Compound enzyme, co-immobilized enzyme and application of co-immobilized enzyme

Abstract

The invention relates to the technical field of biology, in particular to a compound enzyme, a co-immobilized enzyme and application thereof. The invention provides a compound enzyme, a co-immobilized enzyme, application of the co-immobilized enzyme and a preparation method of glassine, the preparation method comprises the following steps: by taking 1-C-(beta-D-xylopyranosyl)-acetone as an initial substrate, selectively synthesizing high-purity S-glassine in one step under the action of carbonyl reductase, introducing formate dehydrogenase to regenerate an NADH (Nicotinamide Adenine Dinucleotide) system, taking NAD (Nicotinamide Adenine Dinucleotide) and sodium formate as substrates, and synthesizing the high-purity S-glassine in one step under the action of carbonyl reductase. NADH and carbon dioxide are circularly generated, accumulation of intermediate products is reduced, finally, 380g / L of products are stably generated, and the reaction conversion rate is gt; the enzyme activity retention rate of the immobilized enzyme is more than 85% after the immobilized enzyme is recycled for 15 times, and compared with the free enzyme amount, the cost is reduced by more than 5 times.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a composite enzyme, a co-immobilized enzyme and applications thereof. Background Art

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

[0003] At present, the methods for synthesizing bosine are mostly concentrated on the synthesis by organic chemistry. In the chemical synthesis technology, there is a patent document that discloses a method for preparing bosine by Knoevenagel condensation of xylose and acetylacetone in an alkaline solution and then reducing it with sodium borohydride. Although this synthetic route has only two chemical reactions, it is difficult to separate and purify the product because water is used as the reaction solvent. Another patent document reports a method for synthesizing bosine by a one-pot method promoted by a rare earth metal complex. The characteristic of this method is that a rare earth metal complex is used as a catalyst. The invention can catalyze the glycosidation of ethyl acetoacetate of xylose, and at the same time catalyze the hydrolysis, decarboxylation and carbonyl reduction of the ester group, with a final yield of up to 80%. However, due to the use of a homogeneous metal catalyst, the metal residue of the product will inevitably increase. Secondly, in the chemical-enzymatic synthesis technology, a patent document discloses a chemical-enzymatic method for synthesizing bosone, wherein acetylacetone or acetoacetate ester compounds, xylose and an alkaline substance are mixed in a solvent and heated to react to obtain a first intermediate product, and then the first intermediate product is mixed with a dehydrogenase, a coenzyme, a reducing agent and isopropanol in a phosphate buffer to react to obtain bosone.

[0004] In the field of enzyme engineering technology, there are currently patent documents that disclose a method for preparing bosense using a one-pot method using biological enzymes. The process mainly uses xylose and isopropanol as substrates, and generates bosense under the catalysis of isopropanol dehydrogenase, bosense synthase, carbonyl reductase and coenzyme nicotinamide adenine dinucleotide, and the final yield can reach 89.5%. Compared with the chemical synthesis, the yield is significantly improved, the reaction conditions are mild, and it is more environmentally friendly; another related patent uses a dual enzyme coupling method to prepare high-purity S-bosense, using β-acetone xyloside as a substrate for hydrogenation to generate S-bosense, 1000mM substrate (190g / L), the reaction conversion rate can reach 99%, wherein the alcohol dehydrogenase uses isopropanol and NADP + Acetone and NADPH are produced, but the presence of isopropanol and acetone poses a great challenge to the activity and tolerance of the enzyme.

[0005] Comprehensively considering the existing technologies for synthesizing bosera, it can be seen that the enzymatic method has better advantages than the chemical method, but the free enzyme is still unstable and the enzyme tolerance is low, which increases the difficulty of the catalytic process; secondly, the free enzyme can only be used once, which increases the cost of enzyme production and the cost of wastewater and waste residue treatment, and increases the difficulty of subsequent separation and purification of the enzyme and product.

[0006] Relevant studies have shown that enzyme immobilization can separate the enzyme from the reaction system and achieve enzyme recycling, while increasing the enzyme's tolerance to the environment and its stability in the catalytic process.

