Bacillus subtilis alcohol dehydrogenase mutant with improved specific activity and application thereof

Abstract

The invention discloses a bacillus subtilis alcohol dehydrogenase mutant with improved specific activity and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. Amino acids at 49th, 266th and 292th sites of (2, 3)-butanediol dehydrogenase (BsBDHA) of bacillus subtilis are subjected to single mutation and combined mutation to obtain a mutant of which the specific activity and the catalytic efficiency for catalyzing (R)-1-phenyl-1, 2-glycol are remarkably improved, the specific activity reaches 2.44 U / mg or above, and the catalytic efficiency reaches 0.96 mM <-1 > s <-1 > or above; compared with BsBDHAI49L / V266L in the prior art, the (2, 3)-butanediol dehydrogenase mutant disclosed by the invention is improved by 23% or more; the catalytic efficiency of the mutant BsBDHAI49L / V266L is improved by 2.46 times or more compared with that of the mutant BsBDHAI49L / V266L in the prior art; and the conversion rate of 2-hydroxyacetophenone in 12 hours reaches 95.3%.

Description

Technical Field

[0001] This invention relates to a Bacillus subtilis alcohol dehydrogenase mutant with increased specific activity and its applications, belonging to the fields of genetic engineering and enzyme engineering technology. Background Technology

[0002] 2-Hydroxyacetophenone (2-HAP) is used to synthesize a variety of key intermediates and bioactive compounds, such as (R)-2-phenylglycine (a precursor to the novel antibiotic pyrazoline pyrimidine) and hydrazine-Schiff bases (antibacterial and anticancer active compounds). 2-HAP can participate in the synthesis of a wide range of organic compounds, and its unique structure makes it widely applicable in pharmaceutical synthesis, fragrances, and cosmetics.

[0003] To date, various chemical-based methods, such as photocatalysis, nanocomplex or metal-catalyzed oxidation of phenylethanol, and microwave-assisted dehalogenation of bromoacetophenone, have been reported for the preparation of 2-HAP. However, these methods typically require harsh reaction conditions, hazardous metal catalysts, or expensive ligands. With the rise of the global green and sustainable development trend, biocatalytic methods utilizing dehydrogenases to oxidize vicinal diols, which exhibit good stereoselectivity and produce few or no byproducts, are increasingly favored for the preparation of 2-HAP.

[0004] (2,3)-Butanediol dehydrogenases (BDHs, EC 1.1.1.4) are ubiquitous in organisms. They catalyze the interconversion between α-hydroxy ketones and vicinal diols, with NAD(P) as the precursor. + Alternatively, NAD(P)H can be used as a coenzyme. Based on stereoselectivity for 2,3-butanediol, BDHs can be divided into three different types: (meso)-, (2S,3S)-, and (2R,3R)-BDHs. The first two belong to the short-chain dehydrogenase / reductase (SDR) family and are composed of approximately 250 amino acid residues; the latter is widely used in the catalytic reaction for the formation of α-hydroxyketones and belongs to the medium-chain dehydrogenase / reductase (MDR) family, consisting of approximately 350 residues.

[0005] However, most existing wild-type BDHs exhibit higher specific activity and catalytic efficiency (kb) for vicinal diols containing bulky aromatic or aliphatic substituents. cat / K mThe article "Engineering a BsBDHA substrate-binding pocket entrance for the improvement in catalytic performance toward (R)-phenyl-1,2-ethanediol based on the computer-aided design" discovered a wild-type BDH capable of catalyzing the synthesis of 2-HAP from (R)-1-phenyl-1,2-ethanediol (PED), but with low specific activity and catalytic efficiency, at only (0.54 U / mg) and (0.08 mM), respectively. -1 s -1 Based on this, wild-type BDHs were mutated by altering amino acids at positions 49 and 266. The resulting mutants exhibited a maximum specific enzyme activity of 1.98 U / mg and a catalytic efficiency of 0.39 mM. -1 s -1 However, the specific enzyme activity and catalytic efficiency obtained by this technology are still relatively low. Therefore, it is urgent to conduct detailed characterization of novel BDHs or to modify the molecular structure of existing BDHs in order to construct mutants with higher enzyme activity and catalytic efficiency. Summary of the Invention

[0006] To address the issues of low specific enzyme activity and low catalytic efficiency of (2,3)-butanediol dehydrogenase, this invention provides a (2,3)-butanediol dehydrogenase mutant. Compared with the wild-type (2,3)-butanediol dehydrogenase, the mutant enzyme exhibits improved specific enzyme activity and catalytic efficiency. The (2,3)-butanediol dehydrogenase mutant of this invention can be used to synthesize 2-hydroxyacetophenone.

