A (2R, 3R)-butanediol dehydrogenase mutant and its application in the preparation of (2S)-hydroxycyclohexanone

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a (2 R ,3 R )-butanediol dehydrogenase mutant and its preparation (2 S )-hydroxycyclohexanone. The present invention is for wild type Bs BDH was subjected to multiple rounds of mutations, and mutants with significantly improved thermal stability were screened out. cis The catalytic activity of CHD was also significantly improved, among which the mutants with the introduction of non-natural amino acids were more efficient than the wild type. Bs 214.48 times of BDH, it can be seen that the application provides Bs BDH mutants catalyze the oxidation of cis-1,2-cyclohexanediol to prepare (2 S )-hydroxycyclohexanone has great application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a (2 R ,3 R )-Butanediol dehydrogenase mutant and its preparation (2 S )-hydroxycyclohexanone. Background Art

[0002] The steric hindrance of cyclic α-hydroxyketones and their downstream products cyclic β-amino alcohols gives them certain biological activities and they are often used as key building blocks for the synthesis of antiviral and antagonist drugs. For example, the cholecystokinin (CCK-B) receptor antagonist (CI-1015) contains (1 S ,2 S )-trans-2-aminocyclohexanol, which can be used to treat anxiety and panic disorders; the synthesis of phosphodiesterase III inhibitors and antiarrhythmic drugs vernakalant requires (1 R ,2 R )-trans-2-aminocyclohexanol. Therefore, the green and efficient synthesis of optically pure cyclic α-hydroxyketones has good application prospects.

[0003] According to existing literature reports, cyclic α-hydroxyketones and cyclic β-amino alcohols are usually produced using an enzyme cascade catalysis method. First, the epoxy compound is hydrolyzed by epoxide hydrolase to form a vicinal diol; then, the vicinal diol is selectively oxidized by 2,3-butanediol dehydrogenase to form an α-hydroxyketone; finally, the α-hydroxyketone is further oxidized by a transaminase to form a β-amino alcohol. The production methods previously reported in the literature all used Bacillus subtilis as the source ( Bacillus subtilis 2,3-Butanediol dehydrogenase ( Bs BHD) catalyzes the oxidation of vicinal diols to α-hydroxy ketones.

[0004] but Bs The low catalytic activity of BDH towards cyclic vicinal diols severely limits the efficiency of the enzyme cascade catalysis, and Bs BDH has low thermal stability, which leads to serious loss of catalytic activity of the enzyme during use, showing low catalytic activity, and is still a long way from industrial application.

[0005] In Chinese patent CN116042557A, Bs Single-point and double-point mutations were performed on BDH to obtain a product with improved thermal stability. Bs BDH mutants, but they remain unresolved Bs The problem is that BDH has low catalytic activity towards cyclic vicinal diols. Summary of the Invention

[0006] Regarding the existing technology BsThe present invention provides a method for improving the catalytic activity of BDH for cyclic vicinal diols. R ,3 R )-Butanediol dehydrogenase mutant and its preparation (2 S )-hydroxycyclohexanone, the specific technical solution is as follows:

[0007] In a first aspect, the present invention provides a (2 R ,3 R )-butanediol dehydrogenase mutant, obtained by mutating the amino acid sequence shown in SEQ ID NO.1, wherein the mutant form is one of the following:

[0008] (1) Threonine at position 22 mutated to valine, asparagine at position 61 mutated to glycine, glutamine at position 112 mutated to asparagine, alanine at position 230 mutated to arginine, alanine at position 260 mutated to methionine, and isoleucine at position 280 mutated to leucine;

[0009] (2) Threonine at position 22 mutated to valine, glutamine at position 112 mutated to asparagine, alanine at position 230 mutated to arginine, threonine at position 258 mutated to glycine, alanine at position 260 mutated to methionine, and isoleucine at position 280 mutated to leucine;

[0010] (3) Threonine at position 22 mutated to valine, asparagine at position 61 mutated to glycine, alanine at position 230 mutated to arginine, threonine at position 258 mutated to glycine, alanine at position 260 mutated to methionine, and isoleucine at position 280 mutated to leucine;

