Enzyme mutants, combinatorial enzymes for synthesis of s-bone morphogenetic protein and applications thereof

Abstract

The present application relates to the technical field of biology, and particularly relates to an enzyme mutant for synthesizing S-boswax, a combined enzyme and application thereof. The present application utilizes irrational random mutation and rational site-directed mutation, and through multiple rounds of construction and screening of the mutant, a carbonyl reductase mutant with high activity, high stability and high stereoselectivity is obtained. The present application further couples formate dehydrogenase or glucose dehydrogenase to establish a double-enzyme cyclic catalytic system, and achieves a high conversion rate of >95% in a short time, and realizes high yield of >180g / L of boswax, wherein the optical purity of S-boswax is as high as 100%.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to enzyme mutants, combinatorial enzymes, and their applications for the synthesis of S-Bosonic. Background Technology

[0002] Pro-Xylane™, also known as hydroxypropyl tetrahydropyranotriol, is a xylose derivative with anti-aging properties, widely used in the pharmaceutical and cosmetic fields. Studies have shown that Pro-Xylane can directly affect the extracellular matrix in the three layers of the skin, activating or promoting the synthesis of mucopolysaccharides in the skin and significantly increasing the water content of the extracellular matrix. It can also improve the adhesion between the dermis and epidermis by stimulating the synthesis of type IV and VII collagen, promoting the regeneration of damaged tissue, effectively maintaining skin firmness and smoothness, and delaying skin aging. Pro-Xylane exists in several stereoconfigurations: S-Pro-Xylane and R-Pro-Xylane. A higher proportion of S-Pro-Xylane indicates better anti-aging activity; however, the quality of Pro-Xylane products on the market varies greatly, with most being racemic or the S-configuration comprising only a small portion.

[0003] Currently, the main methods for preparing Bosein are chemical synthesis and enzymatic catalysis.

[0004] Foreign chemical synthesis methods involve the condensation reaction of xylose with acetylacetone under alkaline conditions to produce β-acetone xyloside, followed by acidification with a strongly acidic cation exchange resin and reduction of the carbonyl group with borohydride to synthesize Bosein; or the condensation reaction of xylose with acetylacetone followed by reduction of the carbonyl group using a heavy metal catalyst Ru / C. Domestically, improvements have been made to this route. The first step uses xylose as a raw material, employing a strongly basic anion exchange resin instead of ordinary inorganic bases; the second step uses sodium borohydride to reduce the carbonyl group to synthesize Bosein. All of the above chemical synthesis methods inevitably suffer from drawbacks such as low reduction selectivity, high reagent contamination, and difficulties in product separation and purification, which greatly limit the industrial production of Bosein.

[0005] Enzymatic catalysis uses β-pyruvyl xyloside as a raw material and sodium formate, glucose, or isopropanol as co-substrate to prepare Bosein under the catalysis of carbonyl reductase and coenzymes. Compared with chemical methods, the enzymatic method for preparing Bosein has more advantages. Enzymatic methods have high stereoselectivity and avoid the use of polluting and hazardous chemical reagents, greatly improving the greenness of the Bosein production process. At the same time, it simplifies and purifies the product, reducing production costs.

[0006] Currently, some shortcomings remain in the reported enzymatic methods for preparing Bosein. For example, patent CN111876452A discloses a one-pot method for preparing Bosein using biological enzymes, but this method uses enzymes of unknown identity, such as isopropanol dehydrogenase, Bosein synthase, and carbonyl reductase, making it difficult to reproduce. CN113416756A, CN113717997A, and CN 115896199A all mention methods for synthesizing Bosein using a two-enzyme cycle catalysis, but these methods suffer from excessively long reaction times (>24h), low substrate concentrations (<100g / L), and the need to improve the optical purity of the final product (S-type ee value <90%). In summary, existing carbonyl reductases generally suffer from insufficient catalytic ability for β-pyruvate and low stability, which are detrimental to the large-scale production of Bosein. Summary of the Invention

[0007] In view of this, the present invention provides enzyme mutants, combination enzymes, and their applications for the synthesis of S-Bosein. The enzyme mutants provided by the present invention exhibit high activity, strong stability, and high stereoselectivity. Further combination with dehydrogenases achieves highly efficient synthesis of S-Bosein. The synthesis of S-Bosein using the enzyme mutants and combination enzymes provided by the present invention has the advantages of short time, high yield, and high product concentration, wherein the S configuration has an ee value of 100%.

