CN113215122B - Carbonyl reductase mutant and coding gene and application thereof - Google Patents
Carbonyl reductase mutant and coding gene and application thereof Download PDFInfo
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- CN113215122B CN113215122B CN202110650597.4A CN202110650597A CN113215122B CN 113215122 B CN113215122 B CN 113215122B CN 202110650597 A CN202110650597 A CN 202110650597A CN 113215122 B CN113215122 B CN 113215122B
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- 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
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/001—Amines; Imines
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- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01184—Carbonyl reductase (NADPH) (1.1.1.184)
Abstract
The invention relates to a carbonyl reductase mutant and a coding gene and application thereof, wherein the amino acid sequence of the carbonyl reductase mutant is shown as SEQ ID NO.3, and the nucleotide sequence for coding the carbonyl reductase mutant is shown as SEQ ID NO. 4; wherein, the carbonyl reductase mutant coded by SEQ ID NO.4 has high catalytic activity and good heat resistance under the acidic condition, and can be applied to the biosynthesis for preparing adrenaline and analogues thereof; compared with a chemical synthesis method, the carbonyl reduction reaction catalyzed by the method is simple and mild, no waste is discharged, the reaction selectivity is high, the preparation cost is low, and the method has a good application prospect.
Description
Technical Field
The invention relates to the field of biotransformation, in particular to a carbonyl reductase mutant for preparing epinephrine and analogues thereof and a coding gene thereof.
Background
The biotransformation technique is a technique of performing substance transformation using microbial cells or enzymes as a catalyst. The method has the advantages of mild conditions, few side reactions, strong selectivity, low energy consumption, environmental friendliness and the like, and is widely applied to the fields of green chemistry and medicines. The enzyme is used as a common biocatalyst and has a significant effect in the biocatalysis process.
Redox reactions are an important class of chemical reactions. The reaction in which an unsaturated carbonyl compound and its derivative are reduced to a chiral alcohol compound is a very important reaction in medicinal chemistry and organic synthesis. At present, the chiral reduction of unsaturated carbonyl compounds is mainly carried out by chemical methods. The reduction by a chemical method usually needs Pb/C, pure hydrogen and chiral resolution, so that the method is high in cost, complicated and time-consuming in operation and easy to cause environmental pollution.
Carbonyl reductase (also called ketoreductase) and alcohol dehydrogenase, widely exist in animal, plant and microbial cells, are proteins using NAD, NADP, NADH or NADPH as coenzymes, can catalyze both carbonyl reduction and hydroxyl oxidation, and have wide substrate specificity. Carbonyl reductases are further classified into short chain (SDRs), medium chain dehydrogenase/reductase families (MDRs), and aldehyde ketone reductase families (AKRs) according to their typical sequence and structural composition characteristics. In addition, the carbonyl reductase may also have NAD (P) H as a coenzyme, catalyzing the reduction of the carbonyl double bond.
Among them, epinephrine and its analogs (chemical structure is shown in formula a) have important physiological functions, and they can regulate blood pressure, heart rate, heart force, gastrointestinal motility and bronchial smooth tension of human body. The medicine is mainly used for treating bronchial asthma, cardiovascular diseases and other diseases, and has large market capacity. Specifically, in the synthetic route of the epinephrine analog (the compound shown in the formula b), the last step is a process of catalytically hydrogenating carbonyl groups to hydroxyl groups through palladium-carbon, a palladium catalyst used in the process is expensive, the safety of the process is poor, the generated product is a racemic product, a multi-step resolution process is needed, and the total yield is low.
In addition to chemical synthesis, synthesis of adrenal hormones and their analogues can also be catalyzed in the aqueous phase with biological enzymes. However, since the solubility of the starting material in the aqueous phase is low at neutral and alkaline pH, the catalytic efficiency of the enzyme is low at low pH values and the half-life is short. Therefore, there is a need for improvement of the existing carbonyl reductase to improve its catalytic activity and/or stability under acidic conditions, thereby improving the problems of high production cost of adrenal hormone and its analogues in the prior art.
