CN109468293A

CN109468293A - A kind of carbonyl reduction enzyme mutant mut-AcCR (E144A/G152L) and its application and encoding gene

Info

Publication number: CN109468293A
Application number: CN201811412391.2A
Authority: CN
Inventors: 娄文勇; 魏萍; 宗敏华; 郭泽望; 区晓阳
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: 2019-03-15
Anticipated expiration: 2038-11-25
Also published as: CN109468293B

Abstract

The invention discloses a kind of carbonyl reduction enzyme mutant mut-AcCR (E144A/G152L) and its application and encoding genes.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 (E144A/G152L), the mutant for 4 '-chloro-acetophenones specific enzyme activity up to 92.7 U/mg, the Rate activity than unmutated preceding carbonyl reductase improves 17.93 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 (E144A/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 (E144A/ 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 early-stage study of the present invention, clonal expression is a kind of from acetobacter Acetobacter sp.CCTCC M209061 Carbonyl reductase.The carbonyl reductase can be catalyzed a variety of substrates and carry out asymmetric reduction reaction, have good three-dimensional selection Property.But the carbonyl reductase is lower for the enzyme activity of the aromatic carbonyls such as 4 '-chloro-acetophenones, for this kind of substrate Tolerance it is poor, 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 (E144A/ 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 (E144A/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 (E144A/G152L) Using.

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

A kind of carbonyl reduction enzyme mutant mut-AcCR (E144A/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 (E144A/G152L), Gene order is as shown in SEQ ID NO.2.

Further, a kind of above-mentioned carbonyl reduction enzyme mutant mut-AcCR (E144A/G152L) is that reduction will be carbonylated Enzyme AcCR obtains carbonyl reduction enzyme mutant mut-AcCR (E144A/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.Again Using ramachandran map Ramachandran (Ramachandran Plot) and Profile-3D to each amino acid residue structure in homologous modeling result Reasonability and the matching degree of built protein model and protein amino acid sequence are assessed.It determines that model built is reasonable, can use It is analyzed in subsequent experimental.

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 144E and 152G as mutational site.

Further, the variation between mutation front and back carbonyl reductase and 4 '-chloro-acetophenones is analyzed by molecular docking, point Carbonyl reductase AcCR, mutant mut-AcCR (E144A/G152L) and 4 '-chloro-acetophenones molecular docking, analysis are not subjected to Enzyme active sites Ser142, Tyr155 and coenzyme NAD H niacinamide ring C4 distance and effect between substrate 4 '-chloro-acetophenone The variation of power.

Further, the activity of carbonyl reduction enzyme mutant mut-AcCR (E144A/G152L) is measured, carbonyl reduction is measured The zymologic property of enzyme mutant mut-AcCR (E144A/G152L), studies mutant mut-AcCR by way of enzyme activity determination (E144A/G152L) optimum temperature and pH and stability.

The present invention also provides a kind of above-mentioned carbonyl reduction enzyme mutant mut-AcCR (E144A/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 (E144A/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-E144A/G152L and 4 '-chloro-acetophenones dock result figure.

Fig. 2 a, Fig. 2 b are influence comparison diagram of the temperature to mutant mut-E144A/G152L Activity and stabill；

Fig. 3 a, Fig. 3 b are influence comparison diagram of the pH of buffer to mutant mut-E144A/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 4RF2,1ZJY, 1NXQ and 1ZK3 (albumen in database that homology is respectively 53%, 51%, 51% and 51% are chosen in search The title of matter sequence) tertiary structure be template, carry out homologous modeling, and carry out energy minimum, obtain carbonyl reductase The tertiary structure model of AcCR.Further using ramachandran map Ramachandran (Ramachandran Plot) and Profile-3D to homologous modeling As a result the matching degree of each amino acid residue reasonable structure and built protein model and protein amino acid sequence in carries out Assessment.It determines that model built is reasonable, can be used for subsequent experimental analysis.By carbonyl reductase AcCR tertiary structure and coenzyme NAD H into Row docking, the mutantional hotspot of carbonyl reductase is predicted by HotSpot 2.0, chooses the site 144E and 152G as mutation position Point.