[0008] Enzyme mutants, including 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 any of the following sequences:

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

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

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

[0013] In some embodiments, the enzyme mutant includes 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 NO:2 to 13;

[0017] II) An amino acid sequence with 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) is an amino acid sequence that has at least 80% homology with the sequence shown in I) or II).

[0019] This invention marks the first discovery of a carbonyl reductase (CAR) activity derived from a Saccharibacter sp. EH611, Uniprot#A0A6I4YQY2, designated as the wild-type carbonyl reductase CAR, although its catalytic activity is relatively low. This invention utilizes irrational random mutations and rational site-directed mutations to modify this wild-type CAR. Through multiple rounds of mutant construction and screening, high-performance carbonyl reductase mutants, designated CAR-M01 to CAR-M12, were obtained, with their amino acid sequences shown in SEQ ID NO: 2 to 13. Experiments show that the catalytic activity of CAR-M04 to CAR-M12 is significantly higher than that of the wild-type CAR enzyme, greatly increasing the yield of S-Bosein. Compared to the wild-type CAR enzyme, the yield of S-Bosein is increased by more than 2 times, and it also shows a significant advantage compared to the mutants CAR-M01 to CAR-M04.

[0020] The present invention also provides biological materials, including any one of the following: i) to iv)

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

[0022] ii) An expression module containing 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) a host that has been transfected or transformed with the recombinant vector described in iii), or a host whose genome has integrated the nucleic acid described in i) or the expression module described in ii).

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

[0026] In this invention, the host includes eukaryotic hosts and prokaryotic hosts. 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 this invention, the host is *Escherichia coli*, specifically *Escherichia coli* BL21(DE3).

[0027] In this invention, the nucleic acid sequence encoding the wild-type carbonyl reductase CAR was optimized according to the host's codon preference. In some specific embodiments, the codon optimization was performed based on the codon preference of *E. coli*, and the optimized nucleic acid has the sequence shown in SEQ ID NO:14.

[0028] In this invention, the nucleic acids encoding enzyme mutants as shown in SEQ ID NO:2-13 were codon-optimized according to host codon preferences. In some specific embodiments, the codon optimization was performed based on the codon preferences of *E. coli*, and the optimized nucleic acids had any of the following sequences:

[0029] a) Nucleotide sequences as shown in SEQ ID NO:15-26;

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

[0031] c) A nucleotide sequence that has 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 enzyme mutants as shown in SEQ ID NO:2-13 are shown in SEQ ID NO:15-26 in sequence.

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

[0034] The dehydrogenases include formate dehydrogenase and / or glucose dehydrogenase.

[0035] In this invention, the combined enzyme comprises the enzyme mutant and glucose dehydrogenase as described above. In some embodiments, the glucose dehydrogenase is derived from *Bacillus megaterium*; in some specific embodiments, the glucose dehydrogenase is derived from *Bacillus megaterium*, Uniprot#P39483, whose amino acid sequence is shown in SEQ ID NO: 27, and whose encoding gene sequence after codon optimization by *E. coli* is shown in SEQ ID NO: 29. In some embodiments, the enzyme activity ratio of the enzyme mutant to glucose dehydrogenase is 2500–3500:3000–3200; in some specific embodiments, the enzyme activity ratio can specifically be 2500:3000, 2500:3200, 3500:3000, 3500:3200, or any value between any two of the above ranges.

[0036] In this invention, the combined enzyme comprises the enzyme mutant and formate dehydrogenase as described above. Further, the formate dehydrogenase is specifically derived from *Pseudomonas*; in some specific embodiments of this invention, the formate dehydrogenase is derived from *Pseudomonas sp. Strain 101*, whose amino acid sequence is shown in SEQ ID NO: 28; its encoding gene has been optimized using *E. coli* codons, 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 this invention, the enzyme activity ratio can specifically be 3200:4000, 3200:4500, 4000:4000, 4000:4500, or any value between any two of the above ranges.

[0037] This invention also provides the application of any of the following in the preparation of S-Bosonic:

[0038] A. A carbonyl reductase having the amino acid sequence shown in SEQ ID NO: 1;

[0039] B. The enzyme mutant described above in this invention;

[0040] C. The biomaterials described above in this invention;

[0041] D. The present invention is a combination enzyme as described above.