Disclosure of Invention
Aiming at solving the problems of high production cost and high post-treatment difficulty in the process of chemically synthesizing adrenaline and analogues thereof (namely, catalyzing and reducing the compounds shown in the formula I); the invention also provides a method for preparing the carbonyl reductase mutant, and solves the problems of low catalytic activity and poor stability of the biological catalytic enzyme under an acidic condition.
The technical scheme adopted by the invention for solving the technical problems is as follows: a carbonyl reductase mutant gene, the nucleotide sequence of which is shown in SEQ ID NO. 4; the amino acid sequence of the carbonyl reductase mutant coded by the gene is as follows: shown as SEQ ID NO. 3.
The carbonyl reductase mutant gene provided by the invention is derived from a wild type gene of Agrobacterium radiobacter. The gene sequence of the wild type gene subjected to codon optimization aiming at escherichia coli is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 1. Wherein "wild type" refers to a form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that is present in an organism, can be isolated from a source in nature, and has not been intentionally modified by man. The enzyme obtained after the gene expression has low catalytic activity and poor thermal stability for certain substrates.
The invention also provides a recombinant expression vector of the carbonyl reductase mutant gene, which can be constructed by connecting the nucleotide sequence containing the carbonyl reductase mutant gene to various prokaryotic expression vectors or eukaryotic expression vectors by a conventional method in the field. Prokaryotic expression vectors such as pGEX, pMAL, pET series, and eukaryotic expression vectors, more preferably selected from pET series. The plasmid vector used in the invention is pET-24 a.
The invention also provides a genetic engineering bacterium for producing the carbonyl reductase mutant, and the genetic engineering bacterium comprises the carbonyl reductase mutant gene or the recombinant expression vector. The host cell of the genetic engineering bacterium is preferably Escherichia coli (Escherichia coli) BL21(DE 3).
The carbonyl reductase mutant gene, the recombinant expression vector and the genetic engineering bacteria can be applied to preparation of the carbonyl reductase mutant.
Specifically, the invention also provides a method for preparing the carbonyl reductase mutant, which comprises the steps of fermenting and culturing the genetic engineering bacteria, and collecting and preparing the recombinant carbonyl reductase.
The method comprises the step of industrially preparing the carbonyl reductase mutant under the fermentation condition of a certain production tank; the fermentation conditions of the production tank are preferably as follows: more than 30% of DO, and the air flow is 1: 1-2 vvm.
The carbonyl reductase mutant can be applied to catalytic reduction of carbonyl compounds to prepare optical chiral alcohol. Wherein the structural general formula of the carbonyl compound is shown as I:
wherein: r is H, CH3，CH3CH2Or (CH)3)2CH。
Compared with the prior art, the invention has the following advantages and effects:
1. the carbonyl reductase mutant target gene with high activity is obtained by rational design (individual amino acid in protein molecules is changed by site-directed mutagenesis or other methods on the basis of understanding the space structure of the protein) and methods such as overlapping extension PCR, recombination PCR, large primer PCR, circular plasmid PCR and the like, and the nucleotide sequence of the carbonyl reductase mutant target gene is SEQ ID NO.4 on the basis of combining high-throughput enzyme activity screening.
2. The carbonyl reductase mutant coded by SEQ ID NO.4 has higher catalytic activity on the substrate shown in the formula I; when R in the formula I is methyl, the enzyme activity of the carbonyl reductase mutant is 15U/mg protein; the enzyme activity of the wild enzyme on the substrate is less than 1; furthermore, the half-life of this enzyme mutant was greater than 48 hours at 40 ℃ whereas the half-life of the wild-type enzyme was only 16 hours under the same conditions.
3. The carbonyl reductase mutant provided by the invention has high catalytic activity and good heat resistance under acidic conditions, and can be applied to biosynthesis for preparing epinephrine and analogues thereof; compared with a chemical synthesis method, the carbonyl reduction reaction catalyzed by the method is simple and mild, no waste is discharged, the reaction selectivity is high, the preparation cost is low, and the method has a good application prospect.
