A (2R, 3R)-butanediol dehydrogenase mutant with improved thermal stability and its application

Abstract

The present invention discloses a (2R, 3R)-butanediol dehydrogenase mutant with improved thermal stability and its application, relating to the field of genetic engineering. The present invention carries out site-directed mutagenesis modification to BS-BDH sequence, improves its thermal stability, and compared with the wild type, the thermal stability of 21 single-point mutants is improved. The present invention constructs double-point mutants on the basis of single-point mutants, and the thermal stability of double-point mutants is further improved. The inventive method effectively improves the thermal stability of (2R, 3R)-butanediol dehydrogenase derived from Bacillus subtilis, is conducive to alleviating the dilemma that (2R, 3R)-butanediol dehydrogenase is poor in thermal stability and cannot be industrialized, thereby promoting the biocatalytic synthesis of acetoin.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to a (2R, 3R)-butanediol dehydrogenase mutant with improved thermal stability and applications thereof. Background Art

[0002] Acetoin, chemically known as 3-hydroxy-2-butanone, also known as methyl acetyl carbinol, is the smallest unit of an α-hydroxyketone. Due to its strong, creamy, fatty aroma, acetoin is widely used as a food additive in the food processing industry. Furthermore, due to its high calorific value, acetoin can be used as a high-energy fuel in the aerospace industry. Acetoin is also a valuable raw material for chemical synthesis, used in the synthesis of antifreeze, plasticizers, foaming agents, coatings, and other applications, and has a wide range of applications.

[0003] Because acetoin has a chiral carbon atom, it can form two configurations: R-acetoin (R-AC) and S-acetoin (S-AC). Homochiral acetoin offers higher added value and can be used to synthesize optically active drugs. For example, acetoin can be used to synthesize 4-chloro-4,5-dimethyl-1,3-dioxolane-2-one, primarily for modifying penicillin antibiotics to improve efficacy and mitigate side effects. Acetoin can also be used to synthesize ampicillin hydrochloride and to prepare nicotine derivatives of pyrrolidone or tetrahydropyrrole. Furthermore, homochiral acetoin is used in the IT industry as a key component of liquid crystal materials.

[0004] There are three main methods for producing acetoin: chemical synthesis, microbial fermentation and enzymatic conversion.

[0005] There are three main chemical synthesis processes for producing acetoin: partial hydrogenation of diacetyl, selective oxidation of 2,3-butanediol, and hydrolysis of butanone. Chemical synthesis of acetoin has the following drawbacks: First, the yield and yield of the product are low; second, synthesizing optically pure acetoin is difficult, requires expensive catalysts, and can cause environmental pollution; third, the raw materials diacetyl and butanone are derived from petroleum resources. With the shortage of petroleum resources, the cost of raw materials is gradually increasing, which is not in line with the current trend of sustainable development.

[0006] Microbial fermentation involves a series of biochemical reactions that produce the target compound through the microbial metabolism and the catalytic action of enzymes produced during growth. Currently, due to the low yields of acetoin produced by genetically engineered strains, most research efforts focus on screening wild strains for acetoin production and optimizing fermentation media or production processes. While microbial fermentation is environmentally friendly, it produces numerous byproducts, consumes a lot of energy, and has low yields. Furthermore, none of the currently available fermentation strains can produce a fermentation product with an optical purity exceeding 99%, making it unsuitable for producing optically pure acetoin.

[0007] Enzymatic conversion methods can be broadly categorized into two types: whole-cell catalysis and in vitro enzymatic catalysis. 2,3-Butanediol can be oxidized to acetoin by (2R,3R)-butanediol dehydrogenase. Xiao et al. have constructed a whole-cell catalytic system using (2R,3R)-butanediol dehydrogenase from Bacillus subtilis and NADH oxidase from Lactobacillus brevis, achieving an acetoin yield of 36.7 g / L.