[0007] The first objective of this invention is to provide an alcohol dehydrogenase mutant, wherein the amino acid sequence of the alcohol dehydrogenase mutant is mutated at position 292 corresponding to SEQ ID NO.1.

[0008] In one embodiment, the alcohol dehydrogenase is (2,3)-butanediol dehydrogenase.

[0009] In one embodiment, the amino acid sequence of the alcohol dehydrogenase mutant has undergone a mutation at position 292 corresponding to SEQ ID NO.1, as well as mutations at positions 266 and / or 292.

[0010] In one embodiment, glycine at position 292 is mutated to alanine or cysteine; valine at position 266 is mutated to cysteine, isoleucine, or leucine; and isoleucine at position 49 is mutated to leucine.

[0011] In one embodiment, the alcohol dehydrogenase mutant is obtained by mutating glycine at position 292 of the amino acid sequence of wild-type (2,3)-butanediol dehydrogenase to alanine or cysteine.

[0012] Alternatively, based on the mutation of glycine at position 292 to alanine, the valine at position 266 can be further mutated to cysteine, isoleucine, or leucine.

[0013] Alternatively, by mutating glycine at position 292 to alanine and valine at position 266 to leucine, another mutation can be made by mutating isoleucine at position 49 to leucine.

[0014] A second objective of this invention is to provide a gene encoding the aforementioned alcohol dehydrogenase mutant.

[0015] The present invention also provides recombinant plasmids carrying the above-mentioned genes.

[0016] In one embodiment, the vector for the recombinant plasmid is a pET vector.

[0017] The present invention also provides a host cell carrying the above-described gene or any of the above-described recombinant plasmids.

[0018] In one embodiment, the host cell is a bacterium or a fungus.

[0019] In one embodiment, the microbial cells include, but are not limited to, Escherichia coli cells.

[0020] In one embodiment, the *Escherichia coli* includes, but is not limited to, *E. coli* BL21, *E. coli* BL21(DE3), *E. coli* JM109, *E. coli* DH5α, or *E. coli* TOP10.

[0021] A third object of the present invention is to provide a catalyst containing any of the above-described alcohol dehydrogenase mutants or any of the above-described host cells.

[0022] In one embodiment, the catalyst contains microbial cells expressing the mutant and a catalyst carrier; the catalyst carrier may be a cell protectant.

[0023] The present invention also provides a method for synthesizing 2-hydroxyacetophenone, wherein the above-mentioned catalyst is used to catalyze the synthesis of 2-hydroxyacetophenone from (R)-1-phenyl-1,2-ethylenediol.

[0024] In one embodiment, the above reaction is carried out at 10–30°C.

[0025] In one embodiment, 20 mmol / L (R)-PED, 10 mmol / L NAD+, and 75 mg / mL BsBDHA were added to a 100 mL sodium phosphate buffer (50 mmol / L, pH 8.0) system. I49L / V266L / G292A and LcLDH Q88A / I229A 2-hydroxyacetophenone was synthesized by reacting in a stirred reactor at 30℃ and 120 r / min for 24 h.

[0026] The present invention also provides the use of any of the above-mentioned alcohol dehydrogenase mutants, any of the above-mentioned genes, any of the above-mentioned recombinant plasmids, or any of the above-mentioned host cells in the synthesis of 2-hydroxyacetophenone.

[0027] Beneficial effects:

[0028] This invention involves single and combined mutations at amino acid positions 49, 266, and 292 of (2,3)-butanediol dehydrogenase (BsBDHA) in Bacillus subtilis, resulting in mutants with significantly enhanced specific activity and catalytic efficiency for R-PED compared to wild-type BsBDHA. The specific activity reaches over 2.44 U / mg, and the catalytic efficiency reaches 0.96 mM. -1 s -1 above.

