CN112522224A - Alcohol dehydrogenase mutant with improved activity and stereoselectivity, recombinant vector, genetic engineering bacteria and application thereof - Google Patents
Alcohol dehydrogenase mutant with improved activity and stereoselectivity, recombinant vector, genetic engineering bacteria and application thereof Download PDFInfo
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Abstract
The invention discloses an alcohol dehydrogenase mutant with improved activity and stereoselectivity, a recombinant vector, a genetic engineering bacterium and application thereof. The alcohol dehydrogenase mutant comprises mutation by taking wild-type alcohol dehydrogenase shown in SEQ ID NO.1 as a template. The 188 th site of the wild-type alcohol dehydrogenase plays a key role in improving stereoselectivity, and the 93 th site plays a key role in activity. The mutant N188L/Q93L of the alcohol dehydrogenase provided by the invention can catalyze 2, 6-dichloro-3-fluoro acetophenone to prepare (S) -1- (2, 6-dichloro-3-fluoro phenyl) ethanol with high activity and high stereoselectivity, improves the activity and stereoselectivity of various ketones, is more suitable for the requirement of industrial production, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of protein engineering, and in particular relates to an alcohol dehydrogenase mutant with improved activity and stereoselectivity, a recombinant vector, a genetically engineered bacterium and application thereof.
Background
Chiral molecules are molecules having a pair of enantiomers in mirror image relationship to each other. They are similar in physicochemical properties, but have different optical activities in three-dimensional structures, namely racemic form, R form (dextrorotation) and S form (levorotation). A chiral drug is a chiral molecule, often having only one configuration with drug efficacy and the other configuration without drug efficacy or even side effects. Therefore, the optical purity (ee value) of chiral drugs is usually more than 98%. Chiral drugs play an important role in the list of heavy drugs.
The chiral alcohol as an important building block for synthesizing chiral drugs can be obtained by chemical synthesis and biocatalytic synthesis methods. However, the chemical synthesis of chiral alcohols usually involves heavy metals and is harsh, and the synthesis of highly optically pure products is difficult. The biocatalysis method gradually becomes an important way for synthesizing chiral alcohol under the advantages of mild reaction conditions, easy obtaining of high-optical purity products and the like. Biocatalytic methods are further divided into chiral resolution and asymmetric synthesis. Chiral resolution is limited by its reaction mechanism, resulting in a maximum theoretical conversion of 50%. The asymmetric reduction mode does not have the problem that the alcohol dehydrogenase or the microorganism containing the alcohol dehydrogenase is used as a catalyst to reduce the ketone substrate with the latent chirality to generate the corresponding chiral alcohol, and the theoretical conversion rate can reach 100 percent.
The enzymes derived from the Alcohol Dehydrogenase (ADH) family have redox ability on aldehyde group, ketone group, monosaccharide and ketosteroid, and play an important role in the synthesis of chiral compounds, and can be divided into three superfamily, i.e., short-chain dehydrogenase, medium-chain dehydrogenase and aldehyde ketone reductase. Alcohol dehydrogenase is a branch of redox systems, and when a prochiral ketone is asymmetrically reduced to produce a chiral alcohol, reduced Nicotinamide Adenine Dinucleotide (NADH) or Nicotinamide Adenine Dinucleotide Phosphate (NADPH) is used as a coenzyme, and NAD (P) is concomitantly produced+. However, NAD (P) H is very expensive, which requires a further coupling step for enzymatic oxidation to achieve coenzyme recycling. The alcohol dehydrogenase ChADH90 belongs to the short-chain dehydrogenases.
When a chemical reagent is synthesized by an enzyme catalysis method, certain inherent challenges are usually faced, such as low stereoselectivity and regioselectivity of alcohol dehydrogenase, easy generation of substrate inhibition and product inhibition, and the like, which limit the industrial application of the alcohol dehydrogenase. These challenges can now be addressed by employing protein engineering techniques, i.e., directed evolution. Directed evolution can mimic natural evolution, including gene mutation and expression and screening of mutant libraries. Common gene mutation methods include error-prone polymerase chain reaction (epPCR), saturation mutation and DNA shuffling (DNA shuffling).
The error-prone polymerase chain reaction technique is directed to whole protein mutations, and the commonly used method is the shotgun method. The method has strong randomness and aims at the whole gene. This technique does not require analysis of the structure of the enzyme and is the most common approach. In practice, only the concentration of magnesium ions in the PCR system or the PCR conditions need to be changed to generate random errors in the sequence and thus generate a mutation library. Saturation mutagenesis strategy is to generate mutants of all natural amino acid residues at the indicated sites and prepare them into a library of mutations. The investigator first analyzes the protein structure to determine the site to be engineered. Then, the mutation information is encoded into the primer, and finally introduced into the mutant plasmid by PCR technology using the wild type as a template plasmid. The key to the application of saturation mutagenesis technology is the selection of the optimal mutation site. The third mutation method, DNA shuffling technology, was proposed by the Pim Stemmer team in experiments to increase the activity of beta-lactamases. The operation method of the technology is that homologous genes of coding enzyme are firstly enzymolyzed by DNase to generate double-stranded oligonucleotide fragments with the length of 10-50 base pairs, and then the double-stranded oligonucleotide fragments are subjected to PCR amplification to form the full-length mutant genes.
Disclosure of Invention
The invention aims to provide an alcohol dehydrogenase mutant with improved activity and stereoselectivity, a recombinant vector, a genetic engineering bacterium and application thereof, so as to solve the problems that in the prior art, the stereoselectivity and the activity of alcohol dehydrogenase for preparing (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol and the like from ketones such as 2, 6-dichloro-3-fluoroacetophenone and the like are not high, substrate inhibition, product inhibition and the like are easy to generate, and further the industrial application of the alcohol dehydrogenase is limited.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to the first aspect of the present invention, there is provided an alcohol dehydrogenase mutant with improved stereoselectivity, comprising an alcohol dehydrogenase mutant with a wild-type alcohol dehydrogenase ChADH90 shown in SEQ ID NO.1 as a template and with the following mutations at amino acid residue 188: an alcohol dehydrogenase mutant N188A (SEQ ID NO.2) in which asparagine N at position 188 is mutated to alanine A; an alcohol dehydrogenase mutant N188G (SEQ ID NO.3) in which asparagine N at position 188 is mutated to glycine G; an alcohol dehydrogenase mutant N188I (SEQ ID NO.4) in which asparagine N at position 188 is mutated to isoleucine I; an alcohol dehydrogenase mutant N188L (SEQ ID NO.5) in which asparagine N at position 188 is mutated to leucine L; the alcohol dehydrogenase mutant N188T (SEQ ID NO.6) in which asparagine N at position 188 was mutated to threonine T.
According to a second aspect of the present invention, there is provided an alcohol dehydrogenase mutant with improved activity and stereoselectivity, comprising an alcohol dehydrogenase mutant obtained by mutating amino acid position 93 with the amino acid sequence SEQ ID No.5 of alcohol dehydrogenase mutant N188L as a template: an alcohol dehydrogenase mutant N188L/Q93C (SEQ ID NO.7) in which glutamine Q at position 93 is mutated to cysteine C; an alcohol dehydrogenase mutant N188L/Q93G (SEQ ID NO.8) in which glutamine Q at position 93 is mutated to glycine G; an alcohol dehydrogenase mutant N188L/Q93L (SEQ ID NO.9) in which glutamine Q at position 93 is mutated to leucine L; an alcohol dehydrogenase mutant N188L/Q93T (SEQ ID NO.10) in which glutamine Q at position 93 is mutated to threonine T; an alcohol dehydrogenase mutant N188L/Q93S (SEQ ID NO.11) in which glutamine Q at position 93 is mutated to serine S.
According to the present invention, the alcohol dehydrogenase mutant having both improved activity and stereoselectivity further comprises: taking a mutant alcohol dehydrogenase mutant N188A shown in SEQ ID NO.2 as a template, and mutating a glutamine Q at a 93-position amino acid residue into one of alcohol dehydrogenase mutants of cysteine C, glycine G, leucine L, threonine T and serine S; taking a mutant alcohol dehydrogenase mutant N188G shown in SEQ ID NO.3 as a template, and mutating a glutamine Q at a 93-position amino acid residue into one of alcohol dehydrogenase mutants of cysteine C, glycine G, leucine L, threonine T and serine S; taking a mutant alcohol dehydrogenase mutant N188I shown in SEQ ID NO.4 as a template, and mutating a glutamine Q at a 93-position amino acid residue into one of alcohol dehydrogenase mutants of cysteine C, glycine G, leucine L, threonine T and serine S; the alcohol dehydrogenase mutant is one of alcohol dehydrogenase mutants which are obtained by mutating a glutamine Q at a 93-position amino acid residue to cysteine C, glycine G, leucine L, threonine T and serine S by using a mutant alcohol dehydrogenase mutant N188T shown in SEQ ID NO.6 as a template.
