Bacillus subtilis alcohol dehydrogenase mutant with improved catalytic activity

Abstract

The invention discloses a bacillus subtilis alcohol dehydrogenase mutant with improved catalytic activity, and belongs to the field of gene engineering and protein expression. The mutant BsBDHAH42A / V266C / G292A is obtained by performing single mutation and combined mutation on 42,266 and 292 amino acids of (2, 3)-butanediol dehydrogenase (BsBDHA) of bacillus subtilis, the specific activity and catalytic efficiency of the mutant BsBDHAH42A / V266C / G292A are remarkably improved compared with those of wild type BsBDHA for catalyzing (R)-PED, the specific activity reaches 11.6 U / mg, and the catalytic efficiency reaches 2.52 mM <-1 > s <-1 > or above; compared with BsBDHAI49L / V266L in the prior art, the specific activity of the BsBDHAH42A / V266C / G292A mutant disclosed by the invention is improved by 21 times; compared with the mutant BsBDHAI49L / V266L in the prior art, the catalytic efficiency of the mutant BsBDHAI49L / V266L is improved by 31 times; and the conversion rate of catalyzing 2-hydroxyacetophenone in 6 hours reaches 92.1%.

Description

TECHNICAL FIELD

[0001] The present application relates to a Bacillus subtilis alcohol dehydrogenase mutant with improved catalytic activity, belonging to the technical field of genetic engineering and enzyme engineering. BACKGROUND

[0002] 2-hydroxyacetophenone (2-HAP) is not only widely used as a flavor in daily life, but also can be used to synthesize various drug intermediates and compounds with important biological activities, such as 2-hydroxyacetophenone Schiff base (an important antioxidant and antibacterial agent) and (R)-2-phenylglycinol (a precursor of a new antibiotic pyrazolopyrimidine).

[0003] The traditional production of 2-HAP mainly relies on chemical synthesis methods, such as photocatalytic method, nano-complex catalytic method or metal-catalyzed oxidation of phenethyl alcohol method, and microwave-assisted dehalogenation of acetophenone method, etc. These methods usually require the use of peroxide, high-valence iodine compound, metal oxide or oxygen as oxidant, and the reaction conditions are harsh, the process is complex, and dangerous reagents or expensive drugs are used. With the rising tide of global sustainable development, especially in recent domestic policies, the chemical industry is required to carry out technological transformation or eliminate backward production capacity, and the development of more environmentally friendly production methods has become an urgent need.

[0004] The method of biological synthesis can not only realize the efficient utilization of raw materials, but also can realize the reversible waste utilization, so as to reduce or avoid the generation of by-products. Among them, the dehydrogenase-catalyzed vicinal diol oxidation reaction is an effective biological synthesis method. For example, (2,3)-butanediol dehydrogenase (abbreviated as BDH, EC 1.1.1.4) is an enzyme that can realize the mutual transformation between α-hydroxy ketone and vicinal diol, which participates in the reaction with NAD(P) + or NAD(P)H as coenzyme.

[0005] However, the prior art shows that the specific activity and catalytic efficiency (k cat / K m) is relatively low. Although the prior art found that Bacillus subtilis (2,3)-butanediol dehydrogenase (BsBDHA) catalyzes the reaction of (R)-1-phenyl-1,2-ethanediol (Bacillus subtilis (2,3)-butanediol dehydrogenase, BsBDHA) to generate 2-HAP, the specific activity and catalytic efficiency of the enzyme are only 0.54 U / mg and 0.08 mM -1 s -1 On this basis, the prior art mutates the wild-type BDHs, mutates the 49th amino acid and the 266th amino acid, and the specific enzyme activity of the obtained mutant is up to 1.98 U / mg, and the catalytic efficiency is up to 0.39 mM -1 s -1 Although it is improved more than the wild type, it is still at a relatively low level in the enzymatic reaction.

