Enzyme mutant for synthesizing S-glassine, combined enzyme and application of enzyme mutant and combined enzyme

Abstract

The invention relates to the technical field of biology, in particular to an enzyme mutant for synthesizing S-glassine, a combined enzyme and application of the enzyme mutant and the combined enzyme. The carbonyl reductase mutant with high activity, high stability, high stereoselectivity and excellent performance is obtained by utilizing irrational random mutation and rational site-directed mutation and through multiple rounds of construction and screening of the mutant. According to the invention, a double-enzyme circulating catalysis system is established by coupling formate dehydrogenase or glucose dehydrogenase, and the temperature of gt is reached in a short time; the conversion rate is 95%, and the glass color gt is realized; the yield is high and is 180g / L, and the optical purity of S-glassine is as high as 100%.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an enzyme mutant and a combined enzyme for synthesizing S-bosone and applications thereof. Background Art

[0002] Pro-XylaneTM, also known as hydroxypropyl tetrahydropyrantriol, is a xylose derivative with anti-aging function and is widely used in the fields of medicine and cosmetics. Studies have shown that Pro-Xylane can directly affect the extracellular matrix in the three-layer structure of the skin, activate or promote the synthesis of mucopolysaccharides in the skin, and significantly increase the water content of the extracellular matrix. It can also stimulate the synthesis of type IV and type VII collagen, improve the adhesion between the dermis and the epidermis, promote the regeneration of damaged tissues, effectively keep the skin firm and delicate, and delay skin aging. Pro-Xylane has a variety of stereo configurations: S-Pro-Xylane and R-Pro-Xylane. Among them, the higher the proportion of S-Pro-Xylane, the better the anti-aging activity. However, the quality of Pro-Xylane products on the market is uneven, and most of them are racemic or S configuration only accounts for a small part.

[0003] At present, the preparation methods of bosine are mainly chemical synthesis and enzyme catalysis:

[0004] The foreign chemical synthesis method is that xylose reacts with acetylacetone under alkaline conditions to form β-acetone xyloside, which is then acidified with a strong acid cation exchange resin and the carbonyl group is reduced with borohydride to synthesize bosylamine; or xylose first reacts with acetylacetone and then the carbonyl group is reduced with a heavy metal catalyst Ru / C. China has made improvements on this route. The first step is to use xylose as the raw material and use a strong alkaline anion resin instead of ordinary inorganic bases. The second step is to use sodium borohydride to reduce the carbonyl group to synthesize bosylamine. The above chemical synthesis methods inevitably have the disadvantages of low reduction selectivity, high reagent pollution, and difficulty in product separation and purification, which greatly restricts the industrial production of bosylamine.

[0005] The enzyme catalysis method uses β-acetone xyloside as raw material, sodium formate, glucose or isopropanol as co-substrate, and prepares bosera under the catalysis of carbonyl reductase and coenzyme. Compared with the chemical method, the bioenzyme method has more advantages in preparing bosera. The enzyme method has high stereoselectivity and avoids the use of polluting and dangerous chemical reagents, which greatly improves the greenness of the bosera production process, while making the separation and purification of the product simple and efficient, reducing production costs.

[0006] At present, there are still some defects in the reported enzymatic preparation of bosin. For example, patent CN111876452A discloses a method for preparing bosin using a one-pot method of biological enzymes, but the method uses unidentified enzymes such as isopropanol dehydrogenase, bosin synthase, and carbonyl reductase, which is difficult to reproduce. CN113416756A, CN113717997A and CN 115896199 A all mention methods for synthesizing bosin by double enzyme cycle catalysis, but the reaction time is too long (>24h), the substrate concentration is low (<100g / L), and the optical purity of the final product needs to be improved (S-type ee value <90%). In short, the existing carbonyl reductases generally have problems such as insufficient catalytic ability for β-acetone xyloside and low stability, which is not conducive to the large-scale production of bosin. Summary of the invention

[0007] In view of this, the present invention provides an enzyme mutant, a combined enzyme and its application for synthesizing S-Bosone. The enzyme mutant provided by the present invention has high activity, strong stability and high stereoselectivity. It is further used in combination with a dehydrogenase to achieve efficient synthesis of S-Bosone. The synthesis of S-Bosone using the enzyme mutant and combined enzyme provided by the present invention has the advantages of short time, high yield and high product concentration, wherein the S configuration ee value is 100%.

