Application of oxidase mutant of streptomyces coelicolor in synthesis of D-mannose

Abstract

The invention belongs to the technical field of biology, and relates to application of an oxidase mutant of streptomyces coelicolor in synthesis of D-mannose. The invention provides a method for synthesizing D-mannose by taking mannitol as a raw material through enzyme catalysis, and the method specifically comprises the following steps: taking mannitol as a substrate, and generating D-mannose through a mutant catalytic reaction of oxidase, and the molar conversion rate reaches more than 95%.

Description

[0001] This application is a divisional application of Chinese invention patent CN115896206A. The application number of the parent case is 202211588443.8, the application title is "A method for synthesizing D-mannose", the applicant is the Institute of Biology, Hebei Academy of Sciences, and the filing date of the parent case is December 12, 2022. Technical Field

[0002] The present invention belongs to the field of biotechnology, relates to a method for synthesizing D-mannose, and particularly relates to a method for synthesizing D-mannose by using biocatalysis. Background Art

[0003] D-Mannose is a monosaccharide that is widely distributed in nature. A large amount of polysaccharides containing D-mannose units and free D-mannose are contained in plants, plant cell walls, and fruit peels. D-Mannose is widely used in the fields of medicine, food, chemical products, etc. In medicine, it can be used as a drug to prevent urinary tract infections, as a sugar nutrient to deal with diseases such as diabetes and obesity, and has anti-inflammatory and immunomodulatory functions; in the food industry, it is often used as a sweetening additive; in the aquaculture industry, it can prevent broiler chickens from being infected by Salmonella; in chemical synthesis, it can be used to synthesize derivatives such as trifluoromannose and L-ribose.

[0004] At present, the preparation techniques of D-mannose mainly include extraction method, chemical synthesis method, and biological method. The extraction method is a commonly used method for preparing D-mannose. In 2015, Pei Jun et al. invented a method for extracting high-purity D-mannose from coffee grounds (CN105087712A). In this method, the product is easy to separate during the preparation process, the yield reaches more than 60%, and the purity reaches more than 98%. The extraction method for preparing D-mannose has a relatively low cost, but has disadvantages such as the need for a large amount of raw materials and being affected by regions and seasons in production and preparation. The chemical synthesis method mainly uses D-glucose as a substrate and molybdic acid as a catalyst to synthesize D-mannose under high-temperature acidic conditions, and the yield is 30%-36% (Zhao Guanghui, Wang Guanbin, Li Junping, et al. Orthogonal test analysis of catalytic preparation of mannose from glucose [J]. Contemporary Chemical Industry, 2005, 34(1): 39-41.). The yield of the chemical synthesis method for preparing D-mannose is not high, and the operation is cumbersome, which is only suitable for small-scale laboratory preparation.

[0005] The preparation of D-mannose by biological methods can be achieved through biological fermentation or biocatalysis. The biological fermentation method uses microorganisms to ferment polysaccharides or monosaccharides to obtain D-mannose, but the products are mostly mixtures composed of glucose, mannose, and galactose, with problems such as low D-mannose yield and low purity. The research on the synthesis of D-mannose by biocatalysis mainly involves the conversion of fructose and other substances into D-mannose by isomerase. In 2002, Huang Jiawei et al. used D-lyxose isomerase to convert D-fructose into D-mannose, with a conversion rate of 19% (Huang Jiawei, Shi Yue, Zhang Wenli, et al. Study on the enzymatic properties of D-lyxose isomerase [J]. Journal of Food Science and Biotechnology, 2019, 38(1): 83-92.). In 2015, Jiang Bo et al. applied for a patent (CN201510195854.4) in which D-mannose isomerase was used to convert D-fructose into D-mannose. The reaction of synthesizing D-mannose from fructose catalyzed by isomerase is a reversible reaction, and the yield of D-mannose at the reaction equilibrium is low, and it is a mixture of fructose and D-mannose, which is not easy to separate and purify. Therefore, it is difficult to achieve the industrial production of D-mannose. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a method for synthesizing D-mannose, which uses biocatalysis to convert mannitol into D-mannose.

