Mutant double-enzyme catalyst, application and (S)-glassine synthesis method

Abstract

The invention discloses a mutant double-enzyme catalyst, application and an (S)-vitrin synthesis method, and relates to the technical field of bioengineering.Methylglyoxal reductase GRE2 (A) of candida glabrata and phosphite dehydrogenase PTDH of methylotrophicus are used as sources for modification, mutants of the candida glabrata and the phosphite dehydrogenase PTDH are screened, and the mutant double-enzyme catalyst is obtained. The key residues influencing the enzymatic properties of the initial methylglyoxal reductase and the phosphite dehydrogenase are determined, and the methylglyoxal reductase mutant and the phosphite dehydrogenase mutant with high activity are obtained at the same time. The two mutants with the highest utilization efficiency have a good catalytic effect in a reaction for catalyzing beta-acetone xyloside to prepare (S)-bose in a 22T reaction system, the overall catalytic efficiency is improved by about 43%, and the cost is reduced by about 8%.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a mutant dual-enzyme catalyst, its application and a (S)-bosone synthesis method. Background Art

[0002] Pro-Xylane, chemically known as hydroxypropyl tetrahydropyrantriol, is a glycoprotein mixture that can stimulate the production of aminoglucosan (GAGs) in the skin. GAGs are the main components of the extracellular matrix, and hyaluronic acid contained in them is one of the main components of GAGs. Hyaluronic acid has strong water absorption and storage capacity. When injected into the dermis, it can achieve the effect of filling and moisturizing the skin, making the skin plump and elastic, thereby preventing skin aging. It is easy to biodegrade and has little irritation to the human body. It is widely used in skin care products, cosmetics, toiletries and other industries, and has a very broad market prospect.

[0003] (S)-Bosone was initially synthesized mainly using chemical methods. For example, CN113735811A records the synthesis of the first intermediate product from acetylacetone and xylose, followed by ketalization, reduction, and deprotection to obtain (S)-Bosone; CN112812087A records the synthesis of a key intermediate from (2R,3S,4S)-2,3,4-tribenzyloxyglutaraldehyde and acetone, followed by a two-step catalytic reaction to obtain (S)-Bosone. However, the chemical synthesis method has many synthesis steps, complex reaction conditions, high cost, and high pollution.

[0004] In recent years, the bioenzymatic synthesis of (S)-Bose has gradually become the main production method for industrial synthesis, thanks to the advantages of bioenzymatic method such as safety and efficiency, simple catalytic steps, low cost, and green environmental protection. At present, the bioenzymatic method is divided into single enzyme catalysis and multi-enzyme catalysis. For example, CN114507681A records the use of sorbose reductase with β-acetone xyloside, isopropanol, and coenzyme to catalyze the reaction to generate Bose; in 2023, Xiao Yanming et al. used short-chain alcohol dehydrogenase with glucose dehydrogenase, formate dehydrogenase, etc. to catalyze the reaction to obtain (S)-Bose. Single enzyme catalysis has the problem that coenzymes cannot be recycled, which easily causes drug waste and increases production costs; and catalytic production of multiple enzyme preparations will increase the fermentation cost, and the concentration of the catalytic reaction substrate is not high, which is not conducive to large-scale production.

[0005] Therefore, utilizing the dual-enzyme catalytic cycle to solve the efficient circulation of coenzymes, improve enzyme catalytic efficiency, and reduce reaction time are issues that need to be urgently addressed in large-scale production. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the present invention provides a mutant dual enzyme catalyst, application and a method for synthesizing S-configuration boson, specifically providing gene sequences comprising a methylglyoxal reductase mutant and a phosphite dehydrogenase mutant, expression vectors of the methylglyoxal reductase mutant and the phosphite dehydrogenase mutant gene sequences and host cells thereof, and providing a method for preparing (S)-boson from β-acetone xyloside by using a methylglyoxal reductase mutant and a phosphite dehydrogenase mutant to catalyze the synthesis of (S)-boson.

[0007] In order to achieve the purpose of the present invention, the following scheme is proposed: The present invention uses the methylglyoxal reductase GRE2 (A) (GeneID: 2887289) of Nakaseomyces glabratus included in GenBank as the initial methylglyoxal reductase, performs site-directed mutagenesis on the initial methylglyoxal reductase encoding gene, and screens the obtained mutants to determine the key residues affecting the enzymatic properties of the initial methylglyoxal reductase, and obtains a methylglyoxal reductase mutant with high activity. The experimental results show that the methylglyoxal reductase mutant obtained by screening is applied to the catalytic reaction of preparing (S)-bosone with β-acetone xyloside and isopropanol as substrates, and exhibits a good catalytic effect.

