Difunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant and application thereof

Abstract

The invention discloses a difunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant and application thereof, and belongs to the technical field of enzyme engineering and biological catalysis. The invention provides a difunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant and a preparation method of the difunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant. The amino acid sequence of the mutant is characterized in that at least one site of glutamic acid at the 28th site, arginine at the 83rd site, valine at the 126 site, isoleucine at the 161st site, leucine at the 187th site, phenylalanine at the 249th site, leucine at the 319th site and tyrosine at the 343th site of the amino acid sequence shown in SEQ ID No.1 is mutated. According to the method, the conversion efficiency of D-mannose is further improved, and meanwhile, the problem that a large number of by-products exist is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering and biocatalysis, and particularly relates to a bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant and application thereof. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] As a functional sugar, D-mannose possesses diverse physiological activities, including immune system regulation, anti-inflammatory, anti-tumor, and antibacterial properties. It has been widely used in the pharmaceutical, food, and daily chemical industries. With the rapid development of emerging industries such as functional foods, precision medicine, and sustainable feed, market demand for D-mannose continues to rise, offering broad development prospects and enormous potential for application.

[0004] Currently, the production of D-mannose relies primarily on two approaches: direct extraction from plants or microorganisms, but this suffers from low extraction yields and high energy consumption; and chemical isomerization using glucose as a substrate, which produces numerous byproducts and requires complex purification processes. In recent years, enzymatic methods have become a research hotspot due to their environmental and efficiency advantages. However, conventional isomerases (such as glucose isomerase) are limited by thermodynamic equilibrium, and D-mannose conversion rates typically fall below 25%. In contrast, using maltodextrin as a substrate, the synergistic action of α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), the bifunctional enzyme phosphogluconate / phosphogluconate isomerase mutant (PGI / PMI), and mannose-6-phosphate phosphatase (M6PP) enables efficient D-mannose synthesis. To improve conversion efficiency, existing research has optimized some key enzymes. For example, a patented technology provides a method for enhancing the activity of M6PP through site-specific mutations, thereby enhancing the conversion of mannose-6-phosphate to D-mannose. However, the previous step in this pathway, the conversion of glucose-6-phosphate to mannose-6-phosphate, is primarily catalyzed by PGI / PMI, whose catalytic efficiency directly influences the overall reaction process. Currently, research on protein engineering of PGI / PMI remains relatively scarce, with no reports of highly active mutants obtained through rational design or directed evolution. Therefore, optimizing the catalytic performance of PGI / PMI through enzyme molecular engineering techniques is urgently needed to significantly enhance the bioconversion efficiency of maltodextrin to D-mannose.

[0005] In addition, the existing multi-enzyme cascade catalytic system using maltodextrin as a substrate has obvious technical bottlenecks: on the one hand, the reaction path is long and the enzyme system is complex; on the other hand, a large amount of by-products such as glucose and fructose are produced, resulting in low yield of the target product and difficulty in separation and purification, which seriously restricts the economy and feasibility of the industrial production of D-mannose.

[0006] The existence of these key issues makes the development of efficient and highly specific bifunctional enzyme catalysts a key research direction. Summary of the Invention

[0007] In response to the shortcomings of the prior art, the present invention aims to provide a bifunctional enzyme, glucose phosphate isomerase / mannose phosphate isomerase mutant, and its application. The present invention aims to improve the conversion efficiency of D-mannose while overcoming the problem of the presence of a large number of byproducts.

[0008] In order to achieve the above object, the present invention provides the following technical solutions: As a first aspect of the present invention, a bifunctional enzyme phosphoglucose isomerase / mannose phosphate isomerase mutant is provided, wherein the amino acid sequence of the mutant is that at least one of the glutamate at position 28, the arginine at position 83, the valine at position 126, the isoleucine at position 161, the leucine at position 187, the phenylalanine at position 249, the leucine at position 319, and the tyrosine at position 343 of the amino acid sequence shown in SEQ ID No. 1 is mutated.

[0009] Compared to the wild-type enzyme having an amino acid sequence as shown in SEQ ID NO.1, the amino acid single-point mutation of the mutant is selected from at least one of the following: (1) Glutamic acid at position 28 is mutated to serine or aspartic acid; (2) Arginine at position 83 is mutated to lysine or tryptophan; (3) Valine at position 126 is mutated to isoleucine or phenylalanine; (4) Isoleucine at position 161 is mutated to tryptophan or valine; (5) Leucine at position 187 is mutated to methionine or isoleucine; (6) Phenylalanine at position 249 mutated to isoleucine or tyrosine; (7) Leucine at position 319 mutated to phenylalanine or tryptophan; (8) Tyrosine at position 343 mutates to phenylalanine or methionine.