[0007] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0008] The present invention provides the application of an oxidase or a mutant of the oxidase in the enzymatic synthesis of D-mannose using mannitol as a substrate. The sources of the oxidase include: Streptomyces anthocyanicus, Streptomyces coelicolor, Streptomyces lividans, Streptomyces turgidiscabies, Streptomyces ipomoeae, Paenibacillus sp., Paenibacillus lautus, Cytobacillus firmus, or Dermacoccus sp.

[0009] Preferably, the amino acid sequence of the oxidase derived from Streptomyces anthocyanicus is as shown in SEQ ID No. 2;

[0010] The amino acid sequence of the oxidase derived from Streptomyces coelicolor is as shown in SEQ ID No. 4;

[0011] The amino acid sequence of the oxidase derived from Streptomyces lividans is shown in SEQ ID No. 6;

[0012] The amino acid sequence of the oxidase derived from Streptomyces scabies is shown in SEQ ID No. 8;

[0013] The amino acid sequence of the oxidase derived from Streptomyces ipomoeae is shown in SEQ ID No. 10;

[0014] The amino acid sequence of the oxidase derived from Paenibacillus sp. is shown in SEQ ID No. 12;

[0015] The amino acid sequence of the oxidase derived from Paenibacillus lautus is shown in SEQ ID No. 14;

[0016] The amino acid sequence of the oxidase derived from Bacillus firmus is shown in SEQ ID No. 16;

[0017] The amino acid sequence of the oxidase derived from Actinomyces exiguus is shown in SEQ ID No. 18.

[0018] Preferably, the oxidase further comprises a tag linked to the N-terminus and / or C-terminus.

[0019] Preferably, the mutant comprises the following point mutations on the amino acid sequence shown in SEQ ID No. 2: A425V;

[0020] The following point mutations are made on the amino acid sequence shown in SEQ ID No. 4: E295D;

[0021] The following point mutations are made on the amino acid sequence shown in SEQ ID No. 6: N398D.

[0022] The present invention also provides a method for enzymatically synthesizing D-mannose, comprising the following steps: using mannitol as a substrate and performing a catalytic reaction with an oxidase or a mutant of the oxidase, and the reaction product is D-mannose;

[0023] The sources of the oxidase include: Streptomyces anthocyanicus, Streptomyces coelicolor, Streptomyces lividans, Streptomyces turgidiscabies, Streptomyces ipomoeae, Paenibacillus sp., Paenibacillus lautus, Cytobacillus firmus or Dermacoccus sp.

[0024] Preferably, the oxidase or the mutant of the oxidase is added in the form of a crude enzyme solution, a freeze-dried powder of the crude enzyme solution, a pure enzyme or whole cells.

[0025] Preferably, the mannitol concentration is 0.1 mmol / L to 600 mmol / L, the rotation speed of the catalytic reaction is 220 to 300 rpm / min, the temperature of the catalytic reaction is 25 to 55 °C, and the time of the catalytic reaction is 12 to 48 h.

[0026] Preferably, when the oxidase is added in the form of a crude enzyme solution, a freeze-dried powder of the crude enzyme solution or a pure enzyme, the concentration of the oxidase in the reaction system is 0.5 g / L to 10 g / L;

[0027] When the oxidase is added in the form of whole cells, the wet weight of the whole cells is 40 g / L to 80 g / L.

[0028] Preferably, the catalytic reaction is carried out in a phosphate buffer solution with a concentration of 50 to 100 mM and a pH value of 6.5 to 8.5.

[0029] Preferably, when the oxidase is added in the form of a crude enzyme solution, a freeze-dried powder of the crude enzyme solution or a pure enzyme, the reaction system of the catalytic reaction contains catalase in addition to mannitol and the oxidase. Description of the Drawings

[0030] Figure 1 It is the reaction schematic diagram of the oxidase catalyzing mannitol to synthesize D-mannose;

[0031] Figure 2 It is the liquid chromatography detection peak diagram of mannitol and D-mannose standard products;

[0032] Figure 3 It is the liquid chromatography detection peak diagram of the reaction solution. Detailed Embodiments

[0033] A method for catalytic synthesis of chiral D-mannose using bioenzymes may include the following steps (for the reaction principle, see Figure 1 ): Using mannitol as a substrate, it undergoes a catalytic reaction by oxidase and catalase to generate D-mannose;

[0034] Furthermore, the oxidase can be derived from any one of the following microorganisms: Streptomyces anthocyanicus, Streptomyces coelicolor, Streptomyces lividans, Streptomyces turgidiscabies, Streptomyces ipomoeae, Paenibacillus sp., Paenibacillus lautus, Cytobacillus firmus, or Dermacoccus sp.

