A mutant dual-enzyme catalyst, its application and (S)-Bosone synthesis method

Abstract

The present invention discloses a mutant dual-enzyme catalyst, application and (S)-Bosin synthesis method, relating to the field of bioengineering technology, the present invention is based on the methylglyoxal reductase GRE2 (A) of Candida glabrata and the phosphite dehydrogenase PTDH of methylotrophic bacteria as sources for transformation, and its mutants are screened, the key residues affecting the enzymatic properties of the initial methylglyoxal reductase and phosphite dehydrogenase are determined, and methylglyoxal reductase mutants and phosphite dehydrogenase mutants with high activity are obtained. The present invention utilizes the two most efficient mutants to catalyze the reaction of preparing (S)-Bosin from β-acetone xyloside in a 22T reaction system, with good catalytic effect, overall catalytic efficiency is increased by about 43%, and 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 hydroxypropyltetrahydropyrantriol, is a glycoprotein mixture that stimulates the production of glucosamine (GAGs) in the skin. GAGs are a major component of the extracellular matrix, and hyaluronic acid is one of the main components of GAGs. Hyaluronic acid has strong water absorption and storage capabilities. When injected into the dermis, it can achieve a filling and hydrating effect, plumping and elasticizing the skin, thereby preventing skin aging. It is also easily biodegradable and mildly irritating to the human body. It is widely used in skincare, cosmetics, and toiletries, and has a very broad market prospect.

[0003] (S)-Bosone was initially synthesized primarily using chemical methods. For example, CN113735811A describes the synthesis of a first intermediate from acetylacetone and xylose, followed by ketalization, reduction, and deprotection to obtain (S)-Bosone. CN112812087A describes the synthesis of a key intermediate using (2R,3S,4S)-2,3,4-tribenzyloxyglutaraldehyde with acetone, followed by a two-step catalytic reaction to obtain (S)-Bosone. However, chemical synthesis methods suffer from numerous steps, complex reaction conditions, high costs, and significant pollution.

[0004] In recent years, the bioenzymatic synthesis of (S)-Bose has gradually become the most important 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. There is a problem that coenzyme cannot be recycled in single enzyme catalysis, which easily causes waste of drugs and increases production costs; and a variety of enzyme preparations are used for catalytic production, which increases fermentation costs, and the concentration of catalytic reaction substrates 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 the enzyme catalytic efficiency, and reduce the reaction time are issues that need to be urgently addressed in large-scale production. Summary of the Invention

[0006] To address the deficiencies in the prior art, the present invention provides a mutant dual-enzyme catalyst, its application, and a method for synthesizing S-configuration bosonine. Specifically, the present invention provides gene sequences comprising a methylglyoxal reductase mutant and a phosphite dehydrogenase mutant, expression vectors comprising the gene sequences of the methylglyoxal reductase mutant and the phosphite dehydrogenase mutant, and host cells thereof. Furthermore, the present invention provides a method for preparing (S)-bosonine from β-acetone xyloside using the methylglyoxal reductase mutant and the phosphite dehydrogenase mutant.

[0007] In order to achieve the purpose of the present invention, the following scheme is proposed:

[0008] This study used the methylglyoxal reductase GRE2(A) (GeneID: 2887289) from Nakaseomyces glabratus, listed in GenBank, as the initial methylglyoxal reductase. Site-directed mutagenesis of the gene encoding this initial methylglyoxal reductase was performed, and the resulting mutants were screened. Key residues influencing the enzymatic properties of the initial methylglyoxal reductase were identified, and highly active methylglyoxal reductase mutants were obtained. Experimental results demonstrated that the screened methylglyoxal reductase mutants exhibited excellent catalytic activity in the preparation of (S)-bosone using β-acetone xyloside and isopropanol as substrates.

[0009] The methylglyoxal reductase GRE2(A) mutant of the present invention has a protein nucleotide sequence selected from one of the following sequences:

[0010] (1) The nucleotide sequence shown in SEQ ID No. 3: after the amino acids encoded by the nucleotide sequence shown in SEQ ID No. 1, the valine at position 130 is replaced by glycine, the glutamic acid at position 165 is replaced by threonine, and the glycine at position 247 is replaced by arginine.

[0011] (2) The nucleotide sequence shown in SEQ ID No. 4: after the amino acids encoded by the nucleotide sequence shown in SEQ ID No. 1, the valine at position 130 is replaced by glycine, the serine at position 196 is replaced by alanine, and the aspartic acid at position 251 is replaced by methionine.

