CN109355265A

CN109355265A - A kind of carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) and its application and encoding gene

Info

Publication number: CN109355265A
Application number: CN201811412383.8A
Authority: CN
Inventors: 娄文勇; 魏萍; 宗敏华
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2018-11-25
Filing date: 2018-11-25
Publication date: 2019-02-19
Anticipated expiration: 2038-11-25
Also published as: CN109355265B

Abstract

The present invention provides a kind of carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) and its application and encoding gene.Carbonyl reductase AcCR can be catalyzed a variety of latent chiral carbonyl compounds asymmetric reductions, but it is lower for the activity and substrate tolerance of aromatic compound, the present invention is mutated using enzyme molecule transformation means by reductase AcCR is glycosylated, it obtains mutant mut-AcCR (I147V/G152L), mutant for 2- hydroxy acetophenone specific enzyme activity up to 6.4 U/mg, the Rate activity than unmutated preceding carbonyl reductase improves 17.4 times.Substrate tolerable concentration is improved from 50 mmol/L to 200 mmol/L.Carbonyl reduction enzyme mutant of the present invention is widely used in the asymmetric reduction of carbonyls.

Description

A kind of carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) and its application with Encoding gene

Technical field

The invention belongs to enzyme molecules, and field is transformed, and in particular to a kind of carbonyl reduction enzyme mutant mut-AcCR (I144V/ G152L) and its application with encode the mutant gene.

Background technique

Optically pure chiral alcohol and its derivative are the important hands of synthesis of chiral drug, liquid crystal material, flavors and fragrances, pesticide Property intermediate, medicine and other chemical fields occupy critical positions.Chiral alcohol can be synthesized by chemical method and with bioanalysis. Chemical synthesis generally requires the harsh condition such as high temperature and pressure；Using a large amount of organic reagent, environmental pollution is serious；It prepared Journey is complicated, often there is repeatedly protection and deprotection step；The enantioselectivity for the product that more importantly chemical method obtains Property is low.Compared to chemical method, bioanalysis generally reacts at normal temperatures and pressures, and mild condition, equipment requirement is low, and environmental pollution is small, The advantages that Substratspezifitaet is strong, stereoselectivity and regioselectivity with higher, product enantiomeric purity is high.Biocatalysis Synthesis of chiral alcohol in strong momentum in medicine preparation mesosome to play an increasingly important role.Based on Green Chemistry With the principle of sustainable development, it is not only green but also sustainable route of synthesis that biocatalysis, which prepares chiral alcohol,.

Carbonyl reductase is as a kind of efficient, highly selective biocatalyst that can be used for asymmetric reduction reaction, often It is used to prepare chipal compounds with optical activation.Carbonyl reductase is great there are two when catalytic asymmetric reduction is reacted The problem of challenge, and limit it and further apply asymmetric catalysis synthesis.First, the latent hand of many high value chiral alcohols The dissolubility of property substrate is very low or is insoluble in the crude media of enzyme.This disadvantage can improve substrate by using non-aqueous media The method of solubility is addressed, such as uses organic solvent, ionic liquid, the single or double phase reaction medium of supercritical carbon dioxide Carry out biocatalytic reaction.A part of carbonyl reductase is also shown higher using these solvents as medium in high concentration of substrate Enzyme activity.But activity, selectivity and the stability of some carbonyl reductases often will appear in various degree in non-aqueous media Reduction.Therefore, it needs to screen reaction medium when selecting carbonyl reductase non-aqueous reaction medium.Second, due to carbonyl Base reductase has the characteristics that being not suitable with for stringent substrate specificity, coenzyme dependence and physiological environment, makes carbonyl reduction Enzyme can not effectively complete a specific Stereospecific synthesis reaction, such as in high concentration substrate, high temperature, extreme pH Under environment, carbonyl reductase often shows the deficiencies of activity is low, selectivity and stability are poor.

With the development of genomics and proteomics, scientist puts into more sight in protein engineering. Based on directed evolution and design and rational etc., protein sequence is modified, changes its structure, and then influences its catalytic Can, better regio- and stereo-selectivity, higher stability and substrate tolerance are made it have, is provided for the synthesis of chiral alcohol More efficient way.Protein engineering is preferably applied for enzyme molecule transformation, the biology leading with enzyme or cell can be made Catalysis is further developed in terms of other in substrate, medium, reactor etc., and the achievement for obtaining protein engineering obtains more Effectively, it more routinely applies.

