CN109576234A - A kind of leucine -5- hydroxylation enzyme mutant and its application - Google Patents

A kind of leucine -5- hydroxylation enzyme mutant and its application Download PDF

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CN109576234A
CN109576234A CN201811596777.3A CN201811596777A CN109576234A CN 109576234 A CN109576234 A CN 109576234A CN 201811596777 A CN201811596777 A CN 201811596777A CN 109576234 A CN109576234 A CN 109576234A
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leucine
mutant
enzyme
gene
hydroxylase
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CN109576234B (en
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秦慧民
路福平
孙登岳
李玉
毛淑红
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Tiankeda Tianjin Science Park Co ltd
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Tianjin University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)

Abstract

The invention belongs to genetic engineerings and technical field of enzyme engineering, and in particular to a kind of leucine -5- hydroxylation enzyme mutant and its application that activity improves.The present invention obtains the leucine -5- hydroxylase encoding gene in the source NIES-2108 nostoc (Nostoc punctiforme) by molecular biology method, the gene that rite-directed mutagenesis obtains encoding mutant body is carried out using inverse PCR, the gene of encoding mutant body is then subjected to the mutant V77A that heterogenous expression obtains leucine -5- hydroxylase.When the mutant is using leucine and methionine as substrate, relative activity has been respectively increased 69.53%, 23.26% compared with wild type.The variant that the activity that the present invention filters out improves can more efficiently make No. 5 carbon potentials of leucine distal end realize hydroxylating, and can be catalyzed methionine and generate methionine sulfoxide, provide wide prospect for the enzyme law catalysis production of industrial pharmaceutical intermediate.

Description

A kind of leucine -5- hydroxylation enzyme mutant and its application
Technical field:
The invention belongs to genetic engineerings and technical field of enzyme engineering, and in particular to a kind of leucine -5- hydroxyl that activity improves Change enzyme mutant and its application.
Background technique:
Hydroxylated amino acids due to can a variety of chemical materials, medical medicine synthesis in as chiral precursor, intermediate and Therefore final product is widely used in the synthesis of multifunctional bio macromolecular, medicine and fine chemicals.Hydroxy-amino-acid in addition to The intermediate that can be used as field of medicaments pharmaceutical synthesis is also widely used in multiple necks such as feed addictive, food additive, fragrance Domain is alternatively arranged as animal if L- beta-hydroxy alanine is in addition to for the amino acid weight-reducing drinks and sports drink in field of food Feed addictive；L- hydroxy-proline is in fruit juice, nutritious drink, cold drink frequently as nutrition fortifier, flavoring agent；L-β- Hydroxyl Gamma Amino Butyric Acid is also a kind of additive, is usually used in food and field of fodder, in addition, 5- hydroxyl-leucine is 4- methyl dried meat ammonia The important synthesis precursor of acid, it is most important in the synthesis of antibiotic griselimycin.
Currently, the synthetic method of hydroxylating leucine mainly has chemical synthesis and enzyme process biosynthesis.Microorganism conversion is Purpose product is generated by the catalysis of the intracorporal related enzyme system of microorganism.It realizes the hydroxylating of amino acid distal end carbon potential, changes Technique is learned there are many drawbacks, including process route complexity, severe reaction conditions, by-product is more, yield is low etc. etc..And enzyme process is urged Change then because its high specificity, reaction condition are mild, the considerable advantages such as yield height, the exploitation for many pharmaceutical intermediates provides more Add efficient strategy.
Leucine -5- hydroxylase (LEH, the Genbank in the source NIES-2108 nostoc (Nostoc punctiforme) Sequence number RCJ32143.1) or leucine -5- dioxygenase, effect is that No. 5 carbon potential oxidative dehydrogenations of leucine is made to form hydroxyls Base is to realize the key enzyme of leucine distal end C-H functionalization, and can make methionine that sulfonating reaction occur, it is sub- to generate methionine Sulfone.Its open reading frame for containing 795bp encodes 265 amino acid.