[0007] The current technologies for enzymatic synthesis of S-bosone have the problems of low yield and the generation of some organic intermediates during the catalytic process, which will seriously affect the tolerance and activity of the enzyme, thereby increasing the amount of enzyme added and the cost of the enzyme. Summary of the invention

[0008] In view of this, the present invention aims to use a method of co-immobilization of dual enzymes to reduce the process pressure of reaction and purification, and to achieve a method of high-purity S-bosone at low cost. A composite enzyme, a co-immobilized enzyme and its application are provided. The present invention discloses a method of efficiently producing bosone by dual enzyme immobilization, which mainly uses 1-C-(β-D-xylopyranosyl)-acetone as the initial substrate, and selectively synthesizes high-purity S-bosone in the next step under the action of carbonyl reductase, introduces a formate dehydrogenase to regenerate the NADH system, uses NAD and sodium formate as substrates, cyclically generates NADH and carbon dioxide, reduces the accumulation of intermediate products, and finally stably achieves the generation of 380g / L product, the reaction conversion rate is>99%, the ee value is 100%, the immobilized enzyme is recycled 15 times, the enzyme activity retention rate is above 85%, and the cost is reduced by more than 5 times compared with the amount of free enzyme.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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

[0011] The invention also provides a co-immobilized enzyme, comprising the composite enzyme.

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

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

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

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

[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, in terms of mg / g, the mass ratio of the total protein content in the blended solution to the epoxy resin includes (100-300): 1; the immobilization temperature includes 25-40°C; the immobilization time includes 5-16h; and the immobilization rotation speed includes 50-250rpm.

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

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

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

[0021] Step 1: Construct genetically engineered bacteria

[0022] Insert the PYDH coding gene between the NdeI and HindIII sites on the plasmid pET28a to obtain the recombinant plasmid pET28a-PYDH; introduce the recombinant plasmid into competent cells E.coliBL21(DE3) to obtain the engineered bacteria E.coliBL21(DE3) / pET28a-PYDH;

[0023] Inserting the formate dehydrogenase FDH encoding gene between the BamHI and XhoI sites on the plasmid pET28a to obtain the recombinant plasmid pET28a-FDH; introducing the recombinant plasmids into competent cells E. coli BL21 (DE3) to obtain the 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 kanamycin at a final concentration of 2 to 200 μg / mL, and cultured overnight; the inoculation volume of 0.5 to 3% was transferred to LB liquid medium containing 2 to 200 μg / mL kanamycin, and cultured until the bacterial concentration OD 600 to 0.6-0.8; add IPTG with a final concentration of 0.1-0.3 mM to induce culture and obtain induced culture solution; centrifuge and discard the supernatant to obtain wet bacteria;

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

[0027] Take the wet bacteria described in step 2, add PBS buffer solution, mix, and homogenize to obtain the crude enzyme solution of the carbonyl reductase and the formate dehydrogenase.

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

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

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

[0031] The present invention also provides the use of any of the following items in the preparation of boson:

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

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

[0034] (III) The co-immobilized enzyme obtained by the preparation method.

[0035] The present invention also provides a method for preparing bosense, which comprises taking sodium formate, NAD and 1-C-(β-D-xylopyranosyl)-acetone, adding the composite enzyme, the co-immobilized enzyme and / or the co-immobilized enzyme obtained by the preparation method, and performing an enzyme catalytic reaction to obtain bosense.

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

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

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

[0039] In some specific embodiments of the present invention, the Boson comprises S-Boson.

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

[0041] The co-immobilized enzyme prepared by the present invention has higher enzyme activity, mechanical strength, environmental tolerance and cyclic stability than free enzymes. It is applied to the reaction of catalyzing the production of bosogenin, which greatly reduces the production cost of the enzyme, greatly reduces the process difficulty of catalysis and purification, reduces the cost of solid waste treatment, and shows a strong cost reduction advantage.

[0042] The present invention adopts a formate dehydrogenase cyclic regeneration NADH system, takes NAD and sodium formate as substrates, cyclically generates NADH and carbon dioxide, and the carbon dioxide is volatilized, thereby reducing the influence of the accumulation of intermediate products on the enzyme catalytic efficiency and reducing the difficulty of subsequent purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0044] Figure 1 Shows the electrophoresis diagram of PYDH fermentation enzyme solution;

[0045] Figure 2 Shown is the electrophoresis diagram of FDH fermentation enzyme solution;

[0046] Figure 3 shows a co-immobilized enzyme diagram containing carbonyl reductase and formate dehydrogenase;