[0008] A whole-cell catalytic system was constructed using (2R,3R)-butanediol dehydrogenase (BS-BDH) from Bacillus subtilis and NADH oxidase from Lactobacillus brevis. BS-BDH was able to oxidize (2R,3R)-butanediol to acetoin, with an acetoin yield of 36.7 g / L. However, this yield concentration was still low and could not meet industrial production requirements. A whole-cell catalytic system constructed using Paenibacillus polymyxa achieved a maximum acetoin yield of 72.38 g / L. However, the enzymatic activity of the (2R,3R)-butanediol dehydrogenase from Bacillus subtilis was higher than that of Paenibacillus polymyxa, but its thermal stability was poor. Therefore, the low yield may be due to this poor thermal stability. Improving the thermal stability of BS-BDH is beneficial for the production of acetoin by whole-cell catalysis or enzyme catalysis, reducing production costs and increasing yield. Summary of the Invention

[0009] To address the technical problem of low thermal stability and difficulty in industrial application of BS-BDH, the present invention designed a method to improve the thermal stability of BS-BDH based on the Rosetta_ddg computational tool. By modifying the gene through site-directed mutagenesis, a (2R,3R)-butanediol dehydrogenase mutant with improved thermal stability was obtained, providing a certain basis for industrial production.

[0010] To address the low thermal stability of BS-BDH, the present invention uses Rosetta_ddg to calculate ΔΔG (the difference between the Gibbs free energy of the wild-type and mutants). Mutants with a ΔΔG < 0, indicating improved thermal stability, were selected through site-directed mutagenesis. BS-BDH thermal stability was characterized by residual enzyme activity and half-life. Single-point mutations were then combined to identify mutants with significantly improved thermal stability.

[0011] The specific technical solutions are as follows:

[0012] The present invention provides a (2R, 3R)-butanediol dehydrogenase mutant with improved thermal stability, which is obtained by subjecting a wild-type (2R, 3R)-butanediol dehydrogenase from Bacillus subtilis to a single-point mutation or a double-point mutation. The amino acid sequence of the wild-type (2R, 3R)-butanediol dehydrogenase is shown in SEQ ID NO. 2, and the specific single-point mutation is any one of the following:

[0013] (1) The amino acid at position 258 was mutated from threonine to glycine;

[0014] (2) amino acid 22 mutated from threonine to valine;

[0015] (3) amino acid 112 mutated from glutamine to asparagine;

[0016] (4) amino acid 285 mutated from arginine to lysine;

[0017] (5) amino acid 61 mutated from asparagine to glycine;

[0018] (6) amino acid 260 was mutated from alanine to methionine;

[0019] (7) amino acid 230 mutated from alanine to arginine;

[0020] (8) amino acid 137 mutated from phenylalanine to histidine;

[0021] (9) amino acid 252 mutated from glutamine to tryptophan;

[0022] (10) amino acid 130 mutated from serine to valine;

[0023] (11) The amino acid at position 295 was mutated from aspartic acid to asparagine;

[0024] (12) The amino acid at position 8 was mutated from asparagine to glycine;

[0025] (13) amino acid 330 mutated from glycine to glutamic acid;

[0026] (14) amino acid 145 mutated from tyrosine to phenylalanine;

[0027] (15) amino acid 276 was mutated from histidine to asparagine;

[0028] (16) amino acid 263 mutated from threonine to isoleucine;

[0029] (17) amino acid 132 was mutated from aspartic acid to proline;

[0030] (18) amino acid 260 was mutated from alanine to leucine;

[0031] (19) amino acid 263 mutated from threonine to valine;

[0032] (20) amino acid 135 was mutated from leucine to methionine;

[0033] (21) amino acid 154 mutated from serine to alanine;

[0034] The specific double-point mutation is any of the following:

[0035] (a) amino acid 230 mutated from alanine to arginine / amino acid 260 mutated from alanine to methionine;

[0036] (b) amino acid 230 mutated from alanine to arginine / amino acid 61 mutated from asparagine to glycine;