According to a third aspect of the present invention, there is provided a gene encoding the stereoselectivity-enhanced alcohol dehydrogenase mutant as described above, the gene encoding the following protein (i) or (ii): (i) the amino acid sequence shown for the alcohol dehydrogenase mutant as described above; (ii) a protein derived by substituting, deleting or superposing one or more amino acids in the amino acid sequence defined in (i) and having the same function as the protein of (i), said gene encoding a protein having at least 95% homology with the amino acid sequence shown in the above-mentioned alcohol dehydrogenase mutant.
Particularly preferably, the invention provides an alcohol dehydrogenase mutant N188L with improved stereoselectivity, wherein the nucleotide sequence of a coding gene is shown as SEQ ID NO.13, and also provides an alcohol dehydrogenase mutant N188L/Q93L with improved stereoselectivity and enzyme activity, wherein the nucleotide sequence of a coding gene is shown as SEQ ID NO. 14. Experiments prove that the alcohol dehydrogenase mutant shown as SEQ ID NO.13 is the mutant with the best activity in single site mutation, and the alcohol dehydrogenase mutant shown as SEQ ID NO.14 is the mutant with the best activity in double site mutation.
According to a fourth aspect of the present invention, there is provided a recombinant vector comprising a gene encoding the alcohol dehydrogenase mutant having improved stereoselectivity as described above, the recombinant vector being constituted by inserting the gene encoding the alcohol dehydrogenase mutant into a vector plasmid comprising: pET series, pQE series, pRSET series, pGEX series, pBV series, pTrc series, pTwin series, pEZZ series, pKK series, and pUC series.
According to a fifth aspect of the present invention, there is provided a genetically engineered bacterium comprising the above recombinant vector.
According to the sixth aspect of the present invention, there is provided an application of the above alcohol dehydrogenase mutant or the above genetically engineered bacterium in catalyzing a ketone substrate to generate a corresponding chiral alcohol.
Preferably, the use comprises the use of 2, 6-dichloro-3-fluoroacetophenone to prepare (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol.
The optimum temperature of the alcohol dehydrogenase provided by the invention is 30 ℃, and the optimum pH is 6.
Also preferably, the application comprises catalyzing methyl 4-chloroacetoacetate, ethyl 4,4, 4-trifluoroacetoacetate, ethyl acetoacetate, ethyl propionylacetate, ethyl benzoylacetate, ethyl isobutyrylacetate, ethyl levulinate, alpha-chloroacetophenone, alpha-bromoacetophenone, 2 '-hydroxyacetophenone, 3' -nitroacetophenone, 2 '-fluoroacetophenone, 3' -chloroacetophenone, 4 '-methoxypropiophenone, 2, 6-dichloro-3-fluoroacetophenone, 2-chloro-1- (2, 4-dichlorophenyl) ethanone, 4-chlorobenzophenone, acetophenone, 2' -fluoroacetophenone, 2 '-chloroacetophenone, 2' -bromoacetophenone, a, 3' -methylacetophenone, 4 ' -bromoacetophenone, 2, 4-dichloroacetophenone, 3' -chlorophenylacetone, 4-methyl-diphenyl ketone, (4-chlorophenyl) (2-pyridyl) ketone, 2-acetylpyridine, N-boc-3-piperidone, 4-acetylpyridine and N-boc-3-pyrrolidone to generate corresponding chiral alcohol.
According to the present invention, first, an alcohol dehydrogenase ChADH90 gene cloned from a strain Chryseobacterium hisplasmense is selected as a template, the amino acid sequence of the alcohol dehydrogenase ChADH90 is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 12. The spatial structure of alcohol dehydrogenase ChADH90 was analyzed by homology modeling, and the enzyme was found to be composed of four subunits. 2, 6-dichloro-3-fluoroacetophenone was ligated into the alcohol dehydrogenase ChADH 90. The amino acid residue at position 188, which is critical for the stereoselectivity of the alcohol dehydrogenase, was determined on the basis of molecular dynamics simulations. After saturation mutation, the stereoselectivity of each mutant is determined according to the biological reduction reaction. And analyzing and obtaining the 93 rd amino acid residue which plays a key role in the steric hindrance of the alcohol dehydrogenase by taking the mutant with improved stereoselectivity and highest enzyme activity as a template. After the site 93 is subjected to saturation mutation, the stereoselectivity and the enzyme activity of the mutant are determined according to the biological reduction reaction and the enzyme activity analysis. The best double-point mutant ChADH90-N188L/Q93L is determined. According to the substrate tolerance analysis, the mutant has improved tolerance to 2, 6-dichloro-3-fluoro acetophenone. Through the protein engineering strategies, a plurality of mutants with improved catalytic performance of alcohol dehydrogenase are obtained, and the obtaining of the mutants is very beneficial to industrial production.
Further, the invention adopts the mode of asymmetric reduction to synthesize the chiral alcohol. The wild-type alcohol dehydrogenase ChADH90 discovered by the invention can catalyze 20mM 2, 6-dichloro-3-fluoro acetophenone (CFPO) to generate (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol [ (S) -CFPH ] within 24h, and the ee value is 97.2%. (S) -CFPH is an important intermediate of the chiral drug crizotinib. Crizotinib is a potent selective m-epithelial cell transforming factor/anaplastic lymphoma kinase (c-Met/ALK) inhibitor developed by the company Perey. The drug is approved by the FDA in the United states in 2011 to treat non-small cell lung cancer caused by ALK gene rearrangement. In 2017, the global market for crizotinib sold nearly five billion dollars.
When the invention utilizes a saturation mutation strategy to transform the discovered alcohol dehydrogenase ChADH90 to obtain a plurality of mutants with improved catalytic performance of alcohol dehydrogenase, and the mutants are applied to the preparation of chiral alcohol, the ee value of the product of the wild type alcohol dehydrogenase ChADH90 is only 97.2%, the conversion rate under the concentration of 20mM substrate is only 25%, and the enzyme activity is only 1.7U/mg, while the mutant ChADH90-N188L/Q93L formed by double site mutation can completely catalyze 1M CFPO within 6h, and the ee value of the generated (S) -CFPH is improved to 99.9%. However, it should be understood that the alcohol dehydrogenase mutants provided by the present invention are not limited to catalyzing the production of the corresponding chiral alcohols from the above ketones.
In summary, the key invention of the present invention is to provide an alcohol dehydrogenase mutant with improved activity and stereoselectivity. The alcohol dehydrogenase related by the invention takes a wild-type alcohol dehydrogenase shown in SEQ ID NO.1 as a template, determines an amino acid site which plays a key role in stereoselectivity and activity on the basis, and performs one of a plurality of mutants formed by the following mutations: the mutation of the N at the 188 th site is A, G, I, L and T to form mutants N188A, N188G, N188I, N188L and N188T; the Q mutation at position 93 is C, G, L, T and S to form Q93C, Q93G, Q93L, Q93T and Q93S. Among them, the 188 th site plays a key role in improving stereoselectivity, and the 93 th site plays a key role in activity.
The beneficial effects of the invention compared with the prior art comprise: the stereoselectivity of the mutants N188A, N188G, N188I, N188L and N188T obtained by the invention is obviously improved and reaches 99.9 percent (S). The conversion at 20mM substrate concentration was 42%, 30%, 53%, 63% and 41%, respectively. The activity of mutants N188L/Q93C, N188L/Q93G, N188L/Q93L, N188L/Q93T and N188L/Q93S which take ChADH90-N188L as a template is improved from 10.8U/mg to 164U/mg, 132U/mg, 252U/mg, 64U/mg and 199U/mg respectively. In conclusion, the mutants obtained according to the invention have a greater increase compared to the wild-type alcohol dehydrogenase ChADH 90. The alcohol dehydrogenase mutant has high activity, high stereoselectivity and high tolerance to a substrate, can efficiently catalyze ketones such as 2, 6-dichloro-3-fluoro acetophenone and the like to prepare pharmacy-related chiral alcohols such as (S) -1- (2, 6-dichloro-3-fluoro phenyl) ethanol and the like, and has important significance for the production of drug intermediates such as crizotinib and the like. In addition, the mutant has improved activity and stereoselectivity to various ketones, is more suitable for the requirement of industrial production, and has wide application prospect.