[0006] Therefore, it is particularly important to improve the catalytic efficiency of BDHs on (R)-1-phenyl-1,2-ethanediol at the molecular level. Through genetic engineering and enzyme engineering technology, a mutant of Bacillus subtilis alcohol dehydrogenase with improved catalytic activity is obtained, which provides a new solution for the green and efficient production of 2-HAP. SUMMARY

[0007] In order to solve the problems of low specific enzyme activity and low catalytic efficiency of (2,3)-butanediol dehydrogenase, the present application provides a (2,3)-butanediol dehydrogenase mutant, which has improved specific enzyme activity and catalytic efficiency compared with wild-type (2,3)-butanediol dehydrogenase. The (2,3)-butanediol dehydrogenase mutant of the present application can be used for synthesizing 2-hydroxyacetophenone.

[0008] The first object of the present application is to provide an alcohol dehydrogenase mutant, wherein the amino acid sequence of the alcohol dehydrogenase mutant is mutated at the 42nd position corresponding to SEQ ID NO. 1.

[0009] In one embodiment, the alcohol dehydrogenase is (2,3)-butanediol dehydrogenase.

[0010] In one embodiment, the amino acid sequence of the alcohol dehydrogenase mutant is mutated at the 42nd position corresponding to SEQ ID NO. 1, and is further mutated at the 266th and / or 292nd positions.

[0011] In one embodiment, the histidine at the 42nd position is mutated to alanine or threonine; the valine at the 266th position is mutated to cysteine; and the glycine at the 292nd position is mutated to alanine.

[0012] In one embodiment, the alcohol dehydrogenase mutant is further mutated at position 292 from glycine to alanine, based on the mutation of histidine at position 42 to alanine and valine at position 266 to cysteine.

[0013] A second object of the present application is to provide a gene encoding the above-mentioned alcohol dehydrogenase mutant.

[0014] The present application also provides a recombinant plasmid carrying the above-mentioned gene.

[0015] In one embodiment, the vector of the recombinant plasmid is pET vector.

[0016] The present application also provides a host cell carrying the above-mentioned gene or any of the above-mentioned recombinant plasmids.

[0017] In one embodiment, the host cell is a bacterium or a fungus.

[0018] In one embodiment, the bacterium includes, but is not limited to, an Escherichia coli cell.

[0019] In one embodiment, the Escherichia coli includes, but is not limited to, E. coli BL21, E. coli BL21 (DE3), E. coli JM109, E. coli DH5a or E. coli TOP10.

[0020] A third object of the present application is to provide a catalyst containing any of the above-mentioned alcohol dehydrogenase mutants or any of the above-mentioned host cells.

[0021] In one embodiment, the catalyst contains a microbial cell expressing the mutant and a catalyst carrier; the catalyst carrier can be a cell-protecting agent.

[0022] The present application also provides a method for synthesizing 2-hydroxyacetophenone, using the above-mentioned catalyst to catalyze the synthesis of 2-hydroxyacetophenone from (R)-1-phenyl-1,2-ethanediol.

[0023] In one embodiment, the above-mentioned reaction is carried out at 10-30°C.

[0024] In one embodiment, in a 100 mL sodium phosphate buffer (50 mmol / L, pH 8.0) system, 20 mmol / L (R)-PED, 10 mmol / L NAD+ and 75 mg / mL of BsBDHA H42A / V266C / G292A and LcLDH Q88A / I229A are added respectively, and the reaction is carried out in a constant-temperature stirring reactor at 30°C and 120 r / min for 24 h to synthesize 2-hydroxyacetophenone.

[0025] The application also provides the use of any of the above-mentioned alcohol dehydrogenase mutants, or any of the above-mentioned genes, or any of the above-mentioned recombinant plasmids, or any of the above-mentioned host cells in the synthesis of 2-hydroxyacetophenone.

[0026] Beneficial effects:

[0027] The application mutates the amino acids at positions 42, 266 and 292 of (2,3)-butanediol dehydrogenase (BsBDHA) of Bacillus subtilis, and the specific activity of the mutant BsBDHA for catalyzing (R)-PED is significantly improved, and the specific activity is greater than 0.92 U / mg, and the catalytic efficiency is greater than 0.22 0.96 mM -1 s -1 The above.