[0042] The present invention also provides a method for preparing S-Bosein, comprising: reacting β-acetone xyloside as a raw material under the action of a carbonyl reductase or the combination enzyme described in the present invention to obtain S-Bosein;

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

[0044] A) Carbonyl reductase having the amino acid sequence shown in SEQ ID NO: 1;

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

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

[0047] In this invention, the concentration of β-acetone xyloside is 100–1000 mM; in some specific embodiments, the concentration of 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 ranges.

[0048] In this invention, the reactants also include NAD as a co-substrate to regenerate NADH.

[0049] In this invention, when preparing S-Bosoxane using the combined enzyme, the reaction raw materials include at least one of sodium formate, ammonium formate, and glucose, in addition to NAD. In some embodiments, the reaction raw materials further include NAD and sodium formate, or include NAD and ammonium formate. The concentration of NAD is 1–3 mM, specifically 1 mM, 2 mM, or 3 mM. The concentration of sodium formate is 110–2000 mM, specifically 1300 mM, or any value between any two of the above ranges. The concentration of ammonium formate is 110–2000 mM, specifically 1200 mM.

[0050] In other embodiments, the reactants further include NAD and glucose. The concentration of NAD is 1–3 mM, specifically 1 mM, 2 mM, or 3 mM. The concentration of glucose is 36–2000 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, or 2000 mM, or any value within the above two ranges.

[0051] In this 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 ranges;

[0052] And / or, the temperature of the reaction is 25 to 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 solution; specifically, the reaction may be carried out in a PBS buffer, and 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] This invention provides enzyme mutants, combinatorial enzymes, and their applications for the synthesis of S-Bosein. Utilizing both irrational random mutations and rational site-directed mutagenesis, through multiple rounds of mutant construction and screening, this invention yields a carbonyl reductase mutant with high activity, strong stability, and high stereoselectivity, significantly improving the yield of S-Bosein. Furthermore, this invention establishes a dual-enzyme cyclic catalytic system by coupling formate dehydrogenase or glucose dehydrogenase, achieving a high conversion rate of >95% within a short time (approximately 5–10 hours), resulting in a high yield of >180 g / L of S-Bosein, with an optical purity of up to 100%. This invention offers advantages such as short processing time, high yield, and high product concentration. Attached Figure Description

[0057] Figure 1 This diagram shows the synthetic route of the S-Bosonic of the present invention;

[0058] Figure 2 The liquid chromatogram of enzyme cascade system 1 in Example 3 is shown, where A is the liquid chromatogram result of reaction conversion; B is the liquid chromatogram result of R / S Bosine reference standard; and C is the liquid chromatogram result of the product after enzyme reaction of the present invention. The reaction product is only S-Bosine, and the peaks between 3.75 and 5.00 are solvent peaks.

[0059] Figure 3 The mass spectrum of enzyme cascade system 1 in Example 3 is shown;

[0060] Figure 4 The liquid chromatogram of enzyme cascade system 2 in Example 3 is shown;

[0061] Figure 5 The results of CAR mutant colorimetric screening are shown.

[0062] Figure 6 The protein expression results of wild-type CAR (WT) and CAR mutant are shown;

[0063] Figure 7 The liquid chromatogram of Example 6 is shown;

[0064] Figure 8 The liquid chromatogram of Example 7 is shown;

[0065] Figure 9 The liquid chromatogram of Example 8 is shown;

[0066] Figure 10 The liquid chromatogram of Example 9 is shown. Detailed Implementation

[0067] This invention provides enzyme mutants for synthesizing S-Bosone, combinatorial enzymes, and their applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments; those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

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

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

[0070] CAR, carbonylreductase;

[0071] GDH, glucose dehydrogenase;

[0072] FDH stands for formate dehydrogenase.

[0073] In this invention, the wild-type enzyme of the enzyme mutant is carbonyl reductase CAR, derived from a saccharobacter sp. EH611, which has only predictive function (Uniprot#A0A6I4YQY2). This invention tested the carbonyl reduction function of this enzyme and modified it to obtain the enzyme mutant described in this invention, further improving its reduction ability. Mutants with different combinations of mutation points were compared; in this invention, they are numbered M01, M02, M03, M04, M05, M06, M07, M08, M09, M10, M11, and M12. The results show that the above mutants have superior carbonyl reduction ability compared to the wild-type enzyme, resulting in a higher yield of S-Bosein synthesized under their catalysis, making them more suitable for industrial-scale Bosein production.

