A reductase mutant and its use in preparing s-bone morphogenetic protein

Abstract

This invention discloses a reductase mutant and its application in the preparation of S-Bosein. The reductase mutant differs from the amino acid sequence shown in SEQ ID NO:3 in containing one or more amino acid residues at sites Q95, L96, and G190. The reductase mutant of this invention exhibits high substrate conversion and high ee value of S-Bosein when using β-pyruvate as a substrate, making it suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis, specifically to a reductase mutant and its application in the preparation of S-Bosonic. Background Technology

[0002] Pro-Xylane, chemically known as hydroxypropyl tetrahydropyranotriol, also called C-(β-D-xylopyranoside)-2-hydroxypropane, is a xylose derivative with anti-aging activity, commonly used in cosmetics. Developed by L'Oréal over many years, Pro-Xylane is a xylose derivative with cosmetic and anti-wrinkle effects. It is derived from xylose and possesses a wide range of biological activities. Pro-Xylane can directly affect the extracellular matrix in the three layers of the skin, activating or promoting the synthesis of glycosaminoglycans (GAGs) in the skin, improving the adhesion between the dermis and epidermis, promoting the regeneration of damaged tissue, and helping to maintain the elasticity of the dermis. It can effectively keep the skin firm and smooth, delaying skin aging.

[0003] Initially, Bosein was synthesized using chemical methods, such as in US7732414B2, which used xylose as a raw material. Under alkaline conditions, xylose was condensed with acetylacetone to obtain 1-C-(β-D-pyranoxyloyl)-acetone, followed by reduction of the ketone carbonyl group with sodium borohydride to yield a mixture of (R)-Bosein and (S)-Bosein. This method uses sodium borohydride as a reducing agent, making the reaction dangerous and unsuitable for industrial production.

[0004] Subsequently, CN100441588C describes the selective preparation of C-(β-D-xylanopyranoside)-2-propanone using acetic acid and sodium borohydride as a reducing agent, with C-(β-D-xylanopyranoside)-2-(S)-hydroxypropane as a starting material. It also demonstrates that (S)-Bosein exhibits higher activity than a mixture of (R)-Bosein and (S)-Bosein. However, this method still uses sodium borohydride as a reducing agent, posing a hazardous reaction risk. Therefore, developing a highly selective method for preparing (S)-Bosein is crucial.

[0005] Compared to chemical methods, the bioenzymatic synthesis of (S)-Bosein is safer, more efficient, less expensive, exhibits greater stereoselectivity, and is more environmentally friendly. Despite these advantages, current reductase-based methods for (S)-Bosein preparation show relatively low conversion rates and low ee values, indicating that efficient bioenzymatic synthesis methods for (S)-Bosein still require further development. Summary of the Invention

[0006] The technical problem this invention aims to solve is the low conversion rate and low ee value of (S)-Bosein in current reductase preparation methods. This invention provides a reductase and its application in the preparation of (S)-Bosein. When using the reductase of this invention to prepare (S)-Bosein, the substrate conversion rate is high, and the ee value of (S)-Bosein is also high, making it suitable for industrial production.

[0007] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0008] A first aspect of the present invention provides a reductase mutant, characterized in that the reductase mutant differs from the amino acid sequence shown in SEQ ID NO:3 in that it contains one or more amino acid residues at one of the sites Q95, L96 and G190, wherein the positions of 95, 96 and 190 refer to the amino acid residue numbers in SEQ ID NO:3.

[0009] In some embodiments, the reductase mutant differs from the amino acid sequence shown in SEQ ID NO:3 in one or more of the following amino acid residues: K192, G195, V196, and I217, wherein the positions of 192, 195, 196, and 217 refer to the amino acid residue numbers in SEQ ID NO:3.

[0010] In some embodiments, the total number of amino acid residue mutations in the reductase mutant compared to the amino acid sequence shown in SEQ ID NO:3 does not exceed 12.

[0011] In some implementations, the total number of mutations is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0012] In some embodiments, when the reductase mutant prepares S-Bosein using β-pyruvate as a substrate, the conversion rate of β-pyruvate is not less than 60%.

[0013] In some specific embodiments, the conversion rate of the β-acetone xyloside is not less than 70%.

[0014] In some embodiments, the reductase mutant differs from the amino acid sequence shown in SEQ ID NO:3 by one or more amino acid residues from Q95W, Q95M, Q95E, L96A, L96S, L96E, L96G, G190P, and G190A, wherein Q95W indicates that the amino acid at position 95 is mutated from Q to W, and so on.

[0015] In some specific embodiments, the reductase mutant further includes one or more amino acid residues differing from the amino acid sequence shown in SEQ ID NO:3, including K192A, K192D, K192M, G195A, G195L, G195S, V196D, V196T, and I217L.

[0016] In some embodiments, the reductase mutant comprises one of the following combinations of amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO:3:

[0017] (1) Q95W, L96E and G190P;

[0018] (2) Q95W, L96E, G190P, G195A and V196T;

[0019] (3) Q95W, L96E, G190P and I217L;

[0020] (4) Q95W, L96E, G190P and K192M;

[0021] (5) Q95W, L96E, G190P and K192D;

[0022] (6) Q95W, L96E, G190P, G195S and V196T;

[0023] (7) Q95W, L96E, G190P and K192A;

[0024] (8) L96E and G190P;

[0025] (9) L96S and G190P;

[0026] (10) L96A and G190P;

[0027] (11) G190P and G195L;

[0028] (12) Q95W, L96E, G190P, G195L and V196D;

[0029] (13)Q95W, L96E and G190A;

[0030] (14) Q95W and G190P;

[0031] (15) I93W, Q95W, L96G, C206T and F249Y

[0032] (16) L96G and G190A;

[0033] (17) Q95M and G190P;

[0034] (18)G190P;

[0035] (19)Q95E and G190P;

[0036] (20) I93W, Q95W, L96G, C206E and F249C;

[0037] (21) I93W, Q95W, L96E, C206H and F249E;

[0038] (22) G190A and G195L;

[0039] (23)Q95W and L96E.