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

[0048] Figure 5The synthesis of S-bosone and isomer R, S-bosone standard product detection spectrum using co-immobilized dual enzyme resin to catalyze 1-C-(β-D-xylopyranosyl)-acetone;

[0049] Figure 6 1 shows a liquid chromatogram of S-bosine synthesized by the method of Example 6;

[0050] Figure 7 1 is a liquid chromatogram of S-bosine synthesized by the method of Example 7;

[0051] Figure 8 It shows the usage times of the co-immobilized enzyme and the enzyme activity retention;

[0052] Fig. 9 The figure shows the comparison of the reaction effects of immobilized enzyme and liquid enzyme with the same enzyme addition amount. DETAILED DESCRIPTION

[0053] The present invention discloses complex enzymes, co-immobilized enzymes and their applications. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve them. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0054] Abbreviations and English meanings

[0055] NAD: Nicotinamide adenine dinucleotide

[0056] NADH: Nicotinamide adenine dinucleotide reduced

[0057] PYDH: carbonyl reductase

[0058] FDH: Formate dehydrogenase

[0059] The invention discloses a method for efficiently producing bosone by dual enzyme immobilization. The method mainly takes 1-C-(β-D-xylopyranosyl)-acetone as an initial substrate, selectively synthesizes high-purity S-bosone in the next step under the action of carbonyl reductase, introduces a formate 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 228 g / L of products, the reaction conversion rate is greater than 99%, the ee value is 100%, the immobilized enzyme is cyclically used 15 times, the enzyme activity retention rate is above 85%, and compared with the amount of free enzyme, the cost is reduced by more than 5 times.

[0060] The specific process implementation steps (enzymatic synthesis of bosine process roadmap) are as follows:

[0061]

[0062] The present invention provides a method for generating S-bosone by dual enzyme immobilization catalysis, comprising the following steps:

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

[0064] Insert the PYDH encoding gene between the NdeI and HindIII sites on the plasmid pET28a to obtain the recombinant plasmid pET28a-PYDH. Introduce the recombinant plasmid into the competent cell E.coliBL21 (DE3) to obtain the engineered bacteria E.coliBL21 (DE3) / pET28a-PYDH. Insert the formate dehydrogenase FDH encoding gene between the BamHI and XhoI sites on the plasmid pET28a to obtain the recombinant plasmid pET28a-FDH. Introduce the recombinant plasmids into the competent cells E.coliBL21 (DE3) to obtain the engineered bacteria E.coliBL21 (DE3) / pET28a-FDH. The above-mentioned engineered bacteria are extracted and sequenced 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 kanamycin at a final concentration of 2 to 200 μg / mL, cultured overnight at 20 to 37°C and 150 to 300 rpm, and then transferred to LB liquid medium containing 2 to 200 μg / mL kanamycin at an inoculum concentration of 0.5 to 3% by volume, and cultured at 20 to 37°C and 150 to 300 rpm until the bacterial concentration OD 600 to 0.6-0.8, add IPTG with a final concentration of 0.1-0.3 mM to the culture, induce culture at 20-37°C for 15 hours, and obtain induced culture solution. Centrifuge the induced culture solution at 4°C and 4000-10000 rpm for 5-15 minutes, and discard the supernatant; then resuspend the bacteria with 50 mM PBS pH7.5 buffer, centrifuge at 4°C and 4000-10000 rpm for 5-15 minutes, discard the supernatant, collect the wet bacteria as biocatalyst, and store at -20°C for later use.

[0066] Take 3-5 mL of the induced culture medium, centrifuge at 4000-12000 rpm for 5-15 min, discard the supernatant, keep the bacteria, and resuspend them with 1 mL of 20-200 mM PBS pH 7.0-8.5. Ultrasonicate and take 100 μL of the broken bacterial solution for centrifugation, take the supernatant and precipitate respectively, and resuspend the precipitate with 100 μL of 20-200 mM PBS pH 7.0-8.5. Pipette 40μL of uncentrifuged whole bacterial solution, add 4μL 6x Protein Loading Buffer, mix well, and boil for 5-15 minutes. This is the whole bacterial protein; Pipette 40μL of supernatant, add 4μL 6x Protein Loading Buffer, mix well, and boil for 5-15 minutes. This is the supernatant protein; Pipette 40μL of resuspended precipitate, add 4μL 6x Protein Loading Buffer, mix well, and boil for 5-15 minutes. This is the precipitated protein; Finally, take 3-20μL of protein solution and 3-20μL of protein Marker for running on gel.

