Method for preparing D-mannose through catalysis

Abstract

The method for preparing D-mannose through catalysis can effectively reduce the amount of by-products glucose and fructose, and greatly improves the conversion rate of D-mannose. Specifically, engineering bacteria for expressing isoamylase genes and engineering bacteria for expressing alpha-glucan phosphorylase genes, glucophosphate mutase genes, difunctional enzyme glucophosphate isomerase / mannose 6-phosphate isomerase genes and mannose 6-phosphate phosphatase genes are utilized, starch or starch derivatives are used as substrates, and the starch or starch derivatives are used as substrates. A phosphate buffer solution and Mg < 2 + > are added, a preliminary catalysis system is constructed, after the reaction is completed, an incompletely-reacted substrate and maltodisaccharide exist in the reaction system, the incompletely-reacted substrate and maltodisaccharide can be hydrolyzed into glucose by glucoamylase, and the polyphosphoglucokinase can be used for catalyzing the reaction of the polyphosphoglucokinase under the assistance of sodium hexametaphosphate. All glucose is converted into phosphorylated glucose, then D-mannose is generated, and by-products in a system are removed while the yield is increased.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing D-mannose by catalysis. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] D-mannose is a common six-carbon monosaccharide, an isomer of glucose. It appears as white crystals or powder, has a sweet-bitter taste, is soluble in water but poorly soluble in ethanol, and rarely occurs in its free form in nature, mostly as polysaccharides. D-mannose is a rare functional sugar with important applications in biology and medicine. It not only regulates immunity, protects against bacterial and viral invasion, and promotes the normal synthesis of glycoproteins in the body, but also serves as a raw material for the synthesis of several pharmaceutical and chemical raw materials and important carbohydrate drug precursors, such as immunostimulants, anti-tumor drugs, vitamins, and D-mannitol. In recent years, demand for mannose in functional foods, feed additives, and pharmaceuticals has increased significantly, highlighting its potential for economic development.

[0004] Currently, D-mannose production methods primarily include plant extraction, chemical isomerization, and enzymatic methods. Plant extraction, primarily through direct extraction from fruits and other plant materials, is a widely used method. Chemical isomerization, using molybdic acid or molybdate as a catalyst, converts glucose to D-mannose under acidic, high-temperature, and high-pressure conditions. Due to regional raw material restrictions, harsh reaction conditions, complex processes, downstream purification difficulties, and low conversion rates, both extraction and chemical methods have limitations and cannot meet industrial production needs. Enzymatic methods for the production of D-mannose are becoming a growing research trend. During the production of mannose using biochemical methods such as enzyme catalysis, glucose and fructose are major byproducts, posing significant technical challenges in subsequent product separation and purification. In particular, in subsequent product separation steps, ensuring the purity and quality of the mannose product meet predetermined standards requires the use of more sophisticated and complex separation techniques. Therefore, reducing the amount of glucose and fructose byproducts has become a core challenge that needs to be addressed in the industrial production of mannose. Summary of the Invention

[0005] In order to overcome the above problems, the present invention provides a method for catalytically preparing D-mannose.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: The first aspect of the present invention provides a system for catalytically preparing D-mannose, comprising: a phosphate buffer solution, starch or starch derivatives, Mg 2+ As well as engineered bacteria expressing α-glucan phosphorylase (aGP) gene, glucose phosphomutase (PGM) gene, bifunctional enzyme glucose phosphomerase / mannose 6-phosphate isomerase (PGI) gene and mannose 6-phosphate phosphatase (M6PP) gene; engineered bacteria expressing isoamylase (IA) gene, saccharifying enzyme, engineered bacteria expressing defined polyphosphate glucokinase (PPGK) gene and sodium hexametaphosphate.

[0007] In one or more embodiments, the solutes in the phosphate buffer solution are potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4), and the pH is 6.8-7.2, preferably 7.0; the concentration of the solute in the phosphate buffer solution is 1-100 mM, preferably 10 mM.

[0008] In one or more embodiments, the starch derivative includes one or more of partially hydrolyzed starch, starch dextrin, maltodextrin, maltopolysaccharide or maltose.

[0009] In one or more embodiments, the concentration of the starch is 1-500 g / L, preferably 150 g / L.

[0010] In one or more embodiments, the Mg 2+ The concentration is 1~50 mM, preferably 5 mM.