[0074] In this invention, glucose dehydrogenase (GDH) is derived from Bacillus megaterium, Uniprot#P39483. 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 this invention are shown in Table 1.

[0076] Table 1

[0077]

[0078]

[0079]

[0080] Note: Amino acids in bold and underlined text are 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 illustrated below with reference to the embodiments:

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

[0091] Add 75g xylose (500mM), 45g acetylacetone (900mM), and 20g NaOH (1000mM) to 500mL of pure water. React at 55°C for 90min. After the reaction is complete, adjust the pH of the reaction solution to neutral with HCl aqueous solution and remove the solvent to obtain the raw material β-acetone xyloside.

[0092] Example 2: Enzyme Fermentation Production

[0093] The enzymes required for this invention are all produced by synthesizing the corresponding genes, constructing them on specific expression plasmids, and then fermenting them with E. coli; the specific steps include:

[0094] After sequence optimization of the genes corresponding to the above enzymes, they were synthesized by General Biotechnology Co., Ltd. (Chuzhou, Anhui Province) and then subcloned into the pET 28a expression vector. Plasmids with confirmed correct sequences were transformed into E. coli BL21(DE3) competent cells for plate culture and small-scale single-clone liquid culture. Bacteria with correct protein expression were then subjected to stepwise scale-up liquid culture. Specifically, single colonies were transferred to 5 mL of LB medium containing 50 μM kanamycin (37℃) for culture. When the cells reached the logarithmic growth phase, they were inoculated into 250 mL of LB medium containing the same antibiotic. Once the cells reached the logarithmic growth phase, they were transferred to a 5L fermenter for further culture and final protein expression. In the 5L fermenter, when the cell OD reached 20, 0.5 mM isopropyl-β-D-thiopyranogalactopyranoside (IPTG) was added at 25℃ to induce protein expression for 6 hours. Finally, the cells were collected by high-speed centrifugation (4000 rpm, 20 min) to obtain 30-60 g of wet cells with enzyme overexpression. A small amount of cells was first mixed thoroughly with Tris-HCl buffer (50 mM, pH 8.0) on an ice bath. Cells were then lysed using a freeze-thaw cycle, centrifuged at high speed to remove cell walls, and the supernatant was subjected to SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to determine protein expression. LB medium consisted of 1% tryptone, 0.5% yeast extract, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate, and 5% glycerol.

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

[0096] Enzyme cascade system 1: 190 g of β-pyruvate xyloside (1000 mM), 270 g of glucose (1500 mM), and 0.7 g of NAD (1 mM) were added sequentially to 1 L of 100 mM PBS buffer. After adjusting the pH to 7.0 with HCl aqueous solution, 3500 U of CAR and 3200 U of GDH were added to initiate the reaction. The reaction was carried out at pH 6.0 and 35 °C with stirring 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 analysis. See the HPLC chromatogram for details. Figure 2 Testing revealed a Bosein yield of 115.2 g / L (conversion rate of 60%) and an S-type ee value of 100%. Mass spectrometry confirmed the product as Bosein. The mass spectrometry results are shown below. Figure 3 .

[0097] Enzyme cascade system 2: 190 g of β-pyruvate xyloside (1000 mM), 88.4 g of sodium formate (1300 mM), and 2.0 g of NAD (3 mM) were added sequentially to 1 L of 100 mM PBS buffer. After adjusting the pH to 7.0 with HCl aqueous solution, 3200 U of CAR and 4000 U of FDH were added to initiate the reaction. The reaction was carried out at pH 7.0 and 40 °C with stirring 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 analysis. See the HPLC chromatogram for details. Figure 4 Testing revealed that the yield of Bosein was 99.8 g / L (conversion rate of 52%), and the S-type ee value was 100%.