[0010] Preferably, the amino acid of the mutant comprises a combination of mutations at any 2 to 8 sites among the above-mentioned single sites, which significantly improves the catalytic efficiency of the enzyme through a synergistic effect.

[0011] Preferably at least one of the following: (1) Mutations occur simultaneously at glutamic acid at position 28 and arginine at position 83; (2) Mutations occur simultaneously at glutamic acid at position 28 and valine at position 126; (3) Mutations occur simultaneously at glutamic acid at position 28 and isoleucine at position 161; (4) Mutations at both glutamic acid at position 28 and leucine at position 187; (5) Mutations occur simultaneously at glutamic acid at position 28 and phenylalanine at position 249; (6) Mutations at position 28, glutamic acid, position 319, and position 343; (7) Mutations at position 28, glutamic acid, position 83, and position 343; (8) Mutations at position 83, position 161, and position 249 of phenylalanine simultaneously occur; (9) Mutations at position 161: isoleucine, position 187: leucine, and position 249: phenylalanine. (10) Mutations at position 28, glutamic acid, position 83, arginine, position 126, and position 161; (11) Mutations at position 28, glutamic acid, position 83, arginine, position 126, and position 343; (12) Mutations at position 83, arginine at position 161, isoleucine at position 249, and tyrosine at position 343; (13) Mutations at position 83, arginine at position 126, valine at position 249, phenylalanine at position 249, and tyrosine at position 343; (14) Mutations at position 28, glutamic acid, position 126, valine, position 161, isoleucine, position 187, and position 249; (15) Mutations occur simultaneously at arginine at position 83, valine at position 126, isoleucine at position 161, leucine at position 187, and phenylalanine at position 249; (16) Mutations at position 83, arginine at position 126, isoleucine at position 161, leucine at position 187, leucine at position 319, and tyrosine at position 343; (17) Mutations at position 83, arginine at position 126, isoleucine at position 161, leucine at position 187, phenylalanine at position 249, leucine at position 319, and tyrosine at position 343; (18) Mutations occurred simultaneously at glutamic acid at position 28, arginine at position 83, valine at position 126, isoleucine at position 161, leucine at position 187, phenylalanine at position 249, leucine at position 319, and tyrosine at position 343.

[0012] As a second aspect of the present invention, a gene encoding the bifunctional enzyme phosphoglucose isomerase / mannose phosphate isomerase mutant described in the first aspect is provided.

[0013] As a third aspect of the present invention, there is provided a biological material comprising the gene according to the second aspect, wherein the biological material comprises an expression cassette, a vector or a host cell; the host cell is selected from Escherichia coli and Bacillus subtilis.

[0014] As a fourth aspect of the present invention, it provides the use of the biomaterial described in the third aspect in the production of D-mannose.

[0015] As a fifth aspect of the present invention, it is to provide the use of the biomaterial described in the third aspect in any of the following aspects: (1) Improve the conversion efficiency of D-mannose; (2) Inhibit the formation of glucose and fructose by-products.

[0016] As a sixth aspect of the present invention, a multi-stage enzyme-linked catalytic system is provided to achieve efficient synthesis of D-mannose. The multi-stage enzyme-linked catalytic system comprises: a substrate maltodextrin, α-glucan phosphorylase, phosphoglucomutase, the bifunctional enzymes phosphogluconate / phosphogluconate isomerase described in the first aspect, and mannose-6-phosphate phosphatase; or comprises: a substrate maltodextrin, and expression of a gene for α-glucan phosphorylase, a gene for phosphogluconate mutase, a gene for the bifunctional enzymes phosphogluconate / phosphogluconate isomerase, and a gene for mannose-6-phosphate phosphatase.

[0017] α-Glucan phosphorylase is derived from Arabidopsis thaliana The gene number on Uniprot is Q9SD76; phosphoglucomutase is derived from Thermococcus kodakarensis The gene number on Uniprot is Q68BJ6; the bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase is derived from Dictyoglomus turgidum The gene number on Uniprot is B8E0A4; mannose 6-phosphate phosphatase is derived from Pseudothermotoga hypogea The gene number on Uniprot is A0A0X1KRU1.

[0018] Furthermore, the multi-stage enzyme-linked catalytic system further comprises isoamylase, 4-glucanotransferase (4-GT) and polyphosphoglucokinase (PPGK); or expresses isoamylase gene, 4-glucanotransferase gene and polyphosphoglucokinase gene.

[0019] The isoamylase is derived from Pseudomonas amyloderamosa, the gene number is P10342 in Uniprot; the polyphosphate glucose kinase is derived from Thermobifida halotolerans , the gene number is NI17_011080 in KEGG; the 4-glucanotransferase gene is derived from Thermococcus sibiricus , the gene number is TSIB_0455 in KEGG. The genomic DNA of these genes can be obtained from the official website of ATCC (www.atcc.org).

