, the Catalysis Principles prediction of its corresponding nucleotide sequence (GenBank accession number DQ471957) and this albumen obtains report.Therefore the present invention utilizes the Research Thinking of main utilization " half design and rational " to transform this epoxide hydrolase.L (+)-tartrate generated due to this epoxide hydrolase catalysis and salt thereof can detect with " ammonium meta-vanadate development process ", therefore the present invention devises " the high flux screening method " for this epoxide hydrolase, and the means of introducing " orthogenesis " have been carried out one to this epoxide hydrolase and taken turns screening.This epoxide hydrolase has conserved sequence similarity with four enzymes with high thermal stability belonging to halo acids dehalogenation lytic enzyme superfamily (haloacid dehalogenase-like hydrolases (HAD) superfamily), therefore utilize MSAs to contrast the protein sequence of wild-type epoxide hydrolase involved in the present invention and this four protein sequences, predict and obtain the sudden change favourable to thermostability.Utilize " homology modeling " to set up the protein model of wild-type epoxide hydrolase involved in the present invention, use " virtual sudden change " to predict nucleic acid alternative afterwards, obtain the sudden change favourable to thermostability.Utilize " the high flux screening method " set up in the present invention to carry out saturation mutation to the catastrophe point favourable to heat stability of protein obtained, further increase the thermostability of this epoxide hydrolase.
By to the rite-directed mutagenesis in selected site and combinatorial mutagenesis, the thermostability of gained epoxide hydrolase mutant of the present invention obviously strengthens, 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 & S90R, and 5 combinatorial mutagenesis D8K & I83R & F26V & Q122R & S90, the thermostability of D8K & I83R & F26W & Q122R & S90R is all significantly increased, and mutation combination D8K & I83R & F26W & Q122R & S90R also has remarkable enhancing to the adaptability of pH value.Contrast original epoxide hydrolase, the mutant D8K & I83R & F26W & Q122R Rate activity of & S90R after Ni column purification at 37 DEG C (U/mg) reduces to 75.1, K by 76.5
m(mmol/L) 29.1, K is elevated to by 25.3
cat× 10
3(min
-1) become 6.20 from 5.82.Optimal reactive temperature has been brought up to as 55-60 DEG C by 35-40 DEG C, the 293.2.1min that had 8.5min to bring up to of the transformation period at 50 DEG C.Visible, will not significantly impact the catalysis efficiency of wild-type epoxide hydrolase at saltant type epoxide hydrolase D8K & I83R & F26W & Q122R & S90R on the basis of greatly improving thermostability.
Accompanying drawing explanation
Fig. 1 is epoxide hydrolase EH and four the multiple alignment's result from the enzyme of halo acids dehalogenation lytic enzyme superfamily (haloacid dehalogenase-like hydrolases (HAD) superfamily).
Fig. 2 is epoxide hydrolase EH homology model configuration schematic diagram.
Fig. 3 is the SDS-PAGE electrophorogram of epoxide hydrolase EH and 5 kind of mutant.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.
Fig. 4 is that wild-type epoxide hydrolase and saltant type Q122R & F26W & I83R & D8K & S90R ratio enzyme at different temperatures live (U/mg).
Fig. 5 is the remnant enzyme activity (%) after wild-type oxide compound lytic enzyme and saltant type Q122R & F26W & I83R & D8K & S90R process at different temperatures.
Fig. 6 is the relative activity (%) of the enzyme that wild-type epoxide hydrolase and saltant type Q122R & F26W & I83R & D8K & S90R react at various ph values.
Fig. 7 is the remnant enzyme activity (%) after wild-type oxide compound lytic enzyme and saltant type Q122R & F26W & I83R & D8K & S90R process under different pH condition.
Embodiment
The detection method of corresponding parameter in following examples:
Enzyme activity determination method
By the sodium hydrogen cis-epoxysuccinate substrate (pH 8.0) of 0.9mL 1mol/L after 37 DEG C of insulation 5min, add 0.1mol enzyme liquid and react 60min at 37 DEG C, the content of assaying reaction liquid unresolvable tartaric acid.Under the above-described reaction conditions, per minute produces 1 μm of ol tartrate and is defined as a Ge Meihuo unit, represents with U.Under these conditions, the units alive of the enzyme contained by every milligram of albumen is defined as Rate activity, represents with U/mg.
The detection method of tartaric acid content
The ammonium meta-vanadate getting 2.5mL 1%, in the volumetric flask of 25mL, after adding appropriate above-mentioned reaction solution, then adds the sulfuric acid of 1mL 1mol/L, is settled to 25mL with distilled water, surveys the light absorption value at 530nm place after mixing, and calculates tartaric acid concentration according to the typical curve formulated.