In this seminar early-stage study, the clonal expression one from acetobacter Acetobacter sp.CCTCC M209061 Kind carbonyl reductase.The carbonyl reductase can be catalyzed a variety of substrates and carry out asymmetric reduction reaction, have good three-dimensional choosing Selecting property.But the carbonyl reductase is lower for the enzyme activity of the carbonyls such as 2- hydroxy acetophenone, for the resistance to of this kind of substrate It is poor by property, it only can be only achieved good catalytic effect when concentration of substrate is lower.

Summary of the invention

In order to overcome the disadvantage that the enzyme activity is low and substrate tolerance is poor, the primary purpose of the present invention is that providing a kind of work The carbonyl reduction enzyme mutant mut-AcCR (I147V/ for following trans- Prelog rule that property and substrate tolerance are significantly improved G152L)。

It is another object of the present invention to provide above-mentioned carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) Gene and amino acid sequence.

It is yet a further object of the present invention to provide above-mentioned carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) Using.

The purpose of the present invention is achieved through the following technical solutions.

A kind of carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L), amino acid sequence such as SEQ ID NO.1 institute Show.

The present invention also provides the gene for encoding above-mentioned carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L), Gene order is as shown in SEQ ID NO.2.

Further, a kind of above-mentioned carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) is that reduction will be carbonylated Enzyme AcCR obtains carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) by the means that enzyme molecule is mutated.

Further, the gene order of AcCR is translated into its amino acid sequence by standard method, with the sequence in PDB It is searched in database, chooses 4RF2,1ZJY, 1NXQ and 1ZK3 three-level knot that homology is respectively 53%, 51%, 51% and 51% Structure is template, carries out homologous modeling, and carry out energy minimum, obtains the tertiary structure model of carbonyl reductase AcCR.Into One step is using ramachandran map Ramachandran (Ramachandran Plot) and Profile-3D to each amino acid residue in homologous modeling result The matching degree of reasonable structure and built protein model and protein amino acid sequence is assessed.Determine that model built is reasonable, It can be used for subsequent experimental analysis.

Further, carbonyl reductase AcCR tertiary structure is docked with coenzyme NAD H, it is pre- by HotSpot 2.0 The mutantional hotspot of carbonyl reductase is surveyed, chooses the site 147I and 152G as mutational site.

Further, the variation between mutation front and back carbonyl reductase and 2- hydroxy acetophenone is analyzed by molecular docking. Carbonyl reductase AcCR, mutant mut-AcCR (I147V/G152L) and 2- hydroxy acetophenone are subjected to molecular docking respectively, point Analysis enzyme active sites Ser142, Tyr155 and coenzyme NAD H niacinamide ring C4 be located at substrate to distance between 2- hydroxy acetophenone and The variation of active force.

Further, the activity of carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) is measured, carbonyl reduction is measured The zymologic property of enzyme mutant mut-AcCR (I147V/G152L), studies mutant mut-AcCR by way of enzyme activity determination (I147V/G152L) optimum temperature and pH and stability.

The present invention also provides a kind of above-mentioned carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) in catalysis of carbonyl Application in the asymmetric reduction of compound.

Compared with prior art, the invention has the advantages that and the utility model has the advantages that a kind of carbonyl reduction provided by the invention Enzyme mutant mut-AcCR (I147V/G152L), overcomes that original carbonyl reductase enzyme activity is low and what substrate tolerance was poor lacks Point, enzyme activity and substrate tolerance with higher.

Detailed description of the invention

Fig. 1 a, Fig. 1 b are that AcCR and mut-AcCR and 2- hydroxy acetophenone dock comparative result figure；

Fig. 2 a, Fig. 2 b are influence comparison diagram of the temperature to mutant mut-I147V/G152L Activity and stabill；

Fig. 3 a, Fig. 3 b are influence comparison diagram of the pH of buffer to mutant mut-I147V/G152L Activity and stabill.

Specific embodiment

Below with reference to embodiment and attached drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited In this.It is that those skilled in the art can refer to prior art reality if place is not described in detail especially it need to be pointed out that having below It is existing or understanding.