[0012] (3) The amino acid sequence shown in SEQ ID No. 5: after the amino acids encoded by the amino acid sequence shown in SEQ ID No. 1, the histidine at position 87 is replaced by lysine, the arginine at position 173 is replaced by histidine, and the isoleucine at position 203 is replaced by valine.

[0013] (4) The nucleotide sequence shown in SEQ ID No. 6: after the amino acids encoded by the nucleotide sequence shown in SEQ ID No. 1, histidine at position 87 is replaced by lysine, lysine at position 148 is replaced by arginine, and glycine at position 298 is replaced by proline.

[0014] (5) The nucleotide sequence shown in SEQ ID No. 7: after the amino acids encoded by the nucleotide sequence shown in SEQ ID No. 1, the threonine at position 163 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.

[0015] (6) The nucleotide sequence shown in SEQ ID No. 8: after the amino acids encoded by the nucleotide sequence shown in SEQ ID No. 1, the threonine at position 163 is replaced by serine, the phenylalanine at position 189 is replaced by tryptophan, and the glycine at position 278 is replaced by proline.

[0016] This study used the phosphite dehydrogenase PTDH (GeneID: 72987700) from the methylotrophic bacterium Methylorubrum extorquens, listed in GenBank, as the initial phosphite dehydrogenase. Site-directed mutagenesis of the gene encoding this initial phosphite dehydrogenase was performed, and the resulting mutants were screened. Key residues influencing the enzymatic properties of the initial phosphite dehydrogenase were identified, and highly active phosphite dehydrogenase mutants were obtained. Experimental results demonstrated that the screened phosphite dehydrogenase mutants exhibited a significant promoting effect on the NADPH coenzyme cycle.

[0017] The phosphite dehydrogenase PTDH mutant of the present invention has a protein nucleotide sequence selected from one of the following sequences:

[0018] (7) The nucleotide sequence shown in SEQ ID No. 9: after the amino acids encoded by the nucleotide sequence shown in SEQ ID No. 2, tryptophan at position 133 is replaced by tyrosine, and leucine at position 204 is replaced by alanine.

[0019] (8) The nucleotide sequence shown in SEQ ID No. 10: after the amino acids encoded by the nucleotide sequence shown in SEQ ID No. 2, glycine at position 77 is replaced by proline, and phenylalanine at position 166 is replaced by tryptophan.

[0020] 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.

[0021] 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.

[0022] The mutant sequence shown in the present invention is optimized for 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, using any one of pET22b, pET28a, pET32a, and pCold TF, wherein methylglyoxal reductase is preferably a pET28a plasmid and phosphite dehydrogenase is preferably a pET32a plasmid; the host cell is Escherichia coli BL21 (DE3), which is suitable for efficient expression of exogenous genes.

[0023] The present invention also provides a method for synthesizing (S)-Boseine, comprising the following steps:

[0024] (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;

[0025] (2) The methylglyoxal reductase mutant and the phosphite dehydrogenase mutant described above were added to the reaction system, the reaction temperature was 30°C to 40°C, the substrate was β-acetone xyloside, the substrate concentration was 40g / L to 100g / L, the isopropanol concentration was 40g / L to 100g / L, the potassium phosphite concentration was 20g / L to 50g / L, the NADP salt concentration was 0.05g / L to 0.2g / L, the mass ratio of the methylglyoxal reductase mutant to the substrate was 0.2 to 1:1, and the mass ratio of the phosphite dehydrogenase mutant to the substrate was 0.1 to 0.4:1.

[0026] Furthermore, the methylglyoxal reductase mutant and the phosphite dehydrogenase mutant are prepared by centrifuging the bacteria at 6000 rpm or above 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.

[0027] The bacterial sludge of the methylglyoxal reductase mutant and the phosphite dehydrogenase mutant 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, and 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.

[0028] 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.

[0029] 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.

[0030] The beneficial effects of the present invention are:

[0031] (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.

[0032] (2) The present invention modified the phosphite dehydrogenase PTDH from the methylotrophic bacteria (Methylorubrum extorquens) and screened the corresponding mutants. The key residues that affect the enzymatic properties of the initial phosphite dehydrogenase were determined through software analysis. At the same time, a highly active phosphite dehydrogenase mutant was obtained. Its maximum catalytic activity was 0.97 times higher than that of the original strain, achieving a rapid cycle of NADPH.