Wild type leucine -5- '-hydroxylase gene LEHization is gone to E.coli BL21 (DE3) heterogenous expression by the present invention, on Clearly after Ni affinity chromatography and cation exchange chromatography, characterization analysis is carried out, the results showed that it reacts leucine Optimum temperature is 30 DEG C, optimal pH 7.4, specific enzyme activity 1041.67U/mg, is 30 DEG C to methionine reaction optimum temperature, most Suitable pH is 7.4, and specific enzyme activity 822.92U/mg, there are still gaps for this and industrial application, therefore by the way that wild type molecule is transformed Improve its enzymatic activity, it will help popularization of the leucine -5- hydroxylase in industrial production pharmaceutical intermediate.
Summary of the invention:
In order to solve the above-mentioned technical problem, the present invention will provide a kind of leucine -5- hydroxylation enzyme mutant and its preparation with Using.
Realize that technology path of the invention is summarized as follows:
The bright ammonia in the source NIES-2108 nostoc (Nostoc punctiforme) is obtained by molecular biology method The encoding gene of acid -5- hydroxylase (hereinafter referred to as wild type, WT) carries out rite-directed mutagenesis using inverse PCR and obtains encoding mutant The gene of encoding mutant body is then carried out the mutant V77A that heterogenous expression obtains leucine -5- hydroxylase by the gene of body.
It uses and such as gives a definition in the present invention:
1. the nomenclature of amino acid and DNA nucleic acid sequence
Using the generally acknowledged IUPAC nomenclature of amino acid residue, with three-letter codes form.DNA nucleic acid sequence is using generally acknowledged IUPAC nomenclature.
2. the mark of leucine -5- hydroxylation enzyme mutant
The ammonia being mutated in leucine -5- hydroxylation enzyme mutant is indicated using " amino acid of Original amino acid position replacement " Base acid.Such as Val77Ala, indicate that the amino acid of position 77 is substituted for Ala, position by the Val of wild type leucine -5- hydroxylase Number correspond to SEQ ID No.1 in wild type leucine -5- hydroxylase amino acid sequence number.
In the present invention, LEH indicates that the encoding gene of wild type leucine -5- hydroxylase, mLEH indicate the bright ammonia of mutant The encoding gene of acid -5- hydroxylase V77A, information such as following table.
Leucine -5- hydroxylase Amino acid mutation site Gene mutation site
Wild type —— ——
V77A Val77Ala T230→C
The amino acid sequence of the wild type leucine -5- hydroxylase is as shown in sequence table SEQ ID NO.1；
The nucleotide sequence of the wild type leucine -5- hydroxylase is as shown in sequence table SEQ ID NO.3；
The amino acid sequence of the mutant V77A is as shown in sequence table SEQ ID NO.2；
For the nucleotide sequence of the mutant V77A as shown in sequence table SEQ ID NO.4, which is by large intestine bar The sequence of bacterium codon optimization；
The present invention also provides a kind of recombinant vector, recombinant bacteriums for carrying above-mentioned mutant and/or encoding gene.
Preferably, the expression vector of the recombinant vector is pET28a (+)；
Preferably, the host cell of the recombinant bacterium is e. coli bl21 (DE3)；
Experimental program of the invention is specific as follows:
1, the acquisition of the mutant V77A encoding gene, includes the following steps:
(1) will from nostoc leucine -5- hydroxylase gene after codon optimization (SEQ ID NO.3) structure It is built in pET28a (+) expression vector, obtains LEH-pET28a；
(2) using wild type recombinant vector LEH-pET28a in (1) as template, design primer passes through inverse PCR rite-directed mutagenesis Mutant code gene mLEH is obtained, nucleotide sequence is as shown in SEQ ID NO.4.Primer is as follows:
2, specific as follows the present invention also provides the production method of mutant V77A:
(1) correct mutant recombinant vector (mLEH-pET28a) will be verified to walk around by changing into e. coli bl21 (DE3), it is connected to LB culture medium (containing 50-70 μ g/mL kanamycins), 220r/min is incubated overnight at 37 DEG C；It is connect with 1% Kind amount is connected to LB culture medium (containing 50-80 μ g/mL kanamycins), to bacteria concentration OD600IPTG is added in=0.6-0.8, dense eventually Degree is 1mM, induces 18-20h under 16 DEG C, 180r/min；
(2) thalline were collected by centrifugation, ultrasonication after resuspension, and low-temperature and high-speed, which is centrifuged to obtain supernatant, contains leucine -5- hydroxyl Change enzyme mutant V77A.