Drawings
FIG. 1 is a SDS-PAGE protein gel electrophoresis of wild-type alcohol dehydrogenase ChADH90, wherein M is a protein molecular weight standard reagent, S is a supernatant of a cell disruption solution, P is a precipitate of the cell disruption solution, T is a component of the cell disruption solution passing through a nickel column, and 50, 100, 200 and 500 represent components eluted by imidazole solutions with different concentrations, respectively;
FIG. 2 is a SDS-PAGE protein gel of mutant ChADH 90-N188L;
FIG. 3 is an SDS-PAGE protein gel of mutant ChADH 90-N188L/Q93L.
Detailed Description
The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. The techniques used in the examples are conventional in the art, as specifically described.
The invention selects the alcohol dehydrogenase ChADH90 gene cloned from the strain Chryseobacterium hispalense; the amino acid sequence of the alcohol dehydrogenase ChADH90 is shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene is shown in SEQ ID NO. 12. The invention constructs a mutation library by carrying out point saturation mutation on the screened wild alcohol dehydrogenase ChADH 90. Wherein, after saturation mutation is carried out on the 188 site, five mutants of N188A, N188G, N188I, N188L and N188T are obtained by screening, N188L with the highest conversion rate is selected from the five mutants as a template to carry out second round of point saturation mutation, and five mutants of N188L/Q93C, N188L/Q93G, N188L/Q93L, N188L/Q93T and N188L/Q93S with improved enzyme activity are obtained by screening in a 93 site saturation mutation library.
Example 1 enzyme Activity detection and analytical grade Bioreduction reactions
The corresponding prochiral ketone 2, 6-dichloro-3-fluoro acetophenone of the target product (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol is used as a substrate. The relative activity and coenzyme dependence of the alcohol dehydrogenase ChADH90 on the 2, 6-dichloro-3-fluoro acetophenone are determined by means of enzyme activity detection. Then an enzyme-coupled coenzyme circulating system is constructed for biological reduction. And detecting the ee value of the product to judge the stereoselectivity of the alcohol dehydrogenase.
The coenzyme dependence of the alcohol dehydrogenase is detected by the following steps: first, 5. mu.L of a crude enzyme solution of alcohol dehydrogenase ChADH90 and 182. mu.L of a phosphate buffer (50mM, pH 7.0) were added to the wells of the microplate. Each coenzyme was added 8. mu.L NADPH and NADH (0.2mM) separately in two channels. Incubate at 25 ℃ for 2 min. Placing the ELISA plate into an ELISA reader and setting a program, shaking the system for 5s, and scanning the system at the interval of 10s in an ultraviolet band of 340nm for 2 min. If the curve is flat, the system is proved to be stable. At this time, 5. mu.L of 2, 6-dichloro-3-fluoroacetophenone (200mM) was added to the reaction, and the mixture was shaken and read with a microplate reader. Meanwhile, the pure enzyme concentration was adjusted so that the line of the OD travel of the reaction system was straight in the first 1 min. This process was repeated 3 times to take the average as Δ E and then the error was calculated. For a certain alcohol dehydrogenase, the type of coenzyme corresponding to the pore channel with a larger Δ E is the coenzyme on which the alcohol dehydrogenase depends. Determination of the relative enzyme activity of the alcohol dehydrogenase: the enzyme activity of the alcohol dehydrogenase having the largest Δ E in the above-mentioned enzyme activity detection reaction was defined as 100%. The relative enzyme activity of each of the other alcohol dehydrogenases was (Δ En/Δ Emax) × 100%.
The stereoselectivity detection steps of the alcohol dehydrogenase are as follows: to a 2mL EP tube was added 1mL of phosphate buffer (100mM, pH 7.0), the corresponding 100. mu.L of coenzyme NAD (P) + (0.5mM), 50. mu.L of substrate CFPO (20mM), 0.05g of glucose, 1mg of lyophilized alcohol dehydrogenase and 1mg of Glucose Dehydrogenase (GDH). The reaction was allowed to proceed for 24h by placing the EP tube in a constant temperature shaker at 30 ℃ and 200 rpm. After the reaction is finished, the reaction solution is extracted for 2min on a vortex oscillation instrument by using ethyl acetate with the same volume. The mixture was centrifuged in a centrifuge at 14000rpm for 5 min. The supernatant was aspirated by pipette, filtered through a filter membrane and placed in a liquid sample vial for ee. The ee value detection conditions are as follows: liquid chromatography with AD-H chromatographic column, flow rate 0.7mL/min, ultraviolet detector 210nm wavelength, mobility for n-hexane: 99% isopropyl alcohol: 1, the peak-out time of the R-type product is 20.32min, and the peak-out time of the S-type product is 24.67 min. Enantiomeric excess value: ee (%) - (R-S)/(R + S) × 100%.
Example 2 substrate docking and molecular dynamics simulation
To obtain the spatial structure of the alcohol dehydrogenase ChADH90, the enzyme was homologously modeled. On-line modeling was performed using SWISSMODEL (http:// www.swissmodel.expasy.org /), and the 3D model of alcohol dehydrogenase ChADH90 was obtained using short-chain alcohol dehydrogenase from Ralstonia sp.DSM 6428 (PDB No.: 4BMS) as a template, and the sequence similarity between the two was 45%. Coenzyme NADH was superimposed into the ChADH90 model to form the ChADH90/NADH complex. The energy minimized 2, 6-dichloro-3-fluoroacetophenone was then docked into the ChADH90/NADH complex. And screening the docking result according to the catalytic mechanism of alcohol dehydrogenase, selecting the optimal docking posture by combining docking scoring, and performing subsequent molecular dynamics simulation by taking the optimal docking posture as an initial structure.
The docking process was performed using AutoDock Vina software. The protein model was subjected to removal of water molecules using Pymol 2.5 software prior to docking. The protein and COBE were then saved as pdb files. And (3) opening a protein file by using AutoDock software, and sequentially adding hydrogen, calculating charges and adding atom types to the protein. Pdb file is opened to modify small molecules, including hydrogenation, charge calculation, torque center establishment, etc. The central coordinates of the grid box are set at the catalytic center of the enzyme, and the side length is set
After the operation is finished, the optimal substrate docking position is selected according to the binding affinity of the 2, 6-dichloro-3-fluoro acetophenone and the ChADH90 and the analysis of a catalytic mechanism (judging the configuration of a generated product).
The full atom molecular dynamics simulation of the wild-type ChADH90 and the mutants aims to investigate the structural mechanism of the alcohol dehydrogenase ChADH90 on the activity of the substrate 2, 6-dichloro-3-fluoro acetophenone enzyme, the biological reduction reaction conversion rate and the stereoselectivity improvement. All molecular dynamics simulation operations were performed using the gromaccs 4.6.5 software. The LEaP module in Amber 16 was used to add missing hydrogen and heavy atoms to each docked protein system, which was then immersed in an aqueous environment and neutralized with sodium atoms. The water environment model adopts a TIP3P water box which is a rectangular cube with the minimum filling amount of each side
The Amber FF14SB force field file was used to process the entire system and to generate a topology file and a coordinate file. The force field parameters for the substrate 2, 6-dichloro-3-fluoroacetophenone were obtained using the BCC method in the antechamber package, as shown in the following Table. Eventually each system contains 40,000 atoms. The system first receives a 20,000 step energy minimization process using the steepest descent method. The system was then gradually heated from 0K to 300K using the NVT method. After the system rises to 300K, the system is balanced for 2ns by adopting an NPT method, and then a kinetic simulation of 100ns is carried out.
EXAMPLE 3 construction and screening of mutants
The wild-type ChADH90 was first cultured and the plasmid of this strain was extracted as a template for PCR. The primers were designed on-line using Quickchange (https:// www.agilent.com/store/primer design program. The specific method comprises the following steps: firstly, pasting a text or a base sequence of alcohol dehydrogenase ChADH90 in FASTA format into a text box of a browser; secondly, clicking 'Upload Translated' to automatically translate the base sequence into an amino acid sequence by the system; thirdly, a rectangular frame with check is arranged in front of each amino acid after translation is finished, and the site needing mutation is selected; fourth, the target amino acid to be mutated is selected at the position of "change amino acid(s) to"; finally, clicking the "Design Primers" system will automatically generate Primers and contain information such as primer length, annealing temperature, etc.