[0037] (c) amino acid 112 mutated from glutamine to asparagine / amino acid 230 mutated from alanine to arginine;

[0038] (d) amino acid 230 mutated from alanine to arginine / amino acid 285 mutated from arginine to lysine;

[0039] (e) amino acid 230 was mutated from alanine to arginine / amino acid 258 was mutated from threonine to glycine;

[0040] (f) amino acid 61 is mutated from asparagine to glycine / amino acid 260 is mutated from alanine to methionine;

[0041] (g) amino acid 112 mutated from glutamine to asparagine / amino acid 260 mutated from alanine to methionine;

[0042] (h) amino acid 285 mutated from arginine to lysine / amino acid 260 mutated from alanine to methionine;

[0043] (i) amino acid 112 mutated from glutamine to asparagine / amino acid 61 mutated from asparagine to glycine;

[0044] (j) amino acid position 61 was mutated from asparagine to glycine / amino acid position 285 was mutated from arginine to lysine;

[0045] (k) amino acid position 61 is mutated from asparagine to glycine / amino acid position 258 is mutated from threonine to glycine;

[0046] (1) The amino acid at position 112 was mutated from glutamine to asparagine and the amino acid at position 285 was mutated from arginine to lysine;

[0047] (m) amino acid 112 mutated from glutamine to asparagine / amino acid 258 mutated from threonine to glycine;

[0048] (n) The amino acid at position 285 was mutated from arginine to lysine and the amino acid at position 258 was mutated from threonine to glycine.

[0049] The present invention also provides the use of the (2R, 3R)-butanediol dehydrogenase mutant in the production of 2,3-butanediol / acetoin.

[0050] The present invention also provides a gene encoding the (2R, 3R)-butanediol dehydrogenase mutant.

[0051] The present invention also provides application of the gene in producing 2,3-butanediol / acetoin.

[0052] The present invention also provides an expression vector comprising the encoding gene.

[0053] The present invention also provides application of the expression vector in producing 2,3-butanediol / acetoin.

[0054] The present invention also provides a genetically engineered bacterium for expressing the (2R, 3R)-butanediol dehydrogenase mutant.

[0055] The present invention also provides the use of the genetically engineered bacteria in the production of 2,3-butanediol / acetoin.

[0056] Beneficial effects of the present invention:

[0057] The present invention performs site-directed mutagenesis on the BS-BDH sequence to improve its thermal stability. Compared with the wild type, the thermal stability of 21 single-point mutants is improved. Among them, the thermal stability of the T258G mutant (T258G represents: the 258th amino acid is mutated from threonine to glycine; the same applies to the others.), T22V mutant, Q112N mutant, R285K mutant, N61G mutant, A260M mutant and A230R mutant is significantly improved, and their residual enzyme activity is retained after heat treatment at 50°C for 20 minutes. More than 45%.

[0058] In addition, the present invention constructs double-point mutants based on single-point mutants, and the thermal stability of the double-point mutants is further improved. Among them, Q112N / A260M, A230R / N61G, and N61G / A260M have the most significant improvement effects. The residual enzyme activities of A230R / N61G and N61G / A260M double-point mutants after heat treatment at 50°C for 20 minutes are 93.11% and 85.28%, respectively. Q112N / A260M has the most significant improvement effects. 60M has the best thermal stability, with 98.09% of the residual enzyme activity retained after heat treatment at 50°C for 20 min, basically retaining all enzyme activity. The half-lives of Q112N / A260M (176.82 min), A230R / N61G (129.32 min), and N61G / A260M (149.46 min) are 4.89 times, 3.58 times, and 4.13 times that of the wild-type BS-BDH (36.16 min), respectively.

