Composite enzyme, co-immobilized enzyme and application thereof

Abstract

The present application relates to the technical field of biology, and particularly relates to a complex enzyme, a co-immobilized enzyme and application thereof. The present application provides a complex enzyme, a co-immobilized enzyme and application thereof, and a preparation method of Bose factor, wherein the preparation method takes 1-C-(beta-D-pyranosyl) acetone as an initial substrate, selectively synthesizes high-purity S-Bose factor in one step under the action of a carbonyl reductase, introduces formic acid dehydrogenase to regenerate an NADH system, takes NAD and sodium formate as substrates, cyclically generates NADH and carbon dioxide, reduces the accumulation of intermediate products, and finally stably realizes the generation of 380 g / L products, the reaction conversion rate is greater than 99%, the ee value is 100%, the immobilized enzyme is used for 15 times, the enzyme activity retention rate is above 85%, and compared with the free enzyme, the cost is reduced by more than 5 times.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to complex enzymes, co-immobilized enzymes, and their applications. Background Technology

[0002] Pro-Xylane (CAS: 439685-79-7), chemically known as hydroxypropyltetrahydropyranotriol, was originally extracted and prepared by L'Oréal from beech wood in Western Europe. It can promote the production of mucopolysaccharides, hyaluronic acid and collagen in the skin, improve the adhesion between the dermis and epidermis, promote the regeneration of damaged tissue, maintain the elasticity of the dermis, and prevent skin aging. It is a common anti-aging active ingredient in the cosmetics industry.

[0003] Currently, most methods for synthesizing Bosein focus on organic chemical synthesis. Among these chemical synthesis techniques, patent literature discloses a method that utilizes the Knoevenagel condensation of xylose and acetylacetone in an alkaline solution, followed by reduction with sodium borohydride to prepare Bosein. Although this synthetic route involves only two chemical steps, the use of water as a reaction solvent makes product separation and purification difficult. Another patent report describes a one-pot synthesis of Bosein using rare earth metal complexes as catalysts. The key feature of this method is the use of rare earth metal complexes as catalysts. The glycosylation of xylose with ethyl acetoacetate can catalyze the hydrolysis and decarboxylation of the ester group and the reduction of the carbonyl group, with a final yield of up to 80%. However, due to the use of a homogeneous metal catalyst, the metal residue in the product is inevitably increased. Secondly, in the chemical-enzymatic synthesis technology, a patent document discloses a method for the chemical-enzymatic synthesis of bosine. Acetylacetone or acetoacetate ester compound, xylose and an alkaline substance are mixed in a solvent and heated to react to obtain a first intermediate product. The first intermediate product is then mixed with dehydrogenase, coenzyme, reducing agent and isopropanol in phosphate buffer to react and obtain bosine.

[0004] In the field of enzyme engineering technology, patent literature currently discloses a one-pot method for preparing S-Bosein using biological enzymes. This process mainly uses xylose and isopropanol as substrates, and generates S-Bosein under the catalysis of isopropanol dehydrogenase, S-Bosein synthase, carbonyl reductase, and the coenzyme nicotinamide adenine dinucleotide, achieving a yield of 89.5%, which is significantly higher than that of chemical synthesis. The reaction conditions are mild and more environmentally friendly. Another related patent uses a dual-enzyme coupling method to prepare high-purity S-Bosein, using β-acetone xyloside as a substrate for hydrogenation to generate S-Bosein. With 1000mM substrate (190g / L), the conversion rate can reach 99%. The alcohol dehydrogenase utilizes isopropanol and NADP... + It generates acetone and NADPH, but the presence of isopropanol and acetone poses a significant challenge to the enzyme's activity and tolerance.

[0005] Among the existing technologies for synthesizing Bosein, enzymatic methods have significant advantages over chemical methods. However, free enzymes still exhibit instability and low tolerance, increasing the difficulty of the catalytic process. Furthermore, free enzymes can only be used once, increasing enzyme production costs and wastewater and residue treatment costs, and making subsequent separation and purification of the enzyme and product more challenging.

[0006] Existing studies have shown that enzyme immobilization can facilitate the separation of enzymes from the reaction system, enabling enzyme recycling, while also increasing the enzyme's tolerance to the environment and its stability during catalysis.

[0007] Current enzymatic synthesis technologies for S-Bosein suffer from low yields and the generation of organic intermediates during the catalytic process, which severely impacts enzyme tolerance and activity, leading to increased enzyme dosage and higher enzyme costs. Summary of the Invention

[0008] In view of this, the present invention aims to reduce the process pressure of reaction and purification by using a dual-enzyme co-immobilization method, and to achieve high-purity S-Bosein at low cost. It provides a composite enzyme, a co-immobilized enzyme, and their applications. The present invention discloses a method for efficient production of S-Bosein using dual-enzyme immobilization. It primarily uses 1-C-(β-D-xylanosyl)-acetone as the initial substrate, and selectively synthesizes high-purity S-Bosein in the next step under the action of carbonyl reductase. Formate dehydrogenase is introduced to regenerate the NADH system, using NAD and sodium formate as substrates to cyclically generate NADH and carbon dioxide, reducing the accumulation of intermediate products. Ultimately, a stable production of 380 g / L of product is achieved, with a reaction conversion rate >99%, an ee value of 100%, and the immobilized enzyme can be recycled 15 times with an enzyme activity retention rate of over 85%. Compared with free enzyme, the cost is reduced by more than 5 times.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] The present invention provides a complex enzyme comprising carbonyl reductase and formate dehydrogenase; the enzyme ratio of carbonyl reductase to formate dehydrogenase is (1-3U):(5-50U).

[0011] The present invention also provides co-immobilized enzymes, including the aforementioned composite enzyme.

[0012] The present invention also provides a method for preparing the co-immobilized enzyme, comprising:

[0013] S1. Prepare a mixed solution of carbonyl reductase and formate dehydrogenase;

[0014] S2. Take the blended solution and epoxy resin described in S1, immobilize them, filter, wash, and dry to obtain the co-immobilized enzyme.