The DNA polymerase used is a Taq enzyme with high fidelity, which allows perfect amplification of the target fragment at a long length (around 10 kb). The primer designed by the Quickchange method is subjected to PCR to obtain the full-length recombinant plasmid containing the target fragment, and the length of the full-length recombinant plasmid is about 6kb, so that high-fidelity enzyme is selected for plasmid amplification. The PCR reaction system is as follows: 0.5. mu.L of Taq Plus DNA Polymerase; 1. mu.L dNTP; 12.5 μ L Taq Plus Buffer (Mg2 +); 1 μ L Forward primer (20 μ M); 1 μ L of Reverse primer (20 μ M); 1. mu.L of template DNA (50. mu.g/ml); 8 μ L of ddH 2O. The PCR reaction conditions were as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30 s; annealing at 65 deg.c (-1 deg.c/cycle) for 30 s; extension at 72 ℃ for 60 s; denaturation at 94 ℃ for 30s ] x15 cycles; annealing at 50 ℃ for 30 s; extension at 72 ℃ for 60 s; extension at 72 ℃ for 10min ] x20 cycles.
mu.L of 25. mu.L of PCR amplified by PCR was pipetted into a sterilized PCR tube, and then 1. mu.L of DpnI was added under a parallel bath condition to specifically excise the methylated template DNA strand. After mixing well, the PCR tube was water-bathed at 30 ℃ for 2 h. After water bath, PCR products were transformed to e.coli DH5 α and plasmids were re-extracted and finally transformed to e.coli BL21(DE 3). After transformation, single colonies were picked and inoculated in 4mL LB medium, cultured overnight at 37 ℃ and the species sequenced. After ensuring correct sequence, each mutant was inoculated in 24-well plates containing 1mL of self-induction medium. The well plate was placed in a microplate shaker and incubated at 30 ℃ for 24 h. After the mutants were cultured, the 24-well plate was placed in a microplate reader to detect the OD600nm value. The corresponding amount of bacteria was aspirated from each well according to the OD600nm value to ensure that the bacterial mass of each mutant remained consistent. Then, referring to the procedure of example 1, each mutant was subjected to enzyme activity, conversion rate of biological reduction reaction and stereoselectivity. Wherein, five mutants N188A, N188G, N188I, N188L and N188T are obtained by screening after saturation mutation is carried out on the 188 site, and the ee value of the product is increased from 97.2 percent (S) to 99.9 percent (S). The best mutant ChADH90-N188L is selected and used as a template for the subsequent construction of the mutant with the site saturation mutation at the position 93.
Example 4 purification of wild-type ChADH90 and mutants thereof
The alcohol dehydrogenase purification process comprises the following steps: 1) after the recombinant cells containing alcohol dehydrogenase are crushed, the supernatant is transferred into a precooled centrifugal tube and is subjected to ice bath for standby. The liquid flow rate throughout the purification process was maintained at 1 mL/min. The pre-packed column was equilibrated with a 10mM imidazole solution for a nickel column at ten column volumes. 2) The supernatant was passed through a nickel column, in which the His-tagged alcohol dehydrogenase and a portion of the hetero-protein specifically bound to nickel chloride. 3) The proteins in the nickel column were eluted with ten column volumes of 20mM, 50mM, 100mM, 150mM and 200mM imidazole solutions and the effluent was collected. 4) The nickel column was washed with 500mM imidazole solution and all proteins were eluted. 5) The imidazole solution was diluted with 20% ethanol solution and the growth of infectious microbes was prevented. And finally, remaining the collected eluents with various imidazole concentration gradients for protein electrophoresis detection.
Protein gel was prepared according to the gel preparation procedure of the SDS-PAGE gel preparation kit. Transferring the prepared protein gel and the prepared gel plate to an SDS-PAGE gel electrophoresis tank. Adding Tris-glycine protein electrophoresis buffer. Preparing a protein sample: the eluate during purification was pipetted 10. mu.L, and 10. mu.L of 2xSDS loading buffer was added to the protein sample and mixed well. The protein sample was placed in a boiling water bath for 5 min. Protein molecular weight standard reagents were added to the protein gel with each sample. And (3) turning on a power supply, and regulating the voltage to 120V after the pore channel samples run to the separation gel position at the voltage of 80V until the samples run to the whole separation gel.
The separation gel is taken down and placed in a tray, and protein staining solution is added to ensure that the separation gel is submerged in the protein gel. The tray was shaken slowly for 30min to complete staining of the protein gel. The staining solution was discarded, an appropriate amount of destaining solution was added and the tray was shaken slowly for 3 hours. The tray is continued to be shaken overnight after the replacement of the fresh destaining solution. And taking out the protein gel, and observing the pore channel containing the target protein. After determination of the corresponding imidazole concentration the eluate was transferred to an ultrafiltration tube and the tube was centrifuged at 4000g at 4 ℃ for 20 min. The filtrate was discarded, and repeated centrifugation was carried out three times with a phosphate buffer solution to remove imidazole. Glycerol was added to the pure enzyme to achieve a glycerol concentration of 25%. The pure enzyme was placed in a-40 ℃ freezer for later use.
Wherein, the SDS-PAGE protein gel electrophoresis images of the wild type alcohol dehydrogenase ChADH90, the mutant ChADH90-N188 and the mutant ChADH90-N188L/Q93L are respectively shown in figure 1, figure 2 and figure 3.
EXAMPLE 5 establishment of optimum reaction conditions
The optimal conditions for determining the enzyme-catalyzed reaction include temperature, pH, metal ions, and the like. Among them, the activity and stability of the alcohol dehydrogenase at various temperatures and pH should be considered before determining the temperature and pH of the final reaction.
The optimum temperature of alcohol dehydrogenase is determined as follows. Incubating pure enzyme and each component of enzyme activity detection system at a specified temperature of 20-65 deg.C for 2min, and setting an experimental point every 5 deg.C. The detection system is as follows: 182 μ L of phosphate buffer (50mM pH 7.0); 8 μ L NADPH (0.2 mM); 5 mu L of 1/10xCHADH90 pure enzyme solution; mu.L of 2, 6-dichloro-3-fluoroacetophenone (5 mM). The temperature corresponding to the experimental group with the highest activity is the optimum temperature for the enzyme. The enzyme activity is defined herein as 100% at the optimum temperature. Different temperatures and relative enzyme activities are prepared into temperature-activity curves. The optimum temperature of 30 ℃ is finally obtained.
The method for measuring the temperature stability of alcohol dehydrogenase is as follows. The pure enzyme was incubated at 30-50 ℃ and sampled at the same time intervals, and then the remaining activity of the enzyme was examined at the optimum temperature of the enzyme. Taking the initial activity of the alcohol dehydrogenase as 100%, the sampling was stopped when the residual activity decreased to less than 50% of the initial activity. The time intervals were set at 6h, 12h, 24h, 48h, 72h and 96 h. As a result, the half-life of the enzyme was the longest at 30 ℃.
The optimum pH of the alcohol dehydrogenase is determined as follows. Incubating the alcohol dehydrogenase in 182. mu.L of citrate buffer (pH 4.0-6.0), phosphate buffer (pH 6.0-8.0), Tris-HCl buffer (pH8.0-9.0) and glycine-NaOH buffer (pH 9.0-10.0), respectively, and adding 8. mu.L of NADPH (0.2 mM); 5 μ L of 1/10xCHADH90 pure enzyme; 5 mu L of 2, 6-dichloro-3-fluoro acetophenone (5mM) solution for detection. The detection temperature is the optimum temperature of the alcohol dehydrogenase. The pH corresponding to the buffer having the highest activity was used as the optimum pH for the alcohol dehydrogenase. The enzyme activity-pH curve is prepared by different pH values and relative activities of the enzyme. The optimum pH of the enzyme is 6.
The pH stability of the alcohol dehydrogenase was determined as follows. Appropriate amounts of pure enzyme were incubated in the above buffers at different pH values. The incubation time was 24h and the temperature was 4 ℃. And (3) detecting residual enzyme activity of the incubated pure enzyme, wherein the detection temperature is the optimal temperature of the alcohol dehydrogenase, and the pH is the optimal pH of the alcohol dehydrogenase. The alcohol dehydrogenase activity before incubation was defined as 100%, and the residual enzyme activity and the pH for the enzyme activity-pH curve were prepared. The alcohol dehydrogenase is most stable at the pH corresponding to the point with the highest residual enzyme activity. As a result, it was found that the enzyme was most stable at pH 6.
The metal ion-dependent detection method of alcohol dehydrogenase is as follows: preparing different kinds of metal ion chloride aqueous solution and metal ion chelating agent EDTA with the same concentration. The metal ion species includes Mg2+,K+,Li+,Mn2+,Cu2+,Ni+,Ca2+,Co2 +And Zn2+. The group without EDTA and metal ions was set as blank group, and the enzyme activity of the blank group was set as 100%. The results show that the absence of metal ions improves the enzyme activity.
