Application of oxidase mutant of streptomyces penicillium 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 penicillium 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, of which the application number of the parent case is 202211588443.8, the application name is A method for synthesizing D-mannose, the applicant is the Institute of Biology, Hebei Academy of Sciences, and the application date of the parent case is 2022.12.12. Technical Field

[0002] The invention belongs to the field of biotechnology and relates to a method for synthesizing D-mannose, in particular to a method for synthesizing D-mannose by utilizing biological enzyme catalysis. Background Art

[0003] D-Mannose is a monosaccharide widely distributed in nature. Plants, plant cell walls, and fruit peels contain large amounts of polysaccharides containing D-mannose units and free D-mannose. D-Mannose is widely used in medicine, food, and chemical products. In medicine, it can be used to prevent urinary tract infections and as a carbohydrate nutrient to treat conditions like diabetes and obesity, exhibiting anti-inflammatory and immunomodulatory properties. In the food industry, it is often used as a sweetener. In the livestock industry, it can protect broiler chickens from Salmonella infection. In chemical synthesis, it can be used to synthesize trifluoromannose and derivatives such as L-ribose.

[0004] At present, the main preparation technologies of D-mannose include extraction, chemical synthesis and biological methods. Extraction is a common method for preparing D-mannose. In 2015, Pei Jun et al. invented a method for extracting high-purity D-mannose from coffee grounds (CN105087712A). The product is easy to separate during the preparation process, with a yield of more than 60% and a purity of more than 98%. The extraction method is relatively low in cost for preparing D-mannose, but it has the disadvantages of requiring a large amount of raw materials and being affected by regional and seasonal factors in production. The chemical synthesis method mainly uses D-glucose as a substrate and molybdic acid as a catalyst to synthesize D-mannose under high temperature and acidic conditions, with a yield of 30%-36% (Zhao Guanghui, Wang Guanbin, Li Junping, et al. Orthogonal test analysis of glucose-catalyzed production of mannose [J]. Contemporary Chemical Engineering, 2005, 34(1):39-41.). The yield of D-mannose prepared by chemical synthesis is low and the operation is cumbersome, so it is only suitable for small-scale preparation in the laboratory.

[0005] D-mannose can be prepared biologically by biofermentation or biocatalysis. Biofermentation is the process of using microorganisms to ferment polysaccharides or monosaccharides to obtain D-mannose, but the product is mostly a mixture of glucose, mannose, and galactose, which has the problems of low D-mannose yield and low purity. Research on the synthesis of D-mannose by biocatalysis mainly uses isomerase to convert fructose and other substances into D-mannose. 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 and Biotechnology, 2019, 38(1):83-92.). In 2015, Jiang Bo et al. applied for a patent (CN201510195854.4) for converting D-fructose into D-mannose using D-mannose isomerase. The reaction of fructose to D-mannose catalyzed by isomerase is a reversible reaction. The D-mannose yield at reaction equilibrium is low and is a mixture of fructose and D-mannose, which is difficult to separate and purify. Therefore, it is difficult to achieve industrial production of D-mannose. Summary of the Invention

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

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides use of an oxidase or a mutant of the oxidase in enzymatic synthesis of D-mannose using mannitol as a substrate. The oxidase is derived from 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 cyanidis is shown in SEQ ID No. 2;

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

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

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

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

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

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

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

[0017] The amino acid sequence of the oxidase derived from a symbiotic rare actinomycete is shown in SEQ ID No. 18.

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

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

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

[0021] The following point mutation was made to the amino acid sequence shown in SEQ ID No. 6: N398D.

[0022] The present invention also provides a method for synthesizing D-mannose by enzymatic method, comprising the following steps: using mannitol as a substrate, utilizing an oxidase or a mutant of the oxidase to carry out a catalytic reaction, wherein 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 crude enzyme solution, lyophilized crude enzyme solution powder, 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 hours.

[0026] Preferably, when the oxidase is added in the form of crude enzyme solution, lyophilized crude enzyme solution or 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 of 6.5 to 8.5.

[0029] Preferably, when the oxidase is added in the form of crude enzyme solution, lyophilized crude enzyme solution powder or pure enzyme, the reaction system of the catalytic reaction contains catalase in addition to mannitol and the oxidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the reaction principle diagram of oxidase catalyzing the synthesis of D-mannose from mannitol;

[0031] Figure 2 This is the peak diagram of liquid chromatography detection of mannitol and D-mannose standards;

[0032] Figure 3 This is the peak diagram of the reaction solution detected by liquid chromatography. DETAILED DESCRIPTION

[0033] A method for synthesizing chiral D-mannose using enzyme catalysis may include the following steps (reaction principle see Figure 1 ): Using mannitol as substrate, D-mannose is generated through oxidase and catalase catalysis.

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

[0035] Furthermore, the oxidase may be any one of the following (a1)-(a15):

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

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

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

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

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

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

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

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

[0044] (a9) The amino acid sequence of the oxidase derived from a symbiotic rare actinomycete is 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 any of the proteins defined in (1) to (9);

[0046] Furthermore, the mutants of the oxidase may specifically be as follows (b1)-(b4):

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

[0048] (b2) The amino acid sequence shown in SEQ ID No. 4 was subjected to the following point mutations: E295D;

[0049] (b3) the amino acid sequence shown in SEQ ID No. 6 was subjected to the following point mutation: 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 is catalytically active in the form of a crude enzyme solution, a lyophilized crude enzyme solution powder, a pure enzyme, or whole cells;

[0052] Furthermore, the crude enzyme solution, crude enzyme solution lyophilized powder, and pure enzyme are all prepared according to a method comprising the following steps: expressing the oxidase in a host cell to obtain a recombinant cell; lysing the recombinant cell to obtain the crude enzyme solution, crude enzyme solution lyophilized powder, or pure enzyme;