[0015] In some specific embodiments of the present invention, the enzyme ratio of the carbonyl reductase to the formate dehydrogenase is (1-3U):(5-50U).

[0016] In some specific embodiments of the present invention, the volume ratio of the carbonyl reductase to the formate dehydrogenase is 5:1 to 50:1.

[0017] In some specific embodiments of the present invention, the total protein content in the blend solution to the mass ratio of the epoxy resin, expressed in mg / g, is (100-300):1; the immobilization temperature is 25-40°C; the immobilization time is 5-16 h; and the immobilization rotation speed is 50-250 rpm.

[0018] In some specific embodiments of the present invention, the mass ratio of the blended solution to the epoxy resin includes 120:1 to 10:1.

[0019] In some specific embodiments of the present invention, the pore size of the filter membrane includes 100-200 mesh; the cleaning includes cleaning with 100mM PBS at pH 7.0; and the drying includes drying at 20-25°C for 0.5-3 hours.

[0020] In some specific embodiments of the present invention, the preparation methods of the carbonyl reductase and the formate dehydrogenase include the following steps:

[0021] Step 1: Constructing genetically engineered bacteria

[0022] The PYDH encoding gene was inserted between the NdeI and HindIII sites on plasmid pET28a to obtain the recombinant plasmid pET28a-PYDH; the recombinant plasmid was introduced into competent cells E.coli BL21(DE3) to obtain engineered bacteria E.coli BL21(DE3) / pET28a-PYDH.

[0023] The formate dehydrogenase FDH encoding gene was inserted between the BamHI and XhoI sites on plasmid pET28a to obtain the recombinant plasmid pET28a-FDH; the recombinant plasmid was introduced into competent cells E.coli BL21(DE3) to obtain engineered bacteria E.coli BL21(DE3) / pET28a.

[0024] Step 2: Cultivation of genetically engineered bacteria

[0025] The genetically engineered bacteria E. coli BL21(DE3) / pET28a-PYDH and E. coli BL21(DE3) / pET28a-FDH were inoculated into LB liquid medium containing a final concentration of 2–200 μg / mL kanamycin and cultured overnight. They were then transferred to LB liquid medium containing 2–200 μg / mL kanamycin at an inoculum concentration of 0.5–3% (v / v) and cultured until the bacterial cell concentration reached OD500. 600 Add IPTG to a final concentration of 0.6–0.8; induce culture to obtain an induction culture medium; centrifuge, discard the supernatant, and obtain wet cells;

[0026] Step 3: Prepare crude enzyme solution of engineered bacteria

[0027] Take the wet bacterial cells from step 2, add PBS buffer and mix, homogenize to obtain crude enzyme solutions of the carbonyl reductase and the formate dehydrogenase.

[0028] In some specific embodiments of the present invention, the mass ratio of the wet bacterial cells to the PBS buffer includes 1:19.

[0029] In some specific embodiments of the present invention, the concentration of the PBS buffer includes 0.1 to 2 M, and the pH value includes 6.5 to 8.5.

[0030] In some specific embodiments of the present invention, the homogenization pressure of the homogenization process includes 700 to 900 bar; the number of homogenization processes includes 2 to 5 times.

[0031] This invention also provides the application of any of the following in the preparation of bosonicine:

[0032] (I) the aforementioned complex enzyme; and / or

[0033] (II) the co-immobilized enzyme; and / or

[0034] (III) The co-immobilized enzyme prepared by the above method.

[0035] The present invention also provides a method for preparing Bosein, comprising taking sodium formate, NAD and 1-C-(β-D-xylanosyl)-acetone, adding the composite enzyme, the co-immobilized enzyme and / or the co-immobilized enzyme prepared by the method, and performing an enzyme-catalyzed reaction to obtain Bosein.

[0036] In some specific embodiments of the present invention, the preparation method includes taking 1-C-(β-D-xylanosyl)-acetone, sodium formate and NAD in a molar ratio of (1-2 mol): (1-4 mol): (0.1-0.2 mmol);

[0037] The amount of co-immobilized enzyme added ranges from 20 to 100 U / mL.

[0038] In some specific embodiments of the present invention, the temperature of the enzyme-catalyzed reaction includes 25 to 45°C; the pH value of the enzyme-catalyzed reaction includes 6.0 to 8.0.

[0039] In some specific embodiments of the present invention, the bosonic includes S-bosonic.

[0040] (1) The co-immobilized enzyme resin prepared by the method of the present invention is simple, has an enzyme activity recovery rate of over 90%, and a protein recovery efficiency of 70%. After multiple cycles of co-immobilized enzyme (about 15 times), the remaining enzyme activity is still over 85%. The co-immobilized enzyme activity resin can reach 1550 U / g, which reduces the amount of resin used and can achieve 99% conversion of 1M substrate.

[0041] The co-immobilized enzyme prepared by this invention exhibits higher enzyme activity, mechanical strength, environmental tolerance, and stability for recycling compared to the free enzyme. When applied to the reaction catalyzing the generation of Bosein, it significantly reduces the enzyme production cost, greatly simplifies the catalytic and purification process, and reduces the cost of solid waste treatment, demonstrating a strong cost-reduction advantage.

[0042] This invention employs a formate dehydrogenase cyclic regeneration system for NADH, using NAD and sodium formate as substrates to cyclically generate NADH and carbon dioxide. The carbon dioxide is volatilized, reducing the impact of intermediate product accumulation on enzyme catalytic efficiency and lowering the difficulty of subsequent purification. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0044] Figure 1 Electrophoresis diagram of PYDH fermentation enzyme solution;

[0045] Figure 2 Electrophoresis diagram of FDH fermentation enzyme solution;

[0046] Figure 3 Diagram showing co-immobilized enzymes containing carbonyl reductase and formate dehydrogenase;

[0047] Figure 4 The liquid chromatogram shows the synthesis of S-Bosein from 1-C-(β-D-xylanosyl)-acetone catalyzed by the co-immobilized enzyme prepared in Example 5;

[0048] Figure 5The synthesis of S-Bosein and its isomer R using co-immobilized dual-enzyme resin catalysis of 1-C-(β-D-xylanosyl)-acetone is shown in the chromatogram of S-Bosein standard;

[0049] Figure 6 The liquid chromatogram of S-Bosein synthesized using the method of Example 6 is shown.