Example 6 kinetic parameters of wild-type ChADH90 and its mutants
The kinetic parameters of the alcohol dehydrogenase ChADH90 and its mutant for 2, 6-dichloro-3-fluoroacetophenone were determined by fixing the NADH concentration at 0.5mM under standard conditions, so that the concentration of 2, 6-dichloro-3-fluoroacetophenone varied between 0.1 and 20 mM. Immobilization of 2, 6-dichloro-3-hydroxy-benzene in the determination of the kinetic parameters of ChADH90 for NADHThe concentration of acetophenone was 5mM, the NADH concentration was varied between 0.005 and 0.05 mM. The results show that the K of the wild type, the N188L mutant and the N88L/Q93L mutantmThe values were 5.8, 2.62, 0.41mM, respectively. K of wild type, N188L mutant, N88L/Q93L mutantcatThe values are 13.6, 103, 246S respectively-1K of wild type, N188L mutant, N88L/Q93L mutantcat/KmThe values are 2.34, 39.6 and 600S respectively-1/mM-1。
Example 7 substrate tolerance assay
Substrate tolerance C50 12The substrate concentration at which the enzyme activity decreased by 50% after incubation at 30 ℃ for 12 hours was the concentration value that the alcohol dehydrogenase ChADH90 and its mutant type could tolerate. The experimental setup blank was an alcohol dehydrogenase incubated under the same conditions in 100mM phosphate buffer (pH 6.0). All experiments were performed in triplicate and averaged.
Example 8 reduction of CFPO by wild-type ChADH90 and mutant organisms
Under the condition of unifying the bacterial dose and the coenzyme addition amount, the wild ChADH90 and the mutants are used for carrying out the reaction of preparing the biological reduction 2, 6-dichloro-3-fluoro acetophenone so as to determine the substrate concentration and the reaction efficiency. The system for the biological reduction reaction contained 100mL of phosphate buffer (100mM, pH 6.0), 0.1mM NAD+1.0-17.4g 2, 6-dichloro-3-fluoroacetophenone, 1.0g lyophilized wild-type ChADH90 or respective mutant cells, 1.0g lyophilized Glucose Dehydrogenase (GDH) cells, 1.35-22.7g glucose and 5mL methanol. The alcohol dehydrogenase cells were first resuspended in an appropriate amount of phosphate buffer and placed in a three-necked flask in a 30 ℃ water bath for 10 min. The contents were then added to the flask and the stirring blade was turned on. The pH value of the pH automatic liquid adding instrument connected with 2M sodium carbonate is controlled to be 6.0. The bioreduction reaction was carried out for 24h and samples were taken periodically.
The results show that the ee value of the S-type product generated by reducing CFPO by the wild-type alcohol dehydrogenase ChADH90 is only 97.2%, the conversion rate at the substrate concentration of 20mM is only 25%, and the enzyme activity is only 1.7U/mg. The stereoselectivity of the mutants N188A, N188G, N188I, N188L and N188T obtained by the invention is obviously improved, an S-type product with an ee value of 99.9% is generated, and the conversion rates under the substrate concentration of 20mM are respectively 42%, 30%, 53%, 63% and 41%. The activity of mutants N188L/Q93C, N188L/Q93G, N188L/Q93L, N188L/Q93T and N188L/Q93S which take ChADH90-N188L as a template is improved from 10.8U/mg to 164U/mg, 132U/mg, 252U/mg, 64U/mg and 199U/mg respectively. Therefore, the catalytic performance of the mutant obtained by the invention is greatly improved compared with that of the wild alcohol dehydrogenase ChADH 90.
The above embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and various changes may be made in the above embodiments of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present invention. The invention has not been described in detail in order to avoid obscuring the invention.
SEQUENCE LISTING
<110> university of east China's college of science
<120> alcohol dehydrogenase mutant with improved activity and stereoselectivity, recombinant vector, genetic engineering bacteria and application thereof
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 244
<212> PRT
<213> Chryseobacterium hispalense
<400> 1
Met Asn Phe Thr Asp Lys Asn Val Ile Val Thr Gly Gly Ser Ala Gly
1 5 10 15
Ile Gly Leu Ala Thr Val Lys Thr Phe Ile Glu Lys Gly Ala Asn Val
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Leu Ile Thr Gly Arg Asn Ala Asp Asn Leu Arg Thr Ala Ser Val Asn
35 40 45
Ile Asn Ser Pro Lys Leu Lys Thr Leu Ala Ser Asp Ile Ser Lys Leu
50 55 60
Ala Asp Ile Ala Ile Leu Glu Lys Glu Val Ala Glu Ser Gly Asn Lys
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Ile Asp Val Leu Val Leu Asn Ala Gly Ile Ala Lys Gln Phe Ser Ile
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Glu Glu Thr Thr Glu Gln Val Phe Asp Asp Leu Phe Asn Ile Asn Val
100 105 110
Lys Gly Leu Phe Phe Thr Leu Gln Lys Leu Ile Pro His Leu Ala Glu
115 120 125
Gly Ala Ser Val Ile Leu Ile Ser Ser Gly Val Ser Val Ser Gly Tyr
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Ala Gln Met Gly Ala Tyr Ala Ala Thr Lys Ser Ala Val Asp Ala Ile
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Ala Arg Thr Ala Ala Thr Glu Leu Ala Asp Arg Lys Ile Arg Val Asn
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Thr Val Ala Pro Gly Leu Thr Asp Thr Pro Met Asn His Gln Thr Pro
180 185 190
Glu His Ile Lys Asn Ala Ile Ala Ala Ala Val Pro Leu Lys Arg Ile
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Gly Glu Ala Glu Glu Ile Ala Asn Ala Ile Val Phe Leu Ala Ser Asp
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Glu Ala Ser Tyr Ile Ser Gly Ser Tyr Leu Ser Val Asp Gly Gly Val
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Thr Ile Arg Arg
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Met Asn Phe Thr Asp Lys Asn Val Ile Val Thr Gly Gly Ser Ala Gly
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Ile Gly Leu Ala Thr Val Lys Thr Phe Ile Glu Lys Gly Ala Asn Val
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Leu Ile Thr Gly Arg Asn Ala Asp Asn Leu Arg Thr Ala Ser Val Asn
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Ile Asn Ser Pro Lys Leu Lys Thr Leu Ala Ser Asp Ile Ser Lys Leu
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Ala Asp Ile Ala Ile Leu Glu Lys Glu Val Ala Glu Ser Gly Asn Lys
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Ile Asp Val Leu Val Leu Asn Ala Gly Ile Ala Lys Gln Phe Ser Ile
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Glu Glu Thr Thr Glu Gln Val Phe Asp Asp Leu Phe Asn Ile Asn Val
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Lys Gly Leu Phe Phe Thr Leu Gln Lys Leu Ile Pro His Leu Ala Glu
115 120 125
Gly Ala Ser Val Ile Leu Ile Ser Ser Gly Val Ser Val Ser Gly Tyr
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Ala Gln Met Gly Ala Tyr Ala Ala Thr Lys Ser Ala Val Asp Ala Ile
145 150 155 160
Ala Arg Thr Ala Ala Thr Glu Leu Ala Asp Arg Lys Ile Arg Val Asn
165 170 175
Thr Val Ala Pro Gly Leu Thr Asp Thr Pro Met Ala His Gln Thr Pro
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Glu His Ile Lys Asn Ala Ile Ala Ala Ala Val Pro Leu Lys Arg Ile