[0011] In one or more embodiments, the preparation method of the engineered bacteria expressing the α-glucan phosphorylase gene, the phosphoglucomutase gene, the bifunctional enzyme phosphoglucoisomerase / mannose 6-phosphate isomerase gene, and the mannose 6-phosphate phosphatase gene is as shown in any one of the following A1) to A5): A1) nucleotide sequences of the α-glucan phosphorylase gene, the phosphoglucomutase gene, the bifunctional enzyme phosphoglucoisomerase / mannose 6-phosphate isomerase gene, and the mannose 6-phosphate phosphatase gene were separately transferred into Escherichia coli to obtain four recombinant strains; the recombinant strains were induced for expression and then permeabilized to obtain engineered strains; A2) co-transforming the nucleotide sequences of the α-glucan phosphorylase gene, the phosphoglucomutase gene, the bifunctional enzyme phosphoglucoisomerase / mannose 6-phosphate isomerase gene, and the mannose 6-phosphate phosphatase gene into the same Escherichia coli strain in any permutation and combination to obtain a recombinant strain; inducing expression in the recombinant strain, and then permeabilizing the strain to obtain an engineered strain; A3) co-transforming any two nucleotides from the α-glucan phosphorylase gene, the phosphoglucomutase gene, the bifunctional enzyme phosphoglucoisomerase / mannose 6-phosphate isomerase gene, and the mannose 6-phosphate phosphatase gene into one strain of Escherichia coli in any permutation and combination, and co-transforming the remaining two nucleotides into another strain of Escherichia coli in any permutation and combination, to obtain two recombinant strains; inducing expression in each recombinant strain, and then permeabilizing the recombinant strain to obtain engineered strains; A4) co-transferring any two nucleotides from the α-glucan phosphorylase gene, the phosphoglucomutase gene, the bifunctional enzyme phosphoglucoisomerase / mannose 6-phosphate isomerase gene, and the mannose 6-phosphate phosphatase gene into one strain of Escherichia coli in any permutation and combination, and transferring the remaining two nucleotides into two other strains of Escherichia coli to obtain three recombinant strains; inducing expression in each of the recombinant strains, and then permeabilizing them to obtain engineered strains; A5) Any three nucleotides from the α-glucan phosphorylase gene, the phosphoglucomutase gene, the bifunctional enzyme phosphoglucoisomerase / mannose 6-phosphate isomerase gene, and the mannose 6-phosphate phosphatase gene are co-transformed into one strain of Escherichia coli in any permutation and combination, and the remaining nucleotide is separately transferred into another strain of Escherichia coli to obtain two recombinant strains; the recombinant strains are induced to express, and then permeabilized to obtain engineered strains.

[0012] Preferably: A1) the nucleotides of the α-glucan phosphorylase gene, the glucose phosphomutase gene, the bifunctional enzyme glucose phosphomonomerase / mannose 6-phosphate isomerase gene, and the mannose 6-phosphate phosphatase gene are separately transferred into Escherichia coli to obtain four recombinant bacteria; the recombinant bacteria are induced to express respectively, and then permeabilized to obtain engineered bacteria.

[0013] Preferably, in the system for catalytically preparing D-mannose, the concentration of α-glucan phosphorylase is 0.5% to 3%, preferably 1%; the concentration of phosphoglucomutase is 0.5% to 3%, preferably 1%; the concentration of the bifunctional enzyme phosphoglucoisomerase / mannose 6-phosphate isomerase is 0.5% to 3%, preferably 2%; and the concentration of mannose 6-phosphate phosphatase is 1% to 8%, preferably 4%; wherein the % bacterial cells are mass percentages.

[0014] In one or more embodiments, the 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 turgidumThe gene number on Uniprot is B8E0A4; mannose 6-phosphate phosphatase is derived from Pseudothermogo hypogea The gene number on Uniprot is A0A0X1KRU1.

[0015] In one or more embodiments, the method for preparing an engineered bacterium expressing an isoamylase gene comprises: The nucleotides of the isoamylase gene are respectively transferred into Escherichia coli to obtain recombinant bacteria; the recombinant bacteria are induced to express, and then permeabilized to obtain engineered bacteria expressing the isoamylase gene.

[0016] In one or more embodiments, the isoamylase is derived from Pseudomonas amyloderamos The gene number on Uniprot is P10342.

[0017] In one or more embodiments, the method for preparing the engineered bacteria containing the polyphosphate glucokinase gene comprises: The nucleotides of the defined polyphosphate glucokinase gene are respectively transferred into Escherichia coli to obtain recombinant bacteria; the recombinant bacteria are induced to express, and then permeabilized to obtain engineered bacteria expressing the defined polyphosphate glucokinase gene.

[0018] In one or more embodiments, the polyphosphate glucokinase is derived from: Mycobacterium paratuberculosis , the gene number on Uniprot is Q73W41; Mycobacterium sinensis , the gene number on Uniprot is F5YW32; Mycobacterium bovis , the gene number on Uniprot is A0A0H3MD65; Gordonia polyisoprenivorans , the gene number on Uniprot is H6MZT0; Mycobacterium smegmatis , the gene number on Uniprot is I7G9D5; Thermobifida halotolerans , the gene number in KEGG is NI17_011080; Preferably, the polyphosphoglucokinase is derived from Thermobifida halotolerans The KEGG number of this gene is NI17_011080.

[0019] In one or more embodiments, the concentration of saccharifying enzyme is 0.8-1.2 U / mL, preferably 1 U / mL.

[0020] In one or more embodiments, the concentration of isoamylase is 0.1% to 2%, preferably 0.4%; the concentration of polyphosphate glucokinase is 0.1% to 2%, preferably 1%; wherein % bacterial cells is a mass percentage.

[0021] In one or more embodiments, the final concentration of sodium hexametaphosphate is 30-70 mM, preferably 40 mM.

[0022] The second aspect of the present invention provides a method for catalytically preparing D-mannose, wherein starch or starch derivatives are used as substrates, phosphate buffer solution and Mg are added to the catalytically prepared D-mannose. 2+ , after treating starch or starch derivatives with engineered bacteria expressing isoamylase genes; using engineered bacteria expressing α-glucan phosphorylase genes, glucose phosphomutase genes, bifunctional enzyme glucose phosphomonomerase / mannose 6-phosphate isomerase genes and mannose 6-phosphate phosphatase genes for preliminary catalysis, then adding saccharifying enzymes and engineered bacteria expressing limited polyphosphate glucokinase genes and sodium hexametaphosphate for further catalysis to prepare D-mannose.

[0023] In one or more embodiments, the temperature for treating starch or starch derivatives using engineered bacteria expressing the isoamylase gene is 70-85° C., preferably 80° C.; and the treatment time is 8-16 h, preferably 12 h.

[0024] In one or more embodiments, the catalytic reaction temperature is 40-80°C, preferably 65°C; the reaction time is 10-100 h, preferably 16-30 h.

[0025] The beneficial effects