[0040] A second aspect of the invention provides an isolated nucleic acid molecule that encodes a reductase mutant as described in the first aspect.

[0041] A third aspect of the present invention provides a recombinant expression vector comprising isolated nucleic acid molecules as described in the second aspect.

[0042] In some implementations, the backbone of the recombinant expression vector is either the pET28a plasmid or the pET21a plasmid.

[0043] A fourth aspect of the present invention provides a transformant comprising an isolated nucleic acid molecule as described in the second aspect, or a recombinant expression vector as described in the third aspect.

[0044] In some implementations, the host cells used in the construction of the transformant are Escherichia coli or Bacillus subtilis.

[0045] In some implementations, Escherichia coli is Escherichia coli BL21(DE3).

[0046] A fifth aspect of the present invention provides a method for preparing a reductase mutant as described in the first aspect, comprising culturing a transformant as described in the fourth aspect to obtain a fermentation product.

[0047] In some embodiments, the culture medium used for the culture is selected from LB liquid medium or TB liquid medium.

[0048] In some embodiments, the culture conditions are: shaking culture at a temperature of 37±1℃.

[0049] In some embodiments, the method further includes the step of purifying the fermentation product to obtain the reductase mutant.

[0050] A sixth aspect of the present invention provides an enzyme composition comprising two or more of the following: a reductase mutant as described in the first aspect, a glucose dehydrogenase having the amino acid sequence shown in SEQ ID NO:1, and a reductase having the amino acid sequence shown in SEQ ID NO:3.

[0051] The seventh aspect of the present invention provides the use of a reductase having an amino acid sequence as shown in SEQ ID NO:3, a reductase mutant as described in the first aspect, an isolated nucleic acid molecule as described in the second aspect, a recombinant expression vector as described in the third aspect, a transformant as described in the fourth aspect, or an enzyme composition as described in the sixth aspect in the preparation of S-Bosonic.

[0052] In some embodiments, the preparation of S-Bosein uses β-acetone xyloside as a substrate.

[0053] The reaction route of this invention is shown below:

[0054]

[0055] Compound 1 is β-acetone xyloside. Compound 2 is (S)-Bos-Xylane.

[0056] The eighth aspect of the present invention provides a method for preparing S-Bosoxane, the method comprising: contacting and reacting at least one of a reductase having the amino acid sequence shown in SEQ ID NO:3, a reductase mutant as described in the first aspect, or an enzyme composition as described in the sixth aspect with a substrate to obtain S-Bosoxane.

[0057] In some embodiments, the substrate is β-acetone xyloside.

[0058] In some implementations, the reaction also includes the use of a coenzyme.

[0059] In some embodiments, the reaction further includes a coenzyme regeneration reaction.

[0060] In some embodiments, the coenzyme is any one or both of NADH and NADPH.

[0061] In some specific implementations, the NADPH is β-NADPH.

[0062] In some embodiments, the reaction for regenerating the coenzyme uses any one or both of NAD+ and NADP+, a hydrogen donor, and a coenzyme regenerating enzyme; the hydrogen donor and the coenzyme regenerating enzyme are selected from one or more of the following:

[0063] (1) Glucose and glucose dehydrogenase;

[0064] (2) Formic acid and formic acid dehydrogenase;

[0065] (3) Isopropanol and alcohol dehydrogenase.

[0066] In some embodiments, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:1.

[0067] In some embodiments, the reductase, the reductase mutant, or the enzyme composition is used in the form of liquid enzyme, solid enzyme powder, immobilized enzyme, wet cells, or bacterial powder.

[0068] In some embodiments, the concentration of β-acetone xyloside added in the reaction is 1-500 mg / mL.

[0069] In some specific embodiments, the concentration of β-acetone xyloside added in the reaction is 10-200 mg / mL.

[0070] In some embodiments, the molar ratio of the hydrogen donor to the β-acetone xyloside is 0.5:1 to 16:1.

[0071] In some specific embodiments, the molar ratio of the hydrogen donor to the β-acetone xyloside is 0.68:1, 1.3:1, 5:1, or 16:1.

[0072] In some implementations, the NAD + and / or NADP + The mass ratio of the β-acetone xyloside to the xyloside is 1:1000 to 500:1000.

[0073] In some specific implementations, the NAD + and / or NADP + The mass ratio of the β-acetone xyloside to the xyloside is 19:1000 or 37:1000.

[0074] In some implementations, the pH of the reaction is 6.5-8.5.

[0075] In some implementations, the pH of the reaction is 7.0-8.0.

[0076] In some implementations, the reaction temperature is 25-45°C.

[0077] In some embodiments, the reaction temperature is 30-40°C.

[0078] In some embodiments, when the reductase, the reductase mutant, or the enzyme composition is used in the form of wet cells, the mass ratio of the wet cells to the β-acetone xyloside added in the reaction is (0.1-0.5) g: 1 g.