The measuring method of protein concentration
(0.1g Xylene Brilliant Cyanine G is dissolved in 50mL 95% ethanol to get 5mL coomassie brilliant blue staining liquid, add 100mL 85% phosphoric acid, add water and be settled to 1L), add appropriate protein liquid, survey the light absorption value at 595nm place after mixing, and calculate protein concentration according to the typical curve of specifying.
K
mand K
catthe measuring method of value
Equivalent amounts of enzyme liquid being placed in respectively final concentration is 100mmol/L, 70mmol/L, 45mmol/L, 35mmol/L, 25mmol/L, in the sodium hydrogen cis-epoxysuccinate (pH 7.5) of 20mmol/L, 15mmol/L, 10mmol/L, 20min is reacted at 37 DEG C, the content of assaying reaction liquid unresolvable tartaric acid, and with the two counting backward technique of Lineweaver-Burk, calculate the K of enzyme
mand K
cat, its unit is respectively mmol/L and × 10
3min
-1.
The measuring 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, be dissolved in 100mL 1mol/L cis-form epoxy succinic acid two sodium solution, 30 DEG C of oscillatory reaction 24h, add CaCl
2the aqueous solution, filters and water washing and precipitating, then, crystallization refining, concentrated through sulfuric acid solution, cation and anion exchange post and oven dry, obtains L (+) – tartrate respectively.By the sterling L of acquisition, (+) – tartrate dissolves, and uses polarimeter to measure its specific rotatory power.After testing, (the tartaric optical purity of+) – reaches more than 99% to all wild-type epoxide hydrolase products obtained therefrom L.
The experimental methods of molecular biology of unreceipted actual conditions in following examples, all conveniently condition, carry out with reference to condition described in " Molecular Cloning: A Laboratory guide " (New York:Cold Spring Harbor LaboratoryPress, 2001).
Embodiment 1: the structure of recombinant bacterium
Disclosed in U.S. GenBank database, the nucleotide sequence (accession number DQ471957) of rhodococcus Ml-0004 (Rhodococcusopacus ML-0004) epoxide hydrolase carries out codon optimized, makes it be more suitable for carrying out copying and expressing in engineering bacteria E.coli.Gene order (gene order after optimization is as shown in SEQ ID NO.2) after optimizing is served Hai Shenggong biotechnology Services Co., Ltd making recombinant plasmid pUC57-EH, and epoxide hydrolase gene (EH) is between restriction endonuclease sites Bam H I and Hind III.
Then use Bam H I and Hind III pair of pUC57-EH and pET28a (+) to carry out double digestion respectively, and carry out glue recovery small segment and large fragment respectively with QIAquick GEL Extraction Kit test kit.DNA fragmentation and and plasmid fragments in T4 linked system, 16 DEG C of connections are spent the night, and are connected to by goal gene on pET28a (+), construction recombination plasmid pET28a (+)-EH.
Among the competent cell by thermal shock conversion method recombinant plasmid being proceeded to clone's bacterial strain E.coli DH5 α, increase.Single bacterium colony E.coli DH5 α-pET28a (+)-EH on picking LB flat board, incubated overnight in LB substratum, use alkaline lysis method of extracting plasmid, then adopt thermal shock conversion method to be proceeded to by recombinant plasmid to E.coli BL21 (DE3) competence.
Embodiment 2: the expression and purity of wild-type and saltant type epoxide hydrolase
Inoculation wild type gene engineering bacteria and mutated genes engineering bacteria (by the preparation of embodiment 1 method) are extremely containing (1% peptone in the 50mL LB substratum of 50 μ g/mL kantlex respectively, 0.5% yeast extract, 1% sodium-chlor, pH7.0), 37 DEG C of shaking culture, as cell concentration (OD
600) when reaching 0.6-0.8, add 0.1mM isopropylthio-β-D-galactoside, 28 DEG C of shaking culture 12h.
4 DEG C, the centrifugal 10min of 5000rpm collects thalline, after normal saline flushing three times, lysis buffer (50mmol/L Tris-HCl is added in the ratio of every gram of thalline 5mL, 0.5mol/L NaCl, pH 7.5), the ultrasonic broken born of the same parents of ice precooling, 4 DEG C, the centrifugal 5min of 12000rpm, get supernatant liquor, loading is to Ni-NTA affinity column (being purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), with washing assorted damping fluid (50mmol/L Tris-HCl, 0.5mol/L NaCl, 20mmol/L imidazoles, pH 7.5) wash away impurity composition, with elution buffer (50mmol/L Tris-HCl, 0.5mol/L NaCl, 200mmol/L imidazoles, pH 7.5) wash-out target protein, by the enzyme liquid of collection in 4 DEG C, enough hemodialysis in 10mmol/L Tris-HCl (pH 7.5) damping fluid, after polyoxyethylene glycol is concentrated, with 10mmol/L Tris-HCl (pH 7.5) damping fluid containing 50% glycerine, in-20 DEG C of preservations, and pass through SDS-PAGE (the sodium dodecyl sulfatepolyacrylamide gel electropheresis of 12%, SDS-PAGE) purification effect is detected.