Embodiment 2

The variation between mutation front and back carbonyl reductase and 4 '-chloro-acetophenones is analyzed by molecular docking；Respectively by carbonyl Reductase AcCR, mutant mut-AcCR (E144A/G152L) are docked with 4 '-chloro-acetophenones, analyze enzyme active sites The variation of Ser142, Tyr155 and coenzyme NAD H niacinamide ring C4 distance and active force between substrate 4 '-acetophenone.As a result That sees AcCR shown in Fig. 1 a, Fig. 1 b and mut-E144A/G152L and 4 '-chloro-acetophenones docks result figure.It can be with from Fig. 1 a, Fig. 1 b The ketonic oxygen of catalytic site S142, Y155 and hydrogen atom and 4 '-chloro-acetophenones on 4, NADH niacinamide ring is former after finding out mutation The distance between son reduces obviously, is respectively shortened(22.4%), 0.68With (5.2%), it dashes forward After change, the steric hindrance of substrate and enzyme active center is reduced, more conducively contact of the enzyme with active site.In addition, Ala and Leu are equal For hydrophobic amino acid, it is replaced into the hydrophilic amino acid near enzyme molecule activated centre, increases enzyme molecule active site Hydrophobicity can make hydrophobicity ClPE be easier to enter enzyme molecule activated centre.Mut-E144A/G152L is for 4 '-chloro-acetophenones Enzyme activity is 92.7U/mg, is 17.9 times of AcCR enzyme activity (5.17U/mg) before being mutated.

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-E144A/G152L containing mutant mut-AcCR (E144A/G152L) gene.

Rite-directed mutagenesis the primer: the mutant primer of site E144A is Primer1:5'- ATCTGTCTTCCATTGCCGGACTGAT-3'；

Primer2:5'-ATCAGTCCGGCAATGGAAGACAGAT-3'；The mutant primer of site G152L is Primer3: 5'-ACCCAATGTTGGCCGCCTATAAC-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 in the present invention from recombinant bacterium DH5 α (pGEX-mut-E144A/G152L) and correct recombinant plasmid pGEX- is sequenced Mut-E144A/G152L, and be transformed into E. coli expression strains BL21 (DE3), obtain recombinant strains BL21 (DE3)(pGEX-mut-E144A/G152L)。

Embodiment 4

Picking positive transformant is in LB culture medium of the 4mL containing 100 μ g/mL ammonia benzyls, the mistake under the conditions of 37 DEG C, 180r/min Night culture, take 1mL culture solution be forwarded to 50mL containing 100 μ g/mL ammonia benzyls LB culture medium in, 37 DEG C, 180r/min cultivate to OD₆₀₀To about 1.2, the IPTG of final concentration 0.4mmol/L will be added after near 20 DEG C of cultivation temperature, cultivate 15-18h, 4 DEG C, 8000r/min is centrifuged 5min and collects thallus.4 DEG C, 8000r/min centrifugation 5min, remove supernatant, by 0.85% physiology of thallus Salt water washing is weighed afterwards three times, is dispersed in phosphate buffer (50mmol/L, pH 6.5), it is thin to be configured to 30mg/mL Born of the same parents' suspension, be placed in 4 DEG C it is spare.Using Ultrasonic Cell Disruptor smudge cells, Ultrasonic Cell Disruptor is set as power 350W, and work 3s, Interval 5s, ultrasonic time are set as 20min, and entire shattering process cell suspension is in ice-water bath always, keep low temperature environment, In order to avoid temperature is excessively high, inactivate enzyme denaturation.After ultrasonication, broken suspension is placed in a centrifuge, 4 DEG C, 8000r/min It is centrifuged 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/L 4 '-chloro-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.

Enzyme activity determination the results are shown in Table 1 active portion about mutant mut-AcCR (E144A/G152L) to 4 '-chloro-acetophenones Point.

Embodiment 5

The optimum temperature and pH and steady of mutant mut-AcCR (E144A/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 influence of Fig. 2 a temperature to mutant mut-E144A/G152L Activity and stabill (Fig. 2 b).By Fig. 2 a, Fig. 2 b It is found that the optimal reactive temperature of the mutant is 35 DEG C, it is consistent with the recombination optimum temperature of AcCR before mutation；What is studied In temperature range (25-45 DEG C), the relative activity of the mutant is held in 78% or more, especially when temperature is higher than 35 DEG C When, when temperature is 40-45 DEG C, the relative activity of the enzyme is between 80%-92%, hence it is evident that is better than unmutated AcCR (70- 90%).The thermal stability for studying the mutant enzyme is found within the temperature range of investigated, and is incubated for same time, and temperature is lower, is dashed forward The opposite enzyme activity of variant mut-E144A/G152L is better, and thermal stability is better.