[0059] The method of the present invention effectively improves the thermal stability of (2R, 3R)-butanediol dehydrogenase derived from Bacillus subtilis, helps to alleviate the dilemma of poor thermal stability of (2R, 3R)-butanediol dehydrogenase leading to its inability to be industrially applied, thereby promoting the biocatalytic synthesis of acetoin. DETAILED DESCRIPTION

[0060] Reagents used in upstream genetic engineering: The plasmid extraction kit used in the examples of the present invention was purchased from Hangzhou Aisijin Biotechnology Co., Ltd.; PrimeSTAR Max Premix, DpnI, and competence kit were purchased from Beijing Baori Medical Co., Ltd.; E. coli BL21 (DE3) was purchased from Novagen; DNA marker, low molecular weight standard protein, and agarose gel electrophoresis reagents were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; BCA protein concentration determination kit was purchased from Hefei Lanjieke Technology Co., Ltd.; primer synthesis, sequence sequencing, and full plasmid pET28a-BS-BDH were completed by Qingke Bioengineering Co., Ltd.

[0061] Example 1

[0062] Construction of E. coli expression vector for BS-BDH single-point mutant.

[0063] Calculations using the Rosetta_ddg computing tool yielded 21 single-point mutants with improved thermal stability: T258G, T22V, Q112N, R285K, N61G, A260M, A230R, F137H, Q252W, S130V, D295N, N8G, G330E, Y145F, H276N, T263I, D132P, A260L, T263V, L135M, and S154A. The abbreviations for these mutants are as follows: T258G represents a mutation from threonine to glycine at position 258; the same applies to the others. The above single-point mutants were constructed using site-directed mutagenesis. The specific construction method is as follows:

[0064] (1) BS-BDH whole plasmid PCR: Using the nucleotide sequence shown in SEQ ID NO.1 as a template and the mutant primer sequences in Table 1, PCR was performed to obtain recombinant genes, wherein the recombinant gene is the nucleotide sequence shown in SEQ ID NO.1, wherein ACA is mutated to GGT (T258G), ACG is mutated to GTG (T22V), CAA is mutated to AAC (Q112N), CGT is mutated to AAA (R285K), AAT is mutated to GGT (N61G), GCC is mutated to ATG (A260M), GCA is mutated to CGT (A230R), TTC is mutated to CAT (F137H), CAA is mutated to TGG (Q252W), TCT is mutated to GTG (S130V), G AC mutated to AAC (D295N), AAC mutated to GGT (N8G), GGG mutated to GAA (G330E), TAT mutated to TTT (Y145F), CAT mutated to AAC (H276N), ACC mutated to ATT (T263I), GAT mutated to CCG (D132P), GCC mutated to CTG (A260L), ACC mutated to GTG (T263V), CTT mutated to ATG (L135M), and TCT mutated to GCG (S154A).

[0065] Table 1 Mutant primer sequences

[0066]

[0067]

[0068] The PCR amplification system is shown in Table 2 below:

[0069] Table 2 PCR amplification system

[0070] Element Volume / μL PrimeSTAR Max DNA Polymerase 25 Forward primer 0.5 Reverse primer 0.5 pET28a-BS-BDH 1 <![CDATA[ddH2O]]> Replenish to 50

[0071] PCR amplification conditions are as follows: Table 3: 30 cycles in total:

[0072] Table 3 PCR amplification program

[0073]

[0074] (2) Template digestion:

[0075] The remaining plasmid template was digested with DpnI enzyme. The digestion system is shown in Table 4 below:

[0076] Table 4 DpnI enzyme digestion system

[0077]

[0078]

[0079] Digestion conditions: 1 h at 37°C, 15 min at 70°C.

[0080] (3) Competent cell transformation and screening:

[0081] 10 μL of PCR product was added to 100 μL of E. coli BL21(DE3) competent cells and incubated on ice for 30 minutes. After 30 minutes of incubation, the tube was heat-shocked in a 42°C water bath for 90 seconds and immediately removed and incubated on ice for 3 minutes. In a sterile laboratory, 500 μL of LB medium was added to the competent tube and the tube was incubated at 220 rpm at 37°C for 60 minutes. The revived cells were centrifuged at 12,000 rpm for 60 seconds. In a sterile laboratory, approximately 400 μL of the supernatant was discarded and the remaining liquid was mixed with the cells. The mixture was then evenly spread onto solid LB medium containing kanamycin using a disposable spreader. The cells were incubated in an inverted 37°C incubator overnight. Three to four single colonies were selected from each solid medium for sequencing verification. The 21 mutants listed in Table 1 were obtained.