[0028] In particular, the mutant BsBDHA H42A / V266C / G292A The specific activity is 11.6 U / mg, which is 21 times that of the wild-type BsBDHA; the catalytic efficiency is 2.52 mM -1 s -1 , which is 31 times that of the wild type; the mutant BsBDHA H42A / V266C / G292A The conversion rate of the mutant BsBDHA for catalyzing the synthesis of 2-hydroxyacetophenone reaches 92.1% in 6 hours, which significantly shortens the time for reaching reaction equilibrium.

[0029] The mutant BsBDHA of the application H42A / V266C / G292A The specific activity is 5.85 times that of the prior art BsBDHA I49L / V266L The catalytic efficiency is 6.46 times that of the prior art mutant BsBDHA I49L / V266L . BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 BsBDHA H42A / V266C / G292A catalyzing the synthesis of 2-HAP from (R)-PED is shown in the figure;

[0031] Figure 2 BsBDHA H42A / V266C / G292A and the reaction progress chart of BsBDHA catalyzing the synthesis of 2-HAP from (R)-PED. DETAILED DESCRIPTION

[0032] In order to enable a clearer understanding of the technical content of the application, the following examples are used for detailed description, and the purpose is only to better understand the content of the application and not to limit the protection scope of the application.

[0033] Preparation of experimental materials

[0034] 1. Construction of plasmid pET-Duet-1-bsbdha

[0035] The gene for (2,3)-butanediol dehydrogenase (amino acid sequence as shown in SEQ ID NO.1), with nucleotide sequence as shown in SEQ ID NO.2, was ligated into plasmid pET-Duet-1 to construct plasmid pET-Duet-1-bsbdha.

[0036] 2. Construction of recombinant E. coli / bsbdha

[0037] The plasmid pET-Duet-1-bsbdha obtained in (1) was transformed into E. coli BL21(DE3) to construct the recombinant strain E. coli / bsbdha.

[0038] 3. Recombinant E. coli / LcLDH Q88A / I229A Construction

[0039] Encoding lactate dehydrogenase LcLDH Q88A / I229A The nucleotide sequence (amino acid sequence as shown in SEQ ID NO.13) was ligated into plasmid pET-22b to construct plasmid pET-22b-LcLDH. Q88A / I229A The constructed plasmid pET-22b-LcLDH Q88A / I229A By introducing E. coli BL21, recombinant E. coli / LcLDH was constructed. Q88A / I229A .

[0040] Example 1: Mutation of (2,3)-Butanediol dehydrogenase and construction of recombinant strains

[0041] (1) Preparation of recombinant plasmid pET-Duet-1-bsbdha

[0042] The recombinant strain *E. coli* / bsbdha expressed wild-type (2,3)-butanediol dehydrogenase with the amino acid sequence shown in SEQ ID NO.1, and carried the plasmid pET-Duet-1-bsbdha containing the gene sequence of wild-type (2,3)-butanediol dehydrogenase as shown in SEQ ID NO.2. The recombinant strain *E. coli* / bsbdha was inoculated into 5 mL of LB medium and cultured at 37°C with shaking at 220 rpm for 12 h. After culture, the cells were centrifuged at 13,000 rpm for 1 min and collected. The recombinant plasmid pET-Duet-1-bsbdha was extracted from *E. coli* / bsbdha using the TIANprep Mini Plasmid Mini Kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.).