[0053] Furthermore, the whole cells are prepared according to a method comprising the following steps: expressing the oxidase in a host cell, and the resulting recombinant cell is the whole cell;

[0054] Furthermore, the recombinant cell is prepared according to a method comprising the following steps: introducing a nucleic acid molecule capable of expressing the oxidase into the host cell, and obtaining the recombinant cell expressing the oxidase after induction culture;

[0055] Furthermore, 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 (such as an expression vector based on a T7 promoter expressed in bacteria, specifically pET-28a, etc.), a phage, a yeast plasmid or a retroviral packaging plasmid carrying the gene encoding the oxidase; 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 oxidase coding gene with a small fragment between the restriction sites BamHI and Xho I of the pET-28a vector.

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

[0058] Furthermore, the prokaryotic cell may be a bacterium. The lower eukaryotic cell may be a yeast cell.

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

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

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

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

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

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

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

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

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

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

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

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

[0071] The sequence of the gene encoding the mutant of the oxidase derived from the thermoanaerobic bacterium is any one of the following (e1)-(e3): (e1) compared with SEQ ID No. 5, the presence or absence of the following mutation: A1192G; (e2) a fusion sequence obtained by connecting a tag encoding 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 retains function 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 12-24 h, such as 15 h.

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

[0074] When the oxidase is catalyzed in the form of a crude enzyme solution, a lyophilized crude enzyme solution, or a pure enzyme, the concentration of the oxidase in each reaction system can be 0.5 g / L to 10 g / L, such as 10 g / L. When the oxidase is catalyzed 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 per liter of reaction system is 80 g).

[0075] In the present invention, when the oxidase is in the form of crude enzyme solution, lyophilized crude enzyme solution or pure enzyme to catalyze the reaction, the concentration of the catalase in its respective reaction system can be 0.1 to 5 g (specifically 5 g).

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

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

[0078] The related products may 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 product in synthesizing D-mannose also falls within the protection scope of the present invention.

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

[0081] Example 1 Preparation of Oxidase or Its Mutant Engineering Bacteria

[0082] The oxidase-encoding genes were fully synthesized (codon optimization was performed as needed using Escherichia coli as the host), ligated into the pET28a(+) expression vector with BamHI and XhoI, and sequenced to obtain recombinant vectors. Mutants of the related genes were obtained using site-directed mutagenesis.

[0083] The recombinant expression vector verified to be correct by sequencing is transformed into a microbial host of Escherichia coli BL21 (DE3) to obtain the genetically engineered strain of the present invention. The transformation method is a conventional transformation method in the art, preferably a chemical transformation method.

[0084] The oxidases or their mutants involved in this example are detailed in Table 1.

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

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

[0091] To the reaction system, add 50 mM phosphate buffer (pH 7.2), 300 mM mannitol, 5 g / L catalase, and 10 g / L oxidase lyophilized powder or enzyme solution. Incubate the reaction at 28°C for 14 hours.

[0092] After the reaction, centrifuge 1 mL of the reaction solution at 12,000 rpm for 5 minutes. The supernatant was diluted 20-fold and passed through a 0.22 μm aqueous membrane for liquid chromatography. Liquid chromatography conditions were: Bio-Rad Aminex HPX-87H, 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 concentration: 10 mM (1-2 g / L).

[0093] Liquid chromatography test results Figures 2-3 shown. Figure 2 This is the peak diagram of liquid chromatography detection of mannitol and D-mannose standards. The retention time of mannitol is around 11.5 minutes, and the retention time of D-mannose is around 11.1 minutes. Figure 3 is a gas phase detection diagram of the preparation of D-mannose by oxidase or its mutant crude enzyme, and Table 2 is the result of the preparation of D-mannose by oxidase or its mutant crude enzyme.

[0094] Table 2 Results of preparing D-mannose using crude enzymes of oxidase or its mutants

[0095]

[0096]

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

[0098] The reaction system was sequentially added with 50 mM phosphate buffer (pH 7.2), 300 mM mannitol, 5 g / L catalase, and 80 g / L oxidase whole cells. The reaction system was incubated at 28°C for 14 h.

[0099] After the reaction, 1 mL of the reaction solution was centrifuged (14,000 rpm, 2 min). The supernatant was diluted 20-fold and passed through a 0.22 μm aqueous membrane for liquid chromatography. The detection conditions were: Bio-Rad Aminex HPX-87H, 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 concentration: 10 mM (1-2 g / L). Table 3 shows the results of D-mannose production using whole cells of the oxidase or its mutants.

[0100] Table 3 Results of D-mannose production by whole cells of oxidase or its mutants

[0101]

[0102]

[0103] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

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

2. The application according to claim 1, characterized in that The amino acid sequence of the oxidase from Streptomyces lividans is shown in SEQ ID No.

6.

3. The use according to claim 1 or 2, characterized in that: The oxidase further comprises a tag connected to the N-terminus and / or the C-terminus.

4. The application according to claim 1, characterized in that The mutant comprises: performing the following point mutation on the amino acid sequence shown in SEQ ID No. 6: N398D.

5. A method for enzymatically synthesizing D-mannose, characterized in that: The following steps are involved: Mannitol is used as substrate and the oxidase mutant is used to catalyze the reaction, and the reaction product is D-mannose; The oxidase is derived from Streptomyces lividans.

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

7. The method according to claim 5, characterized in that 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 hours.

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, lyophilized 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, characterized in that 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, lyophilized crude enzyme solution powder or pure enzyme, the reaction system of the catalytic reaction contains catalase in addition to mannitol and the oxidase.

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