[0011] (4) Threonine at position 22 mutated to valine, asparagine at position 61 mutated to glycine, glutamine at position 112 mutated to asparagine, alanine at position 230 mutated to arginine, alanine at position 260 mutated to methionine, isoleucine at position 280 mutated to leucine, and tyrosine at position 293 mutated to serine;

[0012] (5) Threonine at position 22 mutated to valine, asparagine at position 61 mutated to glycine, glutamine at position 112 mutated to asparagine, leucine at position 118 mutated to phenylalanine, alanine at position 230 mutated to arginine, alanine at position 260 mutated to methionine, isoleucine at position 280 mutated to leucine, and tyrosine at position 293 mutated to serine.

[0013] The present invention selects 9 to improve BsSingle-point mutations in BDH catalytic activity and thermal stability were randomly combined by Gibson assembly to obtain combined mutants with significantly improved thermal stability and catalytic activity. On this basis, saturation mutations were performed using semi-rational design, and finally a combination mutant with improved thermal stability and catalytic activity was obtained. cis -CHD) catalytic activity is improved Bs BDH mutants.

[0014] In a second aspect, the present invention provides a non-natural amino acid containing (2 R ,3 R )-butanediol dehydrogenase mutant, obtained by performing the following mutations on the amino acid sequence shown in SEQ ID NO.1:

[0015] Threonine at position 22 mutated to valine, phenylalanine at position 50 mutated to 2,5-dichlorophenylalanine, glutamine at position 112 mutated to asparagine, leucine at position 118 mutated to phenylalanine, alanine at position 230 mutated to arginine, threonine at position 258 mutated to glycine, alanine at position 260 mutated to methionine, isoleucine at position 280 mutated to leucine, and tyrosine at position 293 mutated to serine.

[0016] The present invention found that in wild type Bs In the BDH structure, F50, F115, and I291 form hydrophobic interactions, which seriously hinder the release of the product. Therefore, the present invention Bs Based on the BDH mutant, 2,5-dichlorophenylalanine was introduced into the F50 site to further expand the substrate channel, thereby significantly improving the cis -CHD catalytically active mutants.

[0017] In a third aspect, the present invention provides a method for encoding the above-mentioned (2 R ,3 R )-butanediol dehydrogenase mutant gene.

[0018] In a fourth aspect, the present invention provides a recombinant vector comprising the above-mentioned gene.

[0019] In a fifth aspect, the present invention provides a genetically engineered bacterium comprising the above-mentioned gene.

[0020] In a sixth aspect, the present invention provides the above-mentioned (2 R ,3 R )-Butanediol dehydrogenase mutant catalyzes the oxidation of cis-1,2-cyclohexanediol to prepare (2 S )-hydroxycyclohexanone application.

[0021] In a seventh aspect, the present invention provides a biocatalytic synthesis (2 S)-hydroxycyclohexanone method, using cis-1,2-cyclohexanediol as a substrate, and using the above (2 R ,3 R )-butanediol dehydrogenase mutant as catalyst, forming a reaction system, synthesizing (2 S )-hydroxycyclohexanone.

[0022] Furthermore, the reaction system includes 0.004 mM to 0.03 mM zinc ions.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention is for wild type Bs BDH was subjected to multiple rounds of mutations, and mutants with significantly improved thermal stability were screened out. cis The catalytic activity of -CHD was also significantly improved, among which the mutants with the introduction of unnatural amino acids were more efficient than the wild type in terms of catalytic efficiency. Bs 214.48 times of BDH, it can be seen that the application provides Bs BDH mutants catalyze the oxidation of cis-1,2-cyclohexanediol to prepare (2 S )-hydroxycyclohexanone has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a graph showing the residual enzyme activity assay results for WT and 6M1, 6M2, and 6M3 in Example 1.

[0026] Figure 2 is the WT and 6M1, 6M2, 6M3 in Example 1 T m Value measurement result diagram.

[0027] Figure 3 The crude enzyme solutions of WT, 6M1, 6M2, and 6M3 in Example 1 and the purified enzymes are shown in Table 1. cis -CHD specific enzyme activity assay results.