[0098] Example 4: Construction and Screening of CAR Random Mutant Libraries

[0099] Using the CAR gene as a template, error-prone PCR was performed using the GeneMorph II Random Mutagenesis Kit. The PCR fragments were recovered via gel electrophoresis and inserted into the Nde I and Xho I restriction sites of the vector via homologous recombination. The constructed random mutant library was then transformed into *E. coli* BL21(DE3) competent cells and cultured overnight at 37°C on LB agar containing 50 μg / mL kanamycin. In sterilized 96-well plates, 400 μL of LB agar (containing 50 μg / mL kanamycin) was added to each well and incubated overnight at 37°C and 220 rpm. Then, 20 μL of seed culture was transferred to a 96-well plate containing 600 μL of ILB medium (containing 50 μg / mL kanamycin), and cultured at 37°C and 220 rpm until the OD600 value > 0.8. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM, and the plate was cooled to 25°C to induce expression. Cultured for another 16 h. After culture, the 96-well plate was centrifuged at 4°C and 4000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were retained. 150 μL of lysozyme (1 mg / mL lysozyme, 10 mM Tris-HCl, pH 8) was added to each well of the collected bacterial cells, and digested at 25°C and 220 rpm for 1 h. The reaction system was as follows: 600 μL system, 100 μL enzyme solution, the system contained 100 mM PBS, 30 mM β-pyruvate xyloside, 36 mM glucose, and 2 mM NAD. The reaction was carried out for 4 h, and the supernatant was collected by centrifugation for high-throughput colorimetric detection.

[0100] To address the structural differences between β-pyruvyl xyloside and Bosein, the efficiency of CAR enzyme-catalyzed Bosein synthesis was characterized by a colorimetric reaction involving 2,4-dinitrophenylhydrazine and ketone compounds. The high-throughput detection system was as follows: 200 μL system, 5 μL of reaction supernatant was added to 95 μL of 2,4-dinitrophenylhydrazine colorimetric solution (5 mM), reacted for 10 min, and then 4 M NaOH was added for colorimetric observation (see [link to relevant documentation]). Figure 5 The lighter the color, the stronger the catalytic activity of the mutant; the lighter the color, the more dominant the mutant is represented by a single clone.

[0101] Example 5: Construction and Screening of CAR Combinatorial Mutations

[0102] The mutant strains with superior activity screened in Example 4 were sent for sequencing. Multiple mutation sites were selected for combined mutation. Using the CAR gene as a template, corresponding primers were designed for overlapping PCR. The PCR fragments were recovered by gel extraction and inserted into the Nde I and Xho I restriction sites of the vector via homologous recombination. The recombinant plasmid was then transformed into E. coli BL21(DE3) competent cells and cultured overnight at 37°C on LB solid medium containing 50 μg / mL kanamycin. Positive clones were picked and transferred to 5 mL of LB medium containing 50 μM kanamycin and cultured overnight at 37°C and 220 rpm. Sequencing verification was performed, and strains with correct mutations were cultured in shake flasks (expression diagram shown). Figure 6 Activity test. The test system was as follows: 0.95 g of β-pyruvate xyloside (100 mM), 1.08 g of glucose (120 mM) and 0.07 g of NAD (2 mM) were added sequentially to 50 mL of 100 mM PBS buffer. After adjusting the pH to 7.0 with HCl aqueous solution, 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-Bosein catalyzed by the mutant was 100%. The conversion rate is shown in the table below.

[0103] Table 2

[0104]

[0105]

[0106] Example 6: Preparation of Bosine using the mutant CAR-M09 enzymatic method

[0107] 190 g of β-pyruvate xyloside (1000 mM), 270 g of glucose (1500 mM), and 0.7 g of NAD (1 mM) were added sequentially to 1 L of 100 mM PBS buffer. After adjusting the pH to 7.0 with HCl aqueous solution, 3500 U of CAR and 3200 U of GDH were added to initiate the reaction. The reaction was carried out at pH 6.0 and 35 °C with stirring for 8 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 analysis. See the HPLC chromatogram for details. Figure 7 Testing revealed that the yield of Bosein was 182.4 g / L (conversion rate of 95%), and the S-type ee value was 100%.