[0008] An enzyme mutant comprising at least one mutation site among S56P, H125N, Y71P, G161A, F165A, and Y254D;

[0009] The wild-type amino acid sequence of the enzyme mutant has a sequence shown in any of the following:

[0010] 1) The amino acid sequence shown in SEQ ID NO: 1;

[0011] 2) An amino acid sequence having the same or similar function as 1) obtained by replacing, deleting, adding or modifying one or more amino acids in the amino acid sequence shown in 1);

[0012] 3) An amino acid sequence having at least 80% homology with the sequence shown in 1) or 2).

[0013] In some embodiments, the enzyme mutant comprises S56P, H125N and at least one of the following mutation sites:

[0014] Y71P, G161A, F165A, Y254D.

[0015] Furthermore, the enzyme mutant has:

[0016] I), an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 13;

[0017] II), an amino acid sequence having the same or similar function as I) obtained by substituting, deleting, adding or modifying one or more amino acids in the amino acid sequence shown in I);

[0018] III), an amino acid sequence having at least 80% homology with the sequence shown in I) or II).

[0019] The present invention first discovered that Uniprot#A0A6I4YQY2, derived from a sugar bacillus (Saccharibacter sp.EH611), has carbonyl reductase (CAR) activity, which is recorded as wild-type carbonyl reductase CAR, but the catalytic activity of the enzyme is low. The present invention uses irrational random mutations and rational site-directed mutations to transform the wild type, and after multiple rounds of mutant construction and screening, carbonyl reductase mutants with excellent performance are obtained, recorded as CAR-M01~CAR-M12, and their amino acid sequences are shown in SEQ ID NO: 2~13. Experiments show that the catalytic activity of CAR-M04~CAR-M12 is significantly higher than that of the wild-type CAR enzyme, which greatly increases the yield of S-bosine. Compared with the wild-type CAR enzyme, the yield of S-bosine is increased to more than 2 times, and it also has obvious advantages over the mutants CAR-M01~CAR-M04.

[0020] The present invention also provides a biomaterial comprising any of the following i) to iv):

[0021] i), a nucleic acid encoding the enzyme mutant according to any one of claims 1 to 3;

[0022] ii) an expression module comprising the nucleic acid described in i);

[0023] iii), a recombinant vector containing the nucleic acid described in i) or the expression module described in ii);

[0024] iv), transfecting or transforming a host containing iii) the recombinant vector, or a host having i) the nucleic acid or ii) the expression module integrated into its genome.

[0025] In the present invention, the backbone of the vector includes pET series, pGEX series, pRSF series, pCDF series or pUC series. In a specific embodiment, the backbone of the vector is pET 28a.

[0026] In the present invention, the host includes a eukaryotic host and a prokaryotic host. The eukaryotic host includes yeast; the yeast includes Saccharomyces cerevisiae and Pichia pastoris. The prokaryotic host includes Escherichia coli and Bacillus subtilis. In a specific embodiment of the present invention, the host is Escherichia coli, specifically Escherichia coli BL21 (DE3).

[0027] In the present invention, the nucleic acid sequence encoding the wild-type carbonyl reductase CAR is optimized according to the codon preference of the host. In some specific embodiments, the present invention performs codon optimization according to the codon preference of Escherichia coli, and the optimized nucleic acid has a sequence as shown in SEQ ID NO: 14.