[0020] In the multi-stage enzyme-coupled catalytic system, the substrate maltodextrin is treated by the engineering bacteria expressing the isoamylase gene, the enzyme catalyzes the starch to generate amylose by debranching, then the maltodextrin is converted into D-mannose by the engineering bacteria expressing the α-glucan phosphorylase gene, the phosphoglucomutase gene, the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase gene and the mannose-6-phosphate phosphatase gene, in addition, the engineering bacteria expressing the 4-glucanotransferase gene is added in the expression system, the enzyme catalyzes the polymerization of maltodisaccharide and maltotriose to generate higher degree of polymerization of dextrin, and the engineering bacteria expressing the polyphosphate glucose kinase gene is added, the enzyme can specifically transfer the terminal phosphate group of polyphosphate to glucose molecule to generate glucose-6-phosphate, and the glucose-6-phosphate enters the synthesis pathway of D-mannose again, further improving the yield of D-mannose.

[0021] As a seventh aspect of the present application, there is provided a recombinant microorganism expressing the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant of the first aspect. The microorganism comprises the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant or a gene encoding the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant.

[0022] As an eighth aspect of the present application, there is provided a method for efficiently synthesizing D-mannose, using the biological material of the third aspect or the multi-stage enzyme-coupled catalytic system of the sixth aspect or the recombinant microorganism of the seventh aspect to perform a catalytic reaction to generate D-mannose.

[0023] The beneficial effects achieved by one or more embodiments of the present application are as follows: 1. The present application successfully constructs the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant by bioinformatics analysis and screening of key mutation sites combined with saturation mutation technology, the mutant can be efficiently expressed in Escherichia coli and Bacillus subtilis, not only significantly improves the conversion efficiency of D-mannose, but also effectively inhibits the generation of by-products, and provides a high-efficiency and stable biological catalyst for the industrial production of D-mannose.

[0024] 2. The present application uses the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant to catalyze the conversion of maltodextrin into D-mannose, which not only significantly improves the conversion efficiency of D-mannose, but also effectively inhibits the generation of by-products. Dictyoglomus turgidumThe bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase undergoes single-site and combined site saturation mutations at positions 28 (glutamate), 83 (arginine), 126 (valine), 161 (isoleucine), 187 (leucine), 249 (phenylalanine), 319 (leucine), and 343 (tyrosine) to screen for mutants that improve the conversion efficiency of D-mannose and effectively inhibit the formation of glucose and fructose byproducts. Compared to the wild type, the optimal mutant can increase the yield of D-mannose to 95.05% while effectively inhibiting byproduct formation, reducing the byproduct ratios of glucose and fructose byproducts by 55.87% and 58.10%, respectively. This invention provides an efficient and stable biocatalyst for the industrial production of D-mannose, with important application value.

[0025] 3. As a GRAS (Generally Regarded As Safe) strain, Bacillus subtilis produces no pyrogens or endotoxins, and has no toxic side effects, reducing subsequent isolation and purification costs and making it suitable for producing D-mannose. Currently, there are few technologies or reports demonstrating de novo synthesis of D-mannose using Bacillus subtilis. The mutants constructed in this invention also exhibit excellent catalytic performance in the Bacillus subtilis expression system. Compared to the wild-type, the optimal mutant significantly increased the D-mannose yield to 88.42%. Therefore, this invention has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 This is a process flow chart for the multi-stage enzyme-linked catalytic conversion of D-mannose using maltodextrin as a substrate. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0029] The present invention will be further described below with reference to the embodiments.

[0030] In some embodiments of the present invention, a bifunctional enzyme phosphoglucose isomerase / mannose phosphate isomerase mutant is provided, wherein the amino acid sequence of the mutant is a mutation at at least one of the glutamate at position 28, the arginine at position 83, the valine at position 126, the isoleucine at position 161, the leucine at position 187, the phenylalanine at position 249, the leucine at position 319, and the tyrosine at position 343 of the amino acid sequence shown in SEQ ID No. 1.

[0031] In some embodiments of the present invention, compared to the wild-type enzyme having an amino acid sequence as shown in SEQ ID NO. 1, the amino acid single-point mutation of the mutant is selected from at least one of the following: (1) Glutamic acid at position 28 is mutated to serine or aspartic acid; (2) Arginine at position 83 is mutated to lysine or tryptophan; (3) Valine at position 126 is mutated to isoleucine or phenylalanine; (4) Isoleucine at position 161 is mutated to tryptophan or valine; (5) Leucine at position 187 is mutated to methionine or isoleucine; (6) Phenylalanine at position 249 mutated to isoleucine or tyrosine; (7) Leucine at position 319 mutated to phenylalanine or tryptophan; (8) Tyrosine at position 343 mutates to phenylalanine or methionine.