[0082] Example 2

[0083] Protein expression and purification of BS-BDH single-point mutants.

[0084] LB medium components: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L; LB solid medium components are based on LB liquid medium components plus 15 g / L agar; LB medium needs to be sterilized at 121°C for 20 min and added to a concentration of 100 μL / mL before use.

[0085] The mutants successfully sequenced in Example 1 were streaked and isolated, and single colonies were picked and added to 5 mL of LB liquid culture medium containing 100 μL / mL kanamycin, and cultured in a shaker at 37° C. and 220 rpm for 18 h to obtain seed solution.

[0086] Transfer 500 μL of seed solution to 200 mL of LB liquid medium containing 100 μL / mL kanamycin and culture in a shaker at 37°C at 220 rpm until the OD 600 =0.6-0.8 (about 3-4 h), add 200 μL 1 mol / L IPTG, and induce at low temperature for 18 h in a shaking incubator at 18°C ​​and 220 rpm.

[0087] Collect 100 mL of the induced expression cells, centrifuge at 4000 rpm for 10 min at 4°C, discard the supernatant, and add 10 mL of pH 8.0 Tris-HCl buffer for resuspending.

[0088] The resuspended bacterial liquid was placed on ice and ultrasonically disrupted using the following parameters: total ultrasonication time 15 minutes, on-time 3 seconds, rest time 7 seconds, and disruption power 60%. The disrupted bacterial liquid was centrifuged at 12,000 rpm for 30 minutes at 4°C to obtain the supernatant (i.e., crude enzyme solution) for subsequent purification. A nickel column was equilibrated with ten column volumes of 3mM imidazole Tris-HCl buffer (3mM imidazole, 20mM, pH=8.0 Tris-HCl, 500mM NaCl). The crude enzyme solution obtained in the previous step was passed over the nickel column. During this process, the target protein containing the His tag and some contaminants specifically bound to the nickel column. Contaminants were eluted from the nickel column using ten column volumes of 20mM imidazole Tris-HCl buffer (20mM imidazole, 20mM, pH=8.0 Tris-HCl, 500mM NaCl). Finally, the target protein was eluted with 500 mM imidazole Tris-HCl buffer (500 mM imidazole, 20 mM Tris-HCl, pH 8.0, 500 mM NaCl). The collected target protein was ultrafiltered in a centrifuge at 4°C, 4000 rpm, until the imidazole concentration dropped below 5 mM. The resulting liquid was the pure enzyme solution of the single-point mutant.

[0089] Example 3

[0090] Thermal stability assay of BS-BDH and its mutants.

[0091] The following reactions were used for enzyme characterization:

[0092] Acetoin+NADH 2,3-Butanediol+NAD +

[0093] The reaction is a reversible reaction, with (2R,3R)-butanediol as the substrate and NAD+ as the coenzyme. Under the catalysis of BS-BDH, the substrates NADH and acetoin are generated. Since NADH has an absorbance value at 340nm, the absorbance value at 340nm can be measured to characterize the oxidase activity of BS-BDH.

[0094] Definition of BS-BDH and mutant enzyme activity: The enzyme activity unit is defined as the amount of enzyme required to reduce 1 μmol of NAD+ per minute at room temperature. The specific enzyme activity is defined as enzyme activity per unit protein (U / mg).

[0095] Enzyme concentration determination method: Mix Reagent A and Reagent B in the BCA kit at a 50:1 volume ratio. Add 200 μL of reagent A to each well of a 96-well plate, followed by 20 μL of enzyme solution. Incubate the 96-well plate at 37°C for 30 minutes. Measure the absorbance at 562 nm to determine the enzyme concentration.