[0035] Even further, the oxidase can specifically be any one of the following (a1)-(a15):

[0036] (a1) The amino acid sequence of the oxidase derived from Streptomyces anthocyanicus is shown in SEQ ID No. 2;

[0037] (a2) The amino acid sequence of the oxidase derived from Streptomyces coelicolor is shown in SEQ ID No. 4;

[0038] (a3) The amino acid sequence of the oxidase derived from Streptomyces lividans is shown in SEQ ID No. 6;

[0039] (a4) The amino acid sequence of the oxidase derived from Streptomyces turgidiscabies is shown in SEQ ID No. 8;

[0040] (a5) The amino acid sequence of the oxidase derived from Streptomyces ipomoeae is shown in SEQ ID No. 10;

[0041] (a6) The amino acid sequence of the oxidase derived from Paenibacillus sp. is shown in SEQ ID No. 12;

[0042] (a7) The amino acid sequence of the oxidase derived from Paenibacillus lautus is shown in SEQ ID No. 14;

[0043] (a8) The amino acid sequence of the oxidase derived from Cytobacillus firmus is shown in SEQ ID No. 16;

[0044] (a9) The amino acid sequence of the oxidase derived from symbiotic rare actinomycetes is as shown in SEQ ID No. 18.

[0045] (a10) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of the protein defined in any one of (1)-(9);

[0046] Furthermore, the mutant of the oxidase can specifically be the following (b1)-(b4):

[0047] (b1) Compared with the oxidase derived from Streptomyces lividans shown in SEQ ID No. 2, the following mutations exist: A425V;

[0048] (b2) The following point mutation is performed on the amino acid sequence shown in SEQ ID No. 4: E295D;

[0049] (b3) The following point mutation is performed on the amino acid sequence shown in SEQ ID No. 6: N398D;

[0050] (b4) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of the protein defined in any one of (b1) to (b3).

[0051] In the method, the oxidase catalyzes in the form of crude enzyme solution, freeze-dried powder of crude enzyme solution, pure enzyme or whole cells;

[0052] Furthermore, the crude enzyme solution, freeze-dried powder of crude enzyme solution and pure enzyme are all prepared by the following method: expressing the oxidase in a host cell to obtain recombinant cells; lysing the recombinant cells to obtain the crude enzyme solution, freeze-dried powder of crude enzyme solution or pure enzyme;

[0053] Furthermore, the whole cells are all prepared by the following method: expressing the oxidase in a host cell, and the obtained recombinant cells are the whole cells;

[0054] Still further, the recombinant cells are obtained by the following method: introducing a nucleic acid molecule capable of expressing the oxidase into the host cell, and obtaining the recombinant cells expressing the oxidase after induction culture;

[0055] Even further, the "nucleic acid molecule capable of expressing the oxidase" is introduced into the host cell in the form of a recombinant vector; the recombinant vector is a bacterial plasmid carrying the coding gene of the oxidase (such as an expression vector based on the T7 promoter expressed in bacteria, specifically such as pET-28a, etc.), phage, yeast plasmid or retroviral packaging plasmid; and / or the host cell is a prokaryotic cell or a lower eukaryotic cell;

[0056] In one embodiment of the present invention, the recombinant vector is specifically a recombinant plasmid obtained by replacing the small fragment between the restriction enzyme sites BamHI and Xho I of the pET-28a vector with the coding gene of the oxidase.

[0057] Furthermore, the host cell can be a prokaryotic cell or a lower eukaryotic cell.

[0058] Still further, the prokaryotic cell can specifically be a bacterium. The lower eukaryotic cell can specifically be a yeast cell.

[0059] In one embodiment of the present invention, the host cell is specifically Escherichia coli, and more specifically E. coli BL21(DE3). Correspondingly, for the induction culture, IPTG is added to the culture system to a final concentration of 0.1 - 0.5 mM (specifically 0.2 mM), and induction culture is carried out at 20 - 30 °C for 4 - 8 h (specifically 6 h).