[0050] Figure 7 The liquid chromatogram of S-Bosein synthesized using the method of Example 7 is shown.

[0051] Figure 8 The number of times the co-immobilized enzyme was used and the enzyme activity retention were shown.

[0052] Figure 9 This study compares the reaction effects of immobilized enzymes and liquid enzymes with the same amount of added enzyme. Detailed Implementation

[0053] This invention discloses composite enzymes, co-immobilized enzymes, and their applications. Those skilled in the art can refer to the content of 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 can clearly 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.

[0054] Abbreviations and their English meanings

[0055] NAD: Nicotinamide adenine dinucleotide

[0056] NADH: Reduced nicotinamide adenine dinucleotide

[0057] PYDH: Carbonyl reductase

[0058] FDH: Formate dehydrogenase

[0059] This invention discloses a method for the efficient production of S-Bosein using a dual-enzyme immobilization process. The method primarily uses 1-C-(β-D-xylanosyl)-acetone as the initial substrate. High-purity S-Bosein is selectively synthesized in the next step by a carbonyl reductase. Formate dehydrogenase is introduced to regenerate the NADH system, using NAD and sodium formate as substrates to cyclically generate NADH and carbon dioxide, reducing the accumulation of intermediate products. Ultimately, a stable production of 228 g / L of product is achieved, with a reaction conversion rate >99%, an ee value of 100%, and the immobilized enzyme can be recycled 15 times with an enzyme activity retention rate of over 85%. Compared to free enzyme, the cost is reduced by more than 5 times.

[0060] The specific process implementation steps (enzymatic synthesis of Bosein process route diagram) are shown below:

[0061]

[0062] This invention provides a method for the generation of S-Bosonic by dual-enzyme immobilization catalysis, comprising the following steps:

[0063] 1. Construct engineered bacteria containing carbonyl reductase (PYDH) and formate dehydrogenase (FDH).

[0064] The PYDH encoding gene was inserted between the NdeI and HindIII sites on plasmid pET28a to obtain the recombinant plasmid pET28a-PYDH. This recombinant plasmid was introduced into competent E. coli BL21(DE3) cells to obtain the engineered bacterium E. coli BL21(DE3) / pET28a-PYDH. The formate dehydrogenase FDH encoding gene was inserted between the BamHI and XhoI sites on plasmid pET28a to obtain the recombinant plasmid pET28a-FDH. These recombinant plasmids were then introduced into competent E. coli BL21(DE3) cells to obtain the engineered bacterium E. coli BL21(DE3) / pET28a-FDH. The engineered bacteria were then subjected to plasmid extraction and sequencing to ensure accuracy.

[0065] The genetically engineered bacteria *E. coli* BL21(DE3) / pET28a-PYDH and *E. coli* BL21(DE3) / pET28a-FDH were inoculated into LB liquid medium containing 2–200 μg / mL kanamycin and cultured overnight at 20–37°C and 150–300 rpm. Then, they were transferred at an inoculum concentration of 0.5–3% (v / v) into LB liquid medium containing 2–200 μg / mL kanamycin and cultured at 20–37°C and 150–300 rpm until the bacterial cell concentration reached OD500. 600 To obtain an induction culture medium, add IPTG to a final concentration of 0.1–0.3 mM at 0.6–0.8, and induce culture at 20–37 °C for 15 h. Centrifuge the induction culture medium at 4 °C and 4000–10000 rpm for 5–15 min, discarding the supernatant. Resuspend the bacterial cells in 50 mM PBS pH 7.5 buffer, centrifuge at 4 °C and 4000–10000 rpm for 5–15 min, discard the supernatant, and collect the wet bacterial cells as a biocatalyst, storing them at -20 °C for later use.

[0066] Take 3-5 mL of each induction culture medium, centrifuge at 4000-12000 rpm for 5-15 min, discard the supernatant, keep the bacterial cells, resuspend in 1 mL of 20-200 mM PBS pH 7.0-8.5, sonicate to disrupt, take 100 μL of the disrupted bacterial solution, centrifuge, and take the supernatant and precipitate separately. Resuspend the precipitate in 100 μL of 20-200 mM PBS pH 7.0-8.5. Take 40 μL of the undcentrifuged whole bacterial culture, add 4 μL of 6x Protein Loading Buffer, mix well, and boil for 5–15 min; this is the whole bacterial protein. Take 40 μL of the supernatant, add 4 μL of 6x Protein Loading Buffer, mix well, and boil for 5–15 min; this is the supernatant protein. Take 40 μL of the resuspended precipitate, add 4 μL of 6x Protein Loading Buffer, mix well, and boil for 5–15 min; this is the precipitated protein. Finally, take 3–20 μL of the protein solution and 3–20 μL of the protein marker for gel electrophoresis.

[0067] 2. Preparation of fermentation enzyme broth for carbonyl reductase (PYDH) and formate dehydrogenase (FDH)

[0068] Accurately weigh the wet cells of carbonyl reductase PYDH and sodium formate dehydrogenase FDH, and mix them with the PBS buffer system at a ratio of 1:19 (m:m) to form a bacterial slurry. After the bacterial slurry and PBS buffer system are mixed evenly, homogenize the bacterial slurry solution with a homogenizer at a pressure of 700-900 bar for 2-5 times to obtain fermented PYDH enzyme solution and FDH enzyme solution.