[0028] In the present invention, the nucleic acid encoding the enzyme mutants shown in SEQ ID NOs: 2 to 13 is codon optimized according to the host codon preference. In some specific embodiments, the present invention performs codon optimization according to the codon preference of Escherichia coli, and the optimized nucleic acid has any of the following sequences:

[0029] a), the nucleotide sequence shown in SEQ ID NO: 15 to 26;

[0030] b) a nucleotide sequence having the same or similar function as a) obtained by replacing, deleting, adding or modifying one or more bases in the nucleotide sequence shown in a);

[0031] c) a nucleotide sequence having at least 80% homology to the sequence shown in a) or b).

[0032] In a specific embodiment of the present invention, the nucleic acid sequences encoding the enzyme mutants shown in SEQ ID NOs: 2 to 13 are shown in SEQ ID NOs: 15 to 26, respectively.

[0033] The present invention also provides a combined enzyme, comprising: the enzyme mutant as described above and a dehydrogenase of the present invention;

[0034] The dehydrogenase includes formate dehydrogenase and / or glucose dehydrogenase.

[0035] In the present invention, the combined enzyme includes the enzyme mutant and glucose dehydrogenase as described above. In some embodiments, the source of the glucose dehydrogenase includes Bacillus megaterium; in some specific embodiments, the glucose dehydrogenase is derived from Bacillus megaterium, Uniprot#P39483, and its amino acid sequence is shown in SEQ ID NO: 27, and its coding gene sequence after Escherichia coli codon optimization is shown in SEQ ID NO: 29. In some embodiments, the enzyme mutant and glucose dehydrogenase have an enzyme activity ratio of 2500-3500:3000-3200; in some specific embodiments, the enzyme activity ratio can be 2500:3000, 2500:3200, 3500:3000, 3500:3200, or any value between any two of the above numerical ranges.

[0036] In the present invention, the combined enzyme includes the enzyme mutant and formate dehydrogenase as described above. Further, the specific source of the formate dehydrogenase includes Pseudomonas; in some specific implementations of the present invention, the formate dehydrogenase is derived from Pseudomonas sp. Strain 101, and its amino acid sequence is shown in SEQ ID NO: 28; its encoding gene is optimized by Escherichia coli codon, and the optimized sequence is shown in SEQ ID NO: 30. In some embodiments, the enzyme activity ratio of the enzyme mutant and the formate dehydrogenase is 3200-4000:4000-4500; in some specific embodiments of the present invention, the enzyme activity ratio can be 3200:4000, 3200:4500, 4000:4000, 4000:4500, or any value between any two of the above numerical ranges.

[0037] The present invention also provides the use of any of the following items in the preparation of S-boson:

[0038] A, a carbonyl reductase having an amino acid sequence as shown in SEQ ID NO: 1;

[0039] B. the enzyme mutant described above in the present invention;

[0040] C. the biomaterial as described above in the present invention;

[0041] D. The combined enzyme of the present invention as described above.

[0042] The present invention also provides a method for preparing S-bosone, which comprises: reacting with β-acetone xyloside as a raw material under the action of carbonyl reductase or the combined enzyme of the present invention to obtain S-bosone;

[0043] The carbonyl reductase is selected from any of the following:

[0044] A), a carbonyl reductase having an amino acid sequence as shown in SEQ ID NO: 1;

[0045] B), the enzyme mutant as described above of the present invention;

[0046] C) Enzyme mutants prepared based on the biomaterials described above in the present invention.

[0047] In the present invention, the concentration of the β-acetone xyloside is 100-1000 mM; in some specific embodiments, the concentration of the β-acetone xyloside is 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, or any value between any two of the above numerical ranges.

[0048] In the present invention, the raw materials of the reaction also include NAD, which serves as a cosubstrate to regenerate NADH.