[0067] 2. Preparation of Fermentation Enzyme Broth of Carbonyl Reductase (PYDH) and Formate Dehydrogenase (FDH)

[0068] Accurately weigh wet bacteria of carbonyl reductase PYDH and sodium formate dehydrogenase FDH, mix the bacteria mud with PBS buffer system at a ratio of 1:19 (m:m), after the bacteria mud and PBS buffer system are evenly mixed, homogenize the bacteria mud liquid with a homogenizer, control the homogenization pressure at 700-900 bar, and homogenize 2-5 times to obtain fermented PYDH enzyme liquid and FDH enzyme liquid;

[0069] PBS is preferably prepared with 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 dual enzyme co-immobilized enzyme

[0071] The homogenized PYDH enzyme solution and FDH enzyme solution are 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, and the protein content of the crude enzyme solution is calculated by measuring the standard curve of BSA (bovine serum albumin) by ultraviolet spectrophotometry; epoxy resin is added to the obtained crude enzyme solution of the dual enzyme, and the solution is immobilized in a constant temperature water bath shaker, and the immobilized resin is finally filtered, and the resin is washed with a buffer system until the filtrate is protein-free, and the resin on the filter cake is collected and dried at 20 to 25°C for 0.5 to 3 hours to obtain a co-immobilized enzyme containing PYDH and FDH;

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

[0073] 4. Determination of enzyme activity of dual-enzyme co-immobilized enzyme and co-enzyme solution

[0074] An activity test system containing sodium formate, NAD, and 1-C-(β-D-xylopyranosyl)-acetone is prepared, 2 to 10 mL of the activity test solution is taken, the reaction solution is preheated at 25 to 45° C. for 2 to 30 min, then about 0.01 to 0.5 g of the co-immobilized enzyme prepared above and about 0.1 to 0.5 mL of the mixed enzyme solution are added to the activity test system, the activity test time is 2 to 60 min, 10 to 100 μL of the activity test solution is taken, quenched with 90% acetonitrile, and the upper organic phase is taken for liquid chromatography analysis after centrifugation. The enzyme activity of the co-immobilized enzyme is calculated based on the amount of bosogen generated. The enzyme activity is defined as the amount of resin required to generate 1 μmol of product per minute, which is defined as 1U. Finally, the enzyme activity of the co-immobilized enzyme is expressed by specific enzyme activity U / g, and the enzyme activity of the mixed enzyme solution is expressed by volume enzyme activity U / mL.

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

[0076] 5. Double enzyme co-immobilized enzyme catalysis reaction to produce high-purity S-Bosone

[0077] A reaction solution containing sodium formate, NAD, and 1-C-(β-D-xylopyranosyl)-acetone is prepared, and the co-immobilized enzyme prepared above is added to the reaction solution, and a mechanical stirring paddle is used for stirring, and the reaction temperature and pH are controlled to catalyze the generation of high-purity S-bosone;

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

[0079] 6. Recovery of Co-immobilized Enzyme

[0080] After the reaction is completed, the reaction solution is directly filtered, the co-immobilized enzyme on the filter cake is collected, and it is rinsed with 0.1-2M buffer, and the rinsing volume is 1-20 times the volume of the immobilized enzyme; the washed co-immobilized enzyme is collected, the specific enzyme activity is determined, and the enzyme activity retention rate is calculated, so that it can be used again for the preparation of the bosera reaction, thereby realizing the recycling of the immobilized dual enzyme resin.

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

[0082] Carbonyl reductase PYDH encoding gene Uniprot#:Q1J2J0:

[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 (synthetic) (SEQ ID NO.3):

[0091]

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

[0093] MAKVLCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPGQLLGSVSGELGLRKYLESNGHTLVVTSDKDGPDSVFERELVDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRNVTVAEVTYCNSISVAEHVVMMILSLVRNYLPSHEWARKGGWNIADCVSHAYDLEAMHVGTVAA GRIGLAVLRRLAPFDVHLHYTDRHRLPESVEKELNLTWHATREDMYPVCDVVTLNCPLHPETEHMINDETLKLFKRGAYIVNTARGKLCDRDAVARALESGRLAGYAGDVWFPQPAPKDHPWRTMPYNGMTPHISGTTLTAQARYAAGTREILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAKFKKAV

[0094] Unless otherwise specified, the composite enzyme, co-immobilized enzyme and raw materials and reagents used in their application provided by the present invention can be purchased from the market.