[0069] PBS is preferably prepared using potassium dihydrogen phosphate and dipotassium hydrogen phosphate, with a concentration between 0.1 and 2 M and a pH between 6.5 and 8.5.

[0070] 3. Preparation of co-immobilized enzymes

[0071] Homogenized PYDH and FDH enzyme solutions were mixed at an enzyme activity ratio (U:U) of 2:5 and an enzyme solution volume ratio of 5:1 to 50:1 to obtain a crude enzyme solution containing PYDH and FDH. The protein content of the crude enzyme solution was calculated using a standard curve of BSA (bovine serum albumin) determined by ultraviolet spectrophotometry. Epoxy resin was then added to the crude enzyme solution and immobilized in a constant temperature water bath shaker. Finally, the immobilized resin was filtered and washed with a buffer system until the filtrate was free of protein. The resin on the filter cake was collected and dried at 20–25°C for 0.5–3 hours to obtain a co-immobilized enzyme containing PYDH and FDH.

[0072] The ratio of total protein content (mg) of the crude enzyme solution to the mass (g) of epoxy resin was 300:1 to 100:1, and the mass ratio of crude enzyme solution to epoxy resin was 120:1 to 10:1. The immobilization temperature of the constant temperature water bath shaker was between 25 and 40°C. The immobilization time was between 5 and 16 hours. The rotation speed of the constant temperature water bath shaker was between 50 and 250 rpm. The resin used was an epoxy resin.

[0073] 4. Determination of enzyme activity in co-immobilized enzymes and blended enzyme solutions

[0074] Prepare an activity assay system containing sodium formate, NAD, and 1-C-(β-D-xylanosyl)-acetone. Take 2–10 mL of the assay solution and preheat the reaction solution at 25–45 °C for 2–30 min. Then add approximately 0.01–0.5 g of the co-immobilized enzyme and approximately 0.1–0.5 mL of the blended enzyme solution to the assay system. The assay time is 2–60 min. Take 10–100 μL of the assay solution, quench it with 90% acetonitrile, centrifuge it, and take the upper organic phase for liquid chromatography analysis. Calculate the enzyme activity of the co-immobilized enzyme based on the amount of Bosein generated. Enzyme activity is defined as the amount of resin required to generate 1 μmol of product per minute, defined as 1 U. Finally, express the enzyme activity of the co-immobilized enzyme in terms of specific enzyme activity U / g and the enzyme activity of the blended enzyme solution in terms of volumetric enzyme activity U / mL.

[0075] The concentration of the substrate 1-C-(β-D-xylopyranosyl)-acetone was between 1 and 100 mM; the concentration of sodium formate was between 1 and 500 mM; the amount of NAD added was between 0.1 and 5 mM; and the pH was between 6.0 and 9.0.

[0076] 5. High-purity S-Bosein is generated by co-immobilization of two enzymes catalyzing an enzyme reaction.

[0077] A reaction solution containing sodium formate, NAD, and 1-C-(β-D-xylanosyl)-acetone was prepared, and the co-immobilized enzyme prepared above was added to the reaction solution. The reaction was stirred using a mechanical stirrer, and the reaction temperature and pH were controlled to catalyze the generation of high-purity S-Bosein.

[0078] The concentration of the substrate 1-C-(β-D-xylanopyranosyl)-acetone was between 1 and 2 M; the molar ratio of sodium formate to 1-C-(β-D-xylanopyranosyl)-acetone was between 1:1 and 1:2; the amount of NAD added was between 0.1 and 5 mM; the pH of the reaction was controlled by hydrochloric acid between 6.0 and 8.0; the reaction temperature was controlled between 25 and 45 °C; and the amount of co-immobilized enzyme added was between 20 and 100 U / mL.

[0079] 6. Recovery of co-immobilized enzymes

[0080] After the reaction is complete, the reaction solution is filtered directly, and the co-immobilized enzyme on the filter cake is collected. It is then washed with 0.1-2M buffer, with the washing volume being 1-20 times the volume of the immobilized enzyme. The washed co-immobilized enzyme is collected, its specific enzyme activity is measured, and the enzyme activity retention rate is calculated for reuse in the preparation of the Bosein reaction, thereby realizing the recycling of the immobilized dual-enzyme resin.

[0081] The genes encoding PYDH and FDH are shown below:

[0082] Uniprot#:Q1J2J0: Encoding gene for carbonyl reductase PYDH

[0083] Source: Deinococcusgeothermalis

[0084] PYDH gene sequence (SEQ ID NO.1):

[0085] ATGACCTCAACTGCTCAGATGCCCCACATCCTTGACCTCTTTCGTCTCGACGGACGCCACGCCCTGGTGACTGGCGGCGCGCAGGGGATCGGCTTCGAGATCGCGCGCGGACTGGCCCAGGCTGGAGCGCGCGTGACCATCGCGGACCTGAACCCCGACGTGGGGGAGGGTGCGGCCCGCGAATTGGACGGGACCTTCGAGCGACTGAACGTCACCGACGCGGACGCCGTGGCGGACCTGGCCCGGCGCCTCCCCGATGTGGACGTGCTTGTGAACAACGCGGGAATTGTCCGCAACGCTCCCGCCGAGGACACCCCCGATGACGACTGGCGGGCGGTGCTGAGCGTCAACCTGGACGGGGTGTTCTGGTGCTGCCGCGAGTTCGGCCGAACCATGCTGGCTAGAGGCCGCGGCGCCATCGTCAGCACCGCCAGCATGAGCGGCCTGATCAGCAATCATCCGCAGCCGCAGGCCGCCTACAACGCCTCCAAGGCCGCGGTCATTCACCTCACCCGCAGCCTCGCGGGCGAGTGGGCGAGCCGGGGCGTGCGCGTGAACGCCGTCGCTCCCGGCTACACCGCTACACCCCTTACCCGGCGCGGTCTGGAGACGCCCGAGTGGCGCGAAACCTGGCTGAAGGAAACGCCGCTGGGCCGCCTCGCCGAACCCCGCGAGATTGCGCCCGCCGTGCTCTACCTCGCCAGCGACGCCGCCAGCTTCGTCACCGGGCATACGCTCGTGGTGGACGGTGGGTACACGGTGTGGTGA