Detailed Description
The present invention will be described in further detail with reference to examples, which are illustrative of the present invention and are not to be construed as being limited thereto.
Example 1: establishment of wild carbonyl reductase gene engineering bacteria
The sequence of Agrobacterium radiobacter carbonyl reductase wild type gene sequence (UNIPROT ID: B9JM87) recorded by NCBI is optimized, then a whole gene fragment (the nucleotide sequence is shown in SEQ ID NO. 2) is synthesized manually, the gene is inserted into pET-24a plasmid by NdeI and BamHI endonucleases through a gene synthesis company, and the connected vector is transferred into Escherichia coli BL21(DE3) to establish wild type carbonyl reductase gene engineering bacteria.
Example 2: carbonyl reductase mutant and acquisition of target gene (nucleotide sequence is shown as SEQ ID NO. 4)
A carbonyl reductase mutant, the amino acid sequence of which is the mutated amino acid sequence of the wild-type carbonyl reductase shown in SEQ ID NO. 1. Wherein the amino acid sequence of the carbonyl reductase mutant has at least 1 mutation site in the amino acid sequence shown in SEQ ID NO. 1: 42 th, 65 th, 66 th, 75 th, 110 th, 176 th and 223 th bits. And the 42 th L mutation is S, A; the 65 th V mutation is G; the R mutation at the 66 th site is A; m at position 75 is mutated to V; the 110 th F mutation is A; the 176 th A is mutated into S; the M223 th mutation is Q. The amino acid sequence of the carbonyl reductase mutant has a mutation site in the mutated amino acid sequence, and has more than 90% homology with the mutated amino acid sequence.
Specifically, the gene library of the carbonyl reductase mutant is created by the following method:
obtaining a three-dimensional structure through an online protein structure prediction tool, obtaining the three-dimensional structure (3WDS, homology 68.34%) of carbonyl reductase with the highest structural similarity through PDB, performing structural alignment, and then performing substrate I (R is CH) through Docking3) Simulation of binding to the three-dimensional structure of carbonyl reductase protein, and finally selection of amino acids that are likely to be involved in binding to substrate and NAD and involved in NAD proton transfer as mutant amino acids by Pymol analysis.
On the other hand, the carbonyl reductase was protein engineered using an error-prone PCR random mutagenesis approach. Error-prone PCR carries out target gene amplification through DNA polymerase, and mutation frequency in the amplification process is changed by adjusting reaction conditions (including increasing magnesium ion concentration, adding manganese ions, changing dNTP concentration in a system or applying low-fidelity DNA polymerase and the like), so that mutation is randomly introduced into a target gene at a certain frequency, and a random mutant of a protein molecule is obtained.
This example uses lower fidelity Taq polymerase with Mn2+Substitute for natural cofactor Mg2+Increasing the error-prone probability.
The 50 μ L PCR system was as follows:
sterile double distilled water was added to 50. mu.L. In the mutation of the carbonyl reductase gene, primers on the pET24a plasmid were used, and the primers were located upstream and downstream of the carbonyl reductase gene, respectively.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 50-65 ℃ for 40s and extension at 72 ℃ for 40s for 35 cycles; the extension was continued at 72 ℃ for 10min and cooled to 4 ℃.
The carbonyl reductase gene was PCR amplified and inserted into pET24a plasmid as a gene mutation template according to the above method; amplifying the gene of the carbonyl reductase by error-prone PCR, linking the amplified gene fragment to a pET24a vector, and transferring the connected vector into escherichia coli BL21(DE3) to establish a carbonyl reductase gene mutation library;
escherichia coli BL21(DE3) is used as a host, pET24a plasmid is used as a vector, carbonyl reductase of expansion mutation is expressed, and high-activity mutant strains are screened at high flux by the enzyme activity detection method described in example 5. The mutated high-activity carbonyl reductase is subjected to gene identification, the nucleotide sequence of the mutated high-activity carbonyl reductase is shown as SEQ ID NO.4, and the amino acid sequence of the protein coded by the gene is shown as SEQ ID NO. 3.