(3) high speed centrifugation supernatant is through Ni-NTA affinity chromatography and and gel permeation chromatography (GE-Superdex 200 Increase 10/300 GLTM) after purification, obtain leucine -5- hydroxylation enzyme mutant V77A.
3, the present invention also provides the applications of leucine -5- hydroxylation enzyme mutant V77A；
Further, the leucine -5- hydroxylation enzyme mutant V77A is applied to using leucine as the carbon of distal end 5 of substrate The hydroxylating of position；
Further, the leucine -5- hydroxylation enzyme mutant V77A is applied to using methionine as the sulfonating reaction of substrate；
The utility model has the advantages that
For the present invention by Fixedpoint mutation modified wild type leucine -5- '-hydroxylase gene, the mutant V77A of acquisition is wilder The activity that No. 5 carbon potentials of leucine hydroxylation reaction occurs for raw type improves.Leucine -5- after mutation is hydroxylated V77A pairs of enzyme mutant The specific enzyme activity of substrate leucine is 1765.94U/mg, improves 69.53% compared with wild type；To the ratio enzyme of the methionine of substrate Living is 1017.13U/mg, improves 23.26% compared with wild type.
Leucine -5- hydroxylation enzyme mutant of the invention has the specific enzyme activity of leucine and methionine to be mentioned by a relatively large margin Height produces antibiotic medicine intermediate for biological enzyme efficient catalytic from now on and provides theoretical foundation and technical guarantee.
Detailed description of the invention:
Fig. 1 is wild type and mutant gene agarose gel electrophoresis figure
Wherein, M: standard Marker；1:LEH wild type；2: mutant gene mLEH；
Fig. 2 is leucine -5- hydroxylase wild type and mutant SDS-PAGES
Wherein, M: standard protein Marker；1: wild type；2: mutant V77A；
Fig. 3 is leucine -5- hydroxylase to leucine and methionine catalytic process schematic diagram
Wherein, a: leucine -5- hydroxylase catalysis leucine schematic diagram；B: leucine -5- hydroxylase catalysis methionine shows It is intended to；
Fig. 4 is the reaction product HPLC detection figure that leucine is substrate；
Fig. 5 is that (wherein, control group joined methionine sulfoxide mark for reaction product HPLC detection figure that methionine is substrate Product).
Specific embodiment:
The method of the present invention is described below by specific embodiment.Unless stated otherwise, technology used in the present invention Means are method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, rather than this hair is limited Bright range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, it is not carrying on the back Under the premise of from spirit and scope of the present invention, in these embodiments material component and dosage carry out various changes or change It is dynamic to also belong to protection scope of the present invention.
The present invention discloses a kind of leucine -5- hydroxylation enzyme mutant and its construction method and application, belong to genetic engineering and Technical field of enzyme engineering.Leucine -5- hydroxylase (LEH) progress that nostoc will be derived from is Fixedpoint mutation modified, and acquisition is dashed forward Variant has: I70G, I75A, I75F, V77A, V77I, V77L.By screening active ingredients, the mutant that wherein enzymatic activity improves is 69.53% and 23.26% has been respectively increased compared with wild type to the activity of leucine and methionine in V77A.It is provided by the invention bright Propylhomoserin -5- hydroxylation enzyme mutant facilitates it in the popularization of pharmaceutical intermediate compound probability application.