[0086] PYDH amino acid sequence (SEQ ID NO.2):

[0087] MTSTAQMPHILDLFRLDGRHALVTGGAQGIGFEIARGLAQAGARVTIADLNPDVGEGAARELDGTFERLNVTDADAVADLARRLPDVDVLVNNAGIVRNAPAEDTPDDDWRAVLSVNLDGVFWCCRE FGRTMLARGRGAIVSTASMSGLISNHPQPQAAYNASKAAVIHLTRSLAGEWASRGVRVNAVAPGYTATPLTRRGLETPEWRETWLKETPLGRLAEPREIAPAVLYLASDAASFVTGHTLVVDGGYTVW

[0088] Formate dehydrogenase FDH encoding gene Uniprot#:P33160:

[0089] Source: Pseudomonas sp. (strain101)

[0090] FDH gene sequence (synthesized) (SEQ ID NO.3):

[0091]

[0092] FDH amino acid sequence (SEQ ID NO.4):

[0093] MAKVLCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPGQLLGSVSGELGLRKYLESNGHTLVVTSDKDGPDSVFERELVDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRNVTVAEVTYCNSISVAEHVVMMILSLVRNYLPSHEWARKGGWNIADCVSHAYDLEAMHVGTVAA GRIGLAVLRRLAPFDVHLHYTDRHRLPESVEKELNLTWHATREDMYPVCDVVTLNCPLHPETEHMINDETLKLFKRGAYIVNTARGKLCDRDAVARALESGRLAGYAGDVWFPQPAPKDHPWRTMPYNGMTPHISGTTLTAQARYAAGTREILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAKFKKAV

[0094] Unless otherwise specified, the composite enzymes, co-immobilized enzymes, and raw materials and reagents used in the application of the present invention can all be purchased from the market.

[0095] The present invention will be further illustrated below with reference to the embodiments:

[0096] Example 1: Preparation of wet cells of PYDH and FDH engineered bacteria and their crude enzyme solutions

[0097] (1) Construction of genetically engineered bacteria

[0098] PYDH gene-enhanced bacteria: The PYDH encoding gene (SEQ ID NO.1) was inserted between the NdeI and Hind III sites on plasmid pET28a to obtain the recombinant plasmid pET28a-PYDH. The recombinant plasmid was introduced into competent E. coli BL21(DE3) cells to obtain the engineered bacteria E. coli BL21(DE3) / pET28a-PYDH.

[0099] FDH-expressing engineered bacteria: The formate dehydrogenase FDH encoding gene (SEQ ID NO.3) was inserted between the BamHI and XhoI sites on plasmid pET28a to obtain the recombinant plasmid pET28a-FDH. The recombinant plasmid was then introduced into competent E. coli BL21(DE3) cells to obtain engineered bacteria E. coli BL21(DE3) / pET28a-FDH. Sequencing of the extracted plasmids from the engineered bacteria confirmed that each gene was correctly inserted.

[0100] (2) Preparation of wet cell culture of engineered bacteria

[0101] Wet mycelium is prepared as follows:

[0102] The genetically engineered bacteria E. coli BL21(DE3) / pET28a-PYDH and E. coli BL21(DE3) / pET28a-FDH were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm. Then, they were transferred to LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 1% and cultured at 37°C and 220 rpm until the bacterial cell concentration OD600 reached 0.6-0.8. IPTG was added to the culture to a final concentration of 0.3 mM and induced at 35°C for 15 h to obtain the induction culture medium.

[0103] Take the induction culture medium and conduct the following experiments:

[0104] ①SDS-PAGE electrophoresis:

[0105] Take 5 mL of each induction culture medium, centrifuge at 12000 rpm for 5 min, discard the supernatant, retain the bacterial cells, resuspend in 1 mL of 20 mM PBS (pH 7.0), sonicate to disrupt, take 100 μL of the disrupted bacterial solution, centrifuge, and collect the supernatant and precipitate separately. Resuspend the precipitate in 100 μL of 20 mM PBS (pH 7.0) to obtain the resuspended precipitate. Take 40 μL of the undcentrifuged whole bacterial solution, add 4 μL of 6x Protein Loading Buffer, mix well, and boil for 10 min. This is the whole bacterial protein, denoted as W. Take 40 μL of the supernatant, add 4 μL of 6x Protein Loading Buffer, mix well, and boil for 10 min. This is the supernatant protein, denoted as S. Take 40 μL of the resuspended precipitate, add 4 μL of 6x Protein Loading Buffer, mix well, and boil for 10 min. This is the precipitated protein, denoted as P.

[0106] Finally, take 7 μL of protein solution and 7 μL of protein marker and run them on a gel.

[0107] like Figure 1 , Figure 2 As shown, SDS-PAGE analysis indicated that both the PYDH and FDH coding genes were successfully expressed in the genetically engineered bacteria, with good expression levels. Based on the expression levels of supernatant and precipitated proteins (roughly assessed by band depth), the solubility was approximately 90%.

[0108] The induction culture medium was then centrifuged at 4°C and 8000 rpm for 7 min, and the supernatant was discarded. The cells were then resuspended in 50 mM PBS pH 7.5 buffer, centrifuged at 4°C and 8000 rpm for 7 min, and the supernatant was discarded. The wet cells were collected as a biocatalyst and stored at -20°C for later use.