[0008] The methylglyoxal reductase GRE2 (A) mutant of the present invention has a protein nucleotide sequence selected from one of the following sequences: (1) The nucleotide sequence as shown in SEQ ID No.3: the valine at position 130 of the nucleotide sequence as shown in SEQ ID No.1 is replaced by glycine, the glutamic acid at position 165 is replaced by threonine, and the glycine at position 247 is replaced by arginine.

[0009] (2) The nucleotide sequence as shown in SEQ ID No.4: the valine at position 130 of the nucleotide sequence shown in SEQ ID No.1 is replaced by glycine, the serine at position 196 is replaced by alanine, and the aspartic acid at position 251 is replaced by methionine.

[0010] (3) The amino acid sequence as shown in SEQ ID No.5: the histidine at position 87 of the amino acid sequence shown in SEQ ID No.1 is replaced by lysine, the arginine at position 173 is replaced by histidine, and the isoleucine at position 203 is replaced by valine.

[0011] (4) The nucleotide sequence as shown in SEQ ID No.6: the histidine at position 87 of the nucleotide sequence shown in SEQ ID No.1 is replaced by lysine, the lysine at position 148 is replaced by arginine, and the glycine at position 298 is replaced by proline.

[0012] (5) The nucleotide sequence as shown in SEQ ID No.7: the threonine at position 163 of the nucleotide sequence shown in SEQ ID No.1 is replaced by serine, the aspartic acid at position 195 is replaced by glutamic acid, and the alanine at position 313 is replaced by leucine.

[0013] (6) The nucleotide sequence as shown in SEQ ID No.8: the threonine at position 163 of the nucleotide sequence shown in SEQ ID No.1 is replaced by serine, the phenylalanine at position 189 is replaced by tryptophan, and the glycine at position 278 is replaced by proline.

[0014] The present invention uses the phosphite dehydrogenase PTDH (GeneID: 72987700) of the methylotrophic bacteria (Methylorubrum extorquens) included in GenBank as the initial phosphite dehydrogenase, performs site-directed mutagenesis on the coding gene of the initial phosphite dehydrogenase, and screens the obtained mutants, determines the key residues that affect the enzymatic properties of the initial phosphite dehydrogenase, and obtains phosphite dehydrogenase mutants with high activity. The experimental results show that the phosphite dehydrogenase mutants obtained by screening are applied to the coenzyme cycle of NADPH, which has a good promoting effect.

[0015] The phosphite dehydrogenase PTDH mutant of the present invention has a protein nucleotide sequence selected from one of the following sequences: (7) The nucleotide sequence as shown in SEQ ID No.9: the tryptophan at position 133 of the nucleotide sequence as shown in SEQ ID No.2 is replaced by tyrosine, and the leucine at position 204 is replaced by alanine.

[0016] (8) The nucleotide sequence as shown in SEQ ID No.10: the glycine at position 77 of the nucleotide sequence shown in SEQ ID No.2 is replaced by proline, and the phenylalanine at position 166 is replaced by tryptophan.

[0017] A gene encoding a methylglyoxal reductase mutant, wherein the nucleotide sequence of the gene is selected from any one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0018] A gene encoding a phosphite dehydrogenase mutant, wherein the nucleotide sequence of the gene is selected from any one of SEQ ID No.9 and SEQ ID No.10.

[0019] The mutant sequence shown in the present invention is optimized by Escherichia coli codons by Beijing Qingke Biotechnology Co., Ltd., and then fully synthesized, and a 6xHis fusion expression vector of the mutant gene is constructed, for example, any one of pET22b, pET28a, pET32a, and pCold TF is used, wherein methylglyoxal reductase is preferably a pET28a plasmid, and phosphite dehydrogenase is preferably a pET32a plasmid; the host cell is selected as Escherichia coli BL21 (DE3), which is suitable for efficient expression of exogenous genes.