[0043] (2) Point mutation at amino acid position 42

[0044] The following specific site-directed mutagenesis primers were designed and synthesized:

[0045] H42A-F: 5'-TAgccGAATATCTGGGCGGCCCGATCTTTATT-3' (SEQ ID NO.3)

[0046] H42A-R: 5'-GCCCAGATATTCggcTAAATCACTTCCGCAGATGCC-3' (SEQ ID NO.4)

[0047] V266C-F: 5'-CGTCATCtgcAGCATTTGGGAAAAAGGT-3' (SEQ ID NO.5)

[0048] V266C-R: 5'-AAATGCTgcaGATGACGGTTTCACCGGC-3' (SEQ ID NO.6)

[0049] G292A-F: 5'-AATTATCgccTACCGCGACATCTTCCCGGCTG-3' (SEQ ID NO.7)

[0050] G292A-R: 5'-CGCGGTAggcGATAATTCCTTTTACTGTACGTTCTTTGA-3' (SEQ ID NO.8)

[0051] Using the recombinant plasmid pET-Duet-1-bsbdha extracted in step (1) as a template, and H42A-F and H42A-R as upstream and downstream primers, PCR amplification was performed using the Fast Mutagenesis Kit V2 (purchased from Nanjing Novizan Biotechnology Co., Ltd.). The PCR conditions were as follows: pre-denaturation 95℃, 30s; denaturation 95℃, 15s; annealing 60℃, 15s; extension 72℃, 6min; 30 cycles; full extension 72℃, 5min. After PCR, 1μL of Dpn I enzyme was added to the amplification reaction solution and digested at 37℃ for 2h. After digestion, homologous recombination was performed using Exnase II enzyme at 37℃ for 30min. After homologous recombination, 5μL of the reaction solution was transformed into E. coli BL21(DE3) competent cells, plated on ampicillin-resistant LB plates, and cultured at 37℃ for 12-16h to obtain E. coli / bsbdha. H42A Recombinant bacteria.

[0052] (3) Based on the mutation of amino acid position 42, mutate amino acid position 266.

[0053] Take the E. coli / bsbdha obtained in step (2)H42A The recombinant bacteria were prepared in the same manner as in step (1) to obtain plasmid pET-Duet-l-bsbdha H42A , and the plasmid pET-Duet-l-bsbdha H42A was used as a template, V266C-F / R was used as the upstream and downstream primers for mutation, and the same PCR method as in step (2) was used to obtain the recombinant plasmid pET-Duet-l-bsbdha H42A / V266C , and the plasmid pET-Duet-l-bsbdha H42A / V266C .

[0054] (4) Further mutation of the 292nd amino acid

[0055] The strain E. coli / bsbdha H42A / V266C was prepared in the same manner as in step (1) to obtain plasmid pET-Duet-l-bsbdha H42A / V266C , and the plasmid pET-Duet-l-bsbdha H42A / V266C was used as a template, G292A-F / R was used as the upstream and downstream primers for mutation, and the same PCR method as in step (2) was used to construct the recombinant plasmid pET-Duet-l-bsbdha H42A / V266C / G292A , and the plasmid pET-Duet-l-bsbdha H42A / V266C / G292A .

[0056] The recombinant bacteria obtained in steps (1) to (4) were sent to Shanghai Shengong Biotechnology Co., Ltd. for sequencing to confirm the base sequence.

[0057] Example 2: Induced expression and purification of (2,3)-butanediol dehydrogenase mutants

[0058] (1) Induced expression of mutant enzymes

[0059] The recombinant bacteria E. coli / bsbdha H42A , E. coli / bsbdha H42A / V266C , E. coli / bsbdha H42A / V266C / G292A and the wild-type (2,3)-butanediol dehydrogenase recombinant bacteria E. coli / bsbdha prepared in Example 1 were inoculated into 2 mL of LB medium containing 100 μg / mL ampicillin, and cultured at 37°C and 220 r / min overnight. 2 mL of the culture was transferred into 100 mL of LB medium containing 100 μg / mL ampicillin, and cultured until the OD 600When the ratio of OD600 to OD660 was 0.6-0.8, IPTG (final concentration 0.5 mmol / L) was added, and induction was performed at 16°C for 20 h. The bacterial cells were collected and suspended in a sodium phosphate buffer (Na2HPO4-NaH2PO4, 50 mmol / L, pH 8.5) to a concentration of 100 mg / mL. The bacterial suspension was subjected to ultrasonic wave crushing (power 200 W, ultrasonic crushing for 3 s, interval 8 s, total effective crushing time 15 min) in an ice water bath. The cell crushing solution was then centrifuged (4°C, 12,000 rpm, centrifugation for 15 min) at low temperature to separate the cell debris. The supernatant was filtered through a 0.45 μm cellulose acetate microporous filter to remove impurities, and the crude enzyme solution of the recombinant mutant enzyme was obtained.