[0079] In some specific embodiments, the mass ratio of the wet bacterial cells to the β-acetone xyloside added in the reaction is 0.5 g: 1 g.

[0080] In some embodiments, the reductase, the reductase mutant, or the enzyme composition is used as a liquid enzyme, and the ratio of the mass of the wet bacterial cells to the mass of the β-acetone xyloside added in the reaction is (0.1-0.5) g:1 g, calculated based on the mass of the wet bacterial cells that produce the reductase.

[0081] In some specific embodiments, the mass ratio of the wet bacterial cells to the β-acetone xyloside added in the reaction is 0.2g:1g.

[0082] In some embodiments, the coenzyme regenerating enzyme is expressed in a mass ratio of (0.05-1)g:1g to the hydrogen donor, calculated based on the mass of the wet bacterial cells used.

[0083] In some specific implementations, the mass ratio of the coenzyme regenerating enzyme to the hydrogen donor, calculated based on the mass of the wet bacterial cells used, is 0.08 g:1 g, 0.1:1 g, 0.125:1 g, or 0.8 g:1 g.

[0084] A ninth aspect of the present invention provides a reaction end product system for the catalytic synthesis of bosine from β-pyrrolidone as a substrate, the reaction end product system comprising:

[0085] S-Bosorin;

[0086] β-pyrrolidone xyloside; and

[0087] Any one of the following: a reductase with the amino acid sequence shown in SEQ ID NO:3, a reductase mutant as described in the first aspect, a transformant as described in the fourth aspect, and an enzyme composition as described in the sixth aspect.

[0088] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0089] The reagents and raw materials used in this invention are all commercially available.

[0090] The positive and progressive effects of this invention are as follows: when the reductase mutant of this invention is used to prepare S-Bosein using β-acetone xyloside as a substrate, the substrate conversion rate and the ee value of S-Bosein are relatively high, making it suitable for industrial production. Detailed Implementation

[0091] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0092] Unless otherwise specified, the experimental methods in this invention are conventional methods. For specific gene cloning operations, please refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0093] The reagents used in this invention are as follows:

[0094] TB liquid culture medium: tryptone 10g / L, yeast extract 18g / L, glycerol 4mL / L, KH2PO4 (anhydrous) 2.31g / L, K2HPO4 (trihydrate) 16.43g / L.

[0095] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0096] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 18 g / L, kanamycin 50 μg / mL.

[0097] LB agar plates: Plate culture dishes containing LB solid culture medium.

[0098] Escherichia coli BL21 was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0099] pET28a plasmid was purchased from Novagen.

[0100] IPTG was purchased from Bangtai Biotechnology Co., Ltd.

[0101] BugBuster lysis buffer was purchased from Millipore, 4049820.

[0102] β-NADP + Purchased from BONTAC, Lot NO. BT03D223J021.

[0103] KOD PCR Master Mix was purchased from Toyobo.

[0104] The measurement method of the present invention is as follows:

[0105] In this invention, the chirality and substrate conversion of (S)-Bosein were analyzed by high-performance liquid chromatography (HPLC). The specific analytical method is as follows:

[0106] Chromatographic column: CAPCELL PAK ADME (5μm, 250×4.6mm); mobile phase: water; flow rate: 1.0mL / min; injection volume: 20μL; column temperature: 35℃; run time: 20min.

[0107] Conversion rate calculation:

[0108] The conversion rate of the substrate is calculated as follows: Substrate conversion rate = Amount of substrate converted to product / Total amount of substrate × 100%. The amount of substrate converted to product is calculated based on the amount of product generated. The amount of product generated is calculated using the area normalization method based on the peak area ratio of the product in the HPLC chromatogram.

[0109] ee value calculation:

[0110] The optical purity of (S)-Bosein is evaluated by calculating the enantiomeric excess value (ee value) using the area normalization method.

[0111] The formula for calculating the ee value of (S)-Bosonic is: Among them, A R : Peak area of ​​the R configuration product (R-Bosone), A S Peak area of ​​the S-configuration product (S-Bosine).

[0112] The retention time of acetone xyloside was 14.910 min, that of S-Bosein was 11.763 min, and that of R-Bosein was 13.428 min.

[0113] Enzyme activity assay method:

[0114] 1. Enzyme activity detection

[0115] (1) Preparation of substrate solution: Weigh 1 g of Bosein (MW: 194) or 10 mL of isopropanol (MW: 60) as the substrate and dissolve it in 96 mL of water. After complete dissolution, adjust the pH to 7.0 with NaOH. Pour 960 μL of the prepared solution into a 1 mL quartz cuvette and incubate for 1 min. Add 10 μL of 20 mM β-NADP. + Pour the solution into a cuvette and mix thoroughly with a pipette.

[0116] (2) Add 30 μL of enzyme solution and record the absorbance at 340 nm every 5 seconds.

[0117] (3) Plot a curve with time (min) on the x-axis and absorbance at 340nm on the y-axis, and take the slope K1 of the experimental group.

[0118] (4) Plot the absorbance of different concentrations of NADPH on the x-axis (time (min)) and construct a standard curve, taking the slope K0.

[0119] (5) Enzyme activity calculation

[0120] Enzyme activity is defined as the amount of enzyme that can generate 1 μmol NADPH within 1 minute under specific conditions, which is 1 unit of enzyme activity (U).