[0060] The sequence of the coding gene of the oxidase derived from Streptomyces lividans is SEQ ID No.1 or a fusion sequence obtained by connecting a tag coding sequence at its 5'-end and / or 3'-end or a random and / or site-directed mutagenesis sequence that retains the function and encodes the same protein;

[0061] The sequence of the coding gene of the oxidase derived from Streptomyces coelicolor is SEQ ID No.3 or a fusion sequence obtained by connecting a tag coding sequence at its 5'-end and / or 3'-end or a random and / or site-directed mutagenesis sequence that retains the function and encodes the same protein;

[0062] The sequence of the coding gene of the oxidase derived from Streptomyces lividans is SEQ ID No.5 or a fusion sequence obtained by connecting a tag coding sequence at its 5'-end and / or 3'-end or a random and / or site-directed mutagenesis sequence that retains the function and encodes the same protein;

[0063] The sequence of the coding gene of the oxidase derived from Streptomyces scabies is SEQ ID No.7 or a fusion sequence obtained by connecting a tag coding sequence at its 5'-end and / or 3'-end or a random and / or site-directed mutagenesis sequence that retains the function and encodes the same protein;

[0064] The sequence of the coding gene of the oxidase derived from Streptomyces ipomoeae is SEQ ID No.9 or a fusion sequence obtained by connecting a tag coding sequence at its 5'-end and / or 3'-end or a random and / or site-directed mutagenesis sequence that retains the function and encodes the same protein;

[0065] The sequence of the encoding gene of the oxidase derived from Paenibacillus is SEQ ID No. 11 or a fusion sequence obtained by connecting tag encoding sequences to its 5'-end and / or 3'-end, or a random and / or site-directed mutagenesis sequence that retains its function and encodes the same protein;

[0066] The sequence of the encoding gene of the oxidase derived from Paenibacillus lautus is SEQ ID No. 13 or a fusion sequence obtained by connecting tag encoding sequences to its 5'-end and / or 3'-end, or a random and / or site-directed mutagenesis sequence that retains its function and encodes the same protein;

[0067] The sequence of the encoding gene of the oxidase derived from Bacillus firmus is SEQ ID No. 15 or a fusion sequence obtained by connecting tag encoding sequences to its 5'-end and / or 3'-end, or a random and / or site-directed mutagenesis sequence that retains its function and encodes the same protein;

[0068] The sequence of the encoding gene of the oxidase derived from Actinomycetospora symbiosis is SEQ ID No. 17 or a fusion sequence obtained by connecting tag encoding sequences to its 5'-end and / or 3'-end, or a random and / or site-directed mutagenesis sequence that retains its function and encodes the same protein;

[0069] The sequence of the encoding gene of the mutant of the oxidase derived from Aspergillus welwitschiae is any one of the following (c1)-(c3): (c1) compared with SEQ ID No. 1, there is or only is the following mutation: C1274T; (c2) a fusion sequence obtained by connecting tag encoding sequences to the 5'-end and / or 3'-end of the sequence defined in (c1); (c3) a random and / or site-directed mutagenesis sequence that retains its function and encodes the same protein compared with the sequence defined in (c1) or (c2);

[0070] The sequence of the encoding gene of the mutant of the oxidase derived from Thermoanaerobacter is any one of the following (d1)-(d3): (d1) compared with SEQ ID No. 3, there is or only is the following mutation: G885C; (d2) a fusion sequence obtained by connecting tag encoding sequences to the 5'-end and / or 3'-end of the sequence defined in (d1); (d3) a random and / or site-directed mutagenesis sequence that retains its function and encodes the same protein compared with the sequence defined in (d1) or (d2);

[0071] The coding gene sequence of the mutant of the oxidase derived from Thermoanaerobacter is any one of the following (e1)-(e3): (e1) Compared with SEQ ID No.5, there is or only is the following mutation: A1192G; (e2) A fusion sequence obtained by connecting a tag coding sequence to the 5' end and / or 3' end of the sequence defined in (e1); (e3) A random or / and site-directed mutagenesis sequence that has the same function as and encodes the same protein as the sequence defined in (e1) or (e2).

[0072] In the catalytic reaction, the temperature of the catalytic reaction can be 20-55°C, such as 25-30°C, specifically 28°C. The time of the catalytic reaction can be 12-24h, such as 15h.