When incubation temperature is at 40-45 DEG C, with temperature increase and incubation time extend, mutant mutE144A/ G152L albuminous degeneration speed is accelerated, and residual activity decline is obvious, and stability reduces.When incubation temperature is within the scope of 25-35 DEG C, It is incubated for 12h, opposite enzyme activity can be 80% or more；It is incubated at 25 DEG C for 24 hours, relative activity is still after opposite enzyme activity still has 75%, 36h 60% or so.Compared with unmutated AcCR, the mutant 40-45 DEG C of opposite enzyme activity and thermal stability to a certain extent It increases.Binding molecule docking is as a result, the enhancing of substrate and enzyme molecule hydrogen bond action may be it in the temperature model studied The reason of enclosing interior opposite enzyme activity increase.In addition, the Glu in enzyme molecule sports Leu, the hydrophobic side chain of Leu makes residual in enzyme molecule The hydrophobic binding of base increases, enzyme molecule inner hydrophobic effect enhancing, to keep protein structure more stable.

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, (6.0-8.0) 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%.The difference of buffer used in different pH ranges For citrate-phosphate salt buffer (pH 4.5-8.0), Tris-HCl buffer (pH 8.0-8.5), Glycine-NaOH Buffer (pH 8.5-9.5).Each pH value is arranged two in parallel.As a result pH of buffer is living to mutant mut-E144A/G152L The influence of property (Fig. 3 a) and stability (Fig. 3 b).By Fig. 3 a, Fig. 3 b it is found that mutation front and back, the optimal pH of AcCR simultaneously have not been changed, still It is 6.5, within the scope of 6.0-8.0, the relative activity of mutant mut-E144A/G152L 80% or more, has pH of buffer There is higher catalytic activity.It is studied with respect to the stability within the scope of pH of the enzyme activity greater than 80%, finds it in pH 6.0-8.0 More stable in range, for especially pH between 6.5-7.5,96h still remains about 70% opposite enzyme activity, has good pH steady It is qualitative.

Embodiment 7

Carbonyl reduction enzyme mutant mut-AcCR (E144A/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 4 '-chloro-acetophenones as the 100% of the enzyme Opposite enzyme activity.Comparison of the carbonyl reduction enzyme mutant front and back for different substrate actives that the results are shown in Table 1.Mutant mut- E144A/G152L has not the activity of the substituted acetophenone in 4 ' positions other than the enzyme activity to 4 '-chloro-acetophenones increases With the raising of degree.

Table 1

Table 2

E.e: enantiomeric excess value

Carbonyl reduction enzyme mutant mut-AcCR (E144A/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.Not Mutation carbonyl reductase AcCR is catalyzed the yield measurement of different substrates, and 2mL contains different substrates (50mmol/L), 0.1mmol/L In the phosphate buffer (100mmol/L, pH6.5) of NADH and 150mmol/L isopropanol, it is added separately to the phase to have made marks In the 10mL triangular flask answered, in duplicate.After 35 DEG C of incubation 10min, a certain amount of AcCR mutant.Reaction flask is placed in 35 It is reacted (200rpm) in DEG C gas bath constant temperature oscillator, 25 μ L of timing sampling, is analyzed for GC or HPLC.It the results are shown in Table 2 carbonyls Enzyme mutant mut-AcCR (E144A/G152L) is restored to be catalyzed shown in the asymmetric reduction of latent chiral carbonyl compounds.As a result table Bright, mutant enzyme significantly improves the tolerance of substrate and having for catalysis reaction.Under the concentration of substrate of 200mmol/L (R)- The yield of 3 '-methoxyacetophenones is improved by unmutated preceding 61.6% (50mmol/L substrate) to (200mmol/L substrate) 87.05%, improve nearly 26%；(R) -4 '-methoxybenzene ethyl alcohol yield is improved to 79.5%, and this is anti-for mutation enzymatic The yield answered is compared and improves 29%.

Sequence table

<110>South China Science & Engineering University

<120>a kind of carbonyl reduction enzyme mutant mut-AcCR (E144A/G152L) and its application and encoding gene

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 253

<212> PRT

<213>carbonyl reduction enzyme mutant (mut-AcCR (E144A/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 Ile Ala

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly 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 (E144A/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 ccggactgat tggggaccca 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 (E144A/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 (E144A/G152L) described in claim 1, feature exists In: gene order is as shown in SEQ ID NO.2.

3. a kind of carbonyl reduction enzyme mutant mut-AcCR (E144A/G152L) according to claim 1, it is characterised in that: Carbonyl reduction enzyme mutant mut-AcCR (E144A/ is obtained by the means that enzyme molecule is mutated by carbonylation reductase AcCR G152L)。

4. a kind of carbonyl reduction enzyme mutant mut-AcCR (E144A/G152L) described in claim 1 carbonyls not Application in asymmetric reduction.