[0095] The present invention will be further described below in conjunction with embodiments:

[0096] Example 1 Preparation of wet bacterial cells of PYDH and FDH engineered bacteria and crude enzyme solution

[0097] (1) Construction of genetically engineered bacteria

[0098] Genetic engineering bacteria expressing PYDH: Insert the PYDH coding gene (SEQ ID NO.1) between the NdeI and Hind III sites on the plasmid pET28a to obtain the recombinant plasmid pET28a-PYDH. Introduce the recombinant plasmid into competent cells E.coliBL21(DE3) to obtain the engineered bacteria E.coli BL21(DE3) / pET28a-PYDH.

[0099] Genetic engineering bacteria expressing FDH: The formate dehydrogenase FDH encoding gene (SEQ ID NO.3) was inserted between the BamH I and Xho I sites on the plasmid pET28a to obtain the recombinant plasmid pET28a-FDH. The recombinant plasmids were introduced into competent cells E. coli BL21 (DE3) to obtain the engineered bacteria E. coli BL21 (DE3) / pET28a-FDH. The plasmids extracted from the above-mentioned engineered bacteria were sequenced to show that each gene was correctly inserted.

[0100] (2) Preparation of wet cells of engineered bacteria

[0101] The wet cells were 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 culture medium containing kanamycin at a final concentration of 50 μg / mL, respectively, and cultured at 37 ° C, 220 rpm overnight, and then transferred to LB liquid culture medium containing 50 μg / mL kanamycin at an inoculum concentration of 1% by volume, and cultured at 37 ° C, 220 rpm until the bacterial concentration OD600 reached 0.6-0.8, IPTG was added to the culture at a final concentration of 0.3 mM, and induced culture was carried out at 35 ° C for 15 hours to obtain an induced culture solution.

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

[0104] ①SDS-PAGE electrophoresis:

[0105] Take 5mL of the induced culture solution, centrifuge at 12000rpm for 5min, discard the supernatant, keep the bacteria, resuspend with 20mM PBS pH7.01mL, ultrasonically disrupt, take 100μL of the disrupted bacterial solution and centrifuge, take the supernatant and precipitate respectively, and resuspend the precipitate with 100μL of 20mM PBS pH7.0 to obtain a resuspended precipitate. Take 40μL of the uncentrifuged whole bacterial solution, add 4μL 6x Protein Loading Buffer, mix well, and boil for 10min. This is the whole bacterial protein, recorded as W; take 40μL of the supernatant, add 4μL 6x Protein Loading Buffer, mix well, and boil for 10min. This is the supernatant protein, recorded as S; take 40μL of the resuspended precipitate, add 4μL 6x Protein Loading Buffer, mix well, and boil for 10min. This is the precipitated protein, recorded as P;

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

[0107] like Figure 1 , Figure 2 As shown, SDS-PAGE detection showed that the PYDH encoding gene and the FDH encoding gene were successfully expressed in the genetically engineered bacteria with good expression levels. From the expression levels of supernatant protein and precipitated protein (roughly evaluated by the depth of the band), the solubility was about 90%.

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

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

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

[0111] Example 2 Preparation of co-immobilized enzyme

[0112] The PYDH enzyme solution and the FDH enzyme solution prepared in Example 1 were mixed at an enzyme amount ratio of 1U:50U and an enzyme solution volume ratio of 2.5:10 to obtain a crude enzyme solution containing PYDH and FDH enzymes with a total protein content of 2500mg; 25g of epoxy resin ES-103B was added to the obtained crude enzyme solution of the dual enzyme, and the mass ratio of the crude enzyme solution of the dual enzyme to the epoxy resin was 10:1. The enzymes were immobilized in a constant temperature water bath shaker at 35°C with a shaker speed of 150rpm for 5h. Finally, the immobilized resin was filtered and the resin was washed with a 100mM PBS pH7.0 buffer system until the filtrate was protein-free. The resin on the filter cake was collected and dried at 25°C for 0.5h to obtain a co-immobilized enzyme containing PYDH and FDH enzymes.