Protein band after purifying is single, and purity reaches more than 99%, and its molecular weight is about 28kDa, and the SDS-PAGE electrophorogram of epoxide hydrolase EH and 5 kind of mutant 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 ML-0004 epoxide hydrolase gene
Extract the recombinant plasmid pET-28a (+) (embodiment 1 builds) of the wild-type epoxide hydrolase gene EH after being connected with rare codon optimization, in polymerase chain reaction PCR, introduce misreplication in the mode adding divalent manganesetion.
The PCR reaction system of orthogenesis is: the homemade 5mM MnCl of 1.0ul
2, 0.5ul Taq polysaccharase (Takara Bio Inc., Shiga, Japan), the buffer (10 ×) that 10.0ul supplier provides, 14.0ul 25mM MgCl
2, 4.0ul dNTP Mixture, primer (SEQ ID NO.3) before 1.0ul, primer (SEQ ID NO.4) after 1.0ul, 0.5ul template DNA (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 circulation).
Purified pcr product, uses restriction endonuclease BamH I and Hind III to process 3 hours at 37 DEG C.Reaction system is 1ul BamH I, 1ul Hind III, 2ul 10 × M Buffer and 16ul aqua sterilisa.PCR primer after " double digestion " being processed uses nucleic acid electrophoresis purifying, and the smaller fragment containing mutational site is reclaimed in rubber tapping.
PET-28a (+) empty plasmid obtained after mutant fragments after purifying being connected to same method " double digestion ", carry out enzyme to connect: the enzyme disjunctor system of 20ul comprises 1ul T4 ligase enzyme, the mixed solution of 2ul 10 × T4 ligase enzyme and 17ul Insert Fragment and carrier, wherein the ratio of Insert Fragment and carrier is 5:1.Ligation is preserved and is spent the night at 16 DEG C.
All import connecting product by thermal transition in e. coli bl21 (DE3) competent cell prepared in advance: 20ul is connected product and add in e. coli bl21 (DE3) competent cell that 100ul just thawed, ice bath 30min, accurate thermal shock 45s at 42 DEG C, ice bath 10min, then the LB substratum of 250ul sterilizing is added, at 37 DEG C, cultivate 1 hour in the shaking table of 225rpm.Get the cell after preculture, by EP pipe centrifugal 30min at 3,000 rpm, remove clear liquid with liquid-transfering gun, then the cell of precipitation is picked up gently, use coating method to transfer to completely on the LB agar plate containing 50 μ g/mL kantlex, at 37 DEG C, cultivate 10-12h.
After building mutant library, from mutation library, random picking list bacterium colony carries out heat stability of protein detection experiment.Picking doubtful mutant list bacterium colony, joins in " the shallow bore hole 96 porocyte culture dish " of the 300ulLB substratum containing 50ug/ml kantlex and cultivates 6 hours.By " shallow bore hole 96 porocyte culture dish " called after " motherboard ", be stored in-80 DEG C of refrigerator-freezers as the seed next cultivated and induce.From motherboard, extract 50ul bacterium liquid, join in the 600ul LB substratum containing 50ug/ml kantlex and 0.1M sodium hydrogen cis-epoxysuccinate and cultivate.By " shallow bore hole 96 porocyte culture dish " called after " daughter board ".By daughter board at 37 DEG C, cultivate 2.5 hours in the shaking table of 225rpm, on plate, in each hole, add 50ul 1.2mM IPTG induce.Daughter board is at 28 DEG C subsequently, cultivates 10-16 hour in the shaking table of 225rpm.By the bacterium centrifugal 5min under 4000rpm cultivated and after induction.0.1M sodium acetate buffer and pure 1% ammonium meta-vanadate of 25ul of getting 125ul supernatant liquor and 100ul pH4.8 develop the color.Under 530nm, measure the absorbancy of above-mentioned reaction solution, retain color developing effect in mutant library and, close to the bacterium of wild-type, it is extracted from daughter board, carries out thermal stability determination experiment.
By resuspended for the cell 300ul 50mM phosphoric acid buffer on the daughter board that chooses, re-suspension liquid is processed 15min in 50 DEG C of water-baths, be immediately placed in and make it on ice to recover room temperature.In the re-suspension liquid returning to room temperature, add the 50mM phosphoric acid buffer that 300ul contains 0.2M sodium hydrogen cis-epoxysuccinate, react 1 hour at 37 DEG C.Recentrifuge gets supernatant liquor, ammonium meta-vanadate color developing detection remnant enzyme activity.The saltant type of remnant enzyme activity per-cent higher than wild-type is screened, extract seed liquor to send to Shanghai Sheng Gong limited liability company and check order, and the seed liquor containing mutant enzyme is carried out cultivating and inducing, after separation and purification, measure that it is more alive than enzyme, thermostability, and kinetic parameter.