[0028] Figure 4 This is a diagram showing the enzyme activity assay results of the forward mutants obtained by high-throughput screening in Example 2.

[0029] Figure 5 Schematic diagram of the replacement position of the 6×His tag in Example 3.

[0030] Figure 6 Schematic diagram of protein purification before and after the 6×His tag was repositioned in Example 3; wherein, A: N-terminal 6×His tag, B: C-terminal 6×His tag, 1: bacterial liquid, 2: loading flow-through, 3: washing flow-through, 4: elution flow-through.

[0031] Figure 7 Zn in GMML medium in Example 3 2+ The effect of concentration on enzyme catalytic activity.

[0032] Figure 8 This is a graph showing the effects of the 6×His tag position and culture medium type on enzyme catalytic activity in Example 3.

[0033] Figure 9 Schematic diagram of the product release channel in Example 4.

[0034] Figure 10 This is a graph showing the effect of introducing a non-natural amino acid at position 50 on the enzyme catalytic activity in Example 4.

[0035] Figure 11 Schematic diagram of the product release channel when 3-chlorophenylalanine is introduced into position 50 in Example 4.

[0036] Figure 12 Schematic diagram of the product release channel when 2,5-dichlorophenylalanine is introduced into site 50 in Example 4. DETAILED DESCRIPTION

[0037] In order to make those skilled in the art better understand the present invention, the technical solution of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and is only a part of the embodiments of the present invention, rather than all embodiments.

[0038] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0040] The embodiments of the present invention use E. coli BL21 (DE3) was purchased from Novagen; plasmids such as pET-28a (+) were purchased from Novagen; Bs The gene synthesis, primer synthesis and sequence sequencing of BDH were completed by Qingke Bioengineering Co., Ltd.

[0041] Reagents used in the catalytic reaction: Tris (tris(hydroxymethyl)aminomethane) and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co., Ltd.; NAD+ (nicotinamide adenine dinucleotide) was purchased from Bontai Bioengineering (Shenzhen) Co., Ltd.; cis-1,2-cyclohexanediol was purchased from Shanghai Myrrel Biochemical Technology Co., Ltd.

[0042] Reagents used for the introduction of unnatural amino acids: L-3-chlorophenylalanine was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and L-2,5-dichlorophenylalanine and L-2,5-difluorophenylalanine were purchased from Shanghai MacLean Technology Co., Ltd.

[0043] Definition of enzyme activity unit (U): the amount of enzyme required to generate 1 μmol NADH per minute in an oxidation reaction or consume 1 μmol NADH per minute in a reduction reaction.

[0044] In the following examples, the Bs The amino acid sequence of the wild-type BDH enzyme is shown in SEQ ID NO.1, and its coding sequence is shown in SEQ ID NO.2.

[0045] SEQ ID NO.1:

[0046] MKAARWHNQKDIRIEHIEEPKTEPGKVKIKVKWCGICGSDLHEYLGGPIFIPVDKPHPLTNETAPVTMGHEFSGEVVEVGEGVENYKVGDRVVVEPIFATHGHQGAYNLDEQMGFLGLAGGGGGFSEYVSVDEELLFKLPDELSYEQGALVEPSAVALYAVRSSKLKAGDKAA VFGCGPIGLLVIEALKAAGATDIYAVELSPERQQKAEELGAIIVDPSKTDDVVAEIAERTGGGVDVAFEVTGVPVVLRQAIQSTTIAGETVIVSIWEKGAEIHPNDIVIKERTVKGIIGYRDIFPAVLSLMKEGYFSADKLVTKKIVLDDLIEEGFGALIKEKSQVKILVRPN

[0047] SEQ ID NO.2:

[0048]