It will be further detailed below by specific embodiment.
The acquisition of embodiment 1, leucine -5- hydroxylation enzyme mutant V77A encoding gene mLEH
Obtain mutant V77A: by the gene of the wild type leucine -5- hydroxylase in nostoc source through codon optimization (SEQ ID NO.3) is implemented in pET28a (+) expression vector (Genbank sequence number of wild type leucine -5- hydroxylase afterwards RCJ32143.1), LEH-pET28a is obtained；Using LEH-pET28a plasmid as template, V77A_F (SEQ ID NO.5), V77A_R (SEQ ID NO.6) is primer, carries out inverse PCR rite-directed mutagenesis；The present embodiment experiment uses KOD-Plus mutant kit (spinning (Shanghai) Biotechnology Co., Ltd purchased from Japan):
(1) PCR reaction system is as follows:
Template (53ng/ μ L) 1μL
Primers F (10pmol/ μ L) 1.5μL
Primer R (10pmol/ μ L) 1.5μL
2mM dNTP 5μL
10×Buffer for iPCR 5μL
KOD-Plus 1μL
ddH2O 35μL
PCR reaction condition: 94 DEG C of initial denaturation 2min；98 DEG C of denaturation 10sec；68 DEG C of extension 7.5min；4 DEG C after 8 circulations Lower preservation.Target stripe is detected by 0.8% agarose gel electrophoresis.
(2) template in PCR product is digested
2 μ L Dpn I are added in PCR reaction solution (50 μ L), after mixing gently, is placed at 37 DEG C and reacts 1h.
(3) PCR product recirculation, reaction system are as follows:
Dpn I treated PCR product 5μL
Ligation high 5μL
T4Polynucletide Kinase 1μL
ddH2O 4μL
After mixing gently, it is placed at 16 DEG C and reacts 1h.
(4) 10 μ L circularized plasmid in (3) is taken, by changing in the E.coli JM109 that walks around, picking transformant, upgrading grain is surveyed Sequence verifying, as shown in Figure 1.Through being sequenced, mutational site is mutated according to goal-selling, is obtained and is dashed forward shown in SEQ ID NO.4 Variant encoding gene mLEH, the amino acid sequence of corresponding mutant is as shown in SEQ ID NO.2；- 80 DEG C of preservation plasmids and correlation Bacterium.
The expression of embodiment 2, leucine -5- hydroxylase
Wild type encoding gene LEH and mutant code gene mLEH are constructed respectively on pET28a (+) expression vector The recombinant vector LEH-pET28a and mLEH-pET28a of acquisition are transferred to e. coli bl21 (DE3) respectively, obtain BL21/LEH and BL21/V77A recombinant bacterium.
Above-mentioned 2 kinds of recombinant bacteriums are connected to 5mL LB culture medium (containing 50 μ g/mL kanamycins) respectively, 220r/ at 37 DEG C Min cultivates 12h；It is connected to 100mL LB culture medium (containing 50 μ g/mL kanamycins) with 1% inoculum concentration, to bacteria concentration OD600 =0.6-0.8, is added IPTG, and final concentration of 0.75mM induces 20h under 16 DEG C, 180r/min.
The purifying and purification of embodiment 3, leucine -5- hydroxylase
Thalline were collected by centrifugation at 5000r/min, 15min respectively for the bacterium solution that embodiment 2 is obtained, with solution A (20mM Tris-HCl, pH 8.0,300mM NaCl, 20mM imidazoles, 1.5mM DTT) be resuspended after, be added lysozyme (whole solubility be 200 μ G/mL), protease inhibitors (whole solubility is 1mM) places 30min on ice, and (3s is opened, and 5s is closed, 350W function for ultrasonication on ice Rate), low-temperature and high-speed is centrifuged (4 DEG C, 18000r/min) removal cell fragments and obtains supernatant.