The genetic engineering bacterium obtaining a strain and contain the epoxide hydrolase of high thermal stability is screened by orthogenesis.Learn through gene sequencing, this gene carries mutant Q122R, to be on wild-type epoxide hydrolase subject amino acid chain the 122nd and to be suddenlyd change in order to arginine by glutamine.
Preferably, the Rate activity of the mutant enzyme-1 that mutant Q122R obtains after Ni column purification at 37 DEG C (U/mg) is 75.8, its K
m(mmol/L) be 24.8, its K
cat× 10
3(min
-1) be 5.72.The optimal reactive temperature of mutant enzyme-1 is 35-40 DEG C, and the transformation period at 50 DEG C is 31.6min.What visible orthogenesis was introduced is changed to the arginic alternative effect with raising heat stability of protein by 122 upper glutamine, and does not affect the catalysis characteristics of enzyme.
Embodiment 4: rhodococcus ML-0004 epoxide hydrolase gene multiple alignment (MSAs)
Although be proved to be effective, the mutant library of the required random screening of orthogenesis test is too huge, invention introduces multiple alignment to accelerate the speed of transformation object epoxide hydrolase.In same superfamily, the same amino acid residue on the conserved sequence shared between the protein of Heat stability is good may be distinguish the key between they and the protein of poor heat stability.Derive from the epoxide hydrolase EH of rhodococcus ML-0004 and enzyme (the GenBank accession number No.HAD_AGRTR:P60527.1 from four that belong to halo acids dehalogenation lytic enzyme superfamily (haloacid dehalogenase-like hydrolases (HAD) superfamily) with high thermal stability, DhlS5I:P60527.1, L-DEX:Q53464.1and PH0459:83753572) there is conserved sequence similarity (alignment is as shown in Figure 1).Thinking can to causing high thermal stability at the conserved sequence that these four homologous proteins are shared, therefore intend the structural similarity being found EH and these four thermophilic proteins by the mode of multiple alignment, prediction may improve the amino acid substitute mode of heat stability of protein.
Phenylalanine on 26th site on wild-type epoxide hydrolase is sported the thermostability that α-amino-isovaleric acid F26V can improve epoxide hydrolase by success prediction of the present invention; And the tryptophane on the 83rd site on wild-type type epoxide hydrolase is sported the thermostability that arginine S90R (I83R) can improve epoxide hydrolase.
Embodiment 5: the homology modeling of rhodococcus ML-0004 epoxide hydrolase and virtual sudden change
Owing to up to the present also there is no the protein three-dimensional structure about rhodococcus ML-0004 epoxide hydrolase, therefore the protein three-dimensional structure model being set up rhodococcus ML-0004 epoxide hydrolase by " homology modeling " is needed, and use virtual sudden change means to calculate impact that single-point amino acid mutation causes whole protein structure model is to judge whether this sudden change can impact the structure rigidity of protein, and then change the thermostability of protein.
Utilize the homology MBM in software Discovery Studio3.0, search pattern in whole Protein DataBank database, obtain the highest halohydrin dehalogenase DehIVa of structural similarity (GenBank accession number No.Q51645), it can be used as template, set up epoxide hydrolase protein matter model, use software PROCHECK verification model operability.Through homology modeling and model feasibility analysis, the protein structure model (as shown in Figure 2) that it is template that the present invention establishes with halohydrin dehalogenase DehIVa.
Utilize Discovery Studio3.0 software, simulate and amino-acid residues all on all proteins model is all replaced with L-Ala, found that, before and after virtual sudden change, maximum change occurs protein structure energy is occur in Asp8, Asp 25, Phe26, Glu32, Gly34, Leu35, Asp 43, Glu48, Asp 52, Asp 60, Asp 63, Leu65, Phe89, Ser90, Asp 91, Glu101, Gly106, Ser112, Asp 152, and on Gly161 site.
Consider that the structure of modification on protein surface loop more easily changes the structural stability of protein, therefore carry out virtual saturation mutation by the five amino acid residue be in protein surface loop district.This five amino acid residue is Asp 8, Leu65, Ser90, Asp 91 respectively, and Gly161.It is Methionin D8K that virtual saturation mutation predicts the Aspartic acid mutations in the 8th site; Be the thermostability S90R that arginine can improve protein by the mutant serine in 90 sites.Rite-directed mutagenesis is used to present above-mentioned predicting the outcome.
