CN109468293B

CN109468293B - Carbonyl reductase mutant mut-AcCR (E144A/G152L) and application and coding gene thereof

Info

Publication number: CN109468293B
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: 2021-06-08
Anticipated expiration: 2038-11-25
Also published as: CN109468293A

Abstract

The invention discloses a carbonyl reductase mutant mut-AcCR (E144A/G152L) and application and a coding gene thereof. The carbonyl reductase AcCR can catalyze a plurality of potential chiral carbonyl compounds to be asymmetrically reduced, but the activity and the substrate tolerance of the carbonyl reductase AcCR to aromatic compounds are lower, the glycosylation reductase AcCR is mutated by adopting an enzyme molecule modification means to obtain a mutant mut-AcCR (E144A/G152L), the specific enzyme activity of the mutant to 4' -chloroacetophenone can reach 92.7U/mg, and the specific enzyme activity is improved by 17.93 times compared with that of the carbonyl reductase before mutation. The substrate tolerance concentration is increased from 50mmol/L to 200 mmol/L. The carbonyl reductase mutant of the invention is widely applied to asymmetric reduction of carbonyl compounds.

Description

Carbonyl reductase mutant mut-AcCR (E144A/G152L) and application and coding gene thereof

Technical Field

The invention belongs to the field of enzyme molecule modification, and particularly relates to a carbonyl reductase mutant mut-AcCR (E144A/G152L), application thereof and a gene encoding the mutant.

Background

The optically pure chiral alcohol and the derivatives thereof are important chiral intermediates for synthesizing chiral drugs, liquid crystal materials, essences and fragrances and pesticides, and occupy important positions in the fields of medicines and other chemical industries. Chiral alcohols can be synthesized chemically and biologically. The chemical synthesis method generally requires severe conditions such as high temperature and high pressure; a large amount of organic reagents are adopted, so that the environmental pollution is serious; the preparation process is complex, and multiple protection and deprotection steps are often existed; more importantly, the enantioselectivity of the product obtained by the chemical method is low. Compared with chemical methods, biological methods generally react at normal temperature and normal pressure, and have the advantages of mild conditions, low equipment requirements, small environmental pollution, strong substrate specificity, higher stereoselectivity and regioselectivity, high enantiomeric purity of products and the like. The biocatalytic synthesis of chiral alcohols is playing an increasingly important role in the preparation of pharmaceutical intermediates with a strong momentum. Based on the principles of green chemistry and sustainable development, biocatalytic preparation of chiral alcohols is a green and sustainable synthetic route.

Carbonyl reductase is used as a high-efficiency and high-selectivity biocatalyst for asymmetric reduction reaction, and is commonly used for preparing chiral compounds with optical activity. Carbonyl reductases present two very challenging problems in catalyzing asymmetric reduction reactions and limit their further application to asymmetric synthesis reactions. First, many potentially chiral substrates for high-value chiral alcohols have low solubility or are insoluble in the natural medium of the enzyme. This disadvantage can be overcome by using non-aqueous media to increase the solubility of the substrate, such as organic solvents, ionic liquids, supercritical carbon dioxide, and other single or dual phase reaction media for biocatalytic reactions. Part of carbonyl reductase takes the solvents as a medium, and shows higher enzyme activity at high substrate concentration. However, the activity, selectivity and stability of some carbonyl reductases often decreases to varying degrees in nonaqueous media. Therefore, in selecting a carbonyl reductase non-aqueous reaction medium, it is necessary to screen the reaction medium. Secondly, carbonyl reductase has the characteristics of strict substrate specificity, coenzyme dependence, inadaptation to physiological environment and the like, so that carbonyl reductase cannot always effectively complete a specific stereospecific synthesis reaction, and carbonyl reductase often has the defects of low activity, poor selectivity and stability and the like under the environments of high-concentration substrates, high temperature, extreme pH and the like.