[0032] In some embodiments of the present invention, a gene encoding the bifunctional enzyme phosphoglucose isomerase / mannose phosphate isomerase mutant is provided.

[0033] In some embodiments of the present invention, provided are biological materials comprising a gene encoding the bifunctional enzyme phosphoglucose isomerase / mannose phosphate isomerase mutant, wherein the biological material comprises an expression cassette, a vector or a host cell; the host cell is selected from Escherichia coli and Bacillus subtilis.

[0034] In some embodiments of the present invention, use of the biomaterial in producing D-mannose is provided.

[0035] In some embodiments of the present invention, the biomaterial has the following functions in the production of D-mannose: (1) Improve the conversion efficiency of D-mannose; (2) Inhibit the formation of glucose and fructose by-products.

[0036] In some embodiments of the present invention, a multi-stage enzyme-linked catalytic system is provided to achieve efficient synthesis of D-mannose. The multi-stage enzyme-linked catalytic system comprises: a substrate maltodextrin, α-glucan phosphorylase, phosphoglucomutase, the bifunctional enzymes phosphoglucoside / phosphoglucoside isomerase described in the first aspect, and mannose-6-phosphate phosphatase; or comprises: a substrate maltodextrin, and expression of a gene for α-glucan phosphorylase, a gene for phosphoglucoside mutase, a gene for the bifunctional enzymes phosphoglucoside / phosphoglucoside isomerase, and a gene for mannose-6-phosphate phosphatase.

[0037] Furthermore, the multi-stage enzyme-linked catalytic system further comprises isoamylase, 4-glucanotransferase and polyphosphate glucokinase; or an engineered bacterium expressing isoamylase gene, 4-glucanotransferase gene and polyphosphate glucokinase gene.

[0038] In some embodiments of the present invention, a recombinant microorganism is provided, which expresses the bifunctional enzyme phosphoglucose isomerase / phosphoglucose isomerase mutant, and comprises the mutant or a gene encoding the mutant.

[0039] In some embodiments of the present invention, a method for efficiently synthesizing D-mannose is provided, wherein the biomaterial, or the multi-stage enzyme-linked catalytic system described in the sixth aspect, or the recombinant microorganism described in the seventh aspect is used to carry out a catalytic reaction to produce D-mannose. The process flow chart of the multi-stage enzyme-linked catalytic conversion of D-mannose using maltodextrin as a substrate is shown in FIG. Figure 1 shown.

[0040] In some embodiments, the concentration of isoamylase in the reaction system is 0.1-2 wt%, preferably 0.4 wt%; the concentration of 4-glucanotransferase gene is 0.1-3 wt%, in some embodiments, the concentration is 0.5 wt%, 1 wt%, 1.5 wt%, 2.5 wt%, or any value between 0.5-2.5 wt%, preferably 1 wt%; the concentration of polyphosphate glucose kinase is 0.1-2 wt% or any value between 0.5 wt%, 1 wt%, 1.5 wt%, and 0.5-1.5 wt%, preferably 1.5 wt%; the concentration of alpha-glucan phosphorylase is 0.5-5 wt% or any value between 1 wt%, 2 wt%, 3 wt%, 4 wt%, and 1-4 wt%, preferably 1 wt%; the concentration of phosphoglucomutase is 0.5-5 wt% or any value between 1 wt%, 1.5 wt%, 2 wt%, 3 wt%, and 1-3 wt%, preferably 1 wt%; the concentration of bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant is 0.5-5 wt% or any value between 1 wt%, 1.5 wt%, 2 wt%, 3 wt%, and 1-3 wt%, preferably 2 wt%; the concentration of mannose 6-phosphate phosphatase is 2-10 wt% or any value between 2 wt%, 3 wt%, 4 wt%, 6 wt%, and 2-6 wt%, preferably 4 wt%.

[0041] In one or more embodiments, the temperature for treating maltodextrin with the engineered bacteria expressing isoamylase gene is 70-85°C, preferably 80°C; the treatment time is 8-16 h, preferably 12 h. The concentration of maltodextrin is 1-500 g / L, preferably 150 g / L; the concentration of phosphate is 1-100 mM, preferably 10 mM; the concentration of Mg 2+ The concentration of Mg2+is 1-50 mM, preferably 5 mM; the final concentration of sodium hexametaphosphate is 30-70 mM, preferably 40 mM. The reaction temperature is 40-80°C, preferably 65°C; the reaction time is 8-48 h, preferably 24 h.