[0029] In particular, the mutant BsBDHA I49L / V266L / G292A The specific activity and catalytic efficiency reached 14.81 U / mg and 4.47 mM, respectively. -1 s -1 Compared with wild-type BsBDHA, the efficiency was increased by 27.43 and 55.75 times, respectively. The conversion rate of catalytic synthesis of 2-hydroxyacetophenone reached 72.2% in 1 h, which was 46.1% higher than that of wild-type BsBDHA, and the reaction equilibrium time was significantly shortened.

[0030] The mutant BsBDHA of this invention I49L / V266L / G292A Compared to existing technologies, BsBDHA I49L / V266L The specific activity was increased by 7.5 times; the catalytic efficiency was higher than that of the mutant BsBDHA in the existing technology. I49L / V266L It increased by 11.5 times. Attached Figure Description

[0031] Figure 1 A schematic diagram of the synthesis of 2-HAP via (R)-PED catalysis by BsBDHAI49L / V266L / G292A;

[0032] Figure 2 For BsBDHA I49L / V266L / G292A Schematic diagram of the reaction process for the synthesis of 2-HAP by BsBDHA-catalyzed (R)-PED. Detailed Implementation

[0033] In order to better understand the technical content of the present invention, the following embodiments are provided in detail. The purpose of these embodiments is only to better understand the content of the present invention and not to limit the scope of protection of the present invention.

[0034] Experimental materials

[0035] 1. Construction of plasmid pET-Duet-1-bsbdha

[0036] 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.

[0037] 2. Construction of recombinant E. coli / bsbdha

[0038] The plasmid pET-Duet-1-bsbdha obtained in (1) was transformed into E. coli BL21(DE3) to construct the recombinant strain E. coli / bsbdha.

[0039] 3. Recombinant E. coli / LcLDH Q88A / I229A Construction

[0040] 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 .

[0041] Example 1: Mutation of (2,3)-Butanediol dehydrogenase and construction of recombinant strains

[0042] (1) Preparation of recombinant plasmid pET-Duet-1-bsbdha

[0043] 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 the recombinant strain *E. coli* / bsbdha using the TIANprep Mini Plasmid Mini Kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.).

[0044] (2) Point mutation at amino acid position 292

[0045] The following specific site-directed mutagenesis primers were designed and synthesized:

[0046] I49L-F: 5'-CCGctcTTTATTCCGGTTGACAAACCGCACCC-3' (SEQ ID NO.3)

[0047] I49L-R: 5'-ACCGGAATAAAgagCGGGCCGCCCAGATATTC-3' (SEQ ID NO.4)

[0048] V266C-F: 5'-CGTCATCtgcAGCATTTGGGAAAAAGGT-3' (SEQ ID NO.5)

[0049] V266C-R: 5'-AAATGCTgcaGATGACGGTTTCACCGGC-3' (SEQ ID NO.6)

[0050] V266I-F: 5'-CGTCATCatcAGCATTTGGGAAAAAGGT-3' (SEQ ID NO.7)

[0051] V266I-R: 5'-AAATGCTgatGATGACGGTTTCACCGGC-3' (SEQ ID NO.8)

[0052] V266L-F: 5'-CGTCATCctcAGCATTTGGGAAAAAGGT-3' (SEQ ID NO.9)

[0053] V266L-R: 5'-AAATGCTgagGATGACGGTTTCACCGGC-3' (SEQ ID NO.10)

[0054] G292A-F: 5'-AATTATCgccTACCGCGACATCTTCCCGGCTG-3' (SEQ ID NO.11)

[0055] G292A-R: 5'-CGCGGTAggcGATAATTCCTTTTACTGTACGTTCTTTGA-3' (SEQ ID NO.12)

[0056] Using the recombinant plasmid pET-Duet-1-bsbdha extracted in step (1) as a template, and G292A-F and G292A-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. G292A Recombinant bacteria.

[0057] (3) Based on the mutation of amino acid position 292, mutate amino acid position 266.

[0058] Take the E. coli / bsbdha obtained in step (2) G292A The recombinant bacteria were prepared using the same method as in step (1) to obtain the plasmid pET-Duet-1-bsbdha. G292A Using plasmid pET-Duet-1-bsbdha G292A Using V266C-F / R, V266I-F / R, and V266L-F / R as templates, the upstream and downstream primers were used for mutation. Recombinant plasmids were obtained using the same PCR method as in step (2), and then transformed into E. coli BL21(DE3) competent cells, ultimately obtaining recombinant E. coli / bsbdha. V266C / G292A E. coli / bsbdha V266I / G292A and E. coli / bsbdha V266L / G292A .