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Gly Glu Ala Glu Glu Ile Ala Asn Ala Ile Val Phe Leu Ala Ser Asp
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Glu Ala Ser Tyr Ile Ser Gly Ser Tyr Leu Ser Val Asp Gly Gly Val
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Thr Ile Arg Arg
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Met Asn Phe Thr Asp Lys Asn Val Ile Val Thr Gly Gly Ser Ala Gly
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Ile Gly Leu Ala Thr Val Lys Thr Phe Ile Glu Lys Gly Ala Asn Val
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Leu Ile Thr Gly Arg Asn Ala Asp Asn Leu Arg Thr Ala Ser Val Asn
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Ile Asn Ser Pro Lys Leu Lys Thr Leu Ala Ser Asp Ile Ser Lys Leu
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Ala Asp Ile Ala Ile Leu Glu Lys Glu Val Ala Glu Ser Gly Asn Lys
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Ile Asp Val Leu Val Leu Asn Ala Gly Ile Ala Lys Gln Phe Ser Ile
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Glu Glu Thr Thr Glu Gln Val Phe Asp Asp Leu Phe Asn Ile Asn Val
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Lys Gly Leu Phe Phe Thr Leu Gln Lys Leu Ile Pro His Leu Ala Glu
115 120 125
Gly Ala Ser Val Ile Leu Ile Ser Ser Gly Val Ser Val Ser Gly Tyr
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Ala Gln Met Gly Ala Tyr Ala Ala Thr Lys Ser Ala Val Asp Ala Ile
145 150 155 160
Ala Arg Thr Ala Ala Thr Glu Leu Ala Asp Arg Lys Ile Arg Val Asn
165 170 175
Thr Val Ala Pro Gly Leu Thr Asp Thr Pro Met Gly His Gln Thr Pro
180 185 190
Glu His Ile Lys Asn Ala Ile Ala Ala Ala Val Pro Leu Lys Arg Ile
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Gly Glu Ala Glu Glu Ile Ala Asn Ala Ile Val Phe Leu Ala Ser Asp
210 215 220
Glu Ala Ser Tyr Ile Ser Gly Ser Tyr Leu Ser Val Asp Gly Gly Val
225 230 235 240
Thr Ile Arg Arg
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Met Asn Phe Thr Asp Lys Asn Val Ile Val Thr Gly Gly Ser Ala Gly
1 5 10 15
Ile Gly Leu Ala Thr Val Lys Thr Phe Ile Glu Lys Gly Ala Asn Val
20 25 30
Leu Ile Thr Gly Arg Asn Ala Asp Asn Leu Arg Thr Ala Ser Val Asn
35 40 45
Ile Asn Ser Pro Lys Leu Lys Thr Leu Ala Ser Asp Ile Ser Lys Leu
50 55 60
Ala Asp Ile Ala Ile Leu Glu Lys Glu Val Ala Glu Ser Gly Asn Lys
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Ile Asp Val Leu Val Leu Asn Ala Gly Ile Ala Lys Gln Phe Ser Ile
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Glu Glu Thr Thr Glu Gln Val Phe Asp Asp Leu Phe Asn Ile Asn Val
100 105 110
Lys Gly Leu Phe Phe Thr Leu Gln Lys Leu Ile Pro His Leu Ala Glu
115 120 125
Gly Ala Ser Val Ile Leu Ile Ser Ser Gly Val Ser Val Ser Gly Tyr
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Ala Gln Met Gly Ala Tyr Ala Ala Thr Lys Ser Ala Val Asp Ala Ile
145 150 155 160
Ala Arg Thr Ala Ala Thr Glu Leu Ala Asp Arg Lys Ile Arg Val Asn
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Thr Val Ala Pro Gly Leu Thr Asp Thr Pro Met Ile His Gln Thr Pro
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Glu His Ile Lys Asn Ala Ile Ala Ala Ala Val Pro Leu Lys Arg Ile
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Gly Glu Ala Glu Glu Ile Ala Asn Ala Ile Val Phe Leu Ala Ser Asp
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Glu Ala Ser Tyr Ile Ser Gly Ser Tyr Leu Ser Val Asp Gly Gly Val
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Thr Ile Arg Arg
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Met Asn Phe Thr Asp Lys Asn Val Ile Val Thr Gly Gly Ser Ala Gly
1 5 10 15
Ile Gly Leu Ala Thr Val Lys Thr Phe Ile Glu Lys Gly Ala Asn Val
20 25 30
Leu Ile Thr Gly Arg Asn Ala Asp Asn Leu Arg Thr Ala Ser Val Asn
35 40 45
Ile Asn Ser Pro Lys Leu Lys Thr Leu Ala Ser Asp Ile Ser Lys Leu
50 55 60
Ala Asp Ile Ala Ile Leu Glu Lys Glu Val Ala Glu Ser Gly Asn Lys
65 70 75 80
Ile Asp Val Leu Val Leu Asn Ala Gly Ile Ala Lys Gln Phe Ser Ile
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Glu Glu Thr Thr Glu Gln Val Phe Asp Asp Leu Phe Asn Ile Asn Val
100 105 110
Lys Gly Leu Phe Phe Thr Leu Gln Lys Leu Ile Pro His Leu Ala Glu
115 120 125
Gly Ala Ser Val Ile Leu Ile Ser Ser Gly Val Ser Val Ser Gly Tyr
130 135 140
Ala Gln Met Gly Ala Tyr Ala Ala Thr Lys Ser Ala Val Asp Ala Ile
145 150 155 160
Ala Arg Thr Ala Ala Thr Glu Leu Ala Asp Arg Lys Ile Arg Val Asn
165 170 175
Thr Val Ala Pro Gly Leu Thr Asp Thr Pro Met Leu His Gln Thr Pro
180 185 190
Glu His Ile Lys Asn Ala Ile Ala Ala Ala Val Pro Leu Lys Arg Ile
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Gly Glu Ala Glu Glu Ile Ala Asn Ala Ile Val Phe Leu Ala Ser Asp
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Glu Ala Ser Tyr Ile Ser Gly Ser Tyr Leu Ser Val Asp Gly Gly Val
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Thr Ile Arg Arg
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Met Asn Phe Thr Asp Lys Asn Val Ile Val Thr Gly Gly Ser Ala Gly
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Ile Gly Leu Ala Thr Val Lys Thr Phe Ile Glu Lys Gly Ala Asn Val
20 25 30
Leu Ile Thr Gly Arg Asn Ala Asp Asn Leu Arg Thr Ala Ser Val Asn
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Ile Asn Ser Pro Lys Leu Lys Thr Leu Ala Ser Asp Ile Ser Lys Leu
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Ala Asp Ile Ala Ile Leu Glu Lys Glu Val Ala Glu Ser Gly Asn Lys
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Ile Asp Val Leu Val Leu Asn Ala Gly Ile Ala Lys Gln Phe Ser Ile
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Lys Gly Leu Phe Phe Thr Leu Gln Lys Leu Ile Pro His Leu Ala Glu
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Gly Ala Ser Val Ile Leu Ile Ser Ser Gly Val Ser Val Ser Gly Tyr
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Ala Gln Met Gly Ala Tyr Ala Ala Thr Lys Ser Ala Val Asp Ala Ile
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Ala Arg Thr Ala Ala Thr Glu Leu Ala Asp Arg Lys Ile Arg Val Asn
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Thr Val Ala Pro Gly Leu Thr Asp Thr Pro Met Thr His Gln Thr Pro
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Glu His Ile Lys Asn Ala Ile Ala Ala Ala Val Pro Leu Lys Arg Ile
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Gly Glu Ala Glu Glu Ile Ala Asn Ala Ile Val Phe Leu Ala Ser Asp
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Glu Ala Ser Tyr Ile Ser Gly Ser Tyr Leu Ser Val Asp Gly Gly Val
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Thr Ile Arg Arg
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Met Asn Phe Thr Asp Lys Asn Val Ile Val Thr Gly Gly Ser Ala Gly
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Ile Gly Leu Ala Thr Val Lys Thr Phe Ile Glu Lys Gly Ala Asn Val
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Leu Ile Thr Gly Arg Asn Ala Asp Asn Leu Arg Thr Ala Ser Val Asn
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Ile Asn Ser Pro Lys Leu Lys Thr Leu Ala Ser Asp Ile Ser Lys Leu
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Ala Asp Ile Ala Ile Leu Glu Lys Glu Val Ala Glu Ser Gly Asn Lys