Embodiment 6: rhodococcus ML-0004 epoxide hydrolase gene rite-directed mutagenesis
In rite-directed mutagenesis test, by the phenylalanine residue (Phe of the 26th on the subject amino acid sequence of wild-type rhodococcus ML-0004 epoxide hydrolase, corresponding codon is TTC) rite-directed mutagenesis is α-amino-isovaleric acid (Val, corresponding codon is GTT), make corresponding primer (SEQ ID NO.5, SEQ ID NO.6); Be arginine (Arg, corresponding codon is CGT, CGC, CGA or CGG) by the Isoleucine of the 83rd (Ile, corresponding codon is ATC) rite-directed mutagenesis.CGT, CGC, CGA and CGG are synonym, and the present embodiment designs primer (SEQID NO.7, SEQ ID NO.8) for CGT codon; The asparagicacid residue of the 8th (Asp, corresponding codon is GAC) is sported Methionin (Lys, corresponding codon is AAA or AAG).AAA and AAG is synonym, their equal encodes lysine, and the present embodiment makes primer (SEQ ID NO.9, SEQ ID NO.10) for AAA pass phrase; Be arginine (Arg, corresponding codon is CGT, CGC, CGA or CGG) by the Serine of the 90th (Ser, codon is TCT) rite-directed mutagenesis.CGT, CGC, CGA and CGG are synonym, their equal encode arginine, and the present embodiment, for CGT codon, designs mutant primer (SEQ ID NO.11, SEQ ID NO.12).
The PCR reaction system of rite-directed mutagenesis is: distilled water 40 μ L, 10 × PCR buffer 5 μ L, 10mmol/L dNTPs 1 μ L, the 10mmol/L primer of 2 μ L, carrier pET28a-EH-x 1 μ L, Taq enzyme 1 μ L, 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 circulations, and last 72 DEG C extend 10min.Then by above-mentioned PCR primer transformation of E. coli DH5 α competent cell, containing picking colony on the LB agar plate of 50 μ g/mL kantlex, DNA sequencing is carried out by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, screening positive clone.By correctly sudden change after the epoxide hydrolase main body nucleotide sequence place obtained plasmid extraction out, be transformed in e. coli bl21 (DE3), abduction delivering, according to example 2 separation and purification, and measure its thermostability.
Mutant F26V must the Rate activity (U/mg) at 37 DEG C be 76.2 after Ni column purification, its K
m(mmol/L) be 24.4, its K
cat× 10
3(min
-1) be 5.64, the transformation period at 50 DEG C is 29.5min.Mutant I83R must the Rate activity (U/mg) at 37 DEG C be 75.3 after Ni column purification, its K
m(mmol/L) be 24.5, its K
cat× 10
3(min
-1) be 5.81, the transformation period at 50 DEG C is 10.3min.Mutant D8K must the Rate activity (U/mg) at 37 DEG C be 76.0 after Ni column purification, its K
m(mmol/L) be 24.1, its K
cat× 10
3(min
-1) be 5.66, the transformation period at 50 DEG C is 25.3min.Mutant S90R must the Rate activity (U/mg) at 37 DEG C be 75.3 after Ni column purification, its K
m(mmol/L) be 24.4, its K
cat× 10
3(min
-1) be 5.77, the transformation period at 50 DEG C is 11.5min.
Embodiment 7: the fixed point saturation mutation of saltant type epoxide hydrolase
Design degenerate primer fixed point saturation mutation genetic expression 8,26,83,90, and the gene order of 122 upper amino acids.PCR reaction system is: distilled water 40 μ L, 10 × PCR buffer 5 μ L, 10mmol/L dNTPs 1 μ L, the 10mmol/L primer of 2 μ L, carrier pET28a-EH-x 1 μ L, Taq enzyme 1 μ L, 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 circulations, and last 72 DEG C extend 10min.Successfully obtain the engineering bacteria that a strain contains high thermal stability epoxide hydrolase.Find its recombinant plasmid carried is connected with new mutator gene through order-checking, the codon of 26 encode valine is replaced with tryptophane F26W by this gene.26 site saturation mutations use primer sequence as shown in (SEQ ID NO.13, SEQ ID NO.14).
Mutant F26W must the Rate activity (U/mg) at 37 DEG C be 77.1 after Ni column purification, its K
m(mmol/L) be 24.8, its K
cat× 10
3(min
-1) be 5.7, the transformation period at 50 DEG C is 37.2min.
Embodiment 8: the comparison of wild-type epoxide hydrolase and saltant type epoxide hydrolase Q122R & F26W & I83R & D8K & S90R
By saltant type D8K, F26W, I83R, S90R and Q122R1 utilizes rite-directed mutagenesis to combine, obtain the saltant type Q122R & F26W & I83R & D8K & S90R that a strain has heat-flash stability, the aminoacid sequence of saltant type epoxide hydrolase Q122R & F26W & I83R & D8K & S90R is as shown in SEQ ID NO.15, and the gene of this aminoacid sequence of encoding is as shown in SEQ ID NO.16.