[0096] BS-BDH and mutant enzyme activity assay method: The enzyme reaction system is 200 μL, containing 12.5 mM (2R, 3R)-butanediol, 0.50 mM NAD + The enzymatic reaction begins immediately after adding a certain amount of enzyme solution. The enzyme activity is calculated by calculating the NADH production rate based on the change in absorbance at 340 nm. BS-BDH and its mutants were tested in triplicate.

[0097] Characterization of the thermal stability of BS-BDH and its mutants: The enzyme solution was incubated in a 50°C water bath for 20 minutes. The enzyme solution was removed and assayed using the aforementioned enzyme activity assay. The thermal stability of the enzyme was characterized by the residual enzyme activity (enzyme activity after heat treatment / enzyme activity before heat treatment). Three parallel experiments were performed on BS-BDH and its mutants.

[0098] Table 5 Assay results of BS-BDH and its single point mutants

[0099] mutant Enzyme activity (U / mg) Residual enzyme activity (%) BS-BDH 1.013±0.092 15.40±0.44 T258G 0.903±0.037 55.95±2.24 T22V 4.232±0.058 81.00±4.46 Q112N 0.947±0.014 57.20±1.37 R285K 1.125±0.061 48.48±1.59 N61G 3.867±0.044 47.75±0.86 A260M 2.344±0.017 47.50±3.76 A230R 0.810±0.016 45.82±0.67 F137H 2.535±0.030 42.69±0.90 Q252W 4.225±0.105 40.45±1.93 S130V 3.915±0.075 37.61±0.63 D295N 2.359±0.065 35.61±1.95 N8G 0.867±0.012 33.93±1.49 G330E 1.239±0.034 33.33±1.57 Y145F 0.968±0.01 0 27.47±0.48 H276N 1.020±0.032 26.59±0.83 T263I 1.378±0.103 23.95±4.16 D132P 1.220+0.134 23.24±0.57 A260L 1.592±0.107 22.25±0.72 T263V 3.974±0.113 21.81±1.06 L135M 0.790±0.011 18.11±0.49 S154A 1.236±0.174 17.65±0.00

[0100] The protein obtained above was assayed for oxidase activity using a microplate reader. The assay data are shown in Table 5. As can be seen from Table 5, the thermal stability of the 21 single-point mutants was improved to a certain extent. Only the T258G mutant, Q112N mutant, A230R mutant, N8G mutant, and L135M mutant had slightly decreased oxidase activity compared with the wild-type BS-BDH while having improved stability. The most significant decrease was in the L135M mutant, whose activity was only reduced to 78% of the oxidase activity of BS-BDH. Except for the five mutants mentioned above, the oxidative activity of the remaining mutants remained basically unchanged. Among them, the oxidase activity of the T22V mutant, N61G mutant, Q252W mutant, S130V mutant, T263V mutant and A230R mutant was 3-4 times higher than that of the wild-type BS-BDH, among which the T22V mutant (4.232 U / mg) and S130V mutant (3.915 U / mg) showed the most obvious increase, both increasing by 4.17 times.

[0101] The thermal stability of (2R,3R)-butanediol dehydrogenase was characterized by heat treatment at 50°C for 20 min. The results are shown in Table 5. While the residual enzyme activity of the wild-type BS-BDH was only 15.40% after heat treatment at 50°C for 20 min, the residual enzyme activity of the single-point mutants was higher than 15.40%, indicating that the thermal stability of the single-point mutants was significantly improved. As shown in Table 5, among the 21 mutants, the T258G, T22V, Q112N, R285K, N61G, A260M, and A230R mutants showed significant improvements in thermal stability, retaining over 45% of their residual enzyme activity after heat treatment at 50°C for 20 min. The T22V mutant showed the most significant improvement in thermal stability, retaining 81% of its residual enzyme activity after heat treatment at 50°C for 20 min.