[0109] (3) Preparation of crude enzyme solution from engineered bacteria

[0110] Accurately weigh 10g of carbonyl reductase PYDH and sodium formate dehydrogenase FDH wet cells respectively. Mix the wet cells with PBS buffer at a ratio of 1:19 (m:m). After the wet cells are evenly mixed with 100mM PBS pH7.0 buffer, homogenize the bacterial sludge solution using a homogenizer. The homogenization pressure is controlled at 700-900 bar. Homogenize 3 times to obtain fermented PYDH enzyme solution and FDH enzyme solution.

[0111] Example 2: Preparation of co-immobilized enzymes

[0112] The PYDH enzyme solution and FDH enzyme solution prepared in Example 1 were mixed at an enzyme ratio of 1U:50U, resulting in a volume ratio of 2.5:10, to obtain a crude enzyme solution containing both PYDH and FDH, with a total protein content of 2500mg. Then, 25g of epoxy resin ES-103B was added to the crude enzyme solution, resulting in a mass ratio of 10:1 between the crude enzyme solution and the epoxy resin. The mixture was immobilized in a constant temperature water bath at 35℃ and a shaking speed of 150rpm for 5 hours. Finally, the immobilized resin was filtered and washed with a 100mM PBS pH 7.0 buffer system until the filtrate was free of protein. The resin on the filter cake was collected and dried at 25℃ for 0.5 hours to obtain a co-immobilized enzyme containing both PYDH and FDH enzymes.

[0113] The obtained co-immobilized enzyme was subjected to enzyme activity assay, and the specific enzyme activity was calculated to be 1230.62 U / g. The prepared co-immobilized enzyme is as follows: Figure 3 As shown, the immobilized enzyme is whitish in color and has fine particles (pore size 0.1-0.2 mm). The method for determining the enzyme activity of the co-immobilized enzyme is as follows:

[0114] Prepare an activity assay system containing sodium formate, NAD, and 1-C-(β-D-xylanosyl)-acetone. Take 10 mL of the activity assay solution, preheat the reaction solution at 35 °C for 5 min, and then add about 0.2 g of the co-immobilized enzyme prepared above to the activity assay system. The activity assay time is 25 min. Take 100 μL of the activity assay solution, quench it with 90% acetonitrile, centrifuge it, and take the upper organic phase for liquid chromatography analysis. Calculate the enzyme activity of the co-immobilized enzyme based on the amount of Bosein generated. Enzyme activity is defined as the amount of resin required to generate 1 μmol of product per minute, defined as 1 U. Finally, the enzyme activity of the co-immobilized enzyme is expressed as specific enzyme activity U / g.

[0115] The concentration of the substrate 1-C-(β-D-xylanosyl)-acetone was 100 mM; the concentration of sodium formate was 500 mM; the amount of NAD added was 5 mM; and the pH was 6.0.

[0116] Example 3: Preparation of co-immobilized enzymes

[0117] The PYDH enzyme solution and FDH enzyme solution prepared in Example 1 were mixed at an enzyme amount ratio of 3U:5U and an enzyme solution volume ratio of 7.5:1 to obtain a crude enzyme solution containing PYDH and FDH, with a total protein content of 12g. Then, 80g of epoxy resin ES-103B was added to the crude enzyme solution, so that the mass ratio of the crude enzyme solution to the epoxy resin was 120:1. The mixture was immobilized in a constant temperature water bath shaker at 40℃ and a shaking speed of 150rpm for 5h. Finally, the immobilized resin was filtered and washed with a 100mM PBS pH 7.0 buffer system until the filtrate was free of protein. The resin on the filter cake was collected and dried at 25℃ for 0.5h to obtain a co-immobilized enzyme containing PYDH and FDH enzymes.

[0118] The obtained co-immobilized enzyme was subjected to enzyme activity assay, and the specific activity of this co-immobilized enzyme was calculated to be 1430.23 U / g. The method for determining the enzyme activity of the co-immobilized enzyme is as follows:

[0119] Prepare an activity assay system containing sodium formate, NAD, and 1-C-(β-D-xylanosyl)-acetone. Take 10 mL of the activity assay solution, preheat the reaction solution at 35 °C for 5 min, and then add about 0.2 g of the co-immobilized enzyme prepared above to the activity assay system. The activity assay time is 25 min. Take 100 μL of the activity assay solution, quench it with 90% acetonitrile, centrifuge it, and take the upper organic phase for liquid chromatography analysis. Calculate the enzyme activity of the co-immobilized enzyme based on the amount of Bosein generated. Enzyme activity is defined as the amount of resin required to generate 1 μmol of product per minute, defined as 1 U. Finally, the enzyme activity of the co-immobilized enzyme is expressed as specific enzyme activity U / g.

[0120] The concentration of the substrate 1-C-(β-D-xylanosyl)-acetone was 100 mM; the concentration of sodium formate was 500 mM; the amount of NAD added was 5 mM; and the pH was 6.0.

[0121] Example 4: Preparation of co-immobilized enzymes

[0122] The PYDH enzyme solution and FDH enzyme solution prepared in Example 1 were mixed at an enzyme mass ratio of 2U:27U and an enzyme solution volume ratio of 5:5.4 to prepare a crude enzyme solution containing both PYDH and FDH. A crude enzyme solution with a total protein content of 9g was taken. Then, 30g of epoxy resin ES-103B was added to the prepared crude enzyme solution, so that the mass ratio of the crude enzyme solution to the epoxy resin was 60:1. The solution was immobilized in a constant temperature water bath shaker at 25°C and a shaking speed of 150rpm for 16h. Finally, the immobilized resin was filtered and washed with a 100mM PBS pH 7.0 buffer system until the filtrate was free of protein. The resin on the filter cake was collected and dried at 25°C for 0.5h to obtain a co-immobilized enzyme containing both PYDH and FDH enzymes.