[0033] (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)-bosyltransferase from β-acetone xyloside;

[0034] (4) The methylglyoxal reductase mutant and phosphite dehydrogenase mutant screened in the present invention catalyze the β-acetone xyloside in the preparation of (S)-bosine with 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 optical purity of the obtained (S)-bosine is high, and the ee value is between 99.3% and 99.9%. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The plasmid construction map of wild-type methylglyoxal reductase (pET28a-GRE2(A)) is shown;

[0036] Figure 2 The plasmid construction map of phosphite dehydrogenase (pET32a-PTDH) is shown;

[0037] Figure 3 shows the protein electrophoresis patterns of methylglyoxal reductase mutants (H87K, K148R, G298P) and phosphite dehydrogenase mutants (G77P, F166W);

[0038] Figure 4 shows the enzyme activity reaction curve of phosphite dehydrogenase mutants (G77P, F166W);

[0039] Figure 5 The figure shows the high performance liquid chromatogram of the product obtained by using methylglyoxal reductase mutants (H87K, K148R, G298P) as catalyst. DETAILED DESCRIPTION

[0040] Example 1

[0041] 1. Construction of wild-type methylglyoxal reductase expression strain

[0042] 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.

[0043] 2. Construction of methylglyoxal reductase mutant expression strain

[0044] Site-directed mutagenesis of GRE2(A) yielded the following mutants:

[0045] (1) GRE2(A)-1: V130G, E165T, G247R, the nucleotide sequence is shown in SEQ ID No. 3.

[0046] (2) GRE2(A)-2: V130G, S196A, D251M, the nucleotide sequence is shown in SEQ ID No. 4.

[0047] (3) GRE2(A)-3: H87K, R173H, I203V, the nucleotide sequence is shown in SEQ ID No. 5.

[0048] (4) GRE2(A)-4: H87K, K148R, G298P, the nucleotide sequence is shown in SEQ ID No. 6.

[0049] (5) GRE2(A)-5: T163S, D195E, A313L, the nucleotide sequence is shown in SEQ ID No. 7.

[0050] (6) GRE2(A)-6: T163S, F189W, G278P, the nucleotide sequence is shown in SEQ ID No. 8.

[0051] The above sequences were commissioned to Beijing Qingke Biotechnology Co., Ltd. for E. coli codon optimization of the respective coding nucleotide sequences, followed by full gene synthesis to obtain the corresponding recombinant vectors. After sequencing verification, they were introduced into E. coli BL21 (DE3) to obtain the corresponding expression strains.

[0052] 3. Preparation of methylglyoxal reductase mutant enzyme solution

[0053] 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 the OD600 was about 0.8, and IPTG was added at a final concentration of 0.05 mM 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 subjected to protein electrophoresis to verify the band size and solubility, as shown in FIG. Figure 3As 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 fermentation and cell lysis of Escherichia coli BL21(DE3)pET28a-GRE2(A) induced at 25℃, lane 4 is the supernatant protein of high-speed centrifugation after fermentation and cell lysis of Escherichia coli BL21(DE3)pET28a-GRE2(A) induced at 25℃, lane 5 is the whole-cell protein after fermentation and cell lysis of Escherichia coli BL21(DE3)-pET32a-PDTH induced at 25℃, lane 6 is the supernatant protein of high-speed centrifugation after fermentation and cell lysis of Escherichia coli BL21(DE3)-pET32a-PDTH induced at 25℃. The positions and sizes of the protein bands are correct, and the proteins are well soluble.

[0054] 4. Catalytic ability and ee value of methylglyoxal reductase mutants

[0055] 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+, dilute to 50 mL with phosphate buffer (50 mmol / L, pH 7.0), add 2 g of different methylglyoxal reductase mutant enzyme solution obtained in step 3 respectively, and react for 12 h.

[0056] 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 of ddH20 and filtered with a 0.22 μm filter membrane for liquid phase analysis. The conversion rates and product ee values ​​of different methylglyoxal reductase mutants were determined. The results are shown in Table 1.