[0042] The preparation method of KPB phosphate buffer used in this embodiment is as follows: potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4) are used as solutes, the pH is 6.8-7.2, and the concentration of potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4) is 10 mM or 20 mM.

[0043] Example 1, construction of vector and expression analysis of PGI / PMI in E. coli including the following steps: (a) obtaining the isoamylase gene from Dictyoglomus turgidumThe amino acid sequence of the bifunctional enzyme gene fragment of glucose phosphate isomerase / mannose phosphate isomerase (PGI / PMI) from the genus was codon-optimized and recombined into the pET32a vector to generate the recombinant plasmid pET32a-PGI / PMI. The corresponding amino acid sequence is shown in SEQ ID NO. 1. (b) The recombinant plasmid pET32a-PGI / PMI was transformed into the Escherichia coli expression host BL21(DE3) to obtain a prokaryotic expression strain.

[0044] (c) The recombinant strain containing the recombinant plasmid pET32a-PGI / PMI was inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) for overnight activation to obtain seed solution, which was then transferred to 100 mL of LB medium (containing 100 μg / mL ampicillin) at a 1% inoculum volume and cultured until the OD 600 =0.6-0.8, and IPTG was added to a final concentration of 0.1 mM for protein expression induction. Bacterial cells were harvested by centrifugation, and 20 wt% of the bacterial slurry was weighed and suspended in 20 mM KPB phosphate buffer (pH 7.0). After ultrasonic disruption, the expression of the pET32a-PGI / PMI gene was verified by running on a protein gel. The PGI / PMI protein band was approximately 58.96 kDa.

[0045] (d) PGI / PMI enzyme activity assay: To a 1 mL reaction system, add 50 mM glucose-6-phosphate, 10 mM KPB phosphate buffer (pH 7.0), 5 mM MgCl₂, 5 U / mL mannose-6-phosphate phosphatase, and 10–50 μL of crude PGI / PMI enzyme solution (the amount of enzyme added was adjusted based on the measured amount of D-mannose produced, with the amount of enzyme required to produce 5 mM D-mannose being the appropriate amount). The reaction was incubated at 65°C for 10–20 min. After completion of the reaction, the reaction was boiled for 5 min, centrifuged to remove impurities, and the amount of D-mannose produced was determined by HPLC, and the enzyme activity was calculated. HPLC analysis conditions were as follows: a Thermo Scientific™ high-performance liquid chromatograph with a Shodex SUGAR column, column temperature: 80°C, mobile phase: ddH₂O, flow rate: 0.6 mL / min, and injection volume: 20 μL.

[0046] Example 2, Construction of PGI / PMI Mutant Library The steps include: (a) Bioinformatics analysis was performed on the amino acid sequence and structure of wild-type PGI / PMI to identify the key amino acid residues in the catalytically active center of the enzyme. The amino acid residues near the substrate channel were also analyzed. Finally, eight sites that may affect catalytic activity and substrate-specific binding ability were screened: glutamic acid at position 28, arginine at position 83, valine at position 126, isoleucine at position 161, leucine at position 187, phenylalanine at position 249, leucine at position 319, and tyrosine at position 343.

[0047] (b) Single-site saturation mutagenesis was performed at positions 28, 83, 126, 161, 187, 249, 319, and 343, respectively, to design mutation primers for the specified mutation sites; using the recombinant plasmid pET32a-PGI / PMI provided in Example 1 as a template, the recombinant plasmid PGI / PMI was amplified by whole-plasmid PCR in the presence of high-fidelity Pha enzyme using each mutation primer to obtain a linearized plasmid for the specified mutation site. The amplified product was digested with DpnI enzyme at 37°C for 1 h, and then transformed into Escherichia coli BL21 (DE3) competent cells, coated on LB plates (containing 100 μg / mL ampicillin), and cultured overnight at 37°C to obtain positive clone transformants.

[0048] (c) Obtaining crude enzyme solution from mutants: The activity of the strains with the corresponding mutations verified by sequencing was tested; 1% of the positive clone transformants were inoculated into 500 mL of LB liquid medium containing 100 μg / mL ampicillin and cultured at 37°C and 220 rpm until the OD 600 At 0.6-0.8°C, add IPTG to a final concentration of 0.1 mM. Induce at 25°C for 16 h, then collect the cells by centrifugation. Weigh the bacterial sludge to a 20 wt% ratio and resuspend it in 20 mM KPB phosphate buffer. Disrupt the resuspended sludge with an ultrasonic disruptor to obtain a crude enzyme solution.