Example 3: small-scale production of carbonyl reductase mutants in shake flasks
A single colony of E.coli containing a plasmid of the gene of the carbonyl reductase mutant of interest (i.e., the highly active carbonyl reductase DNA molecule selected in example 2) was inoculated into 100mL of LB medium (10 g/L peptone, 5g/L yeast extract, 10g/L NaCl, pH7.2) containing kanamycin sulfate (100. mu.g/mL). Coli were grown in a shaker at 37 ℃ with shaking at 250rpm and incubated for 16 hours. Switching is carried out according to the proportion of 1: 100, 1mL of the cell culture solution was put into 100mL of LB medium containing kanamycin sulfate, and subjected to shaking culture under the same conditions, and the absorbance of the bacterial solution at 600nm was measured at regular intervals to monitor the cell growth density. When OD of culture600Is 0.6 to 0.8, the expression of the carbonyl reductase gene is induced by adding isopropyl beta-D-thiogalactoside (IPTG) to a final concentration of 1mM, and then culturing is continued overnight (10-16 hours). The cells were collected by centrifugation (10000rpm, 10min, 4 ℃) and the supernatant was discarded. Resuspending the cell pellet at 4 ℃ in 100mM citrate-sodium citrate buffer pH6.0, resuspendedThe bacterial sludge concentration is 200 g/L. After sonication, cell debris was removed by centrifugation (13000rpm, 30min, 4 ℃). The clear lysate supernatant was collected to make a crude enzyme solution, which was stored at-20 ℃.
Example 4: fermentative production of carbonyl reductase mutants
The fermentation method comprises the following steps: a single colony of E.coli containing a plasmid carrying the gene for the carbonyl reductase mutant of interest was inoculated into 120mL of LB medium (peptone 10g/L, yeast extract 5g/L, NaCl10g/L, pH7.2) containing kanamycin sulfate (100 mg/mL). Coli were grown overnight (10-16 hours) at 37 ℃ in a shaker with shaking at 250 rpm. Adding the seed solution into a 15L fermentation tank containing 6L fermentation medium according to the inoculation amount of 2%, maintaining the pH value of the fermentation solution at 7.0-7.2 by adding ammonia water, keeping the temperature of the tank at 37 ℃, controlling the stirring speed at 300-900rpm, controlling the dissolved oxygen at about 30% during the fermentation process, and controlling the air flow at 1: 1-2 vvm, culturing for 8 hours, adding IPTG with the final concentration of 1mmol/L to induce the expression of carbonyl reductase, and then continuing to ferment for 12-16 hours at the tank temperature of 22 ℃. The growth of the culture was maintained during the fermentation by adding a feed solution containing 200g/L glucose, 100g/L yeast extract, pH 7.2. And (3) directly homogenizing and crushing the culture by using a high-pressure homogenizer after the fermentation is finished, and preparing and storing a carbonyl reductase mutant crude enzyme solution at the temperature of-20 ℃ after centrifuging, filtering and ultrafiltering and concentrating.
Example 5: determination of carbonyl reductase enzyme Activity
The carbonyl reductase enzyme activity determination system comprises the following steps:
citric acid-sodium citrate buffer solution (100mM, pH 6.0)
A substrate of formula I: 3mM
Wherein: r is selected from H and CH3，CH3CH2Or (CH)3)2CH；
NADH 3mM
The volume of the mixture was 270. mu.L in total, 30. mu.L (300. mu.L in total) of the crude enzyme solution of carbonyl reductase or a diluted solution thereof was added to the mixture, and the mixture was added to a 96-well plate and the absorbance at 340nm was measured with a microplate reader at 40 ℃.
Definition of enzyme activity: under the above conditions, the amount of enzyme required to catalytically consume 1. mu. mol of NADH per minute was defined as 1 enzyme activity unit.
When R is CH3Then, the enzyme activity of the carbonyl reductase mutant with the amino acid sequence shown as SEQ ID NO.3 is 15U/mg. The enzyme activity of the wild carbonyl reductase on the substrate is less than 1 before mutation.