[0057] The mobile phase for liquid chromatography was 850 mL of chromatography-grade acetonitrile, diluted to 1 L with ddH₂O. The mobile phase was filtered through a 0.22 μm organic filter membrane. The mobile phase was ultrasonically degassed for 30 minutes to remove air bubbles. The resulting mobile phase was used. The liquid chromatograph was a Tongwei Liquid Phase 3030, the detector was an evaporative light detector, and the detection column was a Yuexu NH₂ column with specifications of 250 nm × 4.6 nm, 5 μm. The mobile phase flow rate was 1 mL / min, the injection volume was 10 μL, the detection temperature was 35°C, and the peak detection time was approximately 4 minutes.

[0058] Table 1 Comparison of catalytic abilities of different methylglyoxal reductase mutants

[0059]

[0060] Note: ee value means enantiomeric excess.

[0061] Example 2

[0062] 1. Construction of wild-type phosphite dehydrogenase expression strain

[0063] 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 below. Figure 2 After sequencing verification, the gene was introduced into Escherichia coli BL21 (DE3) to construct an expression strain.

[0064] 2. Construction of phosphite dehydrogenase mutant expression strain

[0065] Site-directed mutagenesis of PTDH resulted in the following mutants:

[0066] (1) PTDH-1: W133Y, L204A, the nucleotide sequence is shown in SEQ ID No. 9.

[0067] (2) PTDH-2: G77P, F166W, the nucleotide sequence is shown in SEQ ID No. 10.

[0068] The above sequences were commissioned to Beijing Qingke Biotechnology Co., Ltd. for E. coli codon optimization of the respective coding nucleotide sequences, followed by full gene synthesis to obtain the corresponding recombinant vectors. After sequencing verification, they were introduced into E. coli BL21 (DE3) to obtain the corresponding expression strains.

[0069] 3. Preparation of phosphite dehydrogenase mutant enzyme solution

[0070] The above recombinant bacteria were inoculated into LB liquid culture medium containing 50 μg / ml of ampicillin, cultured at 37°C and 200 rpm for about 6 hours until the OD600 was about 0.8, and IPTG with a final concentration of 0.05 mM was added for induction, and induced at 22°C for 16 hours. Then, the bacteria were collected by centrifugation at 8000 rpm for 10 minutes. 2.5 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 enzyme solution of the phosphite dehydrogenase mutant corresponding to the above sequence nucleotides, and the mutants (G77P, F166W) were subjected to protein electrophoresis to verify the band size and solubility, as shown in FIG. Figure 3 shown.

[0071] 4. Determination of enzyme activity of phosphite dehydrogenase mutants

[0072] The enzyme activity assay reaction system (2 mL) is: 20 mM MOPS, pH 7.3, 1 mM phosphite, 0.5 mM NADP+, and 400 μl of crude phosphite dehydrogenase enzyme concentrate. After mixing all the contents, the absorbance change at 340 nm was measured. A blank control without the addition of crude phosphite dehydrogenase enzyme concentrate was also performed. The absorbance change of the enzyme activity assay is as follows: Figure 4 Protein concentration was calculated using the Braford method. One enzyme activity was defined as the conversion of 1 μmol of NADP salt to NADPH per minute under the experimental conditions. The overall mutant activity data are shown in Table 2.

[0073] Table 2 Comparison of catalytic abilities of different phosphite dehydrogenase mutants

[0074]

[0075] Test data: 2ml total reaction system, 400μl enzyme solution (9.8mg), absorbance change within 2min is 0.237, NADPH extinction coefficient in diluted buffer solution is 6220L / mol / cm, optical path is 1cm, reaction rate is shown in Figure 4 .

[0076] The calculation formula of enzyme activity determination according to the photometric method is:

[0077]

[0078] : Change in solution absorbance, unitless;

[0079] : molar absorption coefficient of luminescent substance, unit ;

[0080] 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;

[0081] t: reaction time, unit min;

[0082] : reaction volume, unit: ml;

[0083] Substituting the above data, we can obtain: the enzyme activity of the best mutant = 381.03 μmol / min, that is, 381.03 U / 400 μl, which is converted to 952.57 U / ml, and the specific activity is 97.2 U / mg, which has a high enzyme activity.

[0084] Example 3

[0085] This embodiment provides a method for synthesizing (S)-Boseine, comprising the following steps:

[0086] 1. Feeding reaction

[0087] 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 entire system is about 22T. Maintain pH 7.0-7.5 and react at 30°C. 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. After the substrate reaction is completed (such as Figure 5 Then, the temperature is raised to 55-60°C for denaturation for 1-2 hours.