[0123] The obtained co-immobilized enzyme was subjected to enzyme activity assay, and the specific enzyme activity was calculated to be 1540.52 U / g. The method for determining the enzyme activity of the co-immobilized enzyme is as follows:

[0124] Prepare an activity assay system containing sodium formate, NAD, and 1-C-(β-D-xylanosyl)-acetone. Take 10 mL of the activity assay solution, preheat the reaction solution at 35 °C for 5 min, and then add about 0.2 g of the co-immobilized enzyme prepared above to the activity assay system. The activity assay time is 25 min. Take 100 μL of the activity assay solution, quench it with 90% acetonitrile, centrifuge it, and take the upper organic phase for liquid chromatography analysis. Calculate the enzyme activity of the co-immobilized enzyme based on the amount of Bosein generated. Enzyme activity is defined as the amount of resin required to generate 1 μmol of product per minute, defined as 1 U. Finally, the enzyme activity of the co-immobilized enzyme is expressed as specific enzyme activity U / g.

[0125] The concentration of the substrate 1-C-(β-D-xylanosyl)-acetone was 100 mM; the concentration of sodium formate was 500 mM; the amount of NAD added was 5 mM; and the pH was 6.0.

[0126] Example 5: One-step synthesis of S-Bosein from 1-C-(β-D-xylanopyranosyl)-acetone catalyzed by co-immobilized enzyme.

[0127] Prepare a 500 mL reaction solution containing sodium formate, NAD, and 1-C-(β-D-xylanopyranosyl)-acetone. The final concentrations of the substrate 1-C-(β-D-xylanopyranosyl)-acetone are 1 M, sodium formate is 2 M, and NAD is 0.2 mM. Add 10.16 g of the co-immobilized enzyme from Example 2 at a concentration of 25 U / mL. Maintain the reaction temperature at 38°C using a mechanical stirrer. Control the pH at 6.5 with 2 M HCl. The reaction should be completed in 6 hours. Take 50 μL of the reaction solution and add it to 1.95 mL of 90% acetonitrile. Vortex the solution for 1 min until homogeneous. Centrifuge and collect the upper organic phase for liquid chromatography analysis. The yield of Bosein is 190 g / L, the final conversion rate is 99.58%, and the ee value is 100%. The liquid chromatogram is shown below. Figure 4 and Figure 5 As shown, the retention times of 1-C-(β-D-xylanosyl)-acetone and S-Bosein were 5.773 min and 7.302 min, respectively.

[0128] Example 6: One-step synthesis of S-Bosein from 1-C-(β-D-xylanopyranosyl)-acetone catalyzed by co-immobilized enzyme

[0129] Prepare a 1000 mL reaction solution containing sodium formate, NAD, and 1-C-(β-D-xylanopyranosyl)-acetone, wherein the final concentrations of the substrate 1-C-(β-D-xylanopyranosyl)-acetone are 2 M, the final concentrations of sodium formate and NAD are 0.1 mM, and add 13.98 g of the enzyme immobilized in Example 3 at a concentration of 20 U / mL. The reaction temperature is 38 °C, and the mixture is stirred using a mechanical stirrer. The pH is controlled at 6.5 with 2 M HCl, and the reaction is completed after 6 h. Take 50 μL of the reaction solution and add it to 1.95 mL of 90% acetonitrile. Vortex the mixture for 1 min until homogeneous. Centrifuge and collect the upper organic phase for liquid chromatography analysis. Figure 6 As shown, the yield of Boseine was 380 g / L, and the final conversion rate was 99.46%.

[0130] Example 7: One-step synthesis of S-Bosein from 1-C-(β-D-xylanopyranosyl)-acetone catalyzed by co-immobilized enzyme.

[0131] Prepare a 1000 mL reaction solution containing sodium formate, NAD, and 1-C-(β-D-xylanopyranosyl)-acetone. The final concentrations of the substrate 1-C-(β-D-xylanopyranosyl)-acetone are 2 M, sodium formate is 3.8 M, and NAD is 0.1 mM. Add 64.91 g of the co-immobilized enzyme from Example 4 at a concentration of 100 U / mL. Maintain the reaction temperature at 38°C using a mechanical stirrer. Control the pH at 6.5 with 2 M HCl. The reaction should be completed in 6 hours. Take 50 μL of the reaction solution and add it to 1.95 mL of 90% acetonitrile. Vortex the solution in a 8000 rpm mixer for 1 min until homogeneous. Centrifuge and collect the upper organic phase for liquid chromatography analysis. Figure 7 As shown, the yield of Bosein was 380 g / L, and the final conversion rate was 100%.

[0132] Example 8: Recovery of co-immobilized enzymes

[0133] After the reaction in Example 7 was completed, the reaction solution was directly filtered, and the co-immobilized enzyme on the filter cake was collected. The cake was then washed with 100 mM PBS (pH 7.5), with the washing volume being 10 times the volume of the immobilized enzyme. The washed co-immobilized enzyme was collected, and its relative enzyme activity was measured. The initial co-immobilized enzyme activity was taken as 100%, and the relative enzyme activity of each subsequent batch was calculated using the following formula:

[0134] Enzyme activity recovery rate (%) = (Resin specific enzyme activity after Nth cycle / Initial immobilized enzyme activity) * 100

[0135] The relative enzyme activity changes of the co-immobilized enzyme were recorded after each cycle of use, as follows: Figure 8 As shown, even after 15 uses, the enzyme activity retention rate is still above 85%, indicating that the co-immobilized enzyme has good enzyme stability.