Embodiment 1

The gene order of AcCR is translated into its amino acid sequence by standard method, with the sequence in PDB database Search, choosing 4RF2,1ZJY, 1NXQ and 1ZK3 tertiary structure that homology is respectively 53%, 51%, 51% and 51% is mould Plate carries out homologous modeling, and carries out energy minimum, obtains the tertiary structure model of carbonyl reductase AcCR.Further adopt Each amino acid residue structure in homologous modeling result is closed with ramachandran map Ramachandran (Ramachandran Plot) and Profile-3D Rationality and the matching degree of built protein model and protein amino acid sequence are assessed.It determines that model built is reasonable, can be used for Subsequent experimental analysis.Carbonyl reductase AcCR tertiary structure is docked with coenzyme NAD H, carbonyl is predicted by HotSpot2.0 The mutantional hotspot of base reductase, the site 147I and 152G is as mutational site.

Embodiment 2

The variation between mutation front and back carbonyl reductase and 2- hydroxy acetophenone is analyzed by molecular docking；Respectively by carbonyl Reductase AcCR, mutant mut-AcCR (I147V/G152L) are docked with 2- hydroxy acetophenone, analyze enzyme active sites Ser142, Tyr155 and coenzyme NAD H niacinamide ring C4 are located at change of the substrate to distance and active force between 2- hydroxy acetophenone Change.AcCR shown in Fig. 1 a, Fig. 1 b and mut-E144A/G152L docks result figure with 2- hydroxy acetophenone.A, b is followed successively by 2- hydroxyl Benzoylformaldoxime docks result with AcCR, mut-I147V/G152L.It can be seen that catalytic site S142 after being mutated from Fig. 1 a, Fig. 1 b With 4, NADH niacinamide ring on hydrogen atom and the distance between the carbonylic oxygen atoms of 4 '-chloro-acetophenones reduce obvious, contract respectively It is short(20.1%) and(7.1%).These results suggest that the reduction of distance, the hydrogen bond for enhancing enzyme molecule and HAP is made With increasing stability of the enzyme in conjunction with HAP, improve the efficiency that C4 hydrogen atoms of NADH niacinamide ring are transmitted to substrate. Mut-I147V/G152L is 6.4U/mg for the enzyme activity of 2- hydroxy acetophenone, is AcCR enzyme activity (0.37U/mg) before being mutated 17.4 times.

Embodiment 3

Using PrimeSTAR Max DNA Polymerase, obtained by the method for carrying out the full plasmid amplification of pGEX-accr Obtain the plasmid pGEX-mut-I147V/G152L containing mutant mut-AcCR (I147V/G152L) gene.

Rite-directed mutagenesis the primer: the mutant primer of site I1447 is Primer1:5'- ACTGGTAGGGGACCCAATGGGAGCCG-3'；Primer2:5'-CGGCCCTCATTGGG TCCCCTACCAGT-3'；Site The mutant primer of G152L is Primer 3:5'-ACCCAATGTTGGC CGCCTATAAC-3', Primer 4:5'- GTTATAGGCGGCCAACATTGGGT-3'。

PCR amplification system and reaction condition used by rite-directed mutagenesis are as follows:

Polymerase chain reaction (PCR) amplification system

PCR reaction condition:

After reaction, reaction product is handled with restriction enzyme DpnI, acts on the site Gm6A^TC, disappears Template plasmid in change system.Reaction system are as follows:

Prepared endonuclease reaction system is placed at 37 DEG C and is incubated for 15min, that is, completes the digestion process of template plasmid.It will Digestion products directly convert bacillus coli DH 5 alpha.Positive transformant is verified using bacterium colony PCR, plasmid is extracted, is sequenced.

It is extracted from recombinant bacterium DH5 α (pGEX-mut-I147V/G152L) in the present invention and correct recombinant plasmid is sequenced PGEX-mut-I147V/G152L, and be transformed into E. coli expression strains BL21 (DE3), obtain recombinant strains BL21(DE3)(pGEX-mut-I147V/G152L)。