[0108] Example 7: Preparation of Bosein using the mutant CAR-M10 enzymatic method

[0109] 190 g of β-pyruvate xyloside (1000 mM), 88.4 g of sodium formate (1300 mM), and 2.0 g of NAD (3 mM) were added sequentially to 1 L of 100 mM PBS buffer. After adjusting the pH to 7.0 with HCl aqueous solution, 3200 U of CAR and 4000 U of FDH were added to initiate the reaction. The reaction was carried out at pH 7.0 and 40 °C with stirring for 6 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 analysis. See the HPLC chromatogram for details. Figure 8 Testing revealed that the yield of Bosein was 184.3 g / L (conversion rate of 96%), and the S-type ee value was 100%.

[0110] Example 8: Preparation of Bosoxane using the mutant CAR-M11 enzymatic method

[0111] 190 g of β-pyruvate xyloside (1000 mM), 75.7 g of ammonium formate (1200 mM), and 1.3 g of NAD (2 mM) were added sequentially to 1 L of 100 mM PBS buffer. After adjusting the pH to 7.0 with HCl aqueous solution, 4000 U of CAR and 4500 U of FDH were added to initiate the reaction. The reaction was carried out at pH 6.5 and 25 °C with stirring for 5 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 analysis. See the HPLC chromatogram for details. Figure 9 Testing revealed that the yield of Bosein was 186.2 g / L (conversion rate 98%), and the S-type ee value was 100%.

[0112] Example 9: Preparation of Bosine using the mutant CAR-M12 enzymatic method

[0113] 190 g of β-pyruvate xyloside (1000 mM), 360 g of glucose (2000 mM), and 2.0 g of NAD (3 mM) were added sequentially to 1 L of 100 mM PBS buffer. After adjusting the pH to 7.0 with HCl aqueous solution, 2500 U of CAR and 3000 U of GDH were added to initiate the reaction. The reaction was carried out at pH 7.5 and 30 °C with stirring 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 analysis. See the HPLC chromatogram for details. Figure 10 Testing revealed that the yield of Bosein was 188.1 g / L (conversion rate of 99%), and the S-type ee value was 100%.

[0114] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An enzyme mutant, characterized in that, The wild-type amino acid sequence of the enzyme mutant is shown in SEQ ID NO:1; The mutation sites of the enzyme mutant are S56P and H125N, or S56P, H125N and at least one of the following mutation sites: Y71P, G161A, F165A, Y254D.

2. The enzyme mutant according to claim 1, characterized in that, Its amino acid sequence is shown in any one of SEQ ID NO:4~13.

3. A biomaterial, characterized in that, Includes any of the following: i) The nucleic acid encoding the enzyme mutant according to any one of claims 1 to 2; ii) An expression module containing 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) a host that has been transfected or transformed with the recombinant vector described in iii), or a host whose genome has integrated the nucleic acid described in i) or the expression module described in ii).

4. The biomaterial according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid encoding the enzyme mutant of any one of claims 1 to 2 is shown in any one of SEQ ID NO: 17 to 26.

5. Combinatorial enzymes, including: The claims are for the enzyme mutant and dehydrogenase described in any one of claims 1 to 2; The dehydrogenases include formate dehydrogenase and / or glucose dehydrogenase.

6. The combined enzyme according to claim 5, characterized in that, The glucose dehydrogenase is derived from Bacillus megaterium (Bacillus megaterium) Bacillus megaterium The formate dehydrogenase is derived from Pseudomonas ( ); Pseudomonas sp ).

7. The combined enzyme according to claim 5 or 6, characterized in that, The combined enzyme comprises the enzyme mutant with an enzyme activity ratio of 3200~4000:4000~4500 and formate dehydrogenase; Or it may include enzyme mutants with an enzyme activity ratio of 2500~3500:3000~3200 and glucose dehydrogenase.

8. The application of any of the following in the preparation of S-Bosonic: A. The enzyme mutant according to any one of claims 1 to 2; B. The biomaterial as described in claim 3 or 4; C. The combined enzyme according to any one of claims 5 to 7.

9. A method for preparing S-Bosonic, characterized in that, S-Bosein is obtained by reacting β-acetone xyloside as a raw material under the action of carbonyl reductase or the combination enzyme according to any one of claims 5 to 7. The carbonyl reductase is selected from any of the following: A) The enzyme mutant according to any one of claims 1 to 2; B) Enzyme mutants prepared based on the biomaterials described in any one of claims 3 to 4.

10. The method according to claim 9, 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.

11. The method according to claim 9, characterized in that, The pH of the reaction is 6.0~7.5; And / or, the temperature of the reaction 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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