[0020] The present invention also provides a method for synthesizing (S)-Bosone, comprising the following steps: (1) subjecting the host cells described above to ultra-low temperature freeze-thaw disruption to obtain the methylglyoxal reductase mutant and the phosphite dehydrogenase mutant; (2) In the reaction system, the methylglyoxal reductase mutant and the phosphite dehydrogenase mutant as described above are added, the reaction temperature is 30°C to 40°C, the substrate is β-acetone xyloside, the substrate concentration is 40g / L to 100g / L, the isopropanol concentration is 40g / L to 100g / L, the potassium phosphite concentration is 20g / L to 50g / L, the NADP salt concentration is 0.05g / L to 0.2g / L, the mass ratio of the methylglyoxal reductase mutant to the substrate is 0.2 to 1:1, and the mass ratio of the phosphite dehydrogenase mutant to the substrate is 0.1 to 0.4:1.

[0021] Furthermore, the methylglyoxal reductase mutant and the phosphite dehydrogenase mutant are prepared by centrifuging the bacterial cells at a speed of 6000 rpm or more to obtain bacterial sludge, freezing the bacterial sludge in a -25°C freezer for 48 hours to lyse the cells, and then weighing and feeding the reaction.

[0022] The methylglyoxal reductase mutant and phosphite dehydrogenase mutant bacterial sludge can be directly added to the catalytic system to convert the substrate, and the enzyme solution can also be further processed, including but not limited to separation (microfiltration and / or ultrafiltration), decolorization, concentration of the enzyme solution, and even further adding an immobilized carrier to prepare an immobilized enzyme to increase the substrate addition concentration and the number of enzyme reuses.

[0023] Furthermore, NADP+ is added to the reaction system, and the addition of NADP+ and potassium phosphite system realizes the hydrogen donor regeneration cycle. Phosphorous acid is oxidized to phosphoric acid under the catalytic action of the enzyme, accompanied by the generation of NADPH, which acts as a hydrogen donor to further improve the conversion efficiency of the methylglyoxal reductase mutant in catalyzing the reduction of the substrate.

[0024] Furthermore, the reaction is continued for 12 hours at a pH of 7.0 to 7.5, and ventilation and purging are performed. The biocatalytic conversion rate is not less than 96%, and the ee value of the target product is not less than 99.3%, where the ee value represents the enantiomeric excess value.

[0025] The beneficial effects of the present invention are: (1) The present invention transforms the methylglyoxal reductase GRE2 (A) from Nakaseomyces glabratus and screens the corresponding mutants to determine the key residues that affect the enzymatic properties of the initial methylglyoxal reductase. At the same time, a highly active methylglyoxal reductase mutant is obtained, the maximum catalytic activity of which is 1.43 times higher than that of the original strain, achieving efficient catalysis of the production of (S)-bosone using β-acetone xyloside as raw material, and significantly improving the production efficiency of (S)-bosone; (2) The present invention transforms the phosphite dehydrogenase PTDH from the methylotrophic bacteria (Methylorubrum extorquens) and screens the corresponding mutants. The key residues that affect the enzymatic properties of the initial phosphite dehydrogenase are determined through software analysis. At the same time, a highly active phosphite dehydrogenase mutant is obtained. Its maximum catalytic activity is 0.97 times higher than that of the original strain, achieving rapid circulation of NADPH. (3) Provided are a methylglyoxal reductase mutant and a phosphite dehydrogenase mutant having a gene nucleotide sequence as shown in any one of SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 and SEQ ID No.10, as well as an expression vector or recombinant bacteria containing the gene sequence, and use thereof in catalyzing the reaction of preparing (S)-bosone from β-acetone xyloside; (4) The methylglyoxal reductase mutant and phosphite dehydrogenase mutant dual enzyme catalyzed β-acetone xyloside in the preparation of (S)-bosone has good catalytic efficiency. Compared with the original dual enzyme catalysis, in the 22T reaction system, the overall catalytic rate is increased by 43%, the reaction time is reduced by about 5 hours, and at the same time, the input of NADP disodium salt is reduced by about 62%, saving 8% of the cost. The obtained (S)-bosone has high optical purity, and the ee value is between 99.3% and 99.9%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The plasmid construction map of wild-type methylglyoxal reductase (pET28a-GRE2 (A)) is shown; Figure 2 The plasmid construction map of phosphite dehydrogenase (pET32a-PTDH) is shown; Figure 3 The protein electrophoresis diagrams of methylglyoxal reductase mutants (H87K, K148R, G298P) and phosphite dehydrogenase mutants (G77P, F166W) are shown; Figure 4 shows the enzyme activity reaction curve of phosphite dehydrogenase mutants (G77P, F166W); Figure 5 The figure shows the high performance liquid chromatogram of the products obtained by using methylglyoxal reductase mutants (H87K, K148R, G298P) as catalysts. DETAILED DESCRIPTION