And (4) conclusion: compared with wild carbonyl reductase, the carbonyl reductase mutant with the amino acid sequence shown in SEQ ID NO.3 has higher catalytic reduction activity on carbonyl compounds (epinephrine and derivatives thereof) shown in the formula I.
Example 6: effect of temperature on carbonyl reductase stability
20ml of crude enzyme solution of wild carbonyl reductase and crude enzyme solution of carbonyl reductase mutant prepared in example 3 or example 4 were added to 100mM phosphate buffer solutions with pH7.5 at different temperatures (20-60 ℃ C., 10 ℃ C. apart), and the solutions were incubated for 4, 8, 12, 16, 24, and 48 hours, and then cooled in an ice bath, and the residual enzyme activity was measured.
The time when the residual enzyme activity is about 50 percent of the original enzyme activity is the half-life period, thereby determining the temperature stability of the carbonyl reductase mutant. The carbonyl reductase mutant detects the enzyme activity by using the system in the embodiment 5, and the test substrate of the wild carbonyl reductase is shown as a formula II.
The half-lives of the wild-type carbonyl reductase and carbonyl reductase mutant at different temperatures are given in table 1.
TABLE 1 half-lives of wild carbonyl reductases and carbonyl reductase mutants at different temperatures
| Temperature of heat preservation | Wild carbonyl reductase | Carbonyl reductase mutant |
| 20℃ | ＞48h | ＞48h |
| 30℃ | 24-48h | ＞48h |
| 40℃ | 16h | ＞48h |
| 50℃ | ＜4h | 8h |
| 60℃ | ＜4h | 4h |
From table 1, it follows: the half life of the wild carbonyl reductase at 40 ℃ is about 16 h; the half-life period of the carbonyl reductase mutant at 40 ℃ is more than 48h, and the thermal stability is higher.
In addition, it should be noted that the specific embodiments described in the present specification may differ in the shape of the components, the names of the components, and the like. All equivalent or simple changes of the structure, the characteristics and the principle of the invention which are described in the patent conception of the invention are included in the protection scope of the patent of the invention. Various modifications, additions and substitutions for the specific embodiments described may be made by those skilled in the art without departing from the scope of the invention as defined in the accompanying claims.
Sequence listing
<110> Tianjin Fa Mo xi biomedical science and technology Co., Ltd
<120> carbonyl reductase mutant and coding gene and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 261
<212> PRT
<213> wild-type carbonyl reductase amino acid sequence SEQ ID NO.1(Agrobacterium radiobacter)
<400> 1
Met Glu Ala Ser Leu Ser Glu Phe Ala Gly Lys Ser Val Val Val Thr
1 5 10 15
Gly Gly Ala Ser Gly Ile Gly Ala Ala Ile Thr Arg Thr Phe His Ala
20 25 30
Glu Gly Ala Arg Val Thr Ile Leu Asp Leu Asp Ala Gly Arg Ala Ala
35 40 45
Ala Leu Ala Asp Glu Leu Gly Asp Asn Ala Phe Ser Gly Gly Ile Asp
50 55 60