Ni affinity chromatography: taking 4 open columns (Open-Column), and 1mL Ni-NTA resin (QIAGEN) respectively is added.With 20mL solution A balances resin.High speed centrifugation supernatant is placed at 4 DEG C in 1mL resin and combines 40-60min.Mixed liquor is excessively open Column, the resin for being combined with albumen will be trapped.Resin is rinsed with 20mL solution A.Finally use 15mL solution B (20mM Tris- HCl, pH 8.0,300mM NaCl, 300mM imidazoles, 2mM DTT) elute lower albumen.
Gel permeation chromatography: 200 Increase 10/ of GE-AKTA Pure 25L tomographic system and GE-Superdex is used 300 GLTMIt realizes gel filtration chromatography, purifies albumen further.By above-mentioned 15mL eluent super filter tube (Millipore-Amicon-Ultra-15-MWCO-10kD) 500 μ L are concentrated into, by pumping loading.Finally containing 150mM Lower target protein is eluted under the solution C (20mM Tris-HCl, pH 8.0,150mM NaCl, 1mM DTT) of NaCl, it is as wild Type enzyme solution and mutant enzyme solution.
By 12% native polyacrylamide gel electrophoresis (SDS-PAGE), identifies albumen size and purity, obtain egg White molecular weight about 31KDa, consistent with DNAMAN prediction, purity of protein is 95% or more, as shown in Figure 2.
Embodiment 4, leucine -5- hydroxylase enzyme activity determination
1, enzyme concentration measures
Using the concentration of BCA method measurement albumen, kit is purchased from Suo Laibao company.
(1) it prepares working solution: according to standard items and sample size, adding 1 volume Cu reagent (50:1) by 50 volume BCA reagents It is configured to BCA working solution, is mixed well.
(2) dilution standard product: taking 10 μ L BSA standard items to be diluted to 100 μ L (sample is generally available PBS dilution) with PBS, Make final concentration of 0.5mg/ml.Standard items are added in the protein standard sample wells of 96 orifice plates by 0,2,4,6,8,12,16,20 μ L, PBS is added to supply to 20 μ L.
(3) sample is made suitably to dilute, in the sample well for adding 20 μ L to 96 orifice plates.
(4) 200 μ L BCA working solutions, 37 DEG C of placement 15-30min are added in each hole.562nm light absorption is measured with microplate reader OD562, protein concentration is calculated according to standard curve.
2, activity determination method
The protein concentration of wild type enzyme solution and mutant enzyme solution that embodiment 3 obtains is diluted to 0.2mg/mL, 1mL respectively The reaction system of enzyme activity determination is as follows:
Blank control group is to contain the above-mentioned reaction system for inactivating enzyme solution；
Reaction system is placed at 30 DEG C and is incubated for 10min, is measured using succinic acid kit (Megazyme, Irland) In 10min under 340nm reaction solution absorbance change.
Enzymatic activity definition: 1U is defined as consumption (the i.e. generation of succinic acid of 5mM leucine or methionine in per minute Amount) unit be mMmin-1, enzyme activity calculation formula is as follows:
In formula: A0, A1 respectively indicate the absorbance that initial and reaction terminates；
V is reaction volume, is 1mL；
T is the reaction time, is 10min；
ξ600For extinction coefficient, ξ600=18.7 × 103cm-1·M-1。
3, wild type and mutant specific enzyme activity are compared
The enzyme activity measured is specific enzyme activity, wild type and mutant specific enzyme activity such as following table divided by enzyme concentration in reaction system. V77A mutant specific enzyme activity is higher than wild type, and 69.53% and 23.26% has been respectively increased to substrate leucine and methionine.