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

[0114] An activity test system containing sodium formate, NAD, and 1-C-(β-D-xylopyranosyl)-acetone was prepared, 10 mL of the activity test solution was taken, the reaction solution was preheated at 35°C for 5 min, and then about 0.2 g of the co-immobilized enzyme prepared above was added to the activity test system. The activity test time was 25 min, 100 μL of the activity test solution was taken, quenched with 90% acetonitrile, and the upper organic phase was taken for liquid chromatography analysis after centrifugation. The enzyme activity of the co-immobilized enzyme was calculated based on the amount of boson generated. The enzyme activity was defined as the amount of resin required to generate 1 μmol of product per minute, which was defined as 1U. Finally, the enzyme activity of the co-immobilized enzyme was expressed by the specific enzyme activity U / g.

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

[0116] Example 3 Preparation of co-immobilized enzyme

[0117] The PYDH enzyme solution and the 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 prepare a crude enzyme solution containing PYDH and FDH enzymes with a total protein content of 12g; 80g of epoxy resin ES-103B was added to the prepared crude enzyme solution of the dual enzyme, at which time the mass ratio of the crude enzyme solution of the dual enzyme to the epoxy resin was 120:1, and immobilized in a constant temperature water bath shaker at 40°C with a shaker speed of 150rpm for 5h, and finally the immobilized resin was filtered, and the resin was washed with a 100mMPBSpH7.0 buffer system until the filtrate was protein-free, the resin on the filter cake was collected, and dried at 25°C 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 enzyme activity of the co-immobilized enzyme was calculated to be 1430.23 U / g. The method for assaying the enzyme activity of the co-immobilized enzyme is as follows:

[0119] An activity test system containing sodium formate, NAD, and 1-C-(β-D-xylopyranosyl)-acetone was prepared, 10 mL of the activity test solution was taken, the reaction solution was preheated at 35°C for 5 min, and then about 0.2 g of the co-immobilized enzyme prepared above was added to the activity test system. The activity test time was 25 min, 100 μL of the activity test solution was taken, quenched with 90% acetonitrile, and the upper organic phase was taken for liquid chromatography analysis after centrifugation. The enzyme activity of the co-immobilized enzyme was calculated based on the amount of boson generated. The enzyme activity was defined as the amount of resin required to generate 1 μmol of product per minute, which was defined as 1U. Finally, the enzyme activity of the co-immobilized enzyme was expressed by the specific enzyme activity U / g.

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

[0121] Example 4 Preparation of co-immobilized enzyme

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

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

[0124] An activity test system containing sodium formate, NAD, and 1-C-(β-D-xylopyranosyl)-acetone was prepared, 10 mL of the activity test solution was taken, the reaction solution was preheated at 35°C for 5 min, and then about 0.2 g of the co-immobilized enzyme prepared above was added to the activity test system. The activity test time was 25 min, 100 μL of the activity test solution was taken, quenched with 90% acetonitrile, and the upper organic phase was taken for liquid chromatography analysis after centrifugation. The enzyme activity of the co-immobilized enzyme was calculated based on the amount of boson generated. The enzyme activity was defined as the amount of resin required to generate 1 μmol of product per minute, which was defined as 1U. Finally, the enzyme activity of the co-immobilized enzyme was expressed by the specific enzyme activity U / g.

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

[0126] Example 5 One-step synthesis of S-bosone by co-immobilized enzyme catalysis from 1-C-(β-D-xylopyranosyl)-acetone

[0127] Prepare a 500 mL volume of reaction solution containing sodium formate, NAD, and 1-C-(β-D-xylopyranosyl)-acetone, wherein the final concentration of the substrate 1-C-(β-D-xylopyranosyl)-acetone is 1 M, the final concentration of sodium formate is 2 M, and the final concentration of NAD is 0.2 mM. Add 10.16 g of the co-immobilized enzyme in Example 2, the enzyme amount is 25 U / mL, the reaction temperature is 38°C, use a mechanical stirring paddle for stirring, use 2M HCl to control the reaction pH to 6.5, and the reaction is completed after 6 hours. Take 50 μL of the reaction solution in 1.95 mL of 90% acetonitrile, shake in a vortex mixer (vortex mixer speed is 8000 rpm) for 1 min until mixed evenly, take the upper organic phase after centrifugation for liquid chromatography analysis, the boson yield is 190 g / L, the final conversion rate is 99.58%, and the ee value is 100%. The liquid chromatogram is as follows Figure 4 and Figure 5 As shown, the retention times of 1-C-(β-D-xylopyranosyl)-acetone and S-bosyl are 5.773 min and 7.302 min, respectively.