[0073] When the oxidase catalyzes in the form of crude enzyme solution, freeze-dried powder of crude enzyme solution or pure enzyme, the catalytic reaction can be carried out in a phosphate buffer solution with a concentration of 50-100 mM and a pH value of 6.5-8.0, specifically: a phosphate buffer solution with a concentration of 50 mM and a pH value of 7.2; when the oxidase catalyzes in the form of whole cells, the catalytic reaction can be carried out in a phosphate buffer solution with a concentration of 50-100 mM and a pH value of 7.5-8.5, specifically: a phosphate buffer solution with a concentration of 50 mM and a pH value of 7.2.

[0074] When the oxidase catalyzes in the form of crude enzyme solution, freeze-dried powder of crude enzyme solution or pure enzyme, the concentration of the oxidase in its respective reaction system can be 0.5 g / L-10 g / L, such as 10 g / L. When the oxidase catalyzes in the form of whole cells, the concentration of the whole cells in the reaction system is 80 g / L (the wet weight of the whole cells contained in each liter of the reaction system is 80 g).

[0075] In the present invention, when the oxidase catalyzes in the form of crude enzyme solution, freeze-dried powder of crude enzyme solution or pure enzyme, the concentration of the catalase in its respective reaction system can be 0.1-5 g (specifically 5 g).

[0076] The present invention also provides an enzyme and its related products.

[0077] The enzyme provided by the present invention is the oxidase described above.

[0078] The related products can be nucleic acid molecules capable of expressing each enzyme in the enzyme system, or expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing the nucleic acid molecules.

[0079] The application of the enzyme or the related products in the synthesis of D-mannose also belongs to the protection scope of the present invention.

[0080] The following is a detailed description of a method for synthesizing D-mannose provided by the present invention in combination with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0081] Example 1 Preparation of engineered bacteria of oxidase or its mutants

[0082] The coding genes of oxidase were respectively synthesized by total gene synthesis (codon optimization was carried out with Escherichia coli as the host as needed), and the synthesized genes were ligated to the BamHI and Xho I sites of the pET28a(+) expression vector. After correct verification by sequencing, the recombinant vectors were obtained. Related gene mutants were obtained by site-directed mutagenesis.

[0083] The above-mentioned recombinant expression vectors verified by sequencing were transformed into the microbial host of Escherichia coli BL21(DE3) to obtain the gene engineering strains of the present invention. The transformation method is a conventional transformation method in the art, and preferably the chemical transformation method.

[0084] The oxidase or its mutants involved in this example are shown in Table 1 in detail.

[0085] Table 1 Oxidase and its mutants

[0086]

[0087]

[0088] Example 2 Expression of oxidase or its mutants and preparation of crude enzyme

[0089] The recombinant expression vectors of oxidase or mutants constructed in Example 1 were transferred into Escherichia coli BL21(DE3) competent cells and cultured at 37°C for 12 - 16 h. After the transformants grew out, single transformants were inoculated into 5 mL of LB medium containing kanamycin (50 μg / mL) and cultured overnight (12 - 16 h) at 37°C and 200 rmp. Then, they were inoculated into LB medium at a ratio of 1% (volume percentage) and cultured at 37°C and 200 rmp until the OD 600 was about 0.6. IPTG with a final concentration of 0.2 mM was added, and the culture was induced at 25°C for 14 h. The cells were collected by centrifugation at 4°C and 10,000 rpm for 5 min, resuspended and washed once with 50 mM phosphate buffer with a pH value of 7.2, and then the cells were lysed by ultrasonic treatment and freeze-dried enzyme powder was prepared.

[0090] Example 3 Preparation of D-mannose using the crude enzyme of oxidase or mutants

[0091] In the reaction system, add phosphate buffer solution with a concentration of 50 mM and a pH value of 7.2, mannitol with a final concentration of 300 mM, catalase with a final concentration of 5 g / L, and freeze-dried powder or enzyme solution of oxidase with a final concentration of 10 g / L in sequence to form a reaction system. React this reaction system at 28 °C for 14 h.