[0136] Comparison of the reactions of liquid enzyme and immobilized enzyme in Comparative Example 1

[0137] Following the method described in Example 5, a 500 mL volume of reaction solution containing sodium formate, NAD, and 1-C-(β-D-xylanopyranosyl)-acetone was prepared. The final concentrations of the substrate 1-C-(β-D-xylanopyranosyl)-acetone were 1 M, sodium formate 2 M, and NAD 0.2 mM. 10.16 g of the co-immobilized enzyme from Example 2 was added at an enzyme concentration of 25 U / mL. The reaction was carried out at 38°C using a mechanical stirrer. The pH of the reaction was controlled at 6.5 with 2 M HCl. The reaction was completed after 6 h. 50 μL of the reaction solution was added to 1.95 mL of 90% acetonitrile and vortexed for 1 min in a vortex mixer (8000 rpm) until homogeneous. After centrifugation, the upper organic phase was used for liquid chromatography analysis. The initial conversion rate was 99.58%. After the reaction, the immobilized enzyme was recovered in accordance with the method described in Example 8. The recovery was repeated three times for the addition reaction. The control liquid enzyme used was a blend of PYDH and FDH enzyme solution. Its enzyme activity was measured according to the activity assay system provided in Example 2. 0.1 mL of the blended enzyme solution was added to the assay system, resulting in a volumetric enzyme activity of 1230 U / mL. The liquid enzyme reaction system was also prepared according to an enzyme addition rate of 25 U / mL, with 10.17 mL of the blended liquid enzyme added. After the reaction, the hourly reaction efficiency of the liquid enzyme and the co-immobilized enzyme was compared, as follows: Figure 9 As shown, with the same amount of enzyme added, the immobilized enzyme exhibits a better reaction rate than the liquid enzyme, possibly indicating improved stability. Since the blended enzyme is highly soluble and no precipitate is formed after the reaction, recovery is not ideal and therefore no recovery treatment is performed.

[0138] Comparative Example 2: The effect of high enzyme activity ratio between carbonyl reductase and formate dehydrogenase

[0139] The PYDH enzyme solution and FDH enzyme solution prepared in Example 1 were mixed at an enzyme amount ratio of 5U:1U and an enzyme solution volume ratio of 125:2 to prepare a crude enzyme solution containing both PYDH and FDH enzymes. The crude enzyme solution with a total protein content of 2500mg was taken. Then, 25g of epoxy resin ES-103B was added to the crude enzyme solution, so that the mass ratio of the crude enzyme solution to the epoxy resin was 10:1. The mixture was immobilized in a constant temperature water bath shaker at 35°C and a shaking speed of 150rpm for 5h. Finally, the immobilized resin was filtered and washed with a 100mM PBS pH 7.0 buffer system until the filtrate was free of protein. The resin on the filter cake was collected and dried at 25°C for 0.5h to obtain a co-immobilized enzyme containing both PYDH and FDH enzymes.

[0140] The obtained co-immobilized enzyme was subjected to enzyme activity determination. The specific enzyme activity of the co-immobilized enzyme in this example was calculated to be 625.35 U / g, while the enzyme activity in Example 2 was 1230.62 U / g. It can be seen that the high enzyme activity ratio of carbonyl reductase to formate dehydrogenase will affect the enzyme activity of the co-immobilized enzyme.

[0141] The method for determining the activity of the co-immobilized enzyme is as follows:

[0142] Prepare an activity assay system containing sodium formate, NAD, and 1-C-(β-D-xylanosyl)-acetone. Take 10 mL of the activity assay solution, preheat the reaction solution at 35 °C for 5 min, and then add about 0.2 g of the co-immobilized enzyme prepared above to the activity assay system. The activity assay time is 25 min. Take 100 μL of the activity assay solution, quench it with 90% acetonitrile, centrifuge it, and take the upper organic phase for liquid chromatography analysis. Calculate the enzyme activity of the co-immobilized enzyme based on the amount of Bosein generated. Enzyme activity is defined as the amount of resin required to generate 1 μmol of product per minute, defined as 1 U. Finally, the enzyme activity of the co-immobilized enzyme is expressed as specific enzyme activity U / g.

[0143] The concentration of the substrate 1-C-(β-D-xylanosyl)-acetone was 100 mM; the concentration of sodium formate was 500 mM; the amount of NAD added was 5 mM; and the pH was 6.0.

[0144] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made 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. Application of complex enzymes in the preparation of S-Bosonic; The complex enzyme includes: Carbonyl reductase and formate dehydrogenase; The amino acid sequence of the carbonyl reductase is shown in SEQ ID NO.2; The amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO.4; The ratio of the amount of carbonyl reductase to the amount of formate dehydrogenase is (1~3U):(5~50U).

2. Application of co-immobilized enzymes in the preparation of S-Bosonic; The co-immobilized enzyme includes the composite enzyme as described in claim 1.

3. The application of the co-immobilized enzyme obtained by the following preparation method in the preparation of S-Bosonica; the preparation method includes the following steps: S1. Prepare a mixed solution of carbonyl reductase and formate dehydrogenase; S2. Take the blended solution and epoxy resin described in S1, immobilize, filter, wash, and dry to obtain the co-immobilized enzyme; the amino acid sequence of the carbonyl reductase is shown in SEQ ID NO.2; The amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO.4; The ratio of the amount of carbonyl reductase to the amount of formate dehydrogenase is (1~3U):(5~50U).

4. The application as described in claim 3, characterized in that, The total protein content in the blend solution and the mass ratio of the epoxy resin, expressed in mg / g, are (100~300):1; the immobilization temperature is 25~40℃; the immobilization time is 5~16 h; and the immobilization rotation speed is 50~250 rpm.

5. A method for preparing S-Bosein, characterized in that, Sodium formate, NAD, and 1-C-(β-D-xylanosyl)-acetone are added to the complex enzyme described in claim 1, the co-immobilized enzyme described in claim 2, and / or the co-immobilized enzyme described in claim 3 or 4, and an enzyme-catalyzed reaction is carried out to obtain S-Bosoxane.

6. The preparation method according to claim 5, characterized in that, The molar ratio of sodium formate, NAD, and 1-C-(β-D-xylanosyl)-acetone is (1~2 mol):(2~3.8 mol):(0.1~0.2 mmol); the amount of co-immobilized enzyme added is 20~100 U / mL.

7. The preparation method according to claim 5 or 6, characterized in that, The temperature of the enzyme-catalyzed reaction ranges from 25 to 45°C; the pH value of the enzyme-catalyzed reaction ranges from 6.0 to 8.0.

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