Embodiment 4

Picking contains the positive transformant of the BL21 (DE3) of mutant in LB culture medium of the 4mL containing 100 μ g/mL ammonia benzyls, It is incubated overnight under the conditions of 37 DEG C, 180r/min, 1mL culture solution is taken to be forwarded to the LB culture medium that 50mL contains 100 μ g/mL ammonia benzyls In, 37 DEG C, 180r/min cultivates to OD₆₀₀To about 1.2, it will be added final concentration 0.4mmol/L's after near 20 DEG C of cultivation temperature IPTG cultivates 15-18h, 4 DEG C, 8000r/min centrifugation 5min collection thallus.4 DEG C, 8000r/min centrifugation 5min, remove supernatant Thallus is weighed afterwards three times with 0.85% brine, is dispersed in phosphate buffer (50mmol/L, pH by liquid 6.5) in, be configured to 30mg/mL cell suspending liquid, be placed in 4 DEG C it is spare.Using Ultrasonic Cell Disruptor smudge cells, Ultrasonic Cell Disruptor It is set as power 350W, work 3s, interval 5s, and ultrasonic time is set as 20min, and entire shattering process cell suspension is in always In ice-water bath, low temperature environment is kept to inactivate enzyme denaturation in order to avoid temperature is excessively high.After ultrasonication, broken suspension is placed in In centrifuge, 4 DEG C, 8000r/min centrifugation 30min, gained supernatant is the thick zyme extract for recombinating carbonyl reductase.

Using Bio-Rad NGC Quest^TM10 mesohigh tomographic systems carry out recombination carbonyl reductase crude enzyme liquid pure Change.By the crude enzyme liquid of extraction with 0.22 μm of membrane filtration be placed on 4 DEG C it is spare.The general step of purifying are as follows: with 5 times of column volumes Buffer A(4.3mM Na₂HPO₄, 1.47mM KH₂PO₄, 137mM NaCl, 2.7mM KCl, pH7.3) and pre-equilibrate Bio- Scale Mini Profinity GST prepacked column (5mL, protein load amount 500mg), flow velocity 5mL/min；After pre-equilibration Start loading, using pump loading, flow velocity 1mL/min, applied sample amount about 500mg albumen；After end of the sample, continue flat with Buffer A The pillar that weighs is suitable with base line is pre-equilibrated to baseline；Then, with Buffer B (4.3mmol/L Na₂HPO₄, 1.47mM KH₂PO₄, 637mM NaCl, 2.7mM KCl, pH7.3) continue to rinse pillar, remove more firm foreign protein；Finally, with washing De- buffer solution B uffer C (50mM Tris-HCl, 2.5g/L glutathione, pH 8.0) wash-out recombinant protein AcCR, is washed De- obtained solution is the single recombinant protein A cCR after isolating and purifying.By there are also the eluents of recombinant protein A cCR to be placed in It analyses in bag, with PEG 20000, is concentrated in 4 DEG C of environment, the recombination enzyme solutions after concentration are saved in 4 DEG C.

Recombinate the determination condition of carbonyl reductase AcCR reduction activation: 0.25mmol/L NADH, 2mL phosphate buffer Appropriate enzyme solution is added in (50mmol/L, pH 6.5), 20mmol/L2- hydroxy acetophenone, 35 DEG C of incubation 5min, detects reaction system Under 340nm ultraviolet wavelength, the variation of light absorption value in 3min.

Enzyme activity definition: under the above conditions, the enzyme activity of 1 μm of ol NADH of catalysis oxidation per minute is 1 enzyme activity list Position, is indicated with U.

The calculation formula of enzyme activity is as follows:

Enzyme activity (U)=EW × V × 10³/(6220×1)

In EW:1min at 340nm absorbance value variable quantity；V: the total volume of reaction solution, mL；6220: Molar Extinction system Number, L/mol/cm；1: optical path length, cm.

The enzyme activity determination of carbonyl reductase different mutants mut-AcCR the results are shown in Table 1.

The 1. carbonyl reduction enzyme mutant of table of comparison before and after to(for) different substrate actives

Embodiment 5

The optimum temperature and pH and steady of mutant mut-AcCR (I147V/G152L) are studied by way of enzyme activity determination It is qualitative.The optimal reactive temperature for recombinating carbonyl reductase AcCR is determined by the enzyme activity of enzyme when measurement different temperatures, that is, is divided Not at 25,30,35,40,45 DEG C, the enzyme activity of reference standard method measurement recombination carbonyl reductase AcCR.The thermostabilization of the enzyme Property measurement, i.e., the enzyme is placed in (25-45 DEG C) incubation 36h of different temperature, timing sampling measures the enzyme not according to standard method With the enzyme activity at time point.The vigor of the enzyme before being incubated for is set as 100% opposite enzyme activity.The reaction of each temperature is arranged two and puts down Row.As a result see Fig. 2 a, Fig. 2 b.Using 2- hydroxy acetophenone as substrate, influence of the temperature to the mutant is studied, finds the enzyme in institute Enzyme activity enzyme activity at 70% or more, 35 DEG C is preferably embodied in examination range, and 30 DEG C and 40 DEG C of enzyme activities are respectively 95% With 98% or so.Therefore, at 30-40 DEG C, enzyme activity can be obtained well to be showed the enzyme, carries out enzymic catalytic reaction ratio in the range Conveniently.Hydrophobic effect is the principal element for stablizing albumen natural structure.Val ratio Ile has stronger hydrophobic effect, more favorably In the stability for improving albumen.The enzyme has good thermal stability, within the scope of 30-40 DEG C, is incubated for 12h, opposite enzyme activity is still It is able to maintain 70% or more, thermal stability is better than unmutated AcCR.