With the development of genomics and proteomics, scientists put more attention on protein engineering. Based on directed evolution, rational design and the like, a protein sequence is modified, the structure of the protein sequence is changed, and the catalytic performance of the protein sequence is further influenced, so that the protein sequence has better regioselectivity, stereoselectivity, higher stability and substrate tolerance, and a more effective mode is provided for the synthesis of chiral alcohol. Protein engineering is better applied to enzyme molecule modification, so that enzyme or cell-dominated biocatalysis can be further developed in other aspects such as substrates, media, reactors and the like, and the achievements obtained by protein engineering can be more effectively and continuously applied.

In earlier researches, carbonyl reductase is cloned and expressed from Acetobacter sp.CCTCC M209061. The carbonyl reductase can catalyze various substrates to carry out asymmetric reduction reaction, and has good stereoselectivity. However, the carbonyl reductase has low enzyme activity on aromatic carbonyl compounds such as 4' -chloroacetophenone and the like, has poor tolerance on such substrates, and can achieve a good catalytic effect only when the substrate concentration is low.

Disclosure of Invention

In order to overcome the defects of low enzyme activity and poor substrate tolerance, the invention mainly aims to provide a carbonyl reductase mutant mut-AcCR (E144A/G152L) which has obviously improved activity and substrate tolerance and conforms to a reverse-Prelog rule.

Another objective of the invention is to provide the gene and amino acid sequence of the carbonyl reductase mutant mut-AcCR (E144A/G152L).

Still another object of the present invention is to provide the use of the above carbonyl reductase mutant mut-AcCR (E144A/G152L).

The purpose of the invention is realized by the following technical scheme.

A carbonyl reductase mutant mut-AcCR (E144A/G152L) has an amino acid sequence shown in SEQ ID NO. 1.

The invention also provides a gene for coding the carbonyl reductase mutant mut-AcCR (E144A/G152L), and the gene sequence is shown as SEQ ID NO. 2.

Furthermore, the carbonyl reductase mutant mut-AcCR (E144A/G152L) is obtained by mutating the carbonyl reductase AcCR through enzyme molecules (E144A/G152L).

Further, the gene sequence of AcCR is translated into the amino acid sequence by a standard method, the sequence is searched in a PDB database, the three-level structures of 4RF2, 1ZJY, 1NXQ and 1ZK3 with homology of 53%, 51% and 51% are selected as templates, homologous modeling is carried out, energy minimization is carried out, and the three-level structure model of the carbonyl reductase AcCR is obtained. And then, evaluating the structural rationality of each amino acid residue in the homologous modeling result and the matching degree of the established protein model and the protein amino acid sequence by adopting a Ramachandran Plot (Ramachandran Plot) and Profile-3D. And the established model is determined to be reasonable and can be used for subsequent experimental analysis.

Furthermore, the tertiary structure of the carbonyl reductase AcCR is butted with coenzyme NADH, the mutation hot spot of the carbonyl reductase is predicted through HotSpot 2.0, and 144E and 152G sites are selected as mutation sites.

Further, the change between carbonyl reductase and 4 ' -chloroacetophenone before and after mutation is analyzed through molecular docking, the carbonyl reductase AcCR, the mutant mut-AcCR (E144A/G152L) and the 4 ' -chloroacetophenone are subjected to molecular docking respectively, and the change of the distance between the active sites Ser142 and Tyr155 of the enzyme and the coenzyme NADH nicotinamide ring C4 located between the substrates of the 4 ' -chloroacetophenone and the acting force are analyzed.

Furthermore, the activity of the carbonyl reductase mutant mut-AcCR (E144A/G152L) is measured, the enzymological property of the carbonyl reductase mutant mut-AcCR (E144A/G152L) is measured, and the optimal temperature, pH and stability of the mutant mut-AcCR (E144A/G152L) are researched by the enzyme activity measurement mode.

The invention also provides application of the carbonyl reductase mutant mut-AcCR (E144A/G152L) in catalyzing asymmetric reduction of carbonyl compounds.

Compared with the prior art, the invention has the following advantages and beneficial effects: the carbonyl reductase mutant mut-AcCR (E144A/G152L) provided by the invention overcomes the defects of low enzyme activity and poor substrate tolerance of the original carbonyl reductase and has higher enzyme activity and substrate tolerance.