[0121] Based on this definition, the formula for calculating liquid enzyme activity is as follows:

[0122]

[0123] Where: K1: slope of experimental group; K0: slope of standard curve; V1: total reaction volume; V0: enzyme solution volume; F: enzyme solution dilution factor.

[0124] 2. Protein concentration detection

[0125] Add 10 μL of enzyme solution to the microplate and dilute to an appropriate concentration. Then, take 20 μL of the diluted enzyme solution and mix it with 200 μL of Bradford reagent. Incubate at room temperature for 2-3 minutes and detect the absorbance at 595 nm.

[0126] Protein concentration (mg / mL) = (Kd × Xd) × dilution factor.

[0127] Kd is the slope of the standard curve, X is the absorbance value, and d is a constant.

[0128] 3. Compared with enzyme activity

[0129] Enzyme activity (U / mg) = Enzyme activity (U / mL) ÷ Protein concentration (mg / mL).

[0130] The specific enzyme activities measured using Boseine as a substrate are shown in Table 10 (Boseine substrate). The specific enzyme activities measured using isopropanol as a substrate are shown in Table 10 (isopropanol substrate).

[0131] Example 1: Preparation of crude enzyme solution

[0132] 1.1 Synthesis of Recombinant Plasmids

[0133] In this embodiment, crude enzyme solutions of glucose dehydrogenase Enz.0 and alcohol dehydrogenase Enz.1 were prepared. The sequence numbers of the two enzymes are shown in Table 1. Based on the sequences in Table 1, the gene fragments of the above enzymes were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and cloned into the expression vector pET28a (kanamycin resistant), respectively, to obtain the recombinant plasmids pET28a-Enz.0 and pET28a-Enz.1. The restriction sites were NdeI and HindIII.

[0134] Table 1. Enzyme sources and sequences

[0135]

[0136] 1.2 Transformation of enzyme genes

[0137] The synthesized recombinant plasmids were transformed into Escherichia coli BL21(DE3) competent cells and plated on LB agar plates containing 50 μg / mL kanamycin (Kan) and cultured overnight at 37°C.

[0138] 1.3 Expression of enzyme genes

[0139] Single colonies from LB agar plates were inoculated into LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C for 4 h. Then, they were inoculated at a 1% (v / v) inoculation rate into TB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C and 250 rpm until the bacterial concentration reached OD500. 600 =0.6~0.8, add IPTG inducer to a final concentration of 0.1mM, induce overnight at 25℃ and 250rpm, then centrifuge at 4000rpm for 20min and collect wet cells.

[0140] The wet bacterial cells were resuspended in 50 mM pH 7.0 PBS buffer at a ratio of 1 g: 20 mL (i.e., homogenization ratio), and then homogenized to obtain Enz.0 crude enzyme solution and Enz.1 crude enzyme solution, which were then stored at -20℃ for later use.

[0141] Example 2: Random Mutation Screening of Enz.1

[0142] 2.1 Construction of random gene mutation libraries

[0143] Using the pET28a-Enz.1 recombinant plasmid from Example 1 as a template, error-prone PCR primers were designed, and a PCR system for generating random mutant gene libraries was constructed (as shown in Table 2).

[0144] Table 2 PCR system (total volume 50 μL)

[0145]

[0146] The upstream primer PF is 5'-ATGACTGATCGTCTGAAGGGCAAAG-3' (SEQ ID NO:5); the downstream primer PR is 5'-TTACTGTGCGGTAAACCCGCCGTCGAC-3' (SEQ ID NO:6). The Taq DNA polymerase described above does not have 3′-5′ proofreading function. Under high magnesium ion concentrations and different dNTP concentrations, random mutations are introduced into the target gene, thereby constructing a random gene mutation library.

[0147] The PCR amplification procedure is shown in Table 3:

[0148] Table 3 PCR amplification program

[0149]

[0150] 2.2 Construction of mutant expression library

[0151] The PCR products obtained in Example 2.1 were subjected to agarose gel electrophoresis and recovered using a gel extraction method. After double digestion with restriction endonucleases NdeI and HindIII, the plasmids were ligated with pET28a plasmids digested with NdeI and HindIII to obtain various pET28a-Enz.1A recombinant plasmids. These plasmids were transformed into *E. coli* BL21(DE3) competent cells and streaked on LB agar plates to construct a mutant expression library. Additionally, *E. coli* BL21(DE3) containing the pET28a-Enz.1 recombinant plasmid were cultured on LB agar plates.

[0152] 2.3 Cultivating mutants

[0153] Add 400 μL of LB liquid medium to each well of a 96-well deep-well plate. Inoculate single colonies corresponding to the mutant expression library from LB agar plates into each well of the 96-well plate (as experimental wells). Simultaneously, inoculate single colonies of *E. coli* BL21(DE3) containing the pET28a-Enz.1 recombinant plasmid from LB agar plates into each well of the 96-well deep-well plate (as control wells). Incubate at 37°C and 600 rpm for 8 h. Then, transfer 100 μL of bacterial culture from each well (including experimental and control wells) of the 96-well deep-well plate to another 96-well plate containing 100 μL of TB liquid medium in each well. Incubate at 37°C and 600 rpm for 5 h. Wait for the bacterial culture in the 96-well plate to develop OD... 600 When the concentration reaches 0.6, add IPTG solution to the 96-well plate to a final concentration of 0.1 mM and induce for 17 h at 25 °C and 600 rpm. Then centrifuge the 96-well plate at 4000 rpm for 20 min and discard the supernatant culture medium.