[0027] Example 1 1. Construction of wild-type methylglyoxal reductase expression strain According to the sequence of SEQ ID No.1, Beijing Qingke Biotechnology Co., Ltd. was commissioned to optimize the protein coding sequence in E. coli, and then the whole gene was synthesized and cloned into the pET28a(+) vector to obtain the expression vector pET28a-GRE2(A) of the control protein. The recombinant plasmid map is shown in Figure 1 After sequencing verification, the gene was introduced into Escherichia coli BL21 (DE3) to construct an expression strain.

[0028] 2. Construction of methylglyoxal reductase mutant expression strain Site-directed mutagenesis of GRE2(A) was performed to obtain the following mutants: (1) GRE2(A)-1: V130G, E165T, G247R, the nucleotide sequence is shown in SEQ ID No. 3.

[0029] (2) GRE2(A)-2: V130G, S196A, D251M, the nucleotide sequence is shown in SEQ ID No.4.

[0030] (3) GRE2(A)-3: H87K, R173H, I203V, the nucleotide sequence is shown in SEQ ID No.5.

[0031] (4) GRE2(A)-4: H87K, K148R, G298P, the nucleotide sequence is shown in SEQ ID No.6.

[0032] (5) GRE2(A)-5: T163S, D195E, A313L, the nucleotide sequence is shown in SEQ ID No.7.

[0033] (6) GRE2(A)-6: T163S, F189W, G278P, the nucleotide sequence is shown in SEQ ID No.8.

[0034] The above sequences were commissioned to Beijing Qingke Biotechnology Co., Ltd. to optimize the E. coli codons for the respective nucleotide sequences, and then synthesize the whole genes to obtain the corresponding recombinant vectors. After sequencing verification, they were introduced into E. coli BL21 (DE3) to obtain the corresponding expression strains.

[0035] 3. Preparation of methylglyoxal reductase mutant enzyme solution The above recombinant bacteria were inoculated into LB liquid culture medium containing 50 μg / ml of kanamycin, cultured at 37°C and 200 rpm for about 6 hours until OD600 reached about 0.8, and IPTG with a final concentration of 0.05 mM was added for induction, and induced at 25°C for 16 hours. Then the bacteria were collected by centrifugation at 8000 rpm for 10 minutes. 10 g of bacteria were resuspended in 100 mL of phosphate buffer (0.1 mol / L, pH 7.0), ultrasonically disrupted, and centrifuged at 12000 rpm for 10 minutes. The supernatant was taken to obtain the methylglyoxal reductase mutant enzyme solution corresponding to the above optimized nucleotide sequence, and the mutants (H87K, K148R, G298P) were tested by protein electrophoresis to verify the band size and solubility, such as Figure 3 As shown, lane 0 is a 14kDa-120kDa protein marker, lane 1 is the whole cell protein after fermentation and cell lysis of the blank control strain of Escherichia coli BL21(DE3), lane 2 is the supernatant protein of high-speed centrifugation after fermentation and cell lysis of the blank control strain of Escherichia coli BL21(DE3), lane 3 is the whole cell protein after induced fermentation and cell lysis of Escherichia coli BL21(DE3)pET28a-GRE2(A) at 25°C, lane 4 is the supernatant protein of high-speed centrifugation after induced fermentation and cell lysis of Escherichia coli BL21(DE3)pET28a-GRE2(A) at 25°C, lane 5 is the whole cell protein after induced fermentation and cell lysis of Escherichia coli BL21(DE3)-pET32a-PDTH at 25°C, lane 6 is the supernatant protein of high-speed centrifugation after induced fermentation and cell lysis of Escherichia coli BL21(DE3)-pET32a-PDTH at 25°C. The positions and sizes of the protein bands are correct, and the proteins are well soluble.

[0036] 4. Catalytic ability and ee value of methylglyoxal reductase mutants Prepare 50 mL of reaction system as follows: add 5 g of β-acetone xyloside, add 25% (m / v) isopropanol, stir at 30°C, add 10 mg of NADP+, make up to 50 mL with phosphate buffer (50 mmol / L, pH 7.0), add 2 g of different methylglyoxal reductase mutant enzyme solutions obtained in step 3 respectively, and react for 12 h.