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Ile Asp Val Leu Val Leu Asn Ala Gly Ile Ala Lys Cys Phe Ser Ile
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Glu Glu Thr Thr Glu Gln Val Phe Asp Asp Leu Phe Asn Ile Asn Val
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Lys Gly Leu Phe Phe Thr Leu Gln Lys Leu Ile Pro His Leu Ala Glu
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Gly Ala Ser Val Ile Leu Ile Ser Ser Gly Val Ser Val Ser Gly Tyr
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Ala Gln Met Gly Ala Tyr Ala Ala Thr Lys Ser Ala Val Asp Ala Ile
145 150 155 160
Ala Arg Thr Ala Ala Thr Glu Leu Ala Asp Arg Lys Ile Arg Val Asn
165 170 175
Thr Val Ala Pro Gly Leu Thr Asp Thr Pro Met Leu His Gln Thr Pro
180 185 190
Glu His Ile Lys Asn Ala Ile Ala Ala Ala Val Pro Leu Lys Arg Ile
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Gly Glu Ala Glu Glu Ile Ala Asn Ala Ile Val Phe Leu Ala Ser Asp
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Glu Ala Ser Tyr Ile Ser Gly Ser Tyr Leu Ser Val Asp Gly Gly Val
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Thr Ile Arg Arg
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Met Asn Phe Thr Asp Lys Asn Val Ile Val Thr Gly Gly Ser Ala Gly
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Ile Gly Leu Ala Thr Val Lys Thr Phe Ile Glu Lys Gly Ala Asn Val
20 25 30
Leu Ile Thr Gly Arg Asn Ala Asp Asn Leu Arg Thr Ala Ser Val Asn
35 40 45
Ile Asn Ser Pro Lys Leu Lys Thr Leu Ala Ser Asp Ile Ser Lys Leu
50 55 60
Ala Asp Ile Ala Ile Leu Glu Lys Glu Val Ala Glu Ser Gly Asn Lys
65 70 75 80
Ile Asp Val Leu Val Leu Asn Ala Gly Ile Ala Lys Gly Phe Ser Ile
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Glu Glu Thr Thr Glu Gln Val Phe Asp Asp Leu Phe Asn Ile Asn Val
100 105 110
Lys Gly Leu Phe Phe Thr Leu Gln Lys Leu Ile Pro His Leu Ala Glu
115 120 125
Gly Ala Ser Val Ile Leu Ile Ser Ser Gly Val Ser Val Ser Gly Tyr
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Ala Gln Met Gly Ala Tyr Ala Ala Thr Lys Ser Ala Val Asp Ala Ile
145 150 155 160
Ala Arg Thr Ala Ala Thr Glu Leu Ala Asp Arg Lys Ile Arg Val Asn
165 170 175
Thr Val Ala Pro Gly Leu Thr Asp Thr Pro Met Leu His Gln Thr Pro
180 185 190
Glu His Ile Lys Asn Ala Ile Ala Ala Ala Val Pro Leu Lys Arg Ile
195 200 205
Gly Glu Ala Glu Glu Ile Ala Asn Ala Ile Val Phe Leu Ala Ser Asp
210 215 220
Glu Ala Ser Tyr Ile Ser Gly Ser Tyr Leu Ser Val Asp Gly Gly Val
225 230 235 240
Thr Ile Arg Arg
<210> 9
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Met Asn Phe Thr Asp Lys Asn Val Ile Val Thr Gly Gly Ser Ala Gly
1 5 10 15
Ile Gly Leu Ala Thr Val Lys Thr Phe Ile Glu Lys Gly Ala Asn Val
20 25 30
Leu Ile Thr Gly Arg Asn Ala Asp Asn Leu Arg Thr Ala Ser Val Asn
35 40 45
Ile Asn Ser Pro Lys Leu Lys Thr Leu Ala Ser Asp Ile Ser Lys Leu
50 55 60
Ala Asp Ile Ala Ile Leu Glu Lys Glu Val Ala Glu Ser Gly Asn Lys
65 70 75 80
Ile Asp Val Leu Val Leu Asn Ala Gly Ile Ala Lys Leu Phe Ser Ile
85 90 95
Glu Glu Thr Thr Glu Gln Val Phe Asp Asp Leu Phe Asn Ile Asn Val
100 105 110
Lys Gly Leu Phe Phe Thr Leu Gln Lys Leu Ile Pro His Leu Ala Glu
115 120 125
Gly Ala Ser Val Ile Leu Ile Ser Ser Gly Val Ser Val Ser Gly Tyr
130 135 140
Ala Gln Met Gly Ala Tyr Ala Ala Thr Lys Ser Ala Val Asp Ala Ile
145 150 155 160
Ala Arg Thr Ala Ala Thr Glu Leu Ala Asp Arg Lys Ile Arg Val Asn
165 170 175
Thr Val Ala Pro Gly Leu Thr Asp Thr Pro Met Leu His Gln Thr Pro
180 185 190
Glu His Ile Lys Asn Ala Ile Ala Ala Ala Val Pro Leu Lys Arg Ile
195 200 205
Gly Glu Ala Glu Glu Ile Ala Asn Ala Ile Val Phe Leu Ala Ser Asp
210 215 220
Glu Ala Ser Tyr Ile Ser Gly Ser Tyr Leu Ser Val Asp Gly Gly Val
225 230 235 240
Thr Ile Arg Arg
<210> 10
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Met Asn Phe Thr Asp Lys Asn Val Ile Val Thr Gly Gly Ser Ala Gly
1 5 10 15
Ile Gly Leu Ala Thr Val Lys Thr Phe Ile Glu Lys Gly Ala Asn Val
20 25 30
Leu Ile Thr Gly Arg Asn Ala Asp Asn Leu Arg Thr Ala Ser Val Asn
35 40 45
Ile Asn Ser Pro Lys Leu Lys Thr Leu Ala Ser Asp Ile Ser Lys Leu
50 55 60
Ala Asp Ile Ala Ile Leu Glu Lys Glu Val Ala Glu Ser Gly Asn Lys
65 70 75 80
Ile Asp Val Leu Val Leu Asn Ala Gly Ile Ala Lys Thr Phe Ser Ile
85 90 95
Glu Glu Thr Thr Glu Gln Val Phe Asp Asp Leu Phe Asn Ile Asn Val
100 105 110
Lys Gly Leu Phe Phe Thr Leu Gln Lys Leu Ile Pro His Leu Ala Glu
115 120 125
Gly Ala Ser Val Ile Leu Ile Ser Ser Gly Val Ser Val Ser Gly Tyr
130 135 140
Ala Gln Met Gly Ala Tyr Ala Ala Thr Lys Ser Ala Val Asp Ala Ile
145 150 155 160
Ala Arg Thr Ala Ala Thr Glu Leu Ala Asp Arg Lys Ile Arg Val Asn
165 170 175
Thr Val Ala Pro Gly Leu Thr Asp Thr Pro Met Leu His Gln Thr Pro
180 185 190
Glu His Ile Lys Asn Ala Ile Ala Ala Ala Val Pro Leu Lys Arg Ile
195 200 205
Gly Glu Ala Glu Glu Ile Ala Asn Ala Ile Val Phe Leu Ala Ser Asp
210 215 220
Glu Ala Ser Tyr Ile Ser Gly Ser Tyr Leu Ser Val Asp Gly Gly Val
225 230 235 240
Thr Ile Arg Arg
<210> 11
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Met Asn Phe Thr Asp Lys Asn Val Ile Val Thr Gly Gly Ser Ala Gly
1 5 10 15
Ile Gly Leu Ala Thr Val Lys Thr Phe Ile Glu Lys Gly Ala Asn Val
20 25 30
Leu Ile Thr Gly Arg Asn Ala Asp Asn Leu Arg Thr Ala Ser Val Asn
35 40 45
Ile Asn Ser Pro Lys Leu Lys Thr Leu Ala Ser Asp Ile Ser Lys Leu
50 55 60
Ala Asp Ile Ala Ile Leu Glu Lys Glu Val Ala Glu Ser Gly Asn Lys
65 70 75 80
Ile Asp Val Leu Val Leu Asn Ala Gly Ile Ala Lys Ser Phe Ser Ile
85 90 95
Glu Glu Thr Thr Glu Gln Val Phe Asp Asp Leu Phe Asn Ile Asn Val
100 105 110
Lys Gly Leu Phe Phe Thr Leu Gln Lys Leu Ile Pro His Leu Ala Glu
115 120 125
Gly Ala Ser Val Ile Leu Ile Ser Ser Gly Val Ser Val Ser Gly Tyr
130 135 140
Ala Gln Met Gly Ala Tyr Ala Ala Thr Lys Ser Ala Val Asp Ala Ile
145 150 155 160
Ala Arg Thr Ala Ala Thr Glu Leu Ala Asp Arg Lys Ile Arg Val Asn
165 170 175
Thr Val Ala Pro Gly Leu Thr Asp Thr Pro Met Leu His Gln Thr Pro
180 185 190
Glu His Ile Lys Asn Ala Ile Ala Ala Ala Val Pro Leu Lys Arg Ile
195 200 205
Gly Glu Ala Glu Glu Ile Ala Asn Ala Ile Val Phe Leu Ala Ser Asp
210 215 220
Glu Ala Ser Tyr Ile Ser Gly Ser Tyr Leu Ser Val Asp Gly Gly Val
225 230 235 240
Thr Ile Arg Arg
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<213> Chryseobacterium hispalense
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<211> 735
<212> DNA
<213> Chryseobacterium hispalense
<400> 13
atgaatttca ccgataaaaa tgtaatcgtt acaggcggaa gcgcaggaat tggattagca 60
accgtaaaaa cgtttattga aaaaggggca aacgttttaa taaccggcag aaacgctgac 120
aatctgcgca cagcatcagt aaacattaac agtccgaaac tgaaaacctt agcttctgat 180
atttctaaat tggcagacat tgcaatattg gaaaaagaag ttgcagaaag cggaaataaa 240
attgatgtgc tcgtactaaa tgcaggaatt gcaaagcaat tttcaataga agaaacaacg 300
gagcaagtgt ttgatgattt gttcaacatc aatgtgaaag gattgttctt tacgttgcaa 360
aaactgattc cgcatttagc agaaggagct tctgttattc ttatttcttc tggagtttca 420
gtgagcggat acgcacaaat gggcgcttac gccgcgacaa aaagtgcagt tgatgcaatt 480
gcacgtactg ctgcaacaga attggcagac agaaaaatcc gggtaaatac tgttgctcca 540
ggacttaccg ataccccgat gctacatcag acaccggaac atattaaaaa tgctattgca 600
gcagcagttc cgctgaaaag aatcggagaa gctgaagaaa tcgcaaacgc cattgtgttc 660