[0154] 2.4 Preparation of supernatant enzyme solution

[0155] Add 50 μL of BugBuster lysis buffer to each well of a 96-well plate after discarding the supernatant culture medium, resuspend the bacterial cells, and lyse at 30 °C and 600 rpm for 1 h. Then add 250 μL of 100 mM ammonium phosphate buffer (pH 9.0) to each well to dilute the enzyme solution. Centrifuge at 4000 rpm for 20 min, discard the precipitate, and obtain the supernatant enzyme solution (including the various Enz.1A supernatant enzyme solutions from the experimental wells and the Enz.1 supernatant enzyme solution from the control wells).

[0156] 2.5 High-throughput screening of mutant enzymes

[0157] Preparation of reaction solution: Weigh 2 mg (S)-Bosein and 10 μL 50 mM β-NADP. + Adjust the pH to 7.0 with sodium hydroxide and bring the volume to 150 μL. Add 50 μL of Enz.1A supernatant or Enz.1 supernatant to the above reaction solution to prepare a total reaction system of 200 μL. Record the absorbance at 340 nm for each well every 1 minute using a microplate reader for a total of 10 minutes. Calculate the NADPH generation rate per unit time to determine the enzyme activity. Based on the enzyme activity, select the dominant mutant for sequencing and perform saturation mutations at key sites.

[0158] Example 3: Screening for saturation mutations of the Enz.1 enzyme

[0159] 3.1 Using the pET28a-Enz.1 recombinant plasmid from Example 1.1 as a template, and with the primers shown in Table 4, the PCR amplification system shown in Table 5 was constructed, and a series of saturation mutations were performed on the above sites.

[0160] Table 4 Primer sequences

[0161]

[0162]

[0163] In this context, N represents any one of the nucleotides A, G, C, and T, M represents A or C, and K represents G or T.

[0164] Table 5. PCR amplification system (total volume 50 μL)

[0165]

[0166]

[0167] Table 6 PCR Amplification Procedure

[0168]

[0169] 3.2 The PCR product was transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 37°C to obtain transformants containing mutant libraries.

[0170] 3.3 Following the steps in Section 1.3 of Example 3.2, various crude Enz.1B enzyme solutions corresponding to the transformants of Example 3.2 were prepared.

[0171] 3.4.1 The enzyme catalytic system shown in Table 7 was constructed, which used glucose and glucose dehydrogenase (Enz.0) to regenerate the reduced coenzyme β-NADPH. The enzyme catalytic system was reacted at 35℃ and 220 rpm for 18 h. After the reaction, the final reaction solution was collected, and after removing enzymes and other impurities, it was analyzed by HPLC. Based on the HPLC results, enzymes with certain conversion rates (denoted as Enz.2 to Enz.56) were screened from various Enz.1B enzymes, as shown in Table 8. Enzymes with lower conversion rates were not included in Table 8. In addition, the enzyme activity and protein concentration of Enz.2 to Enz.56 were measured respectively, and the specific activity (SA) was calculated and listed in Table 8 according to the grade. In Tables 8 and 10, + indicates 1≤SA<2U / mg, ++ indicates 2≤SA<5U / mg, +++ indicates 5≤SA<10U / mg, ++++ indicates 10≤SA<15U / mg, +++++ indicates 15≤SA<25U / mg; ++++++ indicates specific enzyme activity of SA≥25U / mg.

[0172] Table 7 Enzyme catalysis system (total volume 1 mL)

[0173]

[0174]

[0175] In Table 7, β-acetone xyloside was used as the substrate. Enz.0 and Enz.1 crude enzyme solutions were from Example 1, and Enz.1B crude enzyme solution was from Example 3.3. β-NADP + As an oxidative cofactor, β-NADPH is used as a reducing cofactor in the catalytic reaction of substrates when Enz.1 crude enzyme solution or Enz.1B crude enzyme solution is generated.

[0176] Table 8 Screening Results

[0177]

[0178]

[0179]

[0180] 3.4.2 The enzyme catalytic system shown in Table 9 was constructed. Enz.1 mutants were screened that could both catalyze the production of (S)-Bosein using β-pyruvate as a substrate and regenerate the reduced coenzyme β-NADPH using isopropanol. This enzyme catalytic system was reacted at 35℃ and 220 rpm for 18 h. After the reaction, the final reaction solution was collected, and after removing enzymes and other impurities, it was analyzed by HPLC. Based on the HPLC results, enzymes with certain conversion rates (from Enz.2 to Enz.56) were screened from various Enz.1B enzymes, as shown in Table 10. Furthermore, since both Bosein and isopropanol can be used as substrates for either the crude Enz.1 or crude Enz.1B enzyme solutions, the enzyme activity and protein concentration of Enz.2 to Enz.56 were measured for both substrates, and the specific enzyme activity was calculated and listed in Table 10 according to the grade.

[0181] Table 9 Enzyme catalytic system (total volume 1 mL)

[0182] Material Name Added amount β-pyruvyl xyloside 50mg <![CDATA[β-NADP + ]]> 1.859mg Isopropanol 100μL Enz.1 crude enzyme solution or Enz.1B crude enzyme solution 200μL PBS solution (pH 7.0; 50 mM) Adjust the volume to 1000 μL

[0183] Table 10 Screening Results

[0184]

[0185]

[0186]

[0187]

[0188] The results above indicate that, for enzyme catalytic systems that utilize glucose and glucose dehydrogenase (Enz.0) to regenerate reduced coenzymes, and for coenzyme regeneration systems that utilize isopropanol and alcohol dehydrogenase (Enz.1 or its mutants) to regenerate reduced coenzymes, the enzymes with a conversion rate of over 90% include the following: Enz.43, Enz.44, Enz.46, Enz.50, Enz.51, Enz.52, Enz.53, Enz.54, Enz.55, and Enz.56.