[0060] (2) Purification of the mutant enzyme

[0061] The prepared Ni-NTA column was pretreated with the purification buffer, and the crude enzyme solution prepared in step (1) was loaded after equilibration. The nickel column was washed with a buffer containing 75 mmol / L imidazole (Tris-HCl, 20 mmol / L, pH 8.5, 500 mM NaCl) to remove most of the impure proteins. The imidazole concentration in the buffer was then increased to 200 mmol / L (Tris-HCl, 20 mmol / L, pH 8.5, 500 mM NaCl) to elute the target protein. The collected target protein was finally concentrated by a 30 kDa ultrafiltration membrane, and the concentration was determined using a NanoDrop One to obtain the pure enzyme solution of the mutant enzyme BsBDHA H42A , BsBDHA H42A / V266C , BsBDHA H42A / V266C / G292A , and the pure enzyme solution of wild-type BsBDHA.

[0062] Example 3: Determination of the catalytic activity of (2,3)-butanediol dehydrogenase mutants

[0063] The (2,3)-butanediol dehydrogenase mutants BsBDHA H42A / V266C / G292A and wild-type BsBDHA prepared in Example 2 were diluted to a concentration of 0.5 mg / mL with a sodium phosphate buffer (pH 8.5, 50 mmol / L) for enzyme activity determination. The enzyme activity determination system: NAD + concentration 2.5 mmol / L, ZnSO4concentration 0.01 mmol / L, (R)-PED final concentration 10 mmol / L, and pure enzyme solution concentration 0.02 mg / mL. The reaction solution was placed in a 30°C shaking incubator at 220 r / min for 3 min, and then boiled in a boiling water bath for 3 min to terminate the reaction. The OD value at 340 nm was determined by ultraviolet spectrophotometry.

[0064] Enzyme activity unit definition: Under the conditions of this assay, one activity unit (U) is defined as the amount of enzyme required to generate 1 μmol NADH per minute. The blank group was replaced with buffer solution as a control. The results are shown in Table 1. The mutant enzyme BsBDHA... H42A / V266C / G292A Its specific activity was 11.60 U / mg, which is 21.5 times that of wild-type BsBDHA (0.54 U / mg).

[0065] Table 1. Specific activity of different BsBDHA-catalyzed (R)-PEDs

[0066]

[0067] Example 4: Determination of kinetic parameters of (2,3)-butanediol dehydrogenase mutant

[0068] The (2,3)-butanediol dehydrogenase mutant BsBDHA prepared in Example 2 was used. H42A / V266C / G292A In combination with wild-type BsBDHA, following the method in Example 3, (R)-PED at concentrations of 2, 5, 10, 20, 30, and 40 mmol / L was used as the substrate, and 0.02 mg / mL of pure enzyme solution was used as the catalyst. The initial reaction rate of the enzyme was determined. Finally, the kinetic parameter of (R)-PED—the Michaelis constant K—was determined. m Value and maximum reaction rate V max The results were obtained using nonlinear fitting with Origin 9.0. Table 2 shows the results for the mutant enzyme BsBDHA. H42A / V266C / G292A K m The value is 4.53mM, k cat The value is 11.40s -1 Catalytic efficiency k cat / K m 2.52mM -1 s -1 Mutant BsBDHA H42A / V266C / G292A The catalytic efficiency is significantly improved compared to wild-type BsBDHA.