[0128] Example 6 One-step synthesis of S-bosone by co-immobilized enzyme catalysis from 1-C-(β-D-xylopyranosyl)-acetone

[0129] A 1000 mL volume of reaction solution containing sodium formate, NAD, and 1-C-(β-D-xylopyranosyl)-acetone was prepared, wherein the final concentration of the substrate 1-C-(β-D-xylopyranosyl)-acetone was 2 M, the final concentration of sodium formate was 2 M, and the final concentration of NAD was 0.1 mM. 13.98 g of the immobilized enzyme in Example 3 was added, the enzyme addition amount was 20 U / mL, the reaction temperature was 38° C., a mechanical stirring paddle was used for stirring, and the reaction pH was controlled to 6.5 with 2M HCl. The reaction was terminated after 6 h. 50 μL of the reaction solution was taken in 1.95 mL of 90% acetonitrile, and shaken in a vortex mixer (vortex mixer speed was 8000 rpm) for 1 min until mixed evenly. After centrifugation, the upper organic phase was taken for liquid chromatography analysis, as shown in FIG. Figure 6 As shown, the bosone yield was 380 g / L and the final conversion rate was 99.46%.

[0130] Example 7 One-step synthesis of S-bosone by co-immobilized enzyme catalysis from 1-C-(β-D-xylopyranosyl)-acetone

[0131] A reaction solution containing sodium formate, NAD, and 1-C-(β-D-xylopyranosyl)-acetone with a volume of 1000 mL was prepared, wherein the final concentration of the substrate 1-C-(β-D-xylopyranosyl)-acetone was 2 M, the final concentration of sodium formate was 3.8 M, and the final concentration of NAD was 0.1 mM. 64.91 g of the immobilized enzyme in Example 4 was added, the enzyme addition amount was 100 U / mL, the reaction temperature was 38° C., a mechanical stirring paddle was used for stirring, and the reaction pH was controlled to 6.5 with 2M HCl. The reaction was terminated after 6 h. 50 μL of the reaction solution was taken in 1.95 mL of 90% acetonitrile, and the mixture was shaken in a vortex mixer (the speed of the vortex mixer was 8000 rpm) for 1 min until it was evenly mixed. After centrifugation, the upper organic phase was taken for liquid chromatography analysis, as shown in FIG. Figure 7 As shown, the bosone yield was 380 g / L and the final conversion rate was 100%.

[0132] Example 8 Recovery of co-immobilized enzyme

[0133] After the reaction of Example 7 was completed, the reaction solution was directly filtered, the co-immobilized enzyme on the filter cake was collected, and it was rinsed with 100 mM PBS pH 7.5, and the rinsing volume was 10 times the volume of the immobilized enzyme; the washed co-immobilized enzyme was collected, and the specific enzyme activity was determined, with the initial co-immobilized enzyme activity as 100%, and the relative enzyme activity of each batch after use was calculated as follows:

[0134] Enzyme activity recovery rate (%) = (resin specific enzyme activity after N cycles / initial immobilized enzyme activity) * 100

[0135] Record the relative enzyme activity changes of the co-immobilized enzyme after each cycle, as follows Figure 8 As shown, it can be seen that even after 15 times of use, the enzyme activity retention rate is still above 85%, indicating that the co-immobilized enzyme has relatively good enzyme stability.

[0136] Comparative Example 1 Comparison of the reaction between liquid enzyme and immobilized enzyme