[0049] In the present invention, when the combined enzyme is used to prepare S-bosone, the raw materials of the reaction include at least one of sodium formate, ammonium formate, and glucose on the basis of NAD. In some embodiments, the raw materials of the reaction also include NAD and sodium formate, or include NAD and ammonium formate. Among them, the concentration of NAD is 1 to 3mM, specifically 1mM, 2mM, 3mM. The concentration of sodium formate is 110 to 2000mM, specifically 1300mM. , Or any value between any two numerical ranges mentioned above. The concentration of ammonium formate is 110 to 2000mM, specifically 1200mM.

[0050] In other embodiments, the raw materials of the reaction also include NAD and glucose. The concentration of the NAD is 1 to 3 mM, specifically 1 mM, 2 mM, 3 mM. The concentration of the glucose is 36 to 2000 mM, specifically 36 mM, 60 mM, 80 mM, 100 mM, 120 mM, 300 mM, 500 mM, 800 mM, 1000 mM, 1200 mM, 1500 mM, 1800 mM, 2000 mM, or any value within the above two numerical ranges.

[0051] In the present invention, the pH of the reaction is 6.0 to 7.0, specifically 6.0, 6.5, 7.0, or any value within the above two numerical ranges;

[0052] And / or, the reaction temperature is 25-40°C, specifically 25°C, 30°C, 35°C or 40°C, or any value within the above two numerical ranges;

[0053] And / or, the reaction is carried out under stirring conditions;

[0054] And / or, the reaction is carried out in a buffer; the reaction can be specifically carried out in a PBS buffer, more specifically, the reaction is carried out in a 100 mM PBS buffer;

[0055] And / or, the reaction time is 5 to 10 hours; specifically, it can be 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or any value within the above two numerical ranges.

[0056] The present invention provides enzyme mutants, combined enzymes and applications thereof for synthesizing S-Bosone. The present invention utilizes irrational random mutations and rational site-directed mutations, and obtains excellent carbonyl reductase mutants with high activity, strong stability and high stereoselectivity through multiple rounds of mutant construction and screening, which significantly increases the yield of S-Bosone. The present invention also establishes a dual enzyme cycle catalytic system by coupling formate dehydrogenase or glucose dehydrogenase, and achieves a high conversion rate of >95% in a short time (about 5 to 10 hours), achieving a high yield of >180g / L of Bosone, wherein the optical purity of S-Bosone is as high as 100%, with the advantages of short time, high yield and high product concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The synthetic route diagram of S-Bosone of the present invention is shown;

[0058] Figure 2 The liquid chromatography diagram of the enzyme cascade system 1 in Example 3 is shown, wherein A is the liquid chromatography result of the reaction conversion rate; B is the liquid chromatography result of the R / S boson reference substance; C is the liquid chromatography result of the product after the enzyme reaction of the present invention, the reaction product is only S-boson, and the peak between 3.75 and 5.00 is the solvent peak;

[0059] Figure 3 shows the mass spectrum of the enzyme cascade system 1 in Example 3;

[0060] Figure 4 shows the liquid chromatogram of the enzyme cascade system 2 in Example 3;

[0061] Figure 5 Shows the results of chromogenic screening of CAR mutants;

[0062] Figure 6 Shown are the protein expression results of wild-type CAR (WT) and CAR mutants;

[0063] Figure 7 4 shows the liquid chromatogram of Example 6;

[0064] Figure 8 4 shows the liquid chromatogram of Example 7;

[0065] Fig. 9 4 shows the liquid chromatogram of Example 8;

[0066] Fig.10 The liquid chromatogram of Example 9 is shown. DETAILED DESCRIPTION

[0067] The present invention provides enzyme mutants, combined enzymes and applications for synthesizing S-bosone. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0068] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0069] In the present invention, the abbreviations and their English meanings are as follows:

[0070] CAR, carbonylreductase;

[0071] GDH, glucose dehydrogenase;

[0072] FDH, formate dehydrogenase, formate dehydrogenase.