[0037] After the reaction, 200 μL of the reaction solution was sampled and centrifuged at 13000 rpm for 2 min. 100 mL of the supernatant was added to 400 μL ddH20 and filtered with a 0.22 μ filter membrane for liquid phase analysis. The conversion rate and product ee value of different methylglyoxal reductase mutants were determined. The results are shown in Table 1.

[0038] The mobile phase for liquid chromatography detection is: 850mL of chromatography-grade acetonitrile, which is fixed to 1L with ddH20. Use a 0.22um organic filter membrane to filter through the membrane. Ultrasonic degas the mobile phase after passing through the membrane for 30 minutes to remove the bubbles, and the resulting mobile phase is the mobile phase. The liquid chromatograph is Tongwei Liquid Phase 3030, the detector is an evaporative light detector, the detection column is Yuexu NH2 chromatographic column, the specifications are 250nm×4.6nm, 5μm, the mobile phase flow rate is 1mL / min, the injection volume is 10μL, the detection temperature is 35℃, and the peak detection time is about 4min.

[0039] Table 1 Comparison of catalytic abilities of different methylglyoxal reductase mutants Note: ee value means enantiomeric excess.

[0040] Example 2 1. Construction of wild-type phosphite dehydrogenase expression strain According to the sequence of SEQ ID No.2, Beijing Qingke Biotechnology Co., Ltd. was commissioned to optimize the protein coding sequence in E. coli, and then the whole gene was synthesized and cloned into the pET32a(+) vector to obtain the expression vector pET32a-PTDH of the control protein. The recombinant plasmid map is shown in Figure 2 After sequencing verification, the gene was introduced into Escherichia coli BL21 (DE3) to construct an expression strain.

[0041] 2. Construction of phosphite dehydrogenase mutant expression strain Site-directed mutagenesis of PTDH was performed to obtain the following mutants: (1) PTDH-1: W133Y, L204A, the nucleotide sequence is shown in SEQ ID No.9.

[0042] (2) PTDH-2: G77P, F166W, the nucleotide sequence is shown in SEQ ID No.10.

[0043] The above sequences were commissioned to Beijing Qingke Biotechnology Co., Ltd. to optimize the E. coli codons for the respective nucleotide sequences, and then synthesize the whole genes to obtain the corresponding recombinant vectors. After sequencing verification, they were introduced into E. coli BL21 (DE3) to obtain the corresponding expression strains.

[0044] 3. Preparation of phosphite dehydrogenase mutant enzyme solution The above recombinant bacteria were inoculated into LB liquid culture medium containing 50 μg / ml ampicillin, cultured at 37°C, 200rpm for about 6 hours until OD600 reached about 0.8, and induced by adding IPTG with a final concentration of 0.05mM, and induced at 22°C for 16 hours. Then centrifuge at 8000rpm for 10min to collect the bacteria. Resuspend 2.5g of bacteria in 100mL phosphate buffer (0.1mol / L, pH 7.0), ultrasonically disrupt, and centrifuge at 12000rpm for 10min. Take the supernatant to obtain the phosphite dehydrogenase mutant enzyme solution corresponding to the above sequence nucleotides, and perform protein electrophoresis on the mutants (G77P, F166W) to verify the band size and solubility, such as Figure 3 shown.

[0045] 4. Determination of enzyme activity of phosphite dehydrogenase mutants The reaction system for enzyme activity determination is (2mL): 20mM MOPS, pH 7.3, 1mM phosphite, 0.5mM NADP+ and 400μl phosphite dehydrogenase crude enzyme concentrate. After all are mixed, the absorbance change is measured at 340nm, and a blank control without adding phosphite dehydrogenase crude enzyme concentrate is also performed. Figure 4 The protein concentration was calculated using the Braford method, and one enzyme activity was defined as: catalyzing 1 μmol of NADP salt to produce NADPH per minute under experimental conditions. The overall mutant enzyme activity reaction data are shown in Table 2.

[0046] Table 2 Comparison of catalytic abilities of different phosphite dehydrogenase mutants Test data: 2 ml total reaction system, 400 μl enzyme solution (9.8 mg), absorbance change within 2 min is 0.237, NADPH extinction coefficient in diluted buffer solution is 6220 L / mol / cm, light path is 1 cm, reaction rate is Figure 4 .