[0069] Table 2 BsBDHA H42A / V266C / G292A Kinetic parameters of catalytic (R)-PED

[0070]

[0071] Example 5: Substrate profiling of (2,3)-Butanediol dehydrogenase mutant

[0072] The (2,3)-butanediol dehydrogenase BsBDHA and the mutant BsBDHA prepared in Example 2 were used. H42A / V266C / G292A, the reaction system of Example 3 is used, 10 mmol / L (R)-PED is replaced by (R)-1-(4-chlorophenyl)-1,2-ethanediol, (R)-1-(3-chlorophenyl)-1,2-ethanediol and (R)-1-(3-nitrophenyl)-1,2-ethanediol, and other components in the system remain unchanged. The reaction is carried out in a constant temperature shaker at 30°C and 220 r / min for 3 min, and then the optimal mutant BsBDHA is determined H42A / V266C / G292A The specific activities of the mutant enzymes in catalyzing different substrates are shown in Table 3. The mutant enzyme BsBDHA H42A / V266C / G292A The specific activities of the mutant enzymes in catalyzing different substrates are shown in Table 3. The mutant enzyme BsBDHA H42A / V266C / G292A The specific activity of the mutant enzyme in catalyzing (R)-1-(3-nitrophenyl)-1,2-ethanediol is 0.21 U / mg, and the wild type BsBDHA does not show catalytic activity for this substrate.

[0073] Table 3 Specific activities of the mutant relative to the wild type for different substrates

[0074]

[0075]

[0076] Note: "-" means no activity

[0077] Example 6: Application of (2,3)-butanediol dehydrogenase mutant in the synthesis of 2-hydroxyacetophenone

[0078] Preparation of crude enzyme solution: Take the recombinant strain E. coli / LcLDH Q88A / I229A and the recombinant strain E. coli / bsbdha prepared in Example 1 H42A / V266C / G292A , culture and collect the bacterial cells, perform ultrasonic disruption with an ultrasonic power of 40 w, work for 3 s, interval of 8 s, and continue for half an hour. After disruption, the crude enzyme solution of LcLDH Q88A / I229A and BsBDHA H42A / V266C / G292A is collected. The crude enzyme solution is used to catalyze the synthesis of 2-hydroxyacetophenone from (R)-PED, and the specific steps are as follows:

[0079] The synthesis of 2-hydroxyacetophenone is shown in the schematic diagram Figure 1 , 20 mmol / L (R)-PED, 10 mmol / L NAD + and 75 mg / mL of BsBDHA H42A / V266C / G292A and LcLDH Q88A / I229AThe crude enzyme solution was reacted in a constant temperature stirring reactor at 30℃, 120r / min for 24h. The reaction process was monitored by sampling at fixed time points and using liquid chromatography. The conversion rate was calculated by integrating the peak area of the substrate and product by liquid chromatography. The conversion rate calculation formula is: conversion rate % = reduced substrate peak area after reaction / substrate peak area before reaction.

[0080] The results are shown in Figure 2 : at 1h, the conversion rate reached 58.6%, and the conversion rate catalyzed by BsBDHA was only 49.2% in the same reaction time, indicating that the enzyme activity for synthesizing 2-hydroxyacetophenone was improved, and the reaction equilibrium time was significantly shortened. After 6h of reaction, the mutant BsBDHA H42A / V266L / G292A catalyzed the conversion rate of 2-hydroxyacetophenone to reach 92.1%.

[0081] Comparative Example 1: Only the 42nd histidine is mutated

[0082] According to the same strategy of Reference Examples 1-4, the 42nd histidine is respectively single-mutated to obtain mutants BsBDHA H42A , BsBDHA H42C , BsBDHA H42I , BsBDHA H42L , BsBDHA H42S , BsBDHA H42T , and BsBDHA H42V The specific activity and kinetic parameters of the mutants for catalyzing (R)-PED were determined, and the results are shown in Table 4. The specific activity of mutants BsBDHA H42A and BsBDHA H42T was higher than that of the wild type, and the rest of the 42nd mutation showed lower activity or no activity.