Embodiment 6

The enzyme activity of the enzyme determines when recombinating the optimal reaction pH of carbonyl reductase AcCR by measuring different pH, that is, distinguishes In pH 5.0 to 8.0, the enzyme activity of reference standard method measurement recombination carbonyl reductase AcCR.The pH Stability Determination of the enzyme, Will the enzyme be placed in 4 DEG C, (5.0-7.0) is incubated for 96h within the scope of different pH, and timing sampling measures the enzyme according to standard method The enzyme activity of different time points.The enzyme activity of the enzyme before being incubated for is set as 100%.Each pH value is arranged two in parallel.Knot Fruit sees Fig. 3 a, Fig. 3 b.Influence discovery of the pH to mutant mut-I147V/G152L is studied, using 2- hydroxy-acetophenone as substrate When, which has 80% or more relative activity in pH 5.0-7.0；PH is greater than after 7.0, and enzymatic activity significantly reduces； PH mutation enzyme activity in 6.0-6.5 obtains maximum embodiment.The thermostabilization of the mutant enzyme is studied within the scope of pH5.0-7.0 Property, after pH is saved for 24 hours in 5.0-5.5, relative activity retains between 65%-80%；PH is in 6.0-7.0, thermostabilization Property it is more preferable, relative activity is retained in 95% or more, therefore the mutant has good pH stability.

Embodiment 7

Carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) is catalyzed the determination of activity of different substrates: 2mL is contained The phosphate buffer (100mmol/L, pH 6.5) of different substrates (20mmol/L) is added separately to make marks corresponding In 10mL triangular flask, in duplicate, 35 DEG C of incubation 10min, then a certain amount of AcCR mutant after purification, then, reference Standard method measures the enzyme to the enzyme activity of different substrates, using the enzyme to the enzyme activity of 2- hydroxy acetophenone as the enzyme 100% opposite enzyme activity.It the results are shown in Table 1.Mutant mut-I147V/G152L is other than improving 17.4 times to HAP enzyme activity, to being ground The catalytic activity for a variety of substrates studied carefully is significantly improved, for the enzyme activity of straight chain 'beta '-ketoester have 3.0-7.77 times not With the raising of degree.

2. carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) of table is catalyzed the asymmetry of latent chiral carbonyl compounds Reduction

E.e: enantiomeric excess value

Carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) is catalyzed the yield and enantio-selectivity of different substrates Measurement: 2mL is contained to the phosphate-buffered of different substrates (200mmol/L), 0.1mmol/L NADH and 400mmol/L isopropanol In liquid (100mmol/L, pH 6.5), it is added separately in the corresponding 10mL triangular flask to have made marks, in duplicate.35 DEG C be incubated for 10min after, a certain amount of AcCR mutant.Reaction flask is placed in 35 DEG C of gas bath constant temperature oscillators and is reacted (200rpm), 25 μ L of timing sampling are analyzed for GC or HPLC.It the results are shown in Table 2.The result shows that tolerance of the mutant enzyme for substrate Property and catalysis reaction yield have significantly improve.Under the concentration of substrate of 200mmol/L, mutant purifies 2- hydroxy acetophenone The products collection efficiency of generation is improved by unmutated preceding 50.62% (50mmol/L substrate) to (200mmol/L substrate) 90.5%, is improved 34.3%；The yield of catalysis ethyl acetoacetate asymmetric reduction is improved to 97.4%；It is catalyzed acetophenone asymmetric reduction Yield is improved to 76.8%, improves 31.8% compared with the yield of the unmutated enzymatic reaction.