[0049] Example 3: Analysis of catalytic specificity of PGI / PMI single-site mutants using maltodextrin as substrate The steps include: (a) Based on the crude enzyme solution described in Example 2, a mutation site screening system was established: 150 g / L maltodextrin treated with 0.4 wt% IA, 5 mM Mg 2+and 4wt% respectively, and ddH2O was added to 1 mL. The reaction was carried out in a constant temperature reactor at 65°C, the rotation speed was set to 700 rpm, and after 24h of reaction, the sample was taken. Sample treatment: after centrifugation, the supernatant of the reaction ended sample was diluted 10 times, filtered through a 0.22 μm filter membrane, and detected by HPLC. The yields of D-mannose, glucose and fructose were determined by high performance liquid chromatography.

[0050] (b) In one or more embodiments, the isoamylase is derived from Pseudomonas amyloderamosa , the gene number of which is P10342 on Uniprot; the alpha-glucan phosphorylase is derived from Arabidopsis thaliana , the gene number of which is Q9SD76 on Uniprot; the phosphoglucomutase is derived from Thermococcus kodakarensis , the gene number of which is Q68BJ6 on Uniprot; the bifunctional enzyme phosphoglucoisomerase / mannose 6-phosphate isomerase is derived from Dictyoglomus turgidum , the gene number of which is B8E0A4 on Uniprot; the mannose 6-phosphate phosphatase is derived from Pseudothermotoga hypogea , the gene number of which is A0A0X1KRU1 on Uniprot.

[0051] (c) Mutant verification and property analysis: the specific improvement mutants obtained by preliminary screening were identified by sequencing to analyze the key amino acid substitution types. Advantageous mutants with high specificity and high catalytic activity were screened, and the results are shown in Table 1.

[0052] Table 1 Catalytic specificity and catalytic activity analysis of PGI / PMI unit point mutant with maltodextrin as substrate

[0053] The results in Table 1 show that the amino acid residues at positions 28, 83, 126, 161, 187, 249, 319 and 343 have a key regulatory effect on the catalytic activity and substrate specificity of the enzyme. In the multi-enzyme cascade reaction system with maltodextrin as the substrate, the catalytic efficiency of the glucose phosphoisomerase / mannose phosphoisomerase bifunctional mutant with unit point mutation is significantly improved compared with the wild type: the optimal mutant mannose conversion rate is increased from 72.36% to 83.62% compared with the control group.

[0054] Example 4 Catalytic specificity and catalytic activity analysis of PGI / PMI combined site mutant with maltodextrin as substrate The above sites were subjected to combined saturation mutagenesis, and the catalytic specificity and catalytic activity of the PGI / PMI combined site mutants were evaluated using the method described in Example 3. The results are shown in Table 2.

[0055] Table 2 Catalytic specificity and activity analysis of PGI / PMI combined site mutants using maltodextrin as substrate

[0056] The results in Table 2 show that in a multienzyme cascade reaction system using maltodextrin as a substrate, the catalytic efficiency of the bifunctional glucose phosphate isomerase / mannose phosphate isomerase mutant with combined site mutations at amino acids 28, 83, 126, 161, 187, 249, 319, and 343 was significantly improved compared to the wild-type: the optimal mutant increased the mannose conversion rate to 90.50% compared to 72.36% in the control, and the proportions of glucose and fructose byproducts decreased to 55.87% and 58.10%, respectively. This confirms the role of key site mutations in optimizing enzyme function.

[0057] Example 5, Optimization of the ratio of each enzyme in the multi-enzyme reaction system for catalyzing the synthesis of D-mannose: (a) In a 1 mL reaction system, 150 g / L maltodextrin treated with 0.4 wt% IA, 5 mM Mg 2+ A crude enzyme solution consisting of αGP, PGM, PGI / PMI (Mutant 34, hereafter referred to as M34), and M6PP was added to a 10 mM phosphate buffer (pH 7.0) solution, and the volume was adjusted to 1 mL with ddH2O. The reaction was carried out in a thermostated reactor at 65°C and 700 rpm. After 24 hours of reaction, samples were collected. Sample processing: After the reaction, the supernatant was centrifuged and diluted 10-fold, filtered through a 0.22 μm filter, and analyzed by HPLC.

[0058] Table 3 Reaction results of different enzyme ratios

[0059] The results in Table 3 show that the highest D-mannose production rate is 89.76% when the addition amount of aGP is 1wt%, the addition amount of PGM is 1wt%, the addition amount of PGI / PMI (M34) is 2wt%, and the addition amount of M6PP is 4wt%.