[0088] 2. Ceramic membrane sterilization

[0089] Clarify the feed liquid with a ceramic membrane. Adjust the frequency to maintain a pressure of 0.4-0.5 bar. After concentrating to the minimum volume, slowly add water and continue concentrating until the residue contains less than 2g / L of product. Then stop the ceramic filtration. The retentate is retained and can be used in the next batch of feed liquid.

[0090] 3. Ultrafiltration and concentration

[0091] The clarified feed solution from the ceramic membrane is filtered through a 3000 Da membrane, maintaining a pressure of 0.6-0.8 bar. After concentrating to a minimum volume, water is added and concentration is continued until the retentate contains less than 2 g / L of product. Ultrafiltration is then stopped. The retentate is retained and can be used in the next batch of feed solution. The ultrafiltration filtrate is briefly concentrated to a product content of approximately 100 g / L.

[0092] 4. Desalination and activated carbon adsorption

[0093] 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.

[0094] 5. Distillation, crystallization, centrifugation

[0095] Set the evaporation temperature to 70-80°C and the vacuum degree to ≤-0.08 MPa, and concentrate to a product concentration of 750-850 g / L. Transfer the distilled concentrate to a crystallizer, set the refrigeration temperature to 5-10°C, start stirring, and crystallize for 4-6 hours. After crystallization, centrifuge the mother liquor and add a small amount of anhydrous ethanol to rinse the crystallized product while centrifuging. The resulting clear liquid is mother liquor A, which is transferred to a mother liquor temporary storage tank. The solid obtained by centrifugation is the crude product. After drying, the crude product A is obtained, labeled, sampled for purity and content testing, and transferred to cold storage for temporary storage.

[0096] 6. Process description of mother liquor and crude product

[0097] After a certain amount of mother liquor A is collected, the ethanol is removed and the electrodialysis, distillation, crystallization, and centrifugation steps are repeated to obtain mother liquor B and a crude product. The crude product is then dried to obtain crude product B. After a certain amount of mother liquor B is collected, the electrodialysis, distillation, crystallization, and centrifugation steps are repeated. Samples are taken to test the purity of crude products A and B. The crude products should be white powders with a purity of ≥99% to be acceptable. Unacceptable products need to be recrystallized, dissolved in purified water, and concentrated by centrifugation.

[0098] During the entire process, the substrate conversion rate of the enzymatic reaction reached 96.9% after 12 hours of detection, which was about 5.2 hours shorter 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.

[0099] The above embodiments are only used to illustrate the technical ideas and features of the present invention and are not intended to be exclusive 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 are within the scope of protection of the present invention.

[0100] Nucleotide sequence

[0101] SEQ ID No.1

[0102]

[0103] SEQ ID No.2

[0104]

[0105] SEQ ID No.3

[0106]

[0107] SEQ ID No.4

[0108]

[0109] SEQ ID No.5

[0110]

[0111] SEQ ID No.6

[0112]

[0113] SEQ ID No.7

[0114]

[0115] SEQ ID No.8

[0116]

[0117] SEQ ID No.9

[0118]

[0119] SEQ ID No.10

[0120]

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) After the amino acids encoded by the nucleotide sequence shown in SEQ ID No. 1, the valine at position 130 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) After the amino acids are encoded by the nucleotide sequence shown in SEQ ID No. 1, the valine at position 130 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) After the amino acids encoded by the nucleotide sequence shown in SEQ ID No. 1, the histidine at position 87 is replaced by lysine, the arginine at position 173 is replaced by histidine, and the isoleucine at position 203 is replaced by valine; (4) After the amino acids encoded by the nucleotide sequence shown in SEQ ID No. 1, the histidine at position 87 is replaced by lysine, the lysine at position 148 is replaced by arginine, and the glycine at position 298 is replaced by proline; (5) After the amino acids encoded by the nucleotide sequence shown in SEQ ID No. 1, the threonine at position 163 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) After the amino acids encoded by the nucleotide sequence shown in SEQ ID No. 1, the threonine at position 163 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) After the amino acids encoded by the nucleotide sequence shown in SEQ ID No. 2, the tryptophan at position 133 is replaced by tyrosine, and the leucine at position 204 is replaced by alanine; (8) After the amino acids are encoded by the nucleotide sequence shown in SEQ ID No. 2, glycine at position 77 is replaced by proline, and 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. The use of the mutant dual-enzyme catalyst according to claim 1, characterized in that: Used to synthesize (S)-Boseine.

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