[0049] Example 1 Bs Screening of thermostable combined mutants of BDH

[0050] The single point mutations T22V, N61G, Q112N, A230R, T258G, A260M, I280L, R285K, and E335D that improve thermal stability were randomly combined (the thermal stability data of the single point mutations are shown in Table 1). The random combination process used Gibson assembly: (1) The nine sites were divided into three groups according to the principle of site proximity, with T22V, N61G, and Q112N as the first fragment and A2 as the second fragment. 30R, T258G, A260M are the second fragment, I280L, R285K, E335D are the third fragment, and T22V, N61G, Q112N are taken as examples. First, T22V, N61G, Q112N, T22V / N61G, T22V / Q112N, N61G / Q112N, T22V / N61G / Q112N are constructed by site-directed mutagenesis. Considering the wild type, the primers are shown in Table 2. There are 8 possibilities for the first fragment. Similarly, 8 possibilities for the second and third fragments are constructed; (2) PCR is performed on the 8 first fragments using 1_F and 1_R as primers to obtain 8 recombinant fragments. Similarly, PCR is performed on the second and third fragments using 2_F, 2_R, 3_F, and 3_R to obtain recombinant fragments; (3) The recombinant fragments are subjected to Gibson assembly and transformed into E. coli BL21 (DE3), incubated at 37 ° C for 12 hours, single colonies were picked, and colony PCR was performed with 4_F and 4_R. The fragment length was verified by DNA gel electrophoresis, and the band size was consistent with the 1.6 kb determination sequence. The present invention randomly selected 152 mutants or wild types, including 1 wild type, 9 single-point mutations, 29 double-point mutations, 18 triple-point mutations, 38 quadruple-point mutations, 30 five-point mutations, 16 six-point mutations, 8 seven-point mutations, 2 eight-point mutations, and 1 nine-point mutation (the half-life data of some mutants are shown in Table 3).

[0051] Table 1 Thermal stability of single-point mutants

[0052]

[0053] Table 2 Primers used for the construction of thermostable combination mutants

[0054]

[0055] Table 3 Thermal stability of different mutants

[0056]

[0057] PCR amplification system:

[0058] DNA polymerase 25 μL,

[0059] Upstream primer (10 μM) 1 μL,

[0060] Downstream primer (10 μM) 1 μL,

[0061] Template (5 ng / μL) 1 μL,

[0062] ddH2O22 μL.

[0063] PCR amplification conditions:

[0064] 1) Pre-denaturation: 98°C for 3 min;

[0065] 2) Denaturation: 98°C for 10 s; annealing: 60°C for 15 s; extension: 72°C for 1 min; 33 cycles in total;

[0066] 3) Post-extension: 72°C for 5 min;

[0067] 4) Store at 4℃.

[0068] Gibson assembly system:

[0069] First fragment 62 ng

[0070] Second fragment 66 ng

[0071] The third fragment 50 ng

[0072] 5×CE II Buffer 4 μL

[0073] Exnase II 2 μL

[0074] Add ddH2O to 20 μL

[0075] Gibson assembly conditions:

[0076] 1) Reaction: 37°C for 30 min;

[0077] 2) Store at 4℃.

[0078] (2 R ,3 R )-butanediol was used as the substrate and heat inactivation treatment was carried out in a water bath at 37°C for 120 min. The ratio of the specific enzyme activity after heat treatment to the specific enzyme activity before heat treatment was the residual enzyme activity.

[0079] The three combined mutants with the highest half-lives screened out all had six-point mutations: T22V / N61G / Q112N / A230R / A260M / I280L, T22V / Q112N / A230R / T258G / A260M / I280L, and T22V / N61G / A230R / T258G / A260M / I280L, named 6M1, 6M2, and 6M3, respectively. They retained 92.6%, 92.2%, and 87.5% of enzyme activity after treatment in a water bath at 37°C for 120 min, while the wild type had only 10.5% residual enzyme activity under the same heat treatment conditions (e.g., Figure 1 shown). T m The results of the value determination are shown (such as Figure 2 As shown in Figure 3), 6M1, 6M2, and 6M3 increased by 10.1°C, 9.1°C, and 9.4°C, respectively, compared to the wild type. 6M1, 6M2, and 6M3 were purified and tested for their effects on cis -CHD catalytic activity, 6M2 cis The specific enzyme activity of -CHD was better than that of 6M1 and 6M3, at 0.91 U / mg, which was 1.78 times that of the wild type (0.51 U / mg). Figure 3 ), the present invention will use 6M2 as the basis for subsequent substrate binding pocket modification.