Its phenotype is measured:
(1) temperature is on the impact of wild-type and saltant type epoxide hydrolase activity and stability.
The wild-type epoxide hydrolase that purifying obtains and saltant type epoxide hydrolase measure the activity of 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 according to above-mentioned detection method.Result display (see Fig. 4), wild-type epoxide hydrolase 25-60 DEG C (the highest enzyme live more than 60%), be preferably 35-50 DEG C (the highest enzyme live more than 90%) and have higher vigor, its most higher specific activity is 105U/mg.Saltant type epoxide hydrolase 25-70 DEG C (the highest enzyme live more than 60%), be preferably 40-70 DEG C (the highest enzyme live more than 80%), be more preferably 45-65 DEG C (the highest enzyme live more than 90%) and have higher vigor, its most higher specific activity is 119U/mg.The optimum temperuture of saltant type epoxide hydrolase is higher than wild-type epoxide hydrolase, and the most higher specific activity of saltant type epoxide hydrolase is also higher than wild-type epoxide hydrolase.
After the wild-type epoxide hydrolase obtained by purifying in embodiment 2 and saltant type epoxide hydrolase place 30min respectively at 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, under the condition of 37 DEG C, measure the activity of wild-type and saltant type epoxide hydrolase according to above-mentioned detection method, and calculate remaining vigor according to control group.Control group experiment is: wild-type epoxide hydrolase and saltant type epoxide hydrolase-6 are without 30min insulation, and directly survey enzyme in 37 DEG C and live, corresponding enzyme is lived and is defined as 100%.Result display (see Fig. 5), wild-type epoxide hydrolase is after 40 DEG C of insulation 30min, and remaining vigor is after 90%, 45 DEG C of insulation 30min, the only vigor of surplus 40%.But saltant type epoxide hydrolase-6 is after 55 DEG C of insulation 30min, remaining vigor is still that after 100%, 60 DEG C of insulation 30min, the also vigor of surplus 40%, far above wild-type epoxide hydrolase.This illustrates, the temperature stability of saltant type epoxide hydrolase-6 is significantly increased than wild-type epoxide hydrolase;
(2) pH value is on the impact of wild-type and saltant type epoxide hydrolase activity and stability
The wild-type epoxide hydrolase that purifying obtains and saltant type epoxide hydrolase-5 are respectively under different pH values, be 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 the activity of wild-type epoxide hydrolase and saltant type epoxide hydrolase according to above-mentioned detection method.Result shows, and wild-type epoxide hydrolase is less than 6 very low with vigor under the condition being greater than 10 (less than 10% of the highest enzyme work) in pH value; Have higher vigor when pH 7.0-9.0, reach more than 80% of the work of the highest enzyme, during preferred pH 7.0-8.0, reach more than 90% of the work of the highest enzyme, its optimum pH is 8.Saltant type epoxide hydrolase-5 pH value lower than 5 and higher than 12 time live (between 0%-30%) by lower enzyme, its catalysis activity has higher relative enzyme to live (more than 60%) between pH 6.0-11.0, preferred pH 6.0-10 (enzyme lives 80%), optimum pH 7.0-10.0.This illustrates the optimal reaction pH value basic simlarity of wild-type epoxide hydrolase and saltant type epoxide hydrolase-5, and the optimum pH of saltant type epoxide hydrolase-5 is wider, pH value is on catalytic effect impact less (see Fig. 6) of saltant type epoxide-5.
After the wild-type epoxide hydrolase that purifying obtains and saltant type epoxide hydrolase at room temperature place 60min 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, under the condition of pH 8.0, measure the activity of wild-type epoxide hydrolase and saltant type epoxide hydrolase-6 according to above-mentioned detection method, and calculate remaining vigor according to control group.Control group experiment is: wild-type epoxide hydrolase and saltant type epoxide hydrolase-6 are placed without the pH gradient of 60min, and directly survey enzyme in pH 8.0 and live, corresponding enzyme is lived and is defined as 100%.Result shows, wild-type epoxide hydrolase pH value be less than 6.0 and be greater than 9.0 time place 1 hour after substantially lose enzyme live (remnant enzyme activity is below 10%), after pH places 1h 6.0 times, remnant enzyme activity is lower than 40%, after pH places 1h 9.0 times, remnant enzyme activity is lower than 70%, does not occur that obvious vigor declines after only placing 1h under pH 7.0-8.0.Saltant type epoxide hydrolase-5 to be lived kept stable (remaining more than 80%) at the glucose-6-phosphate dehydrogenase of pH 5.0-10.0, preferred pH 6.0-10 (preferably more than 90%).Clearly, the tolerance of saltant type epoxide hydrolase-5 couples of pH will far away higher than wild-type epoxide hydrolase (see Fig. 7).