[0102] Example 4

[0103] Construction of Escherichia coli expression vector of double point mutant and determination of thermal stability of double point mutant.

[0104] To further enhance the thermostability of these mutants, the present invention combines single-point mutants to create even more pronounced double-point mutants. Among the single-point mutants, the T258G, T22V, Q112N, R285K, N61G, A260M, and A230R mutants significantly enhance thermostability. Therefore, these seven single-point mutants were further combined to create even more thermostable mutants.

[0105] Plasmid template extraction method: Plasmid extraction was performed using the AxyPrep Plasmid Extraction Kit. 4 mL of culture medium containing a single-point mutant cultured for at least 10 hours was centrifuged at 12,000 rpm for 1 minute. The supernatant was discarded and collected in a 2 mL centrifuge tube. The cells were resuspended in 250 μL of Buffer S1 and then lysed in 250 μL of Buffer S2 for no more than 5 minutes. The lysate was then neutralized by adding 350 μL of Buffer S3. The cells were centrifuged at 12,000 rpm for 10 minutes. The supernatant was transferred to a preparation tube and centrifuged for 1 minute to allow the plasmid to adhere to the preparation tube. The cells were then washed with 500 μL of Buffer W1 and 700 μL of Buffer W2 to remove impurities. Finally, the plasmid was eluted with 70 μL of water to obtain the template plasmid.

[0106] Using the above-mentioned plasmid extraction method, plasmids of T258G mutant, T22V mutant, Q112N mutant, R285K mutant, N61G mutant and A260M mutant were extracted and used as templates to construct double-point mutants using the primers in Table 1. The construction process was consistent with the single-point mutant construction method in Example 1.

[0107] The constructed E. coli vector of the double-point mutant was used to express and purify the double-point mutant protein according to the same method as in Example 2 to obtain a double-point mutant pure enzyme solution for subsequent thermal stability verification.

[0108] The obtained double-point mutants were characterized for thermostability according to Example 3. Through thermostability characterization, 14 double-point mutants with further improved thermostability compared to the single-point mutants were obtained. The genes encoding the 14 double-point mutants were GCA mutated to CGT (A230R) and GCC mutated to ATG (A260M), GCA mutated to CGT (A230R) and AAT mutated to GGT (N61G), CAA mutated to AAC (Q112N) and GCA mutated to CGT (A230R), GCA mutated to CGT (A230R) and CGT mutated to AAA (R285K), GCA mutated to CGT (A230R) and ACA mutated to GGT (T258G), AAT mutated to GGT (N61G) and GCC mutated to ATG in the nucleotide sequence shown in SEQ ID NO. (A260M), CAA mutated to AAC (Q112N) and GCC mutated to ATG (A260M), CGT mutated to AAA (R285K) and GCC mutated to ATG (A260M), CAA mutated to AAC (Q112N) and AAT mutated to GGT (N61G), AAT mutated to GGT (N61G) and CGT mutated to AAA (R285K), AAT mutated to GGT (N61G) and ACA mutated to GGT (T258G), CGT mutated to AAA (R285K) and ACA mutated to GGT (T258G).

[0109] The results of oxidase activity and thermal stability assays of the double-point mutants are shown in Table 6.

[0110] Table 6 Assay results of BS-BDH and its double-point mutants

[0111] mutant Enzyme activity (U / mg) Residual enzyme activity (%) BS-BDH 1.013±0.092 15.40±0.44 A230R / A260M 4.380±0.065 71.11±0.75 A230R / N61G 2.939±0.028 93.11±0.39 Q112N / A230R 3.708±0.019 73.82±1.04 A230R / R285K 3.855±0.043 61.49±2.69 A230R / T258G 4.800±0.069 65.08±0.79 N61G / A260M 4.231±0.032 85.28±1.00 Q112N / A260M 3.714±0.045 98.09±1.01 R285K / A260M 3.293±0.021 69.12±0.82 Q112N / N61G 3.859±0.023 74.81±0.81 N61G / R285K 4.522±0.076 60.58±0.00 N61G / T258G 4.457±0.102 72.38±0.79 Q112N / R285K 5.086±0.021 75.50±2.16 Q112N / T258G 4.152±0.011 74.47±0.96 R285K / T258G 4.499±0.047 69.66±0.87