[0059] (4) Further mutation of amino acid position 49

[0060] The strain E. coli / bsbdha prepared in step (3) V266L / G292A The plasmid pET-Duet-1-bsbdha was prepared in the same manner as in step (1). V266L / G292A Using plasmid pET-Duet-1-bsbdha V266L / G292A Using I49L-F and I49L-R as templates, and employing the same PCR method as in step (2), a recombinant plasmid was obtained. This plasmid was then transformed into E. coli BL21(DE3) competent cells, ultimately yielding the recombinant strain E. coli / bsbdha. I49L / V266L / G292A .

[0061] The recombinant bacteria obtained in steps (1) to (4) were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing to confirm the base sequence.

[0062] Example 2: Induction of expression and purification of (2,3)-butanediol dehydrogenase mutant

[0063] (1) Induced expression of mutant enzymes

[0064] Recombinant E. coli / bsbdha V266C / G292A E. coli / bsbdha V266I / G292A E. coli / bsbdha V266L / G292A and E. coli / bsbdha I49L / V266L / G292A Single colonies of wild-type E. coli / bsbdha were inoculated into 2 mL of LB medium containing 100 μg / mL ampicillin and incubated overnight at 37°C and 220 rpm. 2 mL of the culture was then transferred to 100 mL of LB medium also containing 100 μg / mL ampicillin and incubated until OD500. 600 When the pH was 0.6–0.8, IPTG (final concentration 0.5 mmol / L) was added, and induction was performed at 16℃ for 20 h. Bacterial cells were collected separately, and a 100 mg / mL bacterial suspension was prepared using sodium phosphate buffer (Na₂HPO₄-NaH₂PO₄, 50 mmol / L, pH 8.5). The bacterial suspension was then sonicated in an ice-water bath (200 W power, 3 s sonication, 8 s interval, total effective disruption time 15 min). The cell disruption solution was then centrifuged at low temperature (4℃, 12,000 rpm, 15 min) to separate cell debris. The supernatant was filtered through a 0.45 μm cellulose acetate microporous membrane to remove impurities, yielding crude enzyme solutions of both the non-mutated and mutant enzymes.

[0065] (2) Purification of mutant enzymes

[0066] The packed Ni-NTA columns were pretreated with purification buffer and equilibrated before loading the crude enzyme solution prepared in step (1). 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 impurities. 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 then concentrated using a 30 kDa ultrafiltration membrane, and the concentration was determined using NanoDrop One to obtain the pure enzyme solution BsBDHA of the mutant enzyme. V266C / G292A 、BsBDHA V266I / G292A 、BsBDHA V266L / G292A 、BsBDHA I49L / V266L / G292A Pure enzyme solution of wild-type BsBDHA.

[0067] Example 3: Determination of the catalytic specific activity of (2,3)-butanediol dehydrogenase mutant

[0068] The (2,3)-butanediol dehydrogenase mutant prepared in Example 2 was diluted with sodium phosphate buffer (pH 8.5, 50 mmol / L) to a concentration of 0.5 mg / mL for enzyme activity assay. Enzyme activity assay system (final concentration): NAD + The concentration of the enzyme was 2.5 mmol / L, the concentration of ZnSO4 was 0.01 mmol / L, the final concentration of (R)-PED was 10 mmol / L, and the concentration of the pure enzyme solution was 0.02 mg / mL. The reaction solution was placed at 30℃ and reacted on a shaker at 220 rpm 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 measured using a UV spectrophotometer.

[0069] 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... V266C / G292A 、BsBDHA V266I / G292A and BsBDHA I49L / V266L / G292A The specific activities were 4.25, 2.44 and 14.81 U / mg, respectively, which were 9.05, 4.52 and 27.43 times that of wild-type BsBDHA (0.54 U / mg).