[0080] Example 2 Improving the catalytic activity of 6M2 through a semi-rational design strategy

[0081] F115, L118 and Y293 and substrate cis -CHD is close, F115, L118 and the substrate form hydrophobic interactions, affecting the binding of the substrate six-membered ring; while Y293 forms a hydrogen bond with the hydroxyl group of the substrate, playing a positioning role for the hydroxyl group. Therefore, in this Example 2, the mutants were determined to be cis -CHD activity was modified by iterative saturation mutagenesis. The specific screening method is as follows: the saturation mutation PCR product was transformed into E. coli In BL21 (DE3) competent cells, the PCR method was consistent with that in Example 1. After incubation for 12 hours, single clones were picked and transferred to 96-well deep-well plates with 800 μL of LB medium per well. The cells were shaken at 37°C for 4 hours, and IPTG was added to induce the cells at a final concentration of 1 mM. The cells were shaken at 18°C ​​for 18 hours, and 100 μL of the bacterial solution was aspirated and the OD was measured at 600 nm. 600 Centrifuge at 220 rpm for 10 min, collect the cells and remove the supernatant, place on ice, add 200 μL of Tris-HCl lysis buffer containing 1% Triton X-ray, pH 8.0 to a 96-well deep-well plate and resuspend the cells. Lyse for 5 min. Pipette 193 μL of lysis buffer and 5 μL of 500 mM cis -CHD, 2 µL 50 mM NAD + Mix well and measure the absorbance change ΔOD at 340 nm 340 ΔOD 340 / OD 600 The ones that are larger than the parent are screened and matched. cis -CHD mutants with improved catalytic activity.

[0082] from Figure 4 It can be seen that the 6M2 / Y293S pair was screened in the first round of screening. cis The catalytic activity of -CHD was slightly improved to 0.61 U / mg. Therefore, a second round of iterative saturation mutagenesis was performed based on 6M2 / Y293S, and the catalytic activity of 6M2 / Y293S / L118F was further improved to 0.80 U / mg.

[0083] Table 4 Primers used for iterative saturation mutagenesis

[0084]

[0085] Example 3 Exploration of methods and conditions for introducing non-natural amino acids

[0086] 1. Effect of His tag position on protein purification

[0087] The non-natural amino acids were introduced into the system using the gene codon expansion technology based on stop codon suppression. Mb PylRS-IPE / tRNA CUA system, through pULTRA- Mb PylRS-IPE and pET-28a(+)- Bs BDH cotransfection E. coli The BL21(DE3) strain was constructed, in which the IPE was the MbPylRS / N311A / C313A / V31I / T56P / A100E mutant.

[0088] Due to the limitation of the efficiency of introducing non-natural amino acids, there are truncated proteins in the induced expressed proteins due to the termination of expression by TAG codon. If the 6×His tag is expressed at the N-terminus of the protein, the truncated expressed protein will be mixed in the system after protein purification. In this experiment, the 6×His tag was moved from the N-terminus to the C-terminus by PCR (e.g. Figure 5 As shown in Figure 3 ), only the protein expressed to the C-terminus contains a 6×His tag. Therefore, protein purification can yield intact protein with the correct incorporation of unnatural amino acids.

[0089] Table 5 Primers used for 6×His tag position change

[0090]

[0091] In addition, pET-28a(+)- Bs The site where the non-natural amino acid needs to be introduced on BDH is mutagenized to TAG, and pET-28a(+)- Bs BDH and pULTRA- Mb 100 ng of each PylRS-IPE plasmid was added to 100 μL of competent cells and the heat shock transformation process was followed. The bacterial solution was finally spread on a culture plate containing Kan (50 μg / mL) and Spe (50 μg / mL) and cultured at 37°C for 12 h. A single colony was picked for culture and preserved with 25% glycerol and stored in a -80°C refrigerator.