Val Arg Asp Arg Gly Phe Val Gln Ala Ala Met Asp Ala Val Ile Ser
65 70 75 80
Gly Gln Gly Gly Ile Asp Ile Leu Cys Ala Asn Ala Gly Val Ser Thr
85 90 95
Met Gln Ala Ser Val Asp Leu Thr Asp Glu Asp Trp Asp Phe Asn Met
100 105 110
Asp Val Asn Ala Lys Gly Val Phe Leu Cys Asn Gln Ile Val Val Arg
115 120 125
His Phe Leu Ala Thr Gly Asn Lys Gly Val Ile Val Asn Thr Ala Ser
130 135 140
Leu Ala Gly Lys Val Gly Ala Pro Leu Leu Ala His Tyr Ser Ala Ser
145 150 155 160
Lys Phe Ala Val Leu Gly Trp Thr Gln Ala Leu Ala Arg Glu Leu Ala
165 170 175
Pro Thr Gly Ile Arg Val Asn Ala Val Cys Pro Gly Phe Val Arg Thr
180 185 190
Gly Met Gln Glu Arg Glu Ile Ile Trp Glu Gly Lys Leu Arg Asn Met
195 200 205
Thr Pro Asp Glu Val Arg Gln Glu Tyr Val Thr Leu Thr Pro Met Gly
210 215 220
Arg Ile Glu Glu Pro Glu Asp Val Ala Val Val Val Arg Phe Leu Ala
225 230 235 240
Ser Asp Gly Ala Arg Phe Met Thr Gly Gln Gly Ile Asn Val Thr Gly
245 250 255
Gly Val Arg Met Asp
260
<210> 2
<211> 783
<212> DNA
<213> wild type carbonyl reductase nucleotide sequence SEQ ID NO.2(Agrobacterium radiobacter)
<400> 2
atggaagcgt ctctgtctga attcgcaggt aaatctgttg ttgttaccgg tggtgcgtcc 60
ggtatcggcg cagctatcac ccgtactttt cacgcggaag gtgcacgcgt tactattctg 120
gacctggatg ctggtcgtgc cgcagcgctg gcagacgaac tgggtgacaa cgcgttttcc 180
ggcggcatcg acgttcgtga tcgtggcttc gttcaggctg caatggacgc ggttatctcc 240
ggccagggcg gtatcgacat cctgtgcgcc aacgcgggcg tatccaccat gcaagccagc 300
gtcgacctga ccgacgaaga ctgggatttc aatatggacg tcaacgcgaa aggtgtattc 360
ctgtgcaacc aaatcgttgt ccgtcacttt ctggctaccg gtaacaaagg cgtgattgtg 420
aacactgcgt ccctggcggg taaagtcggc gcaccgctgc tggcacacta tagcgcgtcc 480
aaattcgccg ttctgggttg gactcaggct ctggctcgtg aactggcgcc aaccggcatc 540
cgtgtcaacg ccgtttgccc gggcttcgtg cgtaccggca tgcaggaacg cgaaattatt 600
tgggagggta aactgcgtaa catgacccct gatgaagtac gtcaggaata cgttactctg 660
accccgatgg gtcgtatcga agaaccggaa gacgtggcgg ttgtggtccg cttcctggca 720
tccgatggcg cgcgtttcat gacgggtcag ggcattaacg tcaccggtgg tgttcgtatg 780
gat 783
<210> 3
<211> 261
<212> PRT
<213> carbonyl reductase mutant amino acid sequence SEQ ID NO.3(carbonyl reductase)
<400> 3
Met Glu Ala Ser Leu Ser Glu Phe Ala Gly Lys Ser Val Val Val Thr
1 5 10 15
Gly Gly Ala Ser Gly Ile Gly Ala Ala Ile Thr Arg Thr Phe His Ala
20 25 30
Glu Gly Ala Arg Val Thr Ile Leu Asp Ser Asp Ala Gly Arg Ala Ala
35 40 45
Ala Leu Ala Asp Glu Leu Gly Asp Asn Ala Phe Ser Gly Gly Ile Asp
50 55 60
Gly Ala Asp Arg Gly Phe Val Gln Ala Ala Val Asp Ala Val Ile Ser
65 70 75 80
Gly Gln Gly Gly Ile Asp Ile Leu Cys Ala Asn Ala Gly Val Ser Thr
85 90 95
Met Gln Ala Ser Val Asp Leu Thr Asp Glu Asp Trp Asp Ala Asn Met
100 105 110
Asp Val Asn Ala Lys Gly Val Phe Leu Cys Asn Gln Ile Val Val Arg
115 120 125
His Phe Leu Ala Thr Gly Asn Lys Gly Val Ile Val Asn Thr Ala Ser
130 135 140
Leu Ala Gly Lys Val Gly Ala Pro Leu Leu Ala His Tyr Ser Ala Ser
145 150 155 160
Lys Phe Ala Val Leu Gly Trp Thr Gln Ala Leu Ala Arg Glu Leu Ser
165 170 175
Pro Thr Gly Ile Arg Val Asn Ala Val Cys Pro Gly Phe Val Arg Thr
180 185 190