[0189] Example 4: Scale-up reaction of (S)-Bosein produced from substrate using wet bacterial cells catalyzed by enzyme mutants.

[0190] 4.1 Coenzyme regeneration system utilizing isopropanol and alcohol dehydrogenase (Enz.1 or its mutant) to achieve reduced coenzyme regeneration

[0191] The wet cells of Enz.1 obtained according to the method of Example 1.3 and the above-screened mutants with a conversion rate of more than 90% Enz.1B (wet cells of mutants with a conversion rate of more than 90% from Enz.2 to Enz.56) catalyzed the production of (S)-Bosein from the substrate β-acetone xyloside, and the reaction conditions are shown in Table 11 below.

[0192] Table 11 Enzyme catalysis system (total volume 10 mL)

[0193]

[0194]

[0195] The compound was added to the reactor and reacted at 200 rpm and 35°C for 8 h to obtain compound (S)-Bosein. The reaction results, as determined by HPLC, are shown in Table 12 below.

[0196] Table 12

[0197] serial number mutation information Conversion rate % ee value % Enz.1 Enz.1 (unmutated enzyme) 10.80％ 99.90％ Enz.43 Enz.1-L96A / G190P 90.80％ 99.90％ Enz.44 Enz.1-L96S / G190P 91.30％ 99.90％ Enz.46 Enz.1-L96E / G190P 93.40％ 99.90％ Enz.50 Enz.1-Q95W / L96E / G190P / K192D 97.30％ 99.90％ Enz.51 Enz.1-Q95W / L96E / G190P / G195S / V196T 96.70％ 99.90％ Enz.52 Enz.1-Q95W / L96E / G190P / K192M 98.90％ 99.90％ Enz.53 Enz.1-Q95W / L96E / G190P / I217L 98.70％ 99.90％ Enz.54 Enz.1-Q95W / L96E / G190P / K192A 94.10％ 99.90％ Enz.55 Enz.1-Q95W / L96E / G190P 98.90％ 99.90％ Enz.56 Enz.1-Q95W / L96E / G190P / G195A / V196T 99.50％ 99.90％

[0198] 4.2 Enzyme catalytic system for regenerating reduced coenzymes using glucose and glucose dehydrogenase (Enz.O)

[0199] The wet cells of Enz.1 obtained according to the method of Example 1.3 and the above-screened mutants with a conversion rate of over 90% (wet cells of mutants with a conversion rate of over 90% from Enz.2 to Enz.56) catalyzed the production of (S)-Bosein from the substrate β-acetone xyloside, and the reaction conditions are shown in Table 13 below:

[0200] Table 13 Enzyme catalysis system (total volume 10 mL)

[0201] Material Name content β-pyruvyl xyloside 1g <![CDATA[β-NADP + ]]> 18.586mg glucose 600mg Enz.0 wet bacteria 0.5g Enz.1 wet cell or Enz.1B wet cell 0.5g PBS solution (pH 7.0; 50 mM) Adjust the volume to 10 mL

[0202] The compound was added to the reactor and reacted at 35°C for 8 hours at 200 rpm to obtain compound (S)-Bosein. The reaction results were analyzed by HPLC, and the conversion results are shown in Table 14 below:

[0203] Table 14

[0204] serial number mutation information Conversion rate % ee value % Enz.1 Enz.1 (unmutated enzyme) 12.80％ 99.90％ Enz.43 Enz.1-L96A / G190P 90.20％ 99.90％ Enz.44 Enz.1-L96S / G190P 93.20％ 99.90％ Enz.46 Enz.1-L96E / G190P 94.3％ 99.90％ Enz.50 Enz.1-Q95W / L96E / G190P / K192D 96.00％ 99.90％ Enz.51 Enz.1-Q95W / L96E / G190P / G195S / V196T 95.40％ 99.90％ Enz.52 Enz.1-Q95W / L96E / G190P / K192M 97.50％ 99.90％ Enz.53 Enz.1-Q95W / L96E / G190P / I217L 99.40％ 99.90％ Enz.54 Enz.1-Q95W / L96E / G190P / K192A 97.20％ 99.90％ Enz.55 Enz.1-Q95W / L96E / G190P 99.30％ 99.90％ Enz.56 Enz.1-Q95W / L96E / G190P / G195A / V196T 99.40％ 99.90％

[0205] The results above indicate that Enz.43, Enz.44, Enz.46, Enz.50, Enz.51, Enz.52, Enz.53, Enz.54, Enz.55, and Enz.56 can also achieve good catalytic effects in enzyme catalytic amplification systems for coenzyme regeneration using isopropanol and alcohol dehydrogenase (Enz.1 or its mutants) and enzyme catalytic amplification systems for coenzyme regeneration using glucose and glucose dehydrogenase (Enz.0).