[0137] According to the implementation of Example 5, a reaction solution containing sodium formate, NAD, and 1-C-(β-D-xylopyranosyl)-acetone with a volume of 500 mL was prepared, wherein the final concentration of the substrate 1-C-(β-D-xylopyranosyl)-acetone was 1 M, the final concentration of sodium formate was 2 M, and the final concentration of NAD was 0.2 mM. 10.16 g of the co-immobilized enzyme in Example 2 was added, the enzyme addition amount was 25 U / mL, the reaction temperature was 38° C., a mechanical stirring paddle was used for stirring, and the reaction pH was controlled to 6.5 with 2M HCl. The reaction was completed after 6 hours. 50 μL of the reaction solution was taken in 1.95 mL of 90% acetonitrile and shaken in a vortex mixer (the vortex mixer speed was 8000 rpm) for 1 min until mixed evenly. After centrifugation, the upper organic phase was taken for liquid chromatography analysis. The initial conversion rate was 99.58%. After the reaction was completed, the immobilized enzyme was recovered in the manner of Example 8, and the recovery was continued three times for addition reaction. The control liquid enzyme used a mixed enzyme solution of PYDH and FDH, and its enzyme activity was measured according to the activity measurement system provided in Example 2. 0.1 mL of the mixed enzyme solution was added to the activity measurement system, and the volume enzyme activity of the obtained mixed solution was 1230 U / mL. The reaction system of the liquid enzyme was also added according to the enzyme addition amount of 25 U / mL. At this time, 10.17 mL of the mixed liquid enzyme was added. After the reaction was completed, the hourly reaction effects of the liquid enzyme and the co-immobilized enzyme were compared, as follows Fig. 9 As shown, it can be seen that with the same amount of enzyme added, the reaction rate of the immobilized enzyme is better than that of the liquid enzyme, and the stability may be improved. Since the mixed liquid enzyme is very soluble, no precipitation is produced after the reaction, and the recovery is not ideal, so no recovery treatment is performed.

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

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

[0140] The obtained co-immobilized enzyme was subjected to enzyme activity assay, and 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] An activity test system containing sodium formate, NAD, and 1-C-(β-D-xylopyranosyl)-acetone was prepared, 10 mL of the activity test solution was taken, the reaction solution was preheated at 35°C for 5 min, and then about 0.2 g of the co-immobilized enzyme prepared above was added to the activity test system. The activity test time was 25 min, 100 μL of the activity test solution was taken, quenched with 90% acetonitrile, and the upper organic phase was taken for liquid chromatography analysis after centrifugation. The enzyme activity of the co-immobilized enzyme was calculated based on the amount of boson generated. The enzyme activity was defined as the amount of resin required to generate 1 μmol of product per minute, which was defined as 1U. Finally, the enzyme activity of the co-immobilized enzyme was expressed by the specific enzyme activity U / g.

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

[0144] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A complex enzyme, characterized in that It includes carbonyl reductase and formate dehydrogenase; the enzyme amount ratio of carbonyl reductase to formate dehydrogenase includes (1-3U):(5-50U).

2. A co-immobilized enzyme, characterized in that: Comprising the complex enzyme as claimed in claim 1.

3. The method for preparing the co-immobilized enzyme according to claim 2, characterized in that: include: S1. preparing a mixed solution of carbonyl reductase and formate dehydrogenase; S2. Take the mixed solution and epoxy resin described in S1, immobilize, filter, wash, and dry to obtain the co-immobilized enzyme.

4. The preparation method according to claim 3, characterized in that: The enzyme amount ratio of the carbonyl reductase to the formate dehydrogenase is (1-3U):(5-50U).

5. The preparation method according to claim 3 or 4, characterized in that: In terms of mg / g, the mass ratio of the total protein content in the blended solution to the epoxy resin is (100-300):1; the immobilization temperature is 25-40°C; the immobilization time is 5-16h; and the immobilization rotation speed is 50-250rpm.

6. Application of any of the following in the preparation of boson: (I), the complex enzyme as claimed in claim 1; and / or (II), the co-immobilized enzyme as claimed in claim 2; and / or (III) The co-immobilized enzyme obtained by the preparation method according to any one of claims 3 to 5.

7. A method for preparing boson, characterized in that: Sodium formate, NAD and 1-C-(β-D-xylopyranosyl)-acetone are added to the composite enzyme according to claim 1, the co-immobilized enzyme according to claim 2 and / or the co-immobilized enzyme prepared by the preparation method according to any one of claims 3 to 5, and an enzyme catalyzed reaction is carried out to obtain bosogen.

8. The preparation method according to claim 7, characterized in that: The molar ratio of sodium formate, NAD and 1-C-(β-D-xylopyranosyl)-acetone includes (1-2 mol): (2-3.8 mol): (0.1-0.2 mmol); The added amount of the co-immobilized enzyme ranges from 20 to 100 U / mL.

9. The preparation method according to claim 7 or 8, characterized in that: The temperature of the enzyme catalyzed reaction is 25-45° C.; the pH value of the enzyme catalyzed reaction is 6.0-8.

0.

10. The preparation method according to any one of claims 7 to 9, characterized in that: The Boson includes S-Boson.

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