[0092] The introduction of unnatural amino acids was performed as follows: 10 μL of the unnatural amino acid introduction strain stored at -80°C was inoculated into 5 mL of LB liquid medium containing Kan (50 μg / mL) and Spe (50 μg / mL), and cultured at 37°C and 220 rpm for 8 h. 5 mL of the bacterial solution was used as seed liquid and inoculated into 100 mL of GMML liquid medium containing Kan (50 μg / mL) and Spe (50 μg / mL), and cultured at 37°C and 220 rpm until the OD 600 The expression was induced by adding IPTG and unnatural amino acids at a final concentration of 1 mM and shaking at 18°C ​​and 220 rpm for 18 h.

[0093] In this study, the 6×His tag was moved from the N-terminus to the C-terminus by PCR (the mutant with NC indicates that the 6×His tag was moved from the N-terminus to the C-terminus), and 3-chlorophenylalanine was introduced at the 115 position. Bs Taking BDH as an example, the protein purification results are as follows Figure 6 As shown in Figure 2, when the 6×His tag is located at the N-terminus, the protein purification solution contains fully expressed Bs For BDH and truncated proteins, when the 6×His tag was moved to the C-terminus, only a protein band around 52 kDa was present in the protein purification solution, which was consistent with the complete expression. Bs BDH molecular weight.

[0094] 2. Zn 2+ Effect of concentration on enzyme catalytic activity

[0095] Protein expression was performed using GMML medium containing different concentrations of ZnCl2 (0, 0.002, 0.004, 0.01, 0.015, 0.02, 0.03, 0.2, 1 mM), and the enzyme catalytic activity was determined.

[0096] like Figure 7 As shown, 6M2 was cultured in LB medium, and the others were cultured in medium containing different concentrations of Zn 2+ When GMML medium was supplemented with 0.015 mM Zn 2+ In addition, in order to determine the location of the 6×His tag and the influence of the culture medium on the catalytic activity of the enzyme, this study compared 6M2 and NC6M2 cultured in LB medium and GMML medium, respectively. The experimental results are as follows: Figure 8 As shown in the figure, the 6×His tag moved from the N-terminus to the C-terminus will lead to a slight decrease in catalytic activity. cis The specific enzyme activity of -CHD was 1.1 U / mg, but the use of GMML medium had a greater impact on the catalytic activity. cis -CHD specific enzyme activity decreased to 0.34 U / mg.

[0097] Example 4 Site-directed introduction of non-natural amino acids to improve catalytic activity

[0098] like Figure 9 As shown, Bs In BDH, sites 50, 115, and 291 form the product release channel, and the size of the product release channel affects the release of the product. Bs In BDH, the distances between F50-F115, F50-I291, and F115-I291 are 3.3 Å, 4.5 Å, and 5.4 Å, respectively, and the channel area formed is relatively small.

[0099] Based on the idea of ​​expanding the product release channel, a halogen-modified phenylalanine analogue was introduced at position 50, and the polarity of the halogen destroyed the hydrophobic interaction between positions 50, 115, and 291. First, position 50 was replaced by the amber codon TAG and F50 was replaced by 3-chlorophenylalanine (3-ClF) by PCR. Figure 10 It can be seen that the catalytic activity of NC6M2 / Y293S / L118F / F50-3-ClF did not improve, but instead decreased by half. Analysis of the substrate binding pocket showed that the reason for the decreased activity may be that the chlorine atom of 3-ClF was not properly oriented towards I291 and F115, but formed a hydrogen bond with the hydroxyl group of S39 ( Figure 11 ).

[0100] Table 6 Primers used for the introduction of unnatural amino acids

[0101]

[0102] Therefore, 2,5-dichlorophenylalanine (2,5-2ClF) was used to replace 3-ClF. The 2-Cl in the 2,5-2ClF structure may form a hydrogen bond with the peptide bond of the 50-position main chain, thereby fixing the side chain conformation of 2,5-2ClF so that the 5-Cl is correctly oriented toward I291 and F115 (e.g. Figure 12 The experimental results show that the channel design successfully improves the catalytic activity, as shown in Figure 10 As shown, NC6M2 / Y293S / L118F / F50-2,5-2ClF cis The catalytic activity of -CHD was increased to 6.98 times that of the parent compound.