[0073] In the present invention, the wild-type enzyme of the enzyme mutant is carbonyl reductase CAR, which is derived from a saccharobacterium (Saccharibacter sp. EH611), and is only a predicted function, Uniprot#A0A6I4YQY2. The present invention experiments the carbonyl reduction function of this enzyme, and transforms it on the basis of it to obtain the enzyme mutant of the present invention, further improving the reducing ability. Comparing the mutants with different mutation point combinations, the present invention is numbered as M01, M02, M03, M04, M05, M06, M07, M08, M09, M10, M11 and M12; the results show that the above mutants have better carbonyl reduction ability than the wild-type enzyme, and the catalytic synthesis of S-bosine has a higher yield, which is more suitable for industrial production of bosine.

[0074] In the present invention, glucose dehydrogenase (GDH) is derived from Bacillus megaterium, Uniprot#P39483, and formate dehydrogenase (FDH) is derived from Pseudomonas sp. Strain 101, Uniprot#P33160.

[0075] The amino acid sequences of the wild-type enzyme and enzyme mutants involved in the present invention are shown in Table 1.

[0076] Table 1

[0077]

[0078]

[0079]

[0080] Note: The amino acids in bold and underlined are the mutated amino acids.

[0081] Table 2

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] Note: The underlined bases are the mutated codons.

[0089] The present invention will be further described below in conjunction with embodiments:

[0090] Example 1: Synthesis of β-acetone xyloside

[0091] 75 g of xylose (500 mM), 45 g of acetylacetone (900 mM) and 20 g of NaOH (1000 mM) were added to 500 mL of pure water, and the reaction was carried out at 55 degrees for 90 min. After the reaction, the pH of the reaction solution was adjusted to neutral with HCl aqueous solution, and the solvent was removed to obtain the raw material β-acetone xyloside.

[0092] Example 2: Fermentation production of enzymes

[0093] The enzymes required by the present invention are all synthesized by the company, constructed on a specific expression plasmid, and then produced by Escherichia coli fermentation; it specifically includes the following steps:

[0094] After the sequence of the gene corresponding to the above enzyme is optimized, it is ordered to General Biotechnology Company for synthesis (Chuzhou, Anhui), and then subcloned into the pET 28a expression vector. The plasmid with the correct sequence is transferred into Escherichia coli BL21 (DE3) competent cells for plate culture and monoclonal small-scale liquid culture. The bacteria with correct protein expression are finally amplified in liquid culture step by step. The specific steps are to transfer a single colony into 5mL LB culture medium (37℃) containing 50μM kanamycin for culture. When the cells grow to the logarithmic phase, they are inoculated into 250mL LB culture medium containing the same antibiotics. When they grow to the logarithmic phase, they are transferred to a 5L culture fermenter for culture and final protein expression. In the 5L fermenter culture, when the cell OD is ~20, 0.5mM isopropyl-β-D-thiogalactopyranoside (IPTG) is added at 25℃ to induce protein expression for 6 hours. Finally, the cells are collected by high-speed centrifugation (4000rpm, 20min) to obtain 30-60g of enzyme-overexpressing wet cells. Take a small amount of cells and mix them evenly with Tris-HCl buffer (50mM, pH8.0) on an ice basin, then break the cells by freeze-thaw method, remove the cell wall by high-speed centrifugation, and run SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to determine protein expression. LB medium consists of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate and 5% glycerol.

[0095] Example 3: Preparation of Bossagene using wild-type CAR enzyme method

[0096] Enzyme cascade system 1: 190 g of β-acetone xyloside (1000 mM), 270 g of glucose (1500 mM) and 0.7 g of NAD (1 mM) were added to 1 L 100 mM PBS buffer, the pH value was adjusted to 7.0 with HCl aqueous solution, and 3500 U CAR and 3200 U GDH were added to start the reaction. The reaction was stirred at pH 6.0 and 35 ° C for 13 hours. A small amount of the reaction solution was quenched with 60% acetonitrile, centrifuged at 12000 rpm for 2 min, and the supernatant was filtered for HPLC detection. For details of the liquid phase spectrum, see Figure 2 . After testing, the yield of bosonine was 115.2g / L (conversion rate was 60%), and the S-type ee value was 100%. The mass spectrometry of the sample confirmed that the product was bosonine. The mass spectrometry results are shown in Figure 3 .