[0060] (b) To further improve the conversion rate, 4-glucanotransferase and polyphosphate glucokinase were added to the reaction system. In a 1 mL reaction system, 150 g / L maltodextrin treated with 0.4 wt% IA, 5 mM Mg 2+and 10 mM phosphate buffer (pH = 7.0), and crude enzyme solutions consisting of αGP, PGM, PGI / PMI, and M6PP were added to make the enzyme concentrations 1wt%, 1wt%, 2wt%, and 4wt%, respectively. The volume was adjusted to 1 mL with ddH2O. The reaction was carried out in a thermostatic reactor at 65°C and the speed was set to 700 rpm. After 24 h of reaction, 500 μL of the mannose reaction solution was taken and the final concentration of phosphate buffer was 10 mM, Mg 2+ The final concentration of the reaction mixture was 5 mM, sodium hexametaphosphate 40 mM, and crude enzyme solutions of 4-glucanotransferase and polyphospho-glucokinase were added. The volume was then made up to 1 mL with water. The reaction was carried out in a thermostated reactor at 65°C and 700 rpm for 8 hours, after which sampling was performed. Sample preparation: After the reaction, the supernatant was centrifuged and diluted 10-fold, filtered through a 0.22 μm filter, and analyzed by HPLC.

[0061] Table 4 Reaction results of different enzyme ratios

[0062] The results in Table 4 show that when the addition amount of 4-GT is 1wt% and the addition amount of PPGK is 1.5wt%, the D-mannose conversion rate can reach a maximum of 97.08%.

[0063] Example 6: Whole-cell catalysis of D-mannose using Bacillus subtilis as the expression host The steps include: (a) Construction of an engineered bacterium expressing the α-glucan phosphorylase gene, the phosphoglucomutase gene, the phosphoglucoside / mannose phosphate isomerase (M34) gene, a bifunctional enzyme selected as having high catalytic specificity and / or activity, and the mannose-6-phosphate phosphatase gene in Bacillus subtilis.

[0064] In this example, α-glucan phosphorylase is derived from Arabidopsis thaliana The gene number on Uniprot is Q9SD76; phosphoglucomutase is derived from Thermococcus kodakarensis The gene number on Uniprot is Q68BJ6; the bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase mutant is derived from the present invention; mannose 6-phosphate phosphatase is derived from Pseudothermotoga hypogea The gene number on Uniprot is A0A0X1KRU1.

[0065] The specific steps were as follows: Using PWB980 as the vector, primers corresponding to each gene were designed, the vector and fragment were amplified, and the vector and fragment were transformed into SCK6 competent cells via PCR ligation. The cells were plated on LB plates containing kanamycin (25 μg / mL), cultured overnight, and transformants were selected for testing. The successfully constructed strains were named PWB980-αGP, PWB980-PGM, PWB980-PGI / PMI (M34), and PWB980-M6PP.

[0066] (b) Using engineered bacteria expressing genes for α-glucan phosphorylase, phosphoglucomutase, a mutant gene for the bifunctional enzyme phosphoglucoisomerase / mannose phosphoglucoisomerase with high catalytic specificity and / or activity, and a gene for mannose-6-phosphate phosphatase, an enzyme capable of whole-cell catalytic production of D-mannose was obtained. The specific steps were as follows: The PWB980-αGP, PWB980-PGM, PWB980-PGI / PMI (M34), and PWB980-M6PP strains constructed in step (a) were inoculated into test tubes containing 5 mL of LB liquid medium containing kanamycin (50 μg / mL) and cultured in a shaker at 37°C and 200 rpm for 16 h. After the culture, the seed culture was transferred to a shake flask containing 400 mL of LB liquid medium containing kanamycin (50 μg / mL) at a 2% inoculum volume. Culture was carried out at 37°C in a shaker at 200 rpm for 48 hours. The cells were harvested by centrifugation at 8000 rpm for 15 minutes. The bacterial sludge was weighed to a 20 wt% concentration and resuspended. The resuspended sludge was disrupted using an ultrasonic disruptor to obtain a crude enzyme solution.

[0067] (c) Based on the crude enzyme solution obtained above, a reaction system was established: 150 g / L maltodextrin treated with 0.4% IA, 5 mM Mg 2+ A mixture of 10 mM phosphate buffer (pH 7.0) was prepared. Crude enzyme solutions consisting of PWB980-αGP, PWB980-PGM, PWB980-PGI / PMI (M34), and PWB980-M6PP were added at enzyme concentrations of 2 wt%, 1 wt%, 1 wt%, and 4 wt%, respectively. The concentrations of 4GT and PPGK were 1.5 wt%, respectively. The volume was adjusted to 1 mL with ddH2O. The reaction was carried out in a thermostated reactor at 65°C and 700 rpm. After 24 hours of reaction, samples were collected and analyzed by HPLC. Using D-mannose conversion as an indicator of catalytic specificity, the D-mannose conversion achieved in this example reached 88.42%.