[0047] According to the calculation formula of the photometric enzyme activity determination: ∆A: change in solution absorbance, no unit; : molar absorption coefficient of the luminescent substance, unit L (mol∙cm); b: optical path, i.e. the distance that light travels through the solution, usually the width of the light-transmitting surface of the cuvette, in cm; t: reaction time, unit: min; : reaction volume, unit: ml; Substituting the above data, we can obtain: the best mutant enzyme activity = 381.03μmol / min, that is, 381.03U / 400μl, which is converted to 952.57U / ml, with a specific activity of 97.2U / mg, which has a higher enzyme activity.

[0048] Example 3 This embodiment provides a method for synthesizing (S)-Bosene, comprising the following steps: 1. Feeding reaction Add 1200kg of the intermediate (β-acetone xyloside) solution to the reactor, and add 16000kg of water. Adjust the pH to 7.0-7.5 with ammonia water, add 2200g of NADP+, add 1920kg of isopropanol, and then add 1000kg of potassium phosphite. Add 600kg of frozen-thawed methylglyoxal reductase mutant (H87K, K148R, G298P) sludge, and add 500kg of frozen-thawed phosphite dehydrogenase mutant sludge (G77P, F166W). At this time, the whole system is about 22T. Keep the pH at 7.0-7.5 and the temperature at 30℃. During the reaction, HPLC should be performed at any time to detect the product concentration and substrate concentration. Continue the reaction for 10-15 hours, and measure the substrate concentration after the reaction is completed (such as Figure 5 As shown), then heat up for denaturation at 55-60°C for 1-2 hours.

[0049] 2. Ceramic membrane sterilization The feed liquid is clarified by ceramic membrane, the frequency conversion is adjusted, the pressure is maintained at 0.4-0.5 bar, and after concentrating to the minimum volume, water is slowly added, and the concentration is continued until the residue contains less than 2g / L of product, and the ceramic filtration is stopped. The retentate is retained and can be applied to the next batch of feed liquid.

[0050] 3. Ultrafiltration and concentration The feed liquid clarified by the ceramic membrane is filtered with a 3000Da membrane, and the pressure is maintained at 0.6-0.8bar. After concentrating to the minimum volume, water is added and the concentration is continued until the retentate contains less than 2g / L of product, and the ultrafiltration is stopped. The retentate is retained and can be applied to the next batch of feed liquid. The ultrafiltration filtrate is simply concentrated to a product content of about 100g / L.

[0051] 4. Desalination and activated carbon adsorption The concentrated liquid is subjected to electrodialysis filtration for desalination, and then to ion exchange resin for desalination. When the conductivity of the clear liquid is lower than 10us / cm, nanofiltration is performed, and then 0.5% to 1% activated carbon is added to the liquid for adsorption and decolorization. After stirring at 50 to 60°C for 2 hours, it is filtered through a closed filter and finally filtered through a 0.22μm plate and frame filter.

[0052] 5. Distillation, crystallization, centrifugation Set the evaporation temperature to 70-80℃, the vacuum degree ≤-0.08MPa, and concentrate to a product concentration of 750-850g / L. Transfer the distilled concentrate to a crystallization tank, set the refrigeration temperature to 5-10℃, start stirring, and crystallize for 4-6h. Centrifuge after the crystallization is completed, and add a small amount of anhydrous ethanol to wash the crystallized product while centrifuging after the mother liquor is centrifuged. The resulting clear liquid is mother liquor A, which is transferred to the mother liquor temporary storage tank. The solid obtained by centrifugation is the crude product. After drying, the crude product A is obtained, labeled, sampled and tested for purity content, and transferred to a cold storage for temporary storage.

[0053] 6. Process description of mother liquor and crude product After a certain amount of mother liquor A is collected, ethanol is removed, and the steps of electrodialysis, distillation and crystallization are repeated. After centrifugation, mother liquor B and crude product are obtained. The crude product is dried to obtain crude product B. After a certain amount of mother liquor B is collected, the above steps of electrodialysis, distillation and crystallization, and centrifugation are repeated. Samples are taken to test the content purity of crude product A and crude product B. The crude product should be a white powder with a content purity of ≥99% to be qualified. Unqualified products need to be recrystallized, dissolved with purified water, and concentrated and crystallized by centrifugation.