[0097] Enzyme cascade system 2: 190 g of β-acetone xyloside (1000 mM), 88.4 g of sodium formate (1300 mM) and 2.0 g of NAD (3 mM) were added to 1 L 100 mM PBS buffer, the pH value was adjusted to 7.0 with HCl aqueous solution, 3200 U CAR and 4000 U FDH were added to start the reaction, the reaction was stirred at pH 7.0 and 40 ° C for 13 hours, a small amount of the reaction solution was quenched with 60% acetonitrile, centrifuged at 12000 rpm for 2 min, and the supernatant was filtered for HPLC detection. For details of the liquid phase spectrum, see Figure 4 The test showed that the boson yield was 99.8 g / L (conversion rate was 52%), and the S-type ee value was 100%.

[0098] Example 4: Construction and screening of CAR random mutation library

[0099] Using the CAR gene as a template, the GeneMorph II Random Mutagenesis Kit was used for error-prone PCR, and the PCR fragments were recovered by gel. The PCR fragments were inserted into the Nde I and Xho I restriction sites of the vector by homologous recombination, and then the constructed random mutation library was transformed into Escherichia coli BL21 (DE3) competent cells and cultured overnight at 37 degrees on LB solid medium containing 50 μg / mL kanamycin. 400 μL LB medium (containing 50 μg / mL kanamycin) was added to each well of the sterilized 96-well plate and cultured overnight at 37 degrees and 220 rpm. Then transfer 20 μL of seed solution to a 96-well plate containing 600 μL LB medium (containing 50 μg / mL kanamycin), culture at 37 degrees, 220 rpm until OD600 value> 0.8, add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration, cool to 25 degrees to induce expression, and continue to culture for 16 hours. After the culture is completed, the 96 deep-well plate is centrifuged at 4 degrees, 4000 rpm for 10 minutes, the supernatant is discarded, and the bacteria are retained. Add 150 μL of lysozyme digestion solution (1 mg / mL lysozyme, 10 mM Tris-HCl, pH 8) to each well of the collected bacteria, and digest for 1 hour at 25 degrees, 220 rpm. The reaction system was as follows: 600 μL system, 100 μL enzyme solution, the system contained 100 mM PBS, 30 mM β-acetone xyloside, 36 mM glucose, 2 mM NAD, reacted for 4 h, and the supernatant was collected by centrifugation for high-throughput colorimetric detection.

[0100] In view of the structural differences between β-acetone xyloside and bosin, the color reaction of 2,4-dinitrophenylhydrazine and ketone compounds was used to characterize the efficiency of CAR enzyme-catalyzed synthesis of bosin. The high-throughput detection system is as follows: 200μL system, add 5μL reaction solution supernatant to 95μL 2,4-dinitrophenylhydrazine colorimetric solution (5mM), react for 10min, add 4M NaOH, and observe the color (see Figure 5 ). The lighter the color, the stronger the catalytic activity of the mutant, and the single clone corresponding to the lighter color is the dominant mutant strain.