Drawings

FIG. 1a and FIG. 1b are graphs showing the results of the coupling of AcCR and mut-E144A/G152L with 4' -chloroacetophenone.

FIGS. 2a and 2b are graphs comparing the effect of temperature on the activity and stability of mutant mut-E144A/G152L;

FIGS. 3a and 3b are graphs comparing the effect of buffer pH on the activity and stability of mutant mut-E144A/G152L.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto. It is noted that those skilled in the art can implement or understand the following embodiments without specific details, with reference to the prior art.

Example 1

Translating the gene sequence of AcCR into an amino acid sequence by a standard method, searching the sequence in a PDB database, selecting three-level structures of 4RF2, 1ZJY, 1NXQ and 1ZK3 (the names of protein sequences in the database) with homology of 53%, 51% and 51% as templates, carrying out homologous modeling, and carrying out energy minimization to obtain a three-level structure model of the AcCR. Further using Ramachandran Plot (Ramachandran Plot) and Profile-3D to evaluate the structural reasonability of each amino acid residue in the homology modeling result and the matching degree of the created protein model and the protein amino acid sequence. And the established model is determined to be reasonable and can be used for subsequent experimental analysis. The carbonyl reductase AcCR tertiary structure is butted with coenzyme NADH, the mutation hot spot of the carbonyl reductase is predicted through HotSpot 2.0, and 144E and 152G sites are selected as mutation sites.

Example 2

Analyzing the change between carbonyl reductase and 4' -chloroacetophenone before and after mutation by molecular docking; respectively docking carbonyl reductase AcCR, mutant mut-AcCR (E144A/G152L) and 4' -chloroacetophenone, and analyzing the positions of enzyme active sites Ser142, Tyr155 and coenzyme NADH nicotinamide ring C4 in substratesThe distance between 4' -acetophenones and the force change. The results are shown in FIG. 1a and FIG. 1b, which show the results of the coupling of AcCR and mut-E144A/G152L with 4' -chloroacetophenone. From FIGS. 1a and 1b, it can be seen that the distances between the hydrogen atoms at the catalytic sites S142 and Y155 and the position 4 of the NADH nicotinamide ring and the carbonyl oxygen atom of 4' -chloroacetophenone are reduced significantly after mutation, respectively

(22.4％)、

And

(5.2%), after mutation, the steric hindrance of the substrate and the active center of the enzyme is reduced, and the contact of the enzyme and the active site is facilitated. In addition, Ala and Leu are both hydrophobic amino acids, and they can replace hydrophilic amino acids near the active center of the enzyme molecule, so as to increase the hydrophobicity of the active site of the enzyme molecule, and make the hydrophobic ClPE enter the active center of the enzyme molecule more easily. The activity of mut-E144A/G152L on 4' -chloroacetophenone enzyme is 92.7U/mg, which is 17.9 times of that of AcCR enzyme before mutation (5.17U/mg).

Example 3

Plasmid pGEX-mut-E144A/G152L containing mutant mut-AcCR (E144A/G152L) gene was obtained by performing pGEX-AcCR whole plasmid amplification using PrimeSTAR Max DNA Polymerase.

Primers used for site-directed mutagenesis: the mutation Primer at position E144A is Primer 1: 5'-ATCTGTCTTCCATTGCCGGACTGAT-3', respectively;

primer 2: 5'-ATCAGTCCGGCAATGGAAGACAGAT-3', respectively; the mutant primer at the position G152L is

Primer3:5'-ACCCAATGTTGGCCGCCTATAAC-3'；

Primer 4：5'-GTTATAGGCGGCCAACATTGGGT-3'。

The PCR amplification system and reaction conditions adopted by site-directed mutagenesis are as follows:

polymerase Chain Reaction (PCR) amplification system

And (3) PCR reaction conditions:

after the reaction is finished, the reaction product is treated by restriction enzyme DpnI and acts on Gm6A ^ TC sites to digest the template plasmid in the system. The reaction system is as follows:

and (3) placing the prepared enzyme digestion reaction system at 37 ℃ for incubation for 15min to complete the enzyme digestion process of the template plasmid. The enzyme digestion product is directly transformed into Escherichia coli DH5 alpha. And (3) verifying positive transformants by colony PCR, extracting plasmids and sequencing.