[0054] During the whole process, the substrate conversion rate of the enzymatic reaction reached 96.9% after 12 hours of detection, which was about 5.2 hours less than the reaction time of the original strain. Compared with the current catalytic time, the efficiency was increased by 43%, and the ee value was measured to be 99.6%. The crude product reached the qualified purity after two concentration and crystallization. According to manual calculation, the entire 22T fermentation reaction and purification process reduced the cost by about 8% compared with the original dual-enzyme catalysis process.

[0055] The above embodiments are only used to illustrate the technical ideas and features of the present invention, and are not intended to be the only or limit the present invention. It should be understood by those skilled in the art that various changes or equivalent substitutions made to the present invention without departing from the scope of the present invention all fall within the scope of protection of the present invention.

[0056] Nucleotide sequence SEQ ID No.1 SEQ ID No.2 SEQ ID No.3 SEQ ID No.4 SEQ ID No.5 SEQ ID No.6 SEQ ID No.7 SEQ ID No.8 SEQ ID No.9 SEQ ID No.10

Claims

1. A mutant dual enzyme catalyst, characterized in that: include: The methylglyoxal reductase mutant, wherein the amino acid site mutation form is any of the following: (1) The valine at position 130 of the nucleotide sequence shown in SEQ ID No. 1 is replaced by glycine, the glutamic acid at position 165 is replaced by threonine, and the glycine at position 247 is replaced by arginine; (2) The valine at position 130 of the nucleotide sequence shown in SEQ ID No. 1 is replaced by glycine, the serine at position 196 is replaced by alanine, and the aspartic acid at position 251 is replaced by methionine; (3) The histidine at position 87 of the nucleotide sequence shown in SEQ ID No. 1 is replaced by lysine, the arginine at position 173 is replaced by histidine, and the isoleucine at position 203 is replaced by valine; (4) The histidine at position 87 of the nucleotide sequence shown in SEQ ID No. 1 is replaced by lysine, the lysine at position 148 is replaced by arginine, and the glycine at position 298 is replaced by proline; (5) The threonine at position 163 of the nucleotide sequence shown in SEQ ID No. 1 is replaced by serine, the aspartic acid at position 195 is replaced by glutamic acid, and the alanine at position 313 is replaced by leucine; (6) The threonine at position 163 of the nucleotide sequence shown in SEQ ID No. 1 is replaced by serine, the phenylalanine at position 189 is replaced by tryptophan, and the glycine at position 278 is replaced by proline; The phosphite dehydrogenase mutant, wherein the amino acid site mutation form is any of the following: (7) replacing the tryptophan at position 133 of the nucleotide sequence shown in SEQ ID No. 2 with tyrosine, and replacing the leucine at position 204 with alanine; (8) The glycine at position 77 of the nucleotide sequence shown in SEQ ID No. 2 is replaced by proline, and the phenylalanine at position 166 is replaced by tryptophan.

2. The mutant dual enzyme catalyst according to claim 1, characterized in that The mass ratio of the methylglyoxal reductase mutant and the phosphite dehydrogenase mutant is 1 to 1.6:

1.

3. A gene encoding the methylglyoxal reductase mutant according to claim 1.

4. The gene according to claim 3, characterized in that , the nucleotide sequence of the gene is selected from any one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:

8.

5. An expression vector or recombinant bacterium comprising the gene according to claim 3.

6. A gene encoding the phosphite dehydrogenase mutant according to claim 1.

7. The gene according to claim 6, characterized in that , the nucleotide sequence of the gene is selected from any one of SEQ ID NO:9 and SEQ ID NO:

10.

8. An expression vector or recombinant bacterium comprising the gene according to claim 6.

9. The use of the mutant dual enzyme catalyst as claimed in claim 1, characterized in that: Used to synthesize (S)-Boseine.

10. A method for synthesizing (S)-Bosone, characterized in that: The reaction temperature is 30-40°C, the substrate is β-acetone xyloside, the substrate concentration is 40 g / L-100 g / L, the isopropanol concentration is 40 g / L-100 g / L, the potassium phosphite concentration is 20 g / L-50 g / L, the NADP salt concentration is 0.05 g / L-0.2 g / L, the mass ratio of the methylglyoxal reductase mutant to the substrate is 0.2-1:1, the mass ratio of the phosphite dehydrogenase mutant to the substrate is 0.1-0.4:1, the pH range is maintained at 7.0-7.5 during the entire reaction process, the reaction is continued for 12 hours, and ventilation and purging are performed.

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