[0101] Example 5: Construction and screening of CAR combination mutations

[0102] The mutant strains with better activity screened in Example 4 were sent for sequencing, and multiple mutation sites were selected for combined mutation. The CAR gene was used as a template to design corresponding primers for overlapping PCR. The PCR fragments were recovered by gel, and the PCR fragments were inserted into the Nde I and Xho I restriction sites of the vector by homologous recombination. The recombinant plasmid was then transformed into Escherichia coli BL21 (DE3) competent cells, and cultured overnight at 37 degrees on LB solid medium containing 50 μg / mL kanamycin. The positive clones were picked and transferred to 5 mL of LB culture medium containing 50 μM kanamycin, and cultured overnight at 37 degrees and 220 rpm. The strains with correct mutations were cultured in shake flasks (see the expression diagram for details). Figure 6 ), activity test. The test system is: 0.95 g of β-acetone xyloside (100 mM), 1.08 g of glucose (120 mM) and 0.07 g of NAD (2 mM) were added to 50 mL of 100 mM PBS buffer, the pH value was adjusted to 7.0 with HCl aqueous solution, and 50 U of CAR mutant and 50 U of GDH were added to start the reaction. The reaction was stirred at pH 7.0 and 30 ° C for 6 hours. The ee value of S-Bose catalyzed by the test mutant was 100%, and the conversion rate is shown in the following table.

[0103] Table 2

[0104]

[0105]

[0106] Example 6: Enzymatic preparation of bosogen using mutant CAR-M09

[0107] 190 g of β-acetone xyloside (1000 mM), 270 g of glucose (1500 mM) and 0.7 g of NAD (1 mM) were added to 1 L 100 mM PBS buffer, and the pH value was adjusted to 7.0 with HCl aqueous solution. 3500 U CAR and 3200 U GDH were added to start the reaction. The reaction was stirred at pH 6.0 and 35 ° C for 8 hours. A small amount of the reaction solution was quenched with 60% acetonitrile, centrifuged at 12000 rpm for 2 minutes, and the supernatant was filtered for HPLC detection. For details of the liquid phase spectrum, see Figure 7 The test showed that the boson yield was 182.4 g / L (conversion rate was 95%), and the S-type ee value was 100%.

[0108] Example 7: Enzymatic preparation of bosogen using mutant CAR-M10

[0109] 190 g of β-acetone xyloside (1000 mM), 88.4 g of sodium formate (1300 mM) and 2.0 g of NAD (3 mM) were added to 1 L 100 mM PBS buffer, and the pH value was adjusted to 7.0 with HCl aqueous solution. 3200 U CAR and 4000 U FDH were added to start the reaction. The reaction was stirred at pH 7.0 and 40 ° C for 6 hours. A small amount of the reaction solution was quenched with 60% acetonitrile, centrifuged at 12000 rpm for 2 minutes, and the supernatant was filtered for HPLC detection. For details of the liquid phase spectrum, see Figure 8 After testing, the boson yield was 184.3 g / L (conversion rate was 96%), and the S-type ee value was 100%.

[0110] Example 8: Enzymatic preparation of bosogen using mutant CAR-M11

[0111] 190 g of β-acetone xyloside (1000 mM), 75.7 g of ammonium formate (1200 mM) and 1.3 g of NAD (2 mM) were added to 1 L 100 mM PBS buffer, and the pH value was adjusted to 7.0 with HCl aqueous solution. 4000 U CAR and 4500 U FDH were added to start the reaction. The reaction was stirred at pH 6.5 and 25 ° C for 5 hours. A small amount of the reaction solution was quenched with 60% acetonitrile, centrifuged at 12000 rpm for 2 minutes, and the supernatant was filtered for HPLC detection. For details of the liquid phase spectrum, see Fig. 9 After testing, the boson yield was 186.2 g / L (conversion rate was 98%), and the S-type ee value was 100%.

[0112] Example 9: Enzymatic preparation of bosogen using mutant CAR-M12

[0113] 190 g of β-acetone xyloside (1000 mM), 360 g of glucose (2000 mM) and 2.0 g of NAD (3 mM) were added to 1 L 100 mM PBS buffer, and the pH value was adjusted to 7.0 with HCl aqueous solution. Then, 2500 U CAR and 3000 U GDH were added to start the reaction. The reaction was stirred at pH 7.5 and 30 ° C for 10 hours. A small amount of the reaction solution was quenched with 60% acetonitrile, centrifuged at 12000 rpm for 2 min, and the supernatant was filtered for HPLC detection. For details of the liquid phase spectrum, see Fig.10 The test showed that the boson yield was 188.1 g / L (conversion rate was 99%), and the S-type ee value was 100%.