ctggcttcag acgaagcttc ttatatttca ggttcatatc tgtcggttga cggcggtgta 720
acgatccgca gataa 735
<210> 14
<211> 735
<212> DNA
<213> Chryseobacterium hispalense
<400> 14
atgaatttca ccgataaaaa tgtaatcgtt acaggcggaa gcgcaggaat tggattagca 60
accgtaaaaa cgtttattga aaaaggggca aacgttttaa taaccggcag aaacgctgac 120
aatctgcgca cagcatcagt aaacattaac agtccgaaac tgaaaacctt agcttctgat 180
atttctaaat tggcagacat tgcaatattg gaaaaagaag ttgcagaaag cggaaataaa 240
attgatgtgc tcgtactaaa tgcaggaatt gcaaagcaat tttcaataga agaaacaacg 300
gagcaagtgt ttgatgattt gttcaacatc aatgtgaaag gattgttctt tacgttgcaa 360
aaactgattc cgcatttagc agaaggagct tctgttattc ttatttcttc tggagtttca 420
gtgagcggat acgcacaaat gggcgcttac gccgcgacaa aaagtgcagt tgatgcaatt 480
gcacgtactg ctgcaacaga attggcagac agaaaaatcc gggtaaatac tgttgctcca 540
ggacttaccg ataccccgat gctacatcag acaccggaac atattaaaaa tgctattgca 600
gcagcagttc cgctgaaaag aatcggagaa gctgaagaaa tcgcaaacgc cattgtgttc 660
ctggcttcag acgaagcttc ttatatttca ggttcatatc tgtcggttga cggcggtgta 720
acgatccgca gataa 735
Claims (9)
1. An alcohol dehydrogenase mutant with improved stereoselectivity, which is characterized by comprising an alcohol dehydrogenase mutant formed by taking a wild-type alcohol dehydrogenase ChADH90 shown in SEQ ID NO.1 as a template and carrying out the following mutations on the 188 th amino acid residue:
the alcohol dehydrogenase mutant N188A with the amino acid sequence shown in SEQ ID NO.2 is obtained by mutating asparagine N at the 188 th site into alanine A;
the alcohol dehydrogenase mutant N188G with the amino acid sequence shown in SEQ ID NO.3 is obtained by mutating asparagine N at the 188 th position into glycine G;
the alcohol dehydrogenase mutant N188I with the amino acid sequence shown in SEQ ID NO.4 has the mutation of asparagine N at the 188 th site into isoleucine I;
the alcohol dehydrogenase mutant N188L with the amino acid sequence shown in SEQ ID NO.5 has the mutation of asparagine N at the 188 th site into leucine L; and
the alcohol dehydrogenase mutant N188T with the amino acid sequence shown in SEQ ID No.6 has the mutation of asparagine N at the 188 th site into threonine T.
2. An alcohol dehydrogenase mutant having increased activity and stereoselectivity, comprising: the alcohol dehydrogenase mutant is formed by taking wild alcohol dehydrogenase ChADH90 shown in SEQ ID NO.1 as a template, mutating asparagine N at the 188 th position into leucine L and mutating amino acid residue at the 93 th position as follows:
the glutamine Q at the 93 th position is mutated into an alcohol dehydrogenase mutant N188L/Q93C of cysteine C, and the amino acid sequence is shown as SEQ ID NO. 7;
the glutamine Q at the 93 th position is mutated into an alcohol dehydrogenase mutant N188L/Q93G of glycine G, and the amino acid sequence is shown as SEQ ID NO. 8;
the glutamine Q at the 93 th position is mutated into an alcohol dehydrogenase mutant N188L/Q93L of leucine L, and the amino acid sequence is shown as SEQ ID NO. 9;
an alcohol dehydrogenase mutant N188L/Q93T with the amino acid sequence shown as SEQ ID NO.10, wherein the glutamine Q at the 93 th position is mutated into threonine T; and
the glutamine Q at the 93 th position is mutated into an alcohol dehydrogenase mutant N188L/Q93S of serine S, and the amino acid sequence is shown as SEQ ID NO. 11.
3. An alcohol dehydrogenase mutant having increased activity and stereoselectivity, comprising: taking a mutant alcohol dehydrogenase mutant N188A shown in SEQ ID NO.2 as a template, and mutating a glutamine Q at a 93-position amino acid residue into one of alcohol dehydrogenase mutants of cysteine C, glycine G, leucine L, threonine T and serine S;
taking a mutant alcohol dehydrogenase mutant N188G shown in SEQ ID NO.3 as a template, and mutating a glutamine Q at a 93-position amino acid residue into one of alcohol dehydrogenase mutants of cysteine C, glycine G, leucine L, threonine T and serine S;
taking a mutant alcohol dehydrogenase mutant N188I shown in SEQ ID NO.4 as a template, and mutating a glutamine Q at a 93-position amino acid residue into one of alcohol dehydrogenase mutants of cysteine C, glycine G, leucine L, threonine T and serine S;
the alcohol dehydrogenase mutant is one of alcohol dehydrogenase mutants which are obtained by mutating a glutamine Q at a 93-position amino acid residue to cysteine C, glycine G, leucine L, threonine T and serine S by using a mutant alcohol dehydrogenase mutant N188T shown in SEQ ID NO.6 as a template.
4. A gene encoding an alcohol dehydrogenase mutant, wherein the gene encodes the following protein (i) or (ii):
(ii) the amino acid sequence of an alcohol dehydrogenase mutant according to any one of claims 1 to 3;
(ii) a protein derived from the amino acid sequence defined in (i) by substitution, deletion or addition of one or more amino acids and having the same function as the protein of (i), said gene encoding a protein having at least 95% homology with the amino acid sequence of the alcohol dehydrogenase mutant of any one of claims 1 to 3.
5. A recombinant vector comprising the gene encoding the alcohol dehydrogenase mutant according to claim 4, wherein the recombinant vector is constituted by inserting the gene encoding the alcohol dehydrogenase mutant on a vector plasmid comprising: pET series, pQE series, pRSET series, pGEX series, pBV series, pTrc series, pTwin series, pEZZ series, pKK series, and pUC series.
6. A genetically engineered bacterium comprising the recombinant vector according to claim 5.
7. The use of the alcohol dehydrogenase mutant according to claim 1, 2 or 3 or the genetically engineered bacterium according to claim 6 in catalyzing a ketone substrate to produce a corresponding chiral alcohol.
8. The use as claimed in claim 7, which comprises the use in the catalytic preparation of (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol from 2, 6-dichloro-3-fluoroacetophenone.
9. The use of claim 7, comprising catalyzing methyl 4-chloroacetoacetate, ethyl 4,4, 4-trifluoroacetoacetate, ethyl acetoacetate, ethyl propionylacetate, ethyl benzoylacetate, ethyl isobutyroacetate, ethyl levulinate, α -chloroacetophenone, α -bromoacetophenone, 2 '-hydroxyacetophenone, 3' -nitroacetophenone, 2 '-fluoroacetophenone, 3' -chloroacetophenone, 3 '-bromoacetophenone, 4' -methoxypropiophenone, 2, 6-dichloro-3-fluoroacetophenone, 2-chloro-1- (2, 4-dichlorophenyl) ethanone, 4-chlorobenzophenone, acetophenone, 2 '-fluoroacetophenone, 2' -chloroacetophenone, ethyl 4-chloroacetophenone, the application of 2 '-bromoacetophenone, 3' -methylacetophenone, 4 '-bromoacetophenone, 2, 4-dichloroacetophenone, 3' -chloropropiophenone, 4-methyl-diphenyl ketone, (4-chlorphenyl) (2-pyridyl) ketone, 2-acetylpyridine, N-boc-3-piperidone, 4-acetylpyridine and N-boc-3-pyrrolidone in the preparation of corresponding chiral alcohol.
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| CN115011573B (en) * | 2022-06-15 | 2023-12-12 | 浙江大学 | Alcohol dehydrogenase mutant and application thereof |
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