(3) kinetics of wild-type epoxide hydrolase and saltant type epoxide hydrolase Q122R & F26W & I83R & D8K & S90R and stereospecificity
According to above-mentioned detection method, the kinetic parameter (Km and Kcat) of the wild-type epoxide hydrolase that mensuration purifying obtains and saltant type epoxide hydrolase and enantiomeric excess value (stereospecificity of reflection enzyme).Wild-type epoxide hydrolase and saltant type epoxide hydrolase-6 all have the characteristic feature of Michaelis enzyme, Km value is respectively 25.3mmol/L and 29.1mmol/L, Kcat value is respectively 5.82 × 103min-1 and 6.2 × 103min-1, and enantiomeric excess value is respectively 99.5% and 99.6%.
Measurement result shows, kinetic property and the stereospecificity of wild-type epoxide hydrolase and saltant type epoxide hydrolase are basically identical.
Other mutant do not illustrated in an embodiment are all prepared with reference to the method for embodiment 1 ~ embodiment 8.
Claims (8)
1. an epoxide hydrolase mutant, it is characterized in that, described epoxide hydrolase mutant such as the 8th, the 26th, the 83rd, the 90th of aminoacid sequence shown in SEQ ID NO.1 and the amino acid of the 122nd is carried out simple point mutation or multipoint mutation obtains.
2. epoxide hydrolase mutant according to claim 1, is characterized in that, described epoxide hydrolase mutant is one of following:
(1) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin;
(2) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane;
(3) as shown in SEQ ID NO.1, the Isoleucine of the 83rd of aminoacid sequence replaces with arginine;
(4) as shown in SEQ ID NO.1, the Serine of the 90th of aminoacid sequence replaces with arginine;
(5) as shown in SEQ ID NO.1, the glutamine of 122 of aminoacid sequence replaces with arginine;
(6) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, and the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously;
(7) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, and the Isoleucine of the 83rd replaces with arginine simultaneously;
(8) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, and the Serine of the 90th replaces with arginine simultaneously;
(9) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, and the glutamine of the 122nd replaces with arginine simultaneously;
(10) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane, and the Isoleucine of the 83rd replaces with arginine simultaneously;
(11) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane, and the Serine of the 90th replaces with arginine simultaneously;
(12) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane, and the glutamine of the 122nd replaces with arginine simultaneously;
(13) as shown in SEQ ID NO.1, the Isoleucine of the 83rd of aminoacid sequence replaces with arginine, and the Serine of the 90th replaces with arginine simultaneously;
(14) as shown in SEQ ID NO.1, the Isoleucine of the 83rd of aminoacid sequence replaces with arginine, and the glutamine of the 122nd replaces with arginine simultaneously;
The Serine of (15) the 90th replaces with arginine, and the glutamine of the 122nd replaces with arginine simultaneously;
(16) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, and the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, and the Isoleucine of the 83rd replaces with arginine simultaneously;
(17) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, and the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, and the Serine of the 90th replaces with arginine simultaneously;
(18) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, and the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, and the glutamine of the 122nd replaces with arginine simultaneously;
(19) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, and the Isoleucine of the 83rd replaces with arginine simultaneously, and the Serine of the 90th replaces with arginine simultaneously;
(20) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, and the Isoleucine of the 83rd replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine simultaneously;
(21) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, and the Serine of the 90th replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine simultaneously;
(22) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane, and the Isoleucine of the 83rd replaces with arginine simultaneously, and the Serine of the 90th replaces with arginine simultaneously;
(23) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane, and the Isoleucine of the 83rd replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine simultaneously;
(24) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane, and the Serine of the 90th replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine simultaneously;
(25) as shown in SEQ ID NO.1, the Isoleucine of the 83rd of aminoacid sequence replaces with arginine, and the Serine of the 90th replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine simultaneously;
(26) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, the Isoleucine of the 83rd replaces with arginine simultaneously, and the Serine of the 90th replaces with arginine simultaneously;
(27) as shown in SEQ ID NO.1, the phenylalanine of the 26th of aminoacid sequence replaces with α-amino-isovaleric acid or tryptophane, the Isoleucine of the 83rd replaces with arginine simultaneously, the Serine of the 90th replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine simultaneously;
(28) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the Isoleucine of the 83rd replaces with arginine simultaneously, the Serine of the 90th replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine simultaneously;
(29) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, the Serine of the 90th replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine simultaneously;
(30) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, the Isoleucine of the 83rd replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine simultaneously;
(31) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, the Isoleucine of the 83rd replaces with arginine simultaneously, and the Serine of the 90th replaces with arginine simultaneously;
(32) as shown in SEQ ID NO.1, the aspartic acid of the 8th of aminoacid sequence replaces with Methionin, the phenylalanine of the 26th replaces with α-amino-isovaleric acid or tryptophane simultaneously, the Isoleucine of the 83rd replaces with arginine simultaneously, the Serine of the 90th replaces with arginine simultaneously, and the glutamine of the 122nd replaces with arginine simultaneously.