[0114] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An enzyme mutant, characterized in that It includes at least one mutation site among S56P, H125N, Y71P, G161A, F165A, and Y254D; The wild-type amino acid sequence of the enzyme mutant has a sequence shown in any of the following: 1) The amino acid sequence shown in SEQ ID NO: 1; 2) An amino acid sequence having the same or similar function as 1) obtained by replacing, deleting, adding or modifying one or more amino acids in the amino acid sequence shown in 1); 3) An amino acid sequence having at least 80% homology with the sequence shown in 1) or 2).

2. The enzyme mutant according to claim 1, characterized in that It includes S56P, H125N and at least one of the following mutation sites: Y71P, G161A, F165A, Y254D.

3. The enzyme mutant according to claim 1, characterized in that It has: I), an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 13; II), an amino acid sequence having the same or similar function as I) obtained by substituting, deleting, adding or modifying one or more amino acids in the amino acid sequence shown in I); III), an amino acid sequence having at least 80% homology with the sequence shown in I) or II).

4. Biomaterial, characterized in that Include any of the following: i), a nucleic acid encoding the enzyme mutant according to any one of claims 1 to 3; ii) an expression module comprising the nucleic acid described in i); iii), a recombinant vector containing the nucleic acid described in i) or the expression module described in ii); iv), transfecting or transforming a host containing iii) the recombinant vector, or a host having i) the nucleic acid or ii) the expression module integrated into its genome.

5. The biomaterial according to claim 4, characterized in that The nucleic acid encoding the enzyme mutant according to any one of claims 1 to 3 has any one of the following sequences: a), the nucleotide sequence shown in SEQ ID NO: 15 to 26; b) a nucleotide sequence having the same or similar function as a) obtained by replacing, deleting, adding or modifying one or more bases in the nucleotide sequence shown in a); c) a nucleotide sequence having at least 80% homology to the sequence shown in a) or b). 6.Combination enzymes, including: The enzyme mutant and dehydrogenase according to any one of claims 1 to 3; The dehydrogenase includes formate dehydrogenase and / or glucose dehydrogenase.

7. The combined enzyme according to claim 6, characterized in that: The glucose dehydrogenase is derived from Bacillus megaterium; the formate dehydrogenase is derived from Pseudomonas sp.

8. The combined enzyme according to claim 6 or 7, characterized in that: The combined enzyme comprises the enzyme mutant and formate dehydrogenase with an enzyme activity ratio of 3200-4000:4000-4500; Or comprising the enzyme mutant and glucose dehydrogenase at an enzyme activity ratio of 2500-3500:3000-3200.

9. The use of any of the following in the preparation of S-boson: A, a carbonyl reductase having an amino acid sequence as shown in SEQ ID NO: 1; B. The enzyme mutant according to any one of claims 1 to 3; C. The biological material according to claim 4 or 5; D. The enzyme combination according to any one of claims 6 to 8.

10. A method for preparing S-boson, characterized in that: Under the action of carbonyl reductase or the combined enzyme according to any one of claims 6 to 8, β-acetone xyloside is used as a raw material to react and obtain S-bosone; The carbonyl reductase is selected from any of the following: A), a carbonyl reductase having an amino acid sequence as shown in SEQ ID NO: 1; B), the enzyme mutant according to any one of claims 1 to 3; C) An enzyme mutant prepared based on the biological material according to any one of claims 4 to 5.

11. The method according to claim 10, characterized in that The raw materials for the reaction also include NAD and at least one of the following: sodium formate, ammonium formate, and glucose.

12. The method according to claim 10, characterized in that The pH of the reaction is 6.0 to 7.5; And / or, the reaction temperature is 25-40°C; And / or, the reaction is carried out under stirring conditions; And / or, the reaction is carried out in Tris, PBS, or MES buffer; And / or, the reaction time is 5 to 10 hours.

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