[0083] Table 4 Specific activity and kinetic parameters of different BsBDHA for catalyzing (R)-PED

[0084]

[0085]

[0086] Note: “-” indicates no activity after mutation

[0087] Part of the sequence of the application:

[0088] BsBDHA wild type amino acid sequence SEQ ID NO. 1:

[0089] MKAARWHNQKDIRIEHIEEPKTEPGKVKIKVKWCGICGSDLHEYLGGPIFIPVDKPHPLTNETAPVTMGHEFSGEVVEVGEGVENYKVGDRVVVEPIFATHGHQGAYNLDEQMGFLGLAGGGGGFSEYVSVDEELLFKLPDELSYEQGALVEPSAVALYAVRSSKLKAGDKAAVFGCGPIGLLVIEALKAAGATDIYAVELSPERQQKAEELGAIIVDPSKTDDVVAEIAERTGGGVDVAFEVTGVPVVLRQAIQSTTIAGETVIVSIWEKGAEIHPNDIVIKERTVKGIIGYRDIFPAVLSLMKEGYFSADKLVTKKIVLDDLIEEGFGALIKEKSQVKILVRPN

[0090] BsBDHA wild type nucleotide sequence SEQ ID NO. 2:

[0091]

[0092] LcLDH Q88A / I229A Amino acid sequence SEQ ID NO. 9:

[0093] MASITDKDHQKVILVGDGAVGSSYAYAMVLQGIAQEIGIVDIFKDKTKGDAIDLSNALPFTSPKKIYSAEYSDAKDADLVVITAGAPAKPGETRLDLVNKNLKILKSIVDPIVDSGFNGIFLVAANPVDILTYATWKLSGFPKNRVVGSGTSLDTARFRQSIAEMVNVDARSVHAYIMGEHGDTEFPVWSHANIGGVTIAEWVKAHPEIKEDKLVKMFEDVRDAAYEIAKLKGATFYGIATALARISKAILNDENAVLPLSVYMDGQYGLNDIYIGTPAVINRNGIQNILEIPLTDHEEESMQKSASQLKKVLTDAFAKNDIETRQ

[0094] Although the present application has been disclosed in its preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and the scope of protection of the present application should be defined by the claims.

Claims

1. An alcohol dehydrogenase mutant, characterized in that, The amino acid sequence of the alcohol dehydrogenase mutant is mutated at position 42 corresponding to SEQ ID NO.

1.

2. The alcohol dehydrogenase mutant of claim 1, wherein, The amino acid sequence of the alcohol dehydrogenase mutant is mutated at position 42 corresponding to SEQ ID NO. 1, and further mutated at position 266 and / or position 292.

3. The alcohol dehydrogenase mutant according to claim 1 or 2, characterized in that, The histidine at position 42 is mutated to alanine or threonine; the valine at position 266 is mutated to cysteine; and the glycine at position 292 is mutated to alanine.

4. A gene encoding the alcohol dehydrogenase mutant of any one of claims 1 to 3.

5. A recombinant plasmid carrying the gene of claim 4.

6. A host cell carrying the gene of claim 4 or the recombinant plasmid of claim 5.

7. The host cell of claim 6, wherein, The host cell is a bacterium or a fungus.

8. A catalyst characterized by, The catalyst contains the alcohol dehydrogenase mutant of any one of claims 1 to 3 or the host cell of any one of claims 6 to 7.

9. A method of synthesizing 2-hydroxyacetophenone, characterized by, The catalyst of claim 8 is used to catalyze the synthesis of 2-hydroxyacetophenone from (R)-1-phenyl-1,2-ethanediol.

10. Use of the alcohol dehydrogenase mutant of any one of claims 1 to 3, the gene of claim 4, the recombinant plasmid of claim 5, or the host cell of any one of claims 6 to 7 in the synthesis of 2-hydroxyacetophenone.

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