[0092] After the reaction is completed, take 1 mL of the reaction solution and centrifuge it (12000 rpm, 5 min). Take the supernatant, dilute it 20 times, and then filter it through a 0.22 μm aqueous membrane for liquid chromatography detection. The liquid chromatography detection conditions are as follows: Bio-Rad Aminex HPX–87H, detector: differential refractive index detector (RID), mobile phase: 5 mM sulfuric acid, flow rate: 0.5 mL / min, column temperature: 40 °C, injection volume: 10 μL, standard product concentration: 10 mM (1 - 2 g / L).

[0093] The results of the liquid chromatography detection are as Figures 2 - 3 shown. Figure 2 It is the liquid chromatography peak diagram of mannitol and D-mannose standard products. The retention time of mannitol is about 11.5 min, and the retention time of D-mannose is about 11.1 min. Figure 3 It is the gas chromatography detection diagram of D-mannose prepared from crude enzyme of oxidase or mutant, and Table 2 shows the results of preparing D-mannose from crude enzyme of oxidase or its mutant.

[0094] Table 2 Results of preparing D-mannose from crude enzyme of oxidase or its mutant

[0095]

[0096]

[0097] Example 4 Preparation of D-mannose using whole cells of oxidase or mutant

[0098] In the reaction system, add phosphate buffer solution with a concentration of 50 mM and a pH value of 7.2, mannitol with a final concentration of 300 mM, catalase with a final concentration of 5 g / L, and whole cells of oxidase with a final concentration of 80 g / L in sequence to form a reaction system. React this reaction system at 28 °C for 14 h.

[0099] After the reaction is completed, take 1 mL of the reaction solution and centrifuge it (14000 rpm, 2 min). Take the supernatant, dilute it 20 times, and then filter it through a 0.22 μm aqueous membrane for liquid chromatography detection. The detection conditions are as follows: Bio-Rad Aminex HPX–87H, detector: differential refractive index detector (RID), mobile phase: 5 mM sulfuric acid, flow rate: 0.5 mL / min, column temperature: 40 °C, injection volume: 10 μL, standard product concentration: 10 mM (1 - 2 g / L). Table 3 shows the results of preparing D-mannose from whole cells of oxidase or its mutant.

[0100] Results of preparing D-mannose from oxidase or its mutants in whole cells in Table 3

[0101]

[0102]

[0103] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Use of a mutant of oxidase in the enzymatic synthesis of D-mannose using mannitol as a substrate, characterized in that, The oxidase is derived from Streptomyces coelicolor.

2. The application according to claim 1, wherein The amino acid sequence of the oxidase derived from Streptomyces coelicolor is shown in SEQ ID No.

4.

3. The application according to claim 1 or 2, characterized in that, The oxidase further includes a tag linked to the N-terminus and / or C-terminus.

4. The application according to claim 1, characterized in that, The mutant includes the following point mutation to the amino acid sequence shown in SEQ ID No. 4: E295D.

5. A method for enzymatically synthesizing D-mannose, characterized in that, Comprising the following steps: Using the mutant of the oxidase to catalyze a reaction with mannitol as the substrate, and the reaction product is D-mannose; The oxidase is derived from Streptomyces coelicolor.

6. The method according to claim 5, characterized in that, The mutant of the oxidase is added in the form of crude enzyme solution, freeze-dried powder of crude enzyme solution, pure enzyme or whole cells.

7. The method according to claim 5, wherein The concentration of the mannitol is 0.1 mmol / L to 600 mmol / L, the rotation speed of the catalytic reaction is 220 to 300 rpm / min, the temperature of the catalytic reaction is 25 to 55 °C, and the time of the catalytic reaction is 12 to 48 h.

8. The method according to claim 5 or 6, characterized in that When the mutant of the oxidase is added in the form of crude enzyme solution, freeze-dried powder of crude enzyme solution or pure enzyme, the concentration of the mutant of the oxidase in the reaction system is 0.5 g / L to 10 g / L; When the mutant of the oxidase is added in the form of whole cells, the wet weight of the whole cells is 40 g / L to 80 g / L.

9. The method according to claim 8, wherein The catalytic reaction is carried out in a phosphate buffer solution with a concentration of 50 to 100 mM and a pH value of 6.5 to 8.

5.

10. The synthesis method according to claim 5 or 6, characterized in that, When the mutant of the oxidase is added in the form of crude enzyme solution, freeze-dried powder of crude enzyme solution or pure enzyme, the reaction system of the catalytic reaction contains catalase in addition to mannitol and the oxidase.

Images