[0112] As can be seen from Table 6, the oxidase activity of the double-point mutant was further improved on the basis of the single-point mutant. Compared with the wild-type oxidase activity (1.013 U / mg), it was basically increased by 3-5 times. Among them, Q112N / R285K had the most obvious improvement effect, with an oxidase activity of 5.086 U / mg, a 5-fold increase in oxidase activity. In addition, the thermal stability of the 14 double-point mutants was further improved compared with the single-point mutants, among which Q112N / A260M, A230R / N61G, and N61G / A260M showed the most significant improvement. The residual enzyme activities of the A230R / N61G and N61G / A260M double-point mutants were retained at 93.11% and 85.28%, respectively, after heat treatment at 50°C for 20 minutes. Q112N / A260M had the best thermal stability, with a residual enzyme activity retention of 98.09% after heat treatment at 50°C for 20 minutes, basically retaining all enzyme activities.

[0113] Example 5

[0114] Half-life determination of BS-BDH and its double-point mutants.

[0115] In order to further understand the improvement effect in detail, the half-life of wild-type BS-BDH and double-point mutants (Q112N / A260M, A230R / N61G, N61G / A260M) with the most significant improvement in thermal stability will be determined.

[0116] Method for determining half-life: The enzyme solution is kept warm in a 45°C water bath, samples are taken every 30 minutes, and the enzyme activity is tested according to the above-mentioned enzyme activity determination method. The half-life can be calculated based on the relationship between the logarithm of the residual enzyme activity and time.

[0117] Table 7 Half-life determination of BS-BDH and double-point mutants

[0118] mutant Half-life (min) BS-BDH 36.16 A230R / N61G 129.32 N61G / A260M 149.46 Q1 12N / A260M 176.82

[0119] As can be seen from Table 7, the half-life of the double point mutants is significantly improved compared with the wild type. The half-life of A230R / N61G is 129.32 min, the half-life of N61G / A260M is 149.46 min, and the half-life of Q112N / A260M is 176.82 min. The half-lives are 3.58 times, 4.13 times, and 4.89 times the half-life of the wild type BS-BDH (36.16 min), respectively. The thermal stability of the double point mutants has been significantly improved.

Claims

1. A (2R,3R)-butanediol dehydrogenase mutant with improved thermal stability, characterized in that It is derived from Bacillus subtilis ( Bacillus subtilis ) by performing a single-point mutation or a double-point mutation on a wild-type (2R, 3R)-butanediol dehydrogenase, wherein the amino acid sequence of the wild-type (2R, 3R)-butanediol dehydrogenase is shown in SEQ ID NO. 2, and the single-point mutation is: the 260th amino acid is mutated from alanine to methionine; The double-point mutation is any one of the following: (a) The amino acid at position 61 was mutated from asparagine to glycine and the amino acid at position 260 was mutated from alanine to methionine; (b) The amino acid at position 112 was mutated from glutamine to asparagine and the amino acid at position 260 was mutated from alanine to methionine.

2. Use of the (2R,3R)-butanediol dehydrogenase mutant according to claim 1 in the production of 2,3-butanediol / acetoin.

3. A gene encoding the (2R,3R)-butanediol dehydrogenase mutant according to claim 1.

4. Use of the gene as claimed in claim 3 in the production of 2,3-butanediol / acetoin.

5. An expression vector comprising the coding gene according to claim 3.

6. Use of the expression vector according to claim 5 in the production of 2,3-butanediol / acetoin.

7. A genetically engineered bacterium expressing the (2R, 3R)-butanediol dehydrogenase mutant according to claim 1.

8. Use of the genetically engineered bacteria according to claim 7 in the production of 2,3-butanediol / acetoin.

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