[0206] The sequences used in this invention are as follows:

[0207] SEQ ID NO:1:

[0208] MYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGLENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKY FVENDIKGNVINMSSVHEVIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQKADVESMIPMGYIGEPEEIAAVAAWLASKEASYVTGITLFADGGMTQYPSFQAGRG

[0209] SEQ ID NO:2:

[0210] ATGTACCCTGATCTTAAAGGCAAAGTTGTTGCTATCACAGGCGCTGCTTCTGGCCTTGGCAAAGCTATGGCTATCCGTTTCGGCAAAGAACAAGCTAAAGTTGTTATCAACTACTACTCTAACAAACAAGATCCTAACGAAGTTAAAGAAGAAGTTATCAAAGCTGGCGGCGAAGCTGTTGTTGTTCAAGGCGATGTTACAAAAGAAGAAGATGTTAAAAACATCGTTCAAACAGCTATCAAAGAATTCGGCACACTTGATATCATGATCAACAACGCTGGCCTTGAAAACCCTGTTCCTTCTCATGAAATGCCTCTTAAAGATTGGGATAAAGTTATCGGCACAAACCTTACAGGCGCTTTCCTTGGCTCTCGTGAAGCTATCAAATACTTCGTTGAAAACGATATCAAAGGCAACGTTATCAACATGTCTTCTGTTCATGAAGTTATCCCTTGGCCTCTTTTCGTTCATTACGCTGCTTCTAAAGGCGGCATCAAACTTATGACAGAAACACTTGCTCTTGAATACGCTCCTAAAGGCATCCGTGTTAACAACATCGGCCCTGGCGCTATCAACACACCTATCAACGCTGAAAAATTCGCTGATCCTAAACAAAAAGCTGATGTTGAATCTATGATCCCTATGGGCTACATCGGCGAACCTGAAGAAATCGCTGCTGTTGCTGCTTGGCTTGCTTCTAAAGAAGCTTCTTACGTTACAGGCATCACACTTTTCGCTGATGGCGGCATGACACAATACCCTTCTTTCCAAGCTGGCCGTGGC

[0211] SEQ ID NO:3:

[0212] MTDRLKGKVALVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDVSDEAGWTKLFDITEEAFGPVTTVVNNAGIVQLKSLEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNKGLGASIINMASIAGIIGDPAMGAYNATKGAVRIMSKSAALDCALKDYDVRVNTVHPGGIKTPGVADLPGFEEMCSQRTKTPMGHIGEPNDIAWICVYLASDESKFATGAEFVVDGGFTAQ

[0213] SEQ ID NO:4:

[0214] ATGACTGATCGTCTGAAGGGCAAAGTAGCCCTGGTAACCGGCGGGACGCTGGGTATCGGTTTGGCAATCGCCGATAAATTTGTAGAGGAGGGTGCGAAAGTAGTTATTACTGGTCGTCACGCGGATGTAGGTGAAAAGGCCGCCAAATCAATCGGCGGCACTGATGTTATTCGCTTTGTCCAGCACGATGTATCCGATGAGGCAGGCTGGACGAAACTGTTCGACATCACCGAGGAGGCATTCGGCCCGGTTACGACCGTCGTGAACAATGCAGGGATTGTACAGCTGAAAAGCCTTGAAGACACTACCACGGAGGAATGGCGTAAACTGCTGTCCGTTAATCTGGATGGTGTTTTTTTCGGCACCCGTCTGGGCATTCAGCGCATGAAAAATAAAGGCTTGGGCGCTAGCATCATCAATATGGCCAGTATTGCGGGGATCATCGGCGATCCGGCAATGGGGGCATACAACGCTACCAAGGGGGCGGTACGTATCATGTCGAAAAGCGCAGCGCTGGATTGCGCACTGAAGGACTACGATGTGCGTGTCAACACAGTACATCCGGGCGGTATCAAGACCCCGGGCGTCGCAGATCTGCCGGGTTTTGAGGAAATGTGTTCACAGCGTACGAAAACCCCTATGGGCCACATTGGCGAACCGAATGACATCGCATGGATCTGTGTGTACCTGGCATCTGACGAATCGAAATTTGCGACGGGTGCAGAATTTGTGGTCGACGGCGGGTTTACCGCACAGT。

Claims

1. A reductase mutant, characterized in that, The amino acid residue difference between the reductase mutant and the amino acid sequence shown in SEQ ID NO: 3 is one of the following combinations: (1) Q95W, L96E and G190P; (2) Q95W, L96E, G190P, G195A and V196T; (3) Q95W, L96E, G190P and I217L; (4) Q95W, L96E, G190P and K192M; (5) Q95W, L96E, G190P and K192D; (6) Q95W, L96E, G190P, G195S and V196T; (7) Q95W, L96E, G190P and K192A; (8) L96E and G190P; (9) L96S and G190P; (10) L96A and G190P; (11) G190P and G195L; (12) Q95W, L96E, G190P, G195L and V196D; (13) Q95W, L96E and G190A; (14) Q95W and G190P; (15) I93W, Q95W, L96G, C206T and F249Y; (16) L96G and G190A; (17) Q95M and G190P; (18) G190P; (19) Q95E and G190P; (20) I93W, Q95W, L96G, C206E and F249C; (21) I93W, Q95W, L96E, C206H and F249E; (22) G190A and G195L; (23) Q95W and L96E.

2. An isolated nucleic acid molecule, characterized in that, The isolated nucleic acid molecule encodes the reductase mutant as described in claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid molecule as described in claim 2.

4. The recombinant expression vector as described in claim 3, characterized in that, The backbone of the recombinant expression vector is pET28a or pET21a.