Sequence table

<110>South China Science & Engineering University

<120>a kind of carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) and its application and encoding gene

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 252

<212> PRT

<213>carbonyl reduction enzyme mutant (mut-AcCR (I147V/G152L))

<400> 1

Met Thr Arg Val Ala Gly Lys Val Ala Ile Val Ser Gly Ala Ala Asn

1 5 10 15

Gly Ile Gly Lys Ala Thr Ala Gln Leu Leu Ala Lys Glu Gly Ala Lys

20 25 30

Val Val Ile Gly Asp Leu Lys Glu Glu Asp Gly Gln Lys Ala Val Ala

35 40 45

Glu Ile Lys Ala Ala Gly Gly Glu Ala Ala Phe Val Lys Leu Asn Val

50 55 60

Thr Asp Glu Ala Ala Trp Lys Ala Ala Ile Glu Gln Thr Leu Lys Leu

65 70 75 80

Tyr Gly Arg Leu Asp Ile Ala Val Asn Asn Ala Gly Ile Ala Tyr Ser

85 90 95

Gly Ser Val Glu Ser Thr Ser Leu Glu Asp Trp Arg Arg Val Gln Ser

100 105 110

Ile Asn Leu Asp Gly Val Phe Leu Gly Thr Gln Val Ala Ile Glu Ala

115 120 125

Met Lys Lys Ser Gly Gly Gly Ser Ile Val Asn Leu Ser Ser Glu Gly

130 135 140

Leu Val Gly Asp Pro Met Leu Ala Ala Tyr Asn Ala Ser Lys Gly Gly

145 150 155 160

Val Arg Leu Phe Thr Lys Ser Ala Ala Leu His Cys Ala Lys Ser Gly

165 170 175

Tyr Lys Ile Arg Val Asn Ser Val His Pro Gly Tyr Ile Trp Thr Pro

180 185 190

Met Val Ala Gly Leu Thr Lys Glu Asp Ala Ala Ala Arg Gln Lys Leu

195 200 205

Val Asp Leu His Pro Ile Gly His Leu Gly Glu Pro Asn Asp Ile Ala

210 215 220

Tyr Gly Ile Leu Tyr Leu Ala Ser Asp Glu Ser Lys Phe Val Thr Gly

225 230 235 240

Ser Glu Leu Val Ile Asp Gly Gly Tyr Thr Ala Gln

245 250

<210> 2

<211> 762

<212> DNA

<213>carbonyl reduction enzyme mutant (mut-AcCR (I147V/G152L))

<400> 2

atgacacgtg tagcaggcaa ggttgccatt gtttctgggg ccgctaatgg cattggcaag 60

gcaaccgcac agcttttggc caaggaaggc gcaaaagttg ttattggtga tttaaaagaa 120

gaagatgggc agaaagctgt tgcagaaatt aaggcagcag gtggtgaagc cgcatttgtc 180

aaactgaatg taacagatga ggctgcatgg aaagccgcta ttgagcaaac gcttaagctt 240

tatgggcggc tggatattgc agtgaacaat gcaggcattg cgtattctgg cagtgtagaa 300

agcacatctc tggaagattg gcggcgcgtt cagtctatca atctggatgg cgtgtttttg 360

ggcacacagg tggctattga ggccatgaag aagtcgggcg gtggatccat tgtcaatctg 420

tcttccattg aaggactggt aggggaccca atgttggccg cctataacgc cagtaaaggt 480

ggggtaaggc tgtttacaaa atctgcggcc ctacattgcg ccaaatctgg atacaaaatt 540

cgggtaaact cagtgcatcc cggctatatc tggacaccta tggtggccgg tttaacaaag 600

gaagatgctg ctgcacgcca aaagctggtg gatctgcacc ccattggcca cttgggtgag 660

cccaacgata ttgcttacgg tattttgtat cttgcctctg atgaatccaa gtttgttaca 720

gggagcgaac tggtcattga tggtgggtac accgcgcaat aa 762

Claims

1. a kind of carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L), it is characterised in that: its amino acid sequence such as SEQ Shown in ID NO.1.

2. encoding the gene of carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) described in claim 1, feature exists In: gene order is as shown in SEQ ID NO.2.

3. carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) according to claim 1, it is characterised in that: by Carbonylation reductase AcCR obtains carbonyl reduction enzyme mutant mut-AcCR (I147V/ by the means that enzyme molecule is mutated G152L)。

4. asymmetry of the carbonyl reduction enzyme mutant mut-AcCR (I147V/G152L) in carbonyls described in claim 1 Application in reduction.