3. the gene of the epoxide hydrolase mutant as claimed in claim 1 of encoding.
4. one kind is carried recombinant vectors or the reconstitution cell of gene as claimed in claim 3.
5. obtain a method for epoxide hydrolase mutant as claimed in claim 1, it is characterized in that, comprise the steps:
(1) by coding epoxide hydrolase gene clone in plasmid pET28a (+), construction recombination plasmid pET28a (+)-EH;
(2) design contains the primer with the sequence representing mutating acid codon base complementrity, adopts and rolls ring pcr amplification plasmid pET28a (+)-EH, obtains the open loop recombinant vectors of the gene order containing coding epoxide hydrolase mutant;
(3) step (2) gained is contained the PCR reaction solution of the gene of correct mutant of encoding through DpnI endonuclease digestion, remove the protoplasm grain do not suddenlyd change, reaction solution direct transform competent E. coli DH5 α, the correct replicon of picking, and extract correct mutant plasmid;
(4) by correct mutant plasmid transformation of E. coli BL21 (DE3), abduction delivering.
6. method according to claim 5, is characterized in that, in step (1), the gene of coding epoxide hydrolase obtains from rhodococcus Rhodococcus opacus ML-0004.
7. method according to claim 5, it is characterized in that, described abduction delivering process is: recombinant bacterium step (4) obtained 37 DEG C of liquid culture in the LB substratum of the kantlex containing 50 μ g/L are spent the night, the LB fermentation broth 37 DEG C that rear access contains the kantlex of 50 μ g/L is cultured to OD600=0.6, be cooled to 28 DEG C of cultivations, the inductor IPTG adding ultimate density 0.1mM induces 8-12h.
8. method according to claim 5, is characterized in that, also comprise step (4) abduction delivering gained epoxide hydrolase and carry out purifying.
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| CN106497844A (en) * | 2016-12-12 | 2017-03-15 | 常茂生物化学工程股份有限公司 | One plant is produced the tartaric genetic engineering bacteriums of L and its construction method and application |
| CN109182241A (en) * | 2018-09-21 | 2019-01-11 | 清华大学 | A kind of engineering bacteria that expressing epoxide hydrolase and its construction method and application |
| CN113373128A (en) * | 2021-05-17 | 2021-09-10 | 深圳市微滴科技顾问有限公司 | Epoxide hydrolase mutant with improved catalytic efficiency and preparation method thereof |
| CN114220492A (en) * | 2021-12-16 | 2022-03-22 | 江南大学 | Method for redesigning enzyme based on isothermal compression coefficient disturbance, application and mutant screened by method |
| CN114657111A (en) * | 2022-03-19 | 2022-06-24 | 中国科学院青岛生物能源与过程研究所 | Cis-epoxy succinate hydrolase cell surface display system and construction and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106497844A (en) * | 2016-12-12 | 2017-03-15 | 常茂生物化学工程股份有限公司 | One plant is produced the tartaric genetic engineering bacteriums of L and its construction method and application |
| CN106497844B (en) * | 2016-12-12 | 2019-05-21 | 常茂生物化学工程股份有限公司 | One plant of genetic engineering bacterium for producing L-TARTARIC ACID and its construction method and application |
| CN109182241A (en) * | 2018-09-21 | 2019-01-11 | 清华大学 | A kind of engineering bacteria that expressing epoxide hydrolase and its construction method and application |
| CN109182241B (en) * | 2018-09-21 | 2021-11-23 | 清华大学 | Engineering bacterium for expressing epoxide hydrolase and construction method and application thereof |
| CN113373128A (en) * | 2021-05-17 | 2021-09-10 | 深圳市微滴科技顾问有限公司 | Epoxide hydrolase mutant with improved catalytic efficiency and preparation method thereof |
| CN114220492A (en) * | 2021-12-16 | 2022-03-22 | 江南大学 | Method for redesigning enzyme based on isothermal compression coefficient disturbance, application and mutant screened by method |
| CN114220492B (en) * | 2021-12-16 | 2023-02-28 | 江南大学 | Method for redesigning enzyme based on isothermal compression coefficient disturbance, application and mutant screened by method |
| CN114657111A (en) * | 2022-03-19 | 2022-06-24 | 中国科学院青岛生物能源与过程研究所 | Cis-epoxy succinate hydrolase cell surface display system and construction and application thereof |
| CN114657111B (en) * | 2022-03-19 | 2024-04-09 | 中国科学院青岛生物能源与过程研究所 | Cis-epoxysuccinic acid hydrolase cell surface display system, construction and application |
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