5. A transformant, characterized in that, The transformant comprises the isolated nucleic acid molecule as described in claim 2, or the recombinant expression vector as described in claim 3 or 4.

6. The transformant as described in claim 5, characterized in that, The host cell used in the construction of the transformant was *Escherichia coli* (E. coli). Escherichia coli ) or Bacillus subtilis ( Bacillus subtilis ).

7. The transformant as described in claim 6, characterized in that, The Escherichia coli mentioned is Escherichia coli BL21(DE3).

8. A method for preparing the reductase mutant as described in claim 1, comprising culturing the transformant as described in any one of claims 5-7 to obtain a fermentation product.

9. The method as described in claim 8, characterized in that, The culture medium used for the culture is selected from LB liquid medium or TB liquid medium, and / or the culture conditions are: shaking culture at a temperature of 37±1℃.

10. The method as described in claim 9, characterized in that, The method further includes the step of purifying the fermentation product to obtain the reductase mutant.

11. An enzyme composition, characterized in that, The enzyme composition comprises the reductase mutant as described in claim 1 and a glucose dehydrogenase having the amino acid sequence shown in SEQ ID NO:

1.

12. The use of the reductase mutant of claim 1, the isolated nucleic acid molecule of claim 2, the recombinant expression vector of claim 3 or 4, the transformant of any one of claims 5-7, or the enzyme composition of claim 11 in the preparation of S-Bosonic.

13. The application as described in claim 12, characterized in that, The preparation of S-Bosein uses β-acetone xyloside as a substrate.

14. A method for preparing S-Bosonic, characterized in that, The method includes: contacting and reacting at least one of the reductase mutant as described in claim 1 or the enzyme composition as described in claim 11 with a substrate to obtain S-Bosein; The substrate is β-acetone xyloside; and / or the reaction further includes the use of a coenzyme.

15. The method as described in claim 14, characterized in that, The reaction also includes a reaction that regenerates the coenzyme.

16. The method as described in claim 15, characterized in that, The coenzyme is any one or both of NADH and NADPH.

17. The method as described in claim 16, characterized in that, The reaction that regenerates the coenzyme uses NAD. + and NADP + Any one or two of the following, a hydrogen donor and a coenzyme regenerating enzyme; wherein the hydrogen donor and the coenzyme regenerating enzyme are selected from one or more of the following: (1) Glucose and glucose dehydrogenase; (2) Formic acid and formic acid dehydrogenase; (3) Isopropanol and alcohol dehydrogenase.

18. The method as described in claim 17, characterized in that, The amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:

1.

19. The method as described in claim 17 or 18, characterized in that, The reductase mutant or the enzyme composition may be used as liquid enzyme, solid enzyme powder, immobilized enzyme, wet cells or powder. And / or, the concentration of β-acetone xyloside added in the reaction is 1-500 mg / mL; And / or, the molar ratio of the hydrogen donor to the β-acetone xyloside is 0.5:1 to 16:1; And / or, the NAD + and / or NADP + The mass ratio of the β-acetone xyloside to the xyloside is 1:1000 to 500:1000; And / or, the pH of the reaction is 6.5-8.5; And / or, the temperature of the reaction is 25-45°C.

20. The method as described in claim 19, characterized in that, The concentration of β-acetone xyloside added in the reaction is 10-200 mg / mL; And / or, the molar ratio of the hydrogen donor to the β-acetone xyloside is 0.68:1, 1.3:1, 5:1 or 16:1; And / or, the mass ratio of NAD+ and / or NADP+ to β-acetone xyloside is 19:1000 or 37:1000; And / or, the pH of the reaction is 7.0-8.0; And / or, the temperature of the reaction is 30-40°C.

21. The method as described in claim 20, characterized in that, When the reductase mutant or the enzyme composition is used in the form of wet bacterial cells, the mass ratio of the wet bacterial cells to the β-acetone xyloside added in the reaction is (0.1-0.5) g: 1 g; And / or, the reductase mutant or the enzyme composition is used in the form of a liquid enzyme, and the ratio of the mass of the wet bacterial cells to the mass of the β-acetone xyloside added in the reaction is (0.1-0.5) g:1 g, calculated based on the mass of the wet bacterial cells that produce the liquid enzyme; And / or, the mass ratio of the coenzyme regenerating enzyme to the hydrogen donor, calculated based on the mass of the wet bacterial cells used, is (0.05-1) g:1 g.

22. The method as described in claim 21, characterized in that, When the reductase mutant or the enzyme composition is used in the form of wet bacterial cells, the mass ratio of the wet bacterial cells to the β-acetone xyloside added in the reaction is 0.5 g: 1 g; And / or, the reductase mutant or the enzyme composition is used in the form of a liquid enzyme, and the ratio of the mass of the wet bacterial cells to the mass of the β-acetone xyloside added in the reaction is 0.2 g: 1 g, calculated based on the mass of the wet bacterial cells that produce the liquid enzyme; And / or, the mass ratio of the coenzyme regenerating enzyme to the hydrogen donor, calculated based on the mass of the wet bacterial cells used, is 0.08 g:1 g, 0.1 g:1 g, 0.125 g:1 g, or 0.8 g:1 g.

23. A reaction end product system for the catalytic synthesis of S-Bosein using β-pyrone xyloside as a substrate, characterized in that, The reaction end product system includes: S-Bosorin; β-pyrrolidone xyloside; and, The reductase mutant as described in claim 1, the transformant as described in claims 5-7, and the enzyme composition as described in claim 11.

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