[0070] Table 1. Specific activity of different BsBDHA-catalyzed (R)-PEDs

[0071]

[0072] Example 4: Determination of kinetic parameters of (2,3)-butanediol dehydrogenase mutant

[0073] The (2,3)-butanediol dehydrogenase mutant prepared in Example 2 was used in accordance with the method in Example 3, with (R)-PED at concentrations of 2, 5, 10, 20, 30, and 40 mmol / L as substrate and 0.02 mg / mL pure enzyme solution as catalyst. The initial reaction rate of the enzyme was measured. 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. V266C / G292A 、BsBDHA V266I / G292A and BsBDHA I49L / V266L / G292A K m The values ​​were 2.03, 1.93, and 2.81 mM, kJ, respectively. cat The values ​​were 3.31, 1.86, and 12.55s, respectively. -1 Catalytic efficiency k cat / K m The values ​​were 1.63, 0.96, and 4.47 mM, respectively. -1 s -1 It is evident that the mutant BsBDHA I49L / V266L / G292A It has the highest catalytic efficiency.

[0074] Table 2 Kinetic parameters of different BsBDHA-catalyzed (R)-PED

[0075]

[0076]

[0077] Example 5: Substrate profiling of (2,3)-Butanediol dehydrogenase mutant

[0078] The (2,3)-butanediol dehydrogenase BsBDHA and the mutant BsBDHA prepared in Example 2 were used. I49L / V266L / G292A Following the reaction system of Example 3, 10 mmol / L (R)-PED was replaced with (R)-1-(4-chlorophenyl)-1,2-ethylenediol, (R)-1-(3-chlorophenyl)-1,2-ethylenediol, and (R)-1-(3-nitrophenyl)-1,2-ethylenediol, while other components remained unchanged. The reaction was carried out in a constant-temperature shaking reactor at 30°C and 220 r / min for 3 min, after which the optimal mutant BsBDHA was determined. I49L / V266L / G292A The specific activities of different substrates were catalyzed, and the results are shown in Table 3. Mutant enzyme BsBDHA V266C / G292AThe specific activities of (R)-1-(4-chlorophenyl)-1,2-ethylenediol and (R)-1-(3-chlorophenyl)-1,2-ethylenediol were 2.89 and 3.97 U / mg, respectively, which were 9.03 and 11.34 times that of wild-type BsBDHA. I49L / V266L / G292A The specific activity of (R)-1-(3-nitrophenyl)-1,2-ethylene glycol was 0.27 U / mg, while wild-type BsBDHA did not show catalytic activity for this substrate.

[0079] Table 3. Specific activity of mutants compared to wild-type for different substrates.

[0080]

[0081] Note: "–" indicates no activity.

[0082] Example 6: Application of (2,3)-Butanediol dehydrogenase mutant in the synthesis of 2-hydroxyacetophenone

[0083] Preparation of crude enzyme solution: Take recombinant strain E. coli / LcLDH Q88A / I229A The recombinant strain E. coli / bsbdha prepared in Example 1 I49L / V266L / G292A Bacterial cells were cultured and collected, then subjected to ultrasonic disruption at a power of 40W for 3 seconds, with an 8-second interval, for a total of half an hour. LcLDH was collected after disruption. Q88A / I229A and BsBDHA I49L / V266L / G292A The crude enzyme solution was used to catalyze the synthesis of 2-hydroxyacetophenone from (R)-PED. The specific steps are as follows:

[0084] A schematic diagram of the catalysis is shown below. Figure 1 As shown, 20 mmol / L (R)-PED and 10 mmol / L NAD were added to a 100 mL sodium phosphate buffer (50 mmol / L, pH 8.0) system, respectively. + and 75 mg / mL BsBDHA I49L / V266L / G292A and LcLDH Q88A / I229A The crude enzyme solution was reacted in a stirred reactor at 30℃ and 120 rpm for 24 h. Samples were taken at fixed time points, and the reaction progress was monitored by liquid chromatography. The conversion rate was calculated by integrating the peak areas of the substrate and product in the liquid chromatography. The formula for calculating the conversion rate is: Conversion rate % = Decrease in substrate peak area / Initial substrate peak area.

[0085] The results are as follows Figure 2 As shown: After 1 hour of reaction, the mutant BsBDHA I49L / V266L / G292AThe conversion rate reached 72.2%, while the conversion rate catalyzed by wild-type BsBDHA within the same reaction time was only 49.2%, indicating that the enzyme activity was improved and the reaction equilibrium time was significantly shortened. After 12 hours of reaction, BsBDHA... I49L / V266L / G292A The catalytic conversion rate reached 95.3%.