In the invention, a recombinant plasmid pGEX-mut-E144A/G152L with correct sequencing is extracted from a recombinant strain DH5 alpha (pGEX-mut-E144A/G152L) and is transformed into an escherichia coli expression strain BL21(DE3) to obtain a recombinant expression strain BL21(DE3) (pGEX-mut-E144A/G152L).

Example 4

Selecting positive transformant, culturing in 4mL LB culture medium containing 100. mu.g/mL ampicillin overnight at 37 deg.C and 180r/min, transferring 1mL culture medium into 50mL LB culture medium containing 100. mu.g/mL ampicillin, culturing at 37 deg.C and 180r/min to OD₆₀₀And (3) adjusting the culture temperature to 20 ℃, adding IPTG with the final concentration of 0.4mmol/L, culturing for 15-18h, and centrifuging at 4 ℃ and 8000r/min for 5min to collect thalli. Centrifuging at 4 deg.C and 8000r/min for 5min, removing supernatant, and collecting thallusAfter washing three times with 0.85% physiological saline, the cells were weighed, uniformly dispersed in phosphate buffer (50mmol/L, pH6.5) to prepare a 30mg/mL cell suspension, and placed at 4 ℃ for use. And (3) crushing cells by using an ultrasonic crusher, setting the power of the ultrasonic crusher to be 350W, working for 3s, and pausing for 5s, setting the ultrasonic time to be 20min, and keeping the cell suspension in an ice-water bath all the time in the whole crushing process to keep a low-temperature environment so as to prevent the enzyme from being denatured and inactivated due to overhigh temperature. After the ultrasonic crushing is finished, the crushed suspension is placed in a centrifuge and centrifuged for 30min at the temperature of 4 ℃ and the speed of 8000r/min, and the obtained supernatant is the crude enzyme extracting solution of the recombinant carbonyl reductase.

Adopts Bio-Rad NGC Quest ^TM10, purifying the crude enzyme liquid of the recombinant carbonyl reductase by a high-pressure chromatography system. The extracted crude enzyme solution is filtered by a 0.22 mu m filter membrane and then placed at 4 ℃ for standby. The general steps of purification are: 5 column volumes of Buffer A (4.3mM Na)₂HPO₄，1.47mM KH₂PO₄137mM NaCl, 2.7mM KCl, pH7.3) pre-equilibrated Bio-Scale Mini definition GST pre-column (5mL, protein loading 500mg) at a flow rate of 5 mL/min; after the pre-equilibrium is finished, starting to load the sample, and loading the sample by adopting a pump at the flow rate of 1mL/min and the loading amount of about 500mg of protein; after the sample loading is finished, the column is balanced by Buffer A until the baseline is equivalent to the baseline in the pre-balancing process; then, Buffer B (4.3mmol/L Na)₂HPO₄，1.47mM KH₂PO₄637mM NaCl, 2.7mM KCl, pH7.3) to remove the more robust contaminating proteins; and finally, eluting the recombinant protein AcCR by using an elution Buffer solution Buffer C (50mM Tris-HCl,2.5g/L glutathione, pH 8.0), wherein the eluted solution is the separated and purified single recombinant protein AcCR. And (3) placing the eluent containing the recombinant protein AcCR into a dialysis bag, concentrating the eluent at the temperature of 4 ℃ by using polyethylene glycol 20000, and storing the concentrated recombinase solution at the temperature of 4 ℃.

Determination conditions of the reducing activity of the recombinant carbonyl reductase AcCR: 0.25mmol/L NADH, 2mL phosphate buffer (50mmol/L, pH6.5), 20 mmol/L4' -chloroacetophenone, incubating for 5min at 35 ℃, adding a proper amount of enzyme solution, and detecting the change of the absorbance value of the reaction system within 3min under the ultraviolet wavelength of 340 nm.