Gly Met Gln Glu Arg Glu Ile Ile Trp Glu Gly Lys Leu Arg Asn Met
195 200 205
Thr Pro Asp Glu Val Arg Gln Glu Tyr Val Thr Leu Thr Pro Gln Gly
210 215 220
Arg Ile Glu Glu Pro Glu Asp Val Ala Val Val Val Arg Phe Leu Ala
225 230 235 240
Ser Asp Gly Ala Arg Phe Met Thr Gly Gln Gly Ile Asn Val Thr Gly
245 250 255
Gly Val Arg Met Asp
260
<210> 4
<211> 783
<212> DNA
<213> carbonyl reductase mutant nucleotide sequence SEQ ID NO.4(carbonyl reductase)
<400> 4
atggaagcta gcctgtctga attcgcgggc aaaagcgtcg ttgttacggg tggtgcttcc 60
ggtatcggtg cggcgatcac ccgtaccttc cacgctgaag gtgcccgtgt gaccatcctg 120
gactccgacg ctggccgcgc ggccgccctg gcagacgaac tgggtgataa cgcgttctcc 180
ggtggcatcg acggtgcaga ccgcggtttc gtacaggcgg ctgtggacgc tgttatctct 240
ggccagggcg gcattgatat cctgtgcgcg aatgctggtg ttagcactat gcaggcatct 300
gtagatctga cggatgaaga ctgggacgct aacatggacg ttaatgcaaa aggcgtattc 360
ctgtgcaacc agatcgtggt gcgtcacttt ctggcgacgg gtaacaaagg cgttatcgta 420
aacactgcaa gcctggcagg taaagtgggt gcaccgctgc tggcgcatta ctccgctagc 480
aagttcgctg ttctgggctg gactcaggcg ctggcgcgcg aactgtctcc taccggtatt 540
cgcgttaacg ctgtttgccc gggcttcgtt cgtactggta tgcaggaacg tgaaatcatc 600
tgggaaggta aactgcgcaa catgacgccg gatgaggttc gtcaggagta tgttactctg 660
accccgcaag gtcgtatcga agaaccggaa gacgtggcag tcgttgtccg tttcctggcg 720
agcgatggtg cacgtttcat gacgggtcag ggcatcaacg ttactggtgg tgttcgtatg 780
gat 783
Claims (9)
1. A carbonyl reductase mutant is characterized in that the amino acid sequence of the carbonyl reductase mutant is shown as SEQ ID NO. 3.
2. A carbonyl reductase mutant gene encoding the carbonyl reductase mutant of claim 1, having a nucleotide sequence as follows: shown as SEQ ID NO. 4.
3. A recombinant expression vector comprising the carbonyl reductase mutant gene of claim 2.
4. The recombinant expression vector of claim 3, wherein the recombinant expression vector is a vector plasmid that is pET-24 a.
5. A genetically engineered bacterium for producing the carbonyl reductase mutant of claim 1, wherein the genetically engineered bacterium comprises the recombinant expression vector of claim 3, and a host cell of the genetically engineered bacterium is Escherichia coli.
6. The carbonyl reductase mutant gene of claim 2, the recombinant expression vector of claim 3 and the use of the genetically engineered bacterium of claim 5 in preparing the carbonyl reductase mutant of claim 1.
7. A method for preparing the carbonyl reductase mutant of claim 1, comprising the steps of: culturing the genetically engineered bacterium of claim 5 to obtain a recombinant carbonyl reductase mutant.
8. The method of claim 7, comprising the step of preparing the carbonyl reductase mutant by fermentation.
9. The application of the carbonyl reductase mutant of claim 1 in preparing optical chiral alcohol by catalytic reduction of carbonyl compounds, wherein the carbonyl compounds have a structural general formula shown in formula I:
wherein: r is H, CH3，CH3CH2Or (CH)3)2CH。
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