[0086] Comparative Example 1: Mutation of only amino acid position 292

[0087] Following the same strategy as in Examples 1-4, a single mutation was performed on glycine at position 292 to obtain the mutant BsBDHA. G292A 、BsBDHA G292C 、BsBDHA G292I 、BsBDHA G292L 、BsBDHA G292S 、BsBDHA G292T and BsBDHA G292V The specific activity of the mutant catalyzed (R)-PED was determined, and the results are shown in Table 4. (BsBDHA) G292A 、BsBDHA G292C Compared to wild-type BsBDHA, the activity was increased, while the remaining mutants at position 292 all showed lower activity or no activity.

[0088] Table 4. Specific activity and kinetic parameters of different BsBDHA-catalyzed (R)-PED processes.

[0089]

[0090] Note: "–" indicates that the mutation has no activity.

[0091] Partial sequence of the present invention:

[0092] BsBDHA wild-type amino acid sequence SEQ ID NO.1:

[0093] MKAARWHNQKDIRIEHIEEPKTEPGKVKIKVKWCGICGSDLHEYLGGPIFIPVDKPHPLTNETAPVTMGHEFSGEVVEVGEGVENYKVGDRVVVEPIFATHGHQGAYNLDEQMGFLGLA GGGGGFSEYVSVDEELLFKLPDELSYEQGALVEPSAVALYAVRSSKLKAGDKAAVFGCGPIGLLVIEALKAAGATDIYAVELSPERQQKAEELGAIIVDPSKTDDVVAEIAERTGGGVDVAFEVTGVPVVLRQAIQSTTIAGETVIVSIWEKGAEIHPNDIVIKERTVKGIIGYRDIFPAVLSLMKEGYFSADKLVTKKIVLDDLIEEGFGALIKEKSQVKILVRPN

[0094] Wild-type nucleotide sequence of BsBDHA SEQ ID NO.2:

[0095]

[0096] LcLDH Q88A / I229A Amino acid sequence SEQ ID NO.13:

[0097] MASITDKDHQKVILVGDGAVGSSYAYAMVLQGIAQEIGIVDIFKDKTKGDAIDLSNALPFTSPKKIYSAEYSDAKDADLVVITAGAPAKPGETRLDLVNKNLKILKSIVDPIVDSGFNGIFLVAANPVDILTYATWKLSGFPKNRVVGSGTSLDTARFRQSIA EMVNVDARSVHAYIMGEHGDTEFPVWSHANIGGVTIAEWVKAHPEIKEDKLVKMFEDVRDAAYEIAKLKGATFYGIATALARISKAILNDENAVLPLSVYMDGQYGLNDIYIGTPAVINRNGIQNILEIPLTDHEEESMQKSASQLKKVLTDAFAKNDIETRQ

[0098] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An alcohol dehydrogenase mutant, characterized in that, The amino acid sequence of the alcohol dehydrogenase mutant was mutated at position 292 of SEQ ID NO.

1.

2. The alcohol dehydrogenase mutant according to claim 1, characterized in that, The amino acid sequence of the alcohol dehydrogenase mutant has undergone a mutation at position 292 corresponding to SEQ ID NO.1, as well as mutations at positions 266 and / or 292.

3. The alcohol dehydrogenase mutant according to claim 1 or 2, characterized in that, The glycine at position 292 is mutated to alanine or cysteine; the valine at position 266 is mutated to cysteine, isoleucine, or leucine; and the isoleucine at position 49 is mutated to leucine.

4. A gene encoding the alcohol dehydrogenase mutant 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 according to claim 6, characterized in that, The host cell is a bacterium or a fungus.

8. A catalyst, characterized in that, The catalyst contains any of the alcohol dehydrogenase mutants of claims 1 to 3 or any of the host cells of claims 6 to 7.

9. A method for synthesizing 2-hydroxyacetophenone, characterized in that, The catalyst of claim 8 is used to catalyze the synthesis of 2-hydroxyacetophenone from (R)-1-phenyl-1,2-ethylene glycol.

10. The 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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