Definition of enzyme activity: under the above conditions, the enzyme activity for catalytic oxidation of 1. mu. mol NADH per minute is 1 enzyme activity unit, denoted by U.

The calculation formula of the enzyme activity is as follows:

enzyme activity (U) ═ EW × V × 10³/(6220×1)

EW: the variation of the absorbance value at 340nm within 1 min; v: total volume of reaction solution, mL; 6220: molar extinction coefficient, L/mol/cm; 1: optical path distance, cm.

The results of the enzyme activity assay are shown in Table 1 for the active fraction of mutant mut-AcCR (E144A/G152L) on 4' -chloroacetophenone.

Example 5

The optimum temperature and pH and stability of the mutant mut-AcCR (E144A/G152L) were investigated by means of enzyme activity assays. The optimal reaction temperature of the recombinant carbonyl reductase AcCR is determined by measuring the enzyme activity of the enzyme at different temperatures, namely measuring the enzyme activity of the recombinant carbonyl reductase AcCR by a reference standard method at the temperature of 25, 30, 35, 40 and 45 ℃. The thermal stability of the enzyme is measured, namely the enzyme is placed at different temperatures (25-45 ℃) and incubated for 36h, samples are taken at regular time, and the enzyme activity of the enzyme at different time points is measured according to a standard method. The activity of the enzyme before incubation was set at 100% relative enzyme activity. Two reactions were set up in parallel for each temperature. The results are shown in FIG. 2a for the effect of temperature on the activity and stability of mutant mut-E144A/G152L (FIG. 2 b). As can be seen from FIGS. 2a and 2b, the optimum reaction temperature of the mutant was 35 ℃ and was consistent with that of the recombinant AcCR before mutation; the relative activity of the mutant is kept above 78% in the temperature range (25-45 ℃) studied, and particularly when the temperature is higher than 35 ℃, the relative activity of the mutant is between 80% and 92% when the temperature is 40-45 ℃, and the relative activity is obviously better than that of the non-mutated AcCR (70-90%). The research on the heat stability of the mutant enzyme shows that the lower the temperature is in the temperature range to be researched and the higher the heat stability is, the better the relative enzyme activity of the mutant mut-E144A/G152L is.

When the incubation temperature is 40-45 ℃, the mutant mutE144A/G152L protein is accelerated in denaturation speed, the residual activity is obviously reduced, and the stability is reduced along with the increase of the temperature and the extension of the incubation time. When the incubation temperature is within the range of 25-35 ℃, the incubation time is 12 hours, and the relative enzyme activity can be more than 80%; after incubation for 24h at 25 ℃, the relative enzyme activity is still 75%, and after 36h, the relative activity is still about 60%. Compared with the non-mutated AcCR, the mutant has improved relative enzyme activity and heat stability at 40-45 ℃. As a result of the docking of binding molecules, the enhanced hydrogen bonding of the substrate to the enzyme molecule may be responsible for its increased relative enzyme activity over the temperature range studied. In addition, Glu in the enzyme molecule is mutated into Leu, the hydrophobic side chain of Leu increases the hydrophobic combination of residues in the enzyme molecule, and the internal hydrophobic effect of the enzyme molecule is enhanced, so that the protein structure is more stable.

The optimum reaction pH of the recombinant carbonyl reductase AcCR is determined by measuring the enzyme activity of the enzyme at different pH values, namely the enzyme activity of the recombinant carbonyl reductase AcCR is measured by referring to a standard method when the pH value is 5.0 to 8.0 respectively. The pH stability of the enzyme is measured, namely the enzyme is incubated for 96 hours at 4 ℃ in different pH ranges (6.0-8.0), samples are taken at regular time, and the enzyme activity of the enzyme at different time points is measured according to a standard method. The relative enzyme activity of the enzyme before incubation was set at 100%. The buffers used in different pH ranges are respectively citric acid-phosphate buffer (pH 4.5-8.0), Tris-HCl buffer (pH 8.0-8.5), and glycine-sodium hydroxide buffer (pH 8.5-9.5). Two for each pH setting. Results the effect of buffer pH on the activity (FIG. 3a) and stability (FIG. 3b) of mutant mut-E144A/G152L. As can be seen from FIGS. 3a and 3b, before and after mutation, the optimum pH of AcCR was not changed and remained at 6.5, the pH of the buffer was in the range of 6.0-8.0, and the relative activity of mutant mut-E144A/G152L was above 80%, which has higher catalytic activity. The stability of the enzyme activity in the pH range of more than 80% is studied, and the enzyme activity is found to be relatively stable in the pH range of 6.0-8.0, particularly the pH is 6.5-7.5, about 70% of the enzyme activity still remains after 96h, and the pH stability is good.