[0103] The kinetic parameters of wild type and NC6M2 / Y293S / L118F / F50-2,5-2ClF were determined as follows: first, the protein was purified and then cis The specific enzyme activity was determined at 14 different final concentrations of -CHD (0.125, 0.25, 0.625, 1.25, 1.875, 2.5, 3.125, 3.75, 5, 7.5, 10, 12.5, 18.75, and 25 mM) in an incubation period of 50 mM NAD. + , 50 mM Tris-HCl pH 8.5 buffer. K m and k cat The results were obtained by fitting the Michaelis-Menten equation and presented as mean ± standard error (n = 3).

[0104] The results of kinetic parameter determination are shown in Table 7. cis The catalytic efficiency of -CHD is 1.020 ± 0.072 s -1 .mM -1 , which is 214.48 times that of NCWT, even higher than the improvement of 6M2 / F115C / L118F (94.10 times), and its k cat The value is 8.81 times that of NC6M2 / Y293S / L118F, which indicates that the introduction of unnatural amino acids indeed expands the substrate channel and is beneficial to the turnover of substrates.

[0105] Table 7 Kinetic parameter determination of NCWT and NC6M2 / Y293S / L118F / F50-2,5-2ClF

[0106]

[0107] 2,5-2FF was also introduced into the 50 position, but as Figure 10 As shown in the figure, the catalytic activity of NC6M2 / Y293S / L118F / F50-2,5-2FF decreased more seriously, which may be because the polarity of the fluorine atom is too large, which in turn destroys the substrate binding pocket.

Claims

1. A (2R, 3R)-butanediol dehydrogenase mutant, characterized in that Obtained by mutation of the amino acid sequence shown in SEQ ID NO.1, wherein the mutation is one of the following: (1) Threonine at position 22 mutated to valine, glutamine at position 112 mutated to asparagine, alanine at position 230 mutated to arginine, threonine at position 258 mutated to glycine, alanine at position 260 mutated to methionine, and isoleucine at position 280 mutated to leucine; (2) Threonine at position 22 mutated to valine, glutamine at position 112 mutated to asparagine, alanine at position 230 mutated to arginine, threonine at position 258 mutated to glycine, alanine at position 260 mutated to methionine, isoleucine at position 280 mutated to leucine, and tyrosine at position 293 mutated to serine; (3) Threonine at position 22 mutated to valine, glutamine at position 112 mutated to asparagine, leucine at position 118 mutated to phenylalanine, alanine at position 230 mutated to arginine, threonine at position 258 mutated to glycine, alanine at position 260 mutated to methionine, isoleucine at position 280 mutated to leucine, and tyrosine at position 293 mutated to serine.

2. A (2R,3R)-butanediol dehydrogenase mutant containing an unnatural amino acid, characterized in that: The amino acid sequence shown in SEQ ID NO.1 was subjected to the following mutations: Threonine at position 22 mutated to valine, phenylalanine at position 50 mutated to 2,5-dichlorophenylalanine, glutamine at position 112 mutated to asparagine, leucine at position 118 mutated to phenylalanine, alanine at position 230 mutated to arginine, threonine at position 258 mutated to glycine, alanine at position 260 mutated to methionine, isoleucine at position 280 mutated to leucine, and tyrosine at position 293 mutated to serine. 3 . A gene encoding the (2R,3R)-butanediol dehydrogenase mutant according to claim 1 .

4. A recombinant vector, characterized in that The recombinant vector comprises the gene according to claim 3.

5. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria comprises the gene according to claim 3.

6. Use of the (2R,3R)-butanediol dehydrogenase mutant according to any one of claims 1 or 2 in catalyzing the oxidation of cis-1,2-cyclohexanediol to produce (2S)-hydroxycyclohexanone.

7. A method for biocatalytic synthesis of (2S)-hydroxycyclohexanone, characterized in that: A reaction system is formed with cis-1,2-cyclohexanediol as a substrate and the (2R,3R)-butanediol dehydrogenase mutant according to claim 1 or 2 as a catalyst to synthesize (2S)-hydroxycyclohexanone.

8. The method according to claim 7, characterized in that The reaction system includes 0.004 mM to 0.03 mM zinc ions.

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