Embodiment 5, leucine -5- hydroxylase identify leucine and methionine catalysate HPLC
No. 5 carbon in leucine -5- hydroxylase specific catalytic leucine distal end generate 5- hydroxyl-leucine, and can be special Anisotropic catalysis methionine brings it about oxidation sulfonating reaction, generates methionine sulfoxide.Its catalytic process schematic diagram such as Fig. 3 institute Show.It adopts mutant enzyme solution prepared with embodiment 3 and establishes enzymic catalytic reaction system (1mL) as shown in the table (wherein, sulfuric acid Asia Iron, Alpha's ketoglutaric acid are the co-factors of enzyme solution catalysis reaction):
Control group (adding inactivating protein) and 1mL reaction system are placed in reaction overnight at 25 DEG C.High-temperature boiling loses albumen Living, removing protein is removed in centrifugation.300 μ L of supernatant is taken, column front derivation is carried out using 2,4- dinitro-fluorobenzene.Product is detected by HPLC, As shown in Figures 4 and 5.
HPLC testing conditions are as follows:
Chromatographic column:C18 Column(5μm,4.6×250mm,Waters,Ireland)；
Mobile phase: 50% methanol, 50mM sodium acetate (pH 6.4)；
Flow velocity: 1mL/min；
Sample volume: 10 μ L；
Ultraviolet detection wavelength: 360nm.
Although the present invention discloses as above with preferable implementation, it is not intended to limit the invention, any to be familiar with this technology People can all do various change and modification, therefore protection scope of the present invention without departing from the spirit and scope of the present invention It should be subject to defined by claims.
Sequence table
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gaatttgcta ccccgcagga atgcgaaatg ctggttaacc aggctgaact gttcaacttc 180
gactgctacc agaacgttaa cccgaaaatc gaacgtatcg gtatcaccgc gttcgaatac 240
aaccgtatct ctaaagctgc ttacttccag gctgttgaac gtaccaccaa actgcgtgac 300
tgcatcatgg ctgcttcttt caacccgctg gaacgtctga tggttaaaat ccgtgaatgc 360
accggtgcta ccgttcgtat cgcttctgaa ccgttctacg gttcttacta cgctggtctg 420
atccgtaaaa tcgaacaggg tactcagctg cacatcgact acgctccgct ggaacagtct 480
aaatgggaaa tcggtactgt tatctaccag ctgtcttgga acctgtacct gaaattctct 540
ccgaacaacc acggtcagac ccgtatctac gaccgtcagt ggcagccggg tgacgaccag 600
tacaaactgg actcttacgg ttacggtgac accgttatcg ctgacgctga cgctatcgct 660
ttccagccgt acgttggtga cgttttcatc ttcaacaccc gtaactacca caccgttgaa 720
ccgatggacg gtcagcgtgt taccttcacc tctgctatcg gtctgctgcc gaacggtgaa 780
atcatcctgt ggtcttaa 798
<210> 5
<211> 23
<212> DNA
<213>artificial sequence ()
<400> 5
gcgttcgaat acaaccgtat ctc 23
<210> 6
<211> 25
<212> DNA
<213>artificial sequence ()
<400> 6
ggtgataccg atacgttcga ttttc 25

Claims (8)

1. a kind of leucine -5- is hydroxylated enzyme mutant, which is characterized in that the amino acid sequence of the mutant such as sequence table SEQ Shown in ID No.2.
2. the encoding gene of the hydroxylation enzyme mutant of leucine -5- described in claim 1.
3. the encoding gene of the hydroxylation enzyme mutant of leucine -5- described in claim 2, which is characterized in that such as sequence table SEQ ID Shown in No.4.
4. a kind of recombinant vector or recombinant bacterial strain comprising gene described in claim 2.
5. the recombinant vector or recombinant bacterial strain stated such as claim 4, which is characterized in that expression vector is pET28a (+), Su Zhuxi Born of the same parents are e. coli bl21 (DE3).
6. the application of recombinant vector or recombinant bacterial strain described in claim 4.
7. leucine -5- described in claim 1 is hydroxylated enzyme mutant using leucine as the hydroxylating of No. 5 carbon potentials in distal end of substrate Application in reaction.
8. leucine -5- described in claim 1 is hydroxylated application of the enzyme mutant in the sulfonating reaction using methionine as substrate.
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