[0068] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant, characterized in that Compared to the wild-type enzyme having an amino acid sequence as shown in SEQ ID NO.1, the amino acid single-point mutation of the mutant is selected from at least one of the following: (1) Glutamic acid at position 28 is mutated to serine or aspartic acid; (2) Arginine at position 83 is mutated to lysine or tryptophan; (3) Valine at position 126 is mutated to isoleucine or phenylalanine; (4) Isoleucine at position 161 is mutated to tryptophan or valine; (5) Leucine at position 187 is mutated to methionine or isoleucine; (6) Phenylalanine at position 249 mutated to isoleucine or tyrosine; (7) Leucine at position 319 mutated to phenylalanine or tryptophan; (8) Tyrosine at position 343 is mutated to phenylalanine or methionine.

2. The bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant according to claim 1, characterized in that The amino acid of the mutant comprises a combination of mutations at any 2 to 8 sites among the above-mentioned single sites.

3. The bifunctional enzyme phosphoglucose isomerase / mannose phosphate isomerase mutant according to claim 1, characterized in that: The mutations of the mutant include at least one of the following: (1) Mutations occur simultaneously at glutamic acid at position 28 and arginine at position 83; (2) Mutations occur simultaneously at glutamic acid at position 28 and valine at position 126; (3) Mutations occur simultaneously at glutamic acid at position 28 and isoleucine at position 161; mutations occur simultaneously at glutamic acid at position 28 and leucine at position 187; (4) Mutations occur simultaneously at glutamic acid at position 28 and phenylalanine at position 249; (5) Mutations at position 28, glutamic acid, position 319, and position 343; (6) Mutations at position 28, glutamic acid, position 83, and position 343; (7) Mutations at position 83, position 161, and position 249 of phenylalanine simultaneously occur; (8) Mutations at position 161: isoleucine, position 187: leucine, and position 249: phenylalanine; (9) Mutations at position 28, glutamic acid, position 83, arginine, position 126, and position 161; (10) Mutations at position 28, glutamic acid, position 83, arginine, position 126, and position 343; (11) Mutations at position 83, arginine at position 161, isoleucine at position 249, and tyrosine at position 343; (12) Mutations at position 83, arginine at position 126, valine at position 249, phenylalanine at position 249, and tyrosine at position 343; (13) Mutations at position 28, glutamic acid, position 126, valine, position 161, isoleucine, position 187, and position 249; (14) Mutations at position 83, arginine at position 126, isoleucine at position 161, leucine at position 187, and phenylalanine at position 249; (15) Mutations at position 83, arginine at position 126, isoleucine at position 161, leucine at position 187, leucine at position 319, and tyrosine at position 343; (16) Mutations at position 83, arginine at position 126, isoleucine at position 161, leucine at position 187, phenylalanine at position 249, leucine at position 319, and tyrosine at position 343; (17) Mutations occurred simultaneously at glutamic acid at position 28, arginine at position 83, valine at position 126, isoleucine at position 161, leucine at position 187, phenylalanine at position 249, leucine at position 319, and tyrosine at position 343.

4. A gene encoding the bifunctional enzyme phosphoglucose isomerase / mannose phosphate isomerase mutant according to claim 1.

5. A biological material comprising the gene according to claim 4, wherein the biological material comprises an expression cassette, a vector or a host cell.

6. Use of the biomaterial according to claim 5 in the production of D-mannose.

7. Use of the biomaterial according to claim 5 in any of the following aspects: (1) Improve the conversion efficiency of D-mannose; (2) Inhibit the formation of glucose and fructose by-products.

8. A multi-stage enzyme-linked catalytic system, characterized in that: The method comprises: a substrate maltodextrin, an α-glucan phosphorylase, a phosphoglucomutase, the bifunctional enzyme phosphoglucoside isomerase / mannose phosphate isomerase according to any one of claims 1 to 3, and a mannose-6-phosphate phosphatase; or the method comprises: a substrate maltodextrin, and expression of an α-glucan phosphorylase gene, an expression of a phosphoglucoside mutase gene, an expression of a bifunctional enzyme phosphoglucoside isomerase / mannose phosphate isomerase gene, and an expression of a mannose-6-phosphate phosphatase gene; The multi-stage enzyme-linked catalytic system further comprises an engineered bacterium expressing an isoamylase gene, a 4-glucanotransferase gene and a polyphosphate glucokinase gene.

9. A recombinant microorganism, characterized in that Expressing the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant according to claim 1; the microorganism comprises the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant according to claim 1 or a gene encoding the mutant.

10. A method for efficiently synthesizing D-mannose, characterized in that: The biomaterial according to claim 5, the multi-stage enzyme-linked catalytic system according to claim 8, or the recombinant microorganism according to claim 9 is used to carry out a catalytic reaction to produce D-mannose.

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