Example 7

Carbonyl reductase mutant mut-AcCR (E144A/G152L) catalyzes the activity assay of different substrates: adding 2mL of phosphate buffer solution (100mmol/L, pH6.5) containing different substrates (20mmol/L) into corresponding 10mL triangular flasks with stoppers which are marked respectively, incubating for 10min at 35 ℃ in duplicate, then determining the enzyme activity of the enzyme to the different substrates by referring to a standard method, and taking the enzyme activity of the enzyme to 4' -chloroacetophenone as 100% relative enzyme activity of the enzyme. The results are shown in Table 1 for comparison of the activity of the carbonyl reductases before and after mutation for different substrates. The mutant mut-E144A/G152L has improved activity to 4 '-acetophenone, and has improved activity to 4' -substituted acetophenone.

TABLE 1

TABLE 2

e.e: enantiomeric excess value

Carbonyl reductase mutant mut-AcCR (E144A/G152L) catalyzes yield and enantioselective determination of different substrates: 2mL of phosphate buffer (100mmol/L, pH6.5) containing the different substrates (200mmol/L), 0.1mmol/L NADH and 400mmol/L isopropanol were added in duplicate to the respective 10mL stoppered flasks which had been labeled. The unmutated carbonyl reductase AcCR catalyzes the determination of the yield of the different substrates, 2mL of phosphate buffer (100mmol/L, pH6.5) containing the different substrates (50mmol/L), 0.1mmol/L NADH and 150mmol/L isopropanol are added in duplicate to the respective 10mL stoppered flasks which have been labeled. After incubation at 35 ℃ for 10min, a certain amount of AcCR mutant was obtained. The reaction flask was placed in a 35 ℃ gas bath thermostat shaker for reaction (200rpm) and 25. mu.L of sample was periodically taken for GC or HPLC analysis. The results are shown in Table 2 for the carbonyl reductase mutant mut-AcCR (E144A/G152L) catalyzing the asymmetric reduction of a potentially chiral carbonyl compound. The results show that the mutant enzyme has significantly improved tolerance to the substrate and catalytic reaction. The yield of (R) -3' -methoxyacetophenone at the substrate concentration of 200mmol/L is improved from 61.6 percent (50mmol/L substrate) before mutation to 87.05 percent (200mmol/L substrate), which is improved by nearly 26 percent; the yield of (R) -4' -methoxyphenethanol was increased to 79.5% compared to 29% when the reaction was catalyzed by the unmutated enzyme.

Sequence listing

<110> university of southern China's science

<120> carbonyl reductase mutant mut-AcCR (E144A/G152L) and application and encoding gene thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 253

<212> PRT

<213> carbonyl reductase 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 reductase 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 carbonyl reductase mutant mut-AcCR (E144A/G152L), characterized in that: the amino acid sequence is shown in SEQ ID NO. 1.

2. A gene encoding the carbonyl reductase mutant mut-AcCR (E144A/G152L) of claim 1, characterized in that: the gene sequence is shown in SEQ ID NO. 2.

3. The carbonyl reductase mutant mut-AcCR (E144A/G152L) of claim 1, wherein: carbonyl reductase mutant mut-AcCR (E144A/G152L) was obtained from the carbonylation reductase AcCR by means of enzymatic molecular mutagenesis.

4. Use of a carbonyl reductase mutant mut-AcCR (E144A/G152L) as claimed in claim 1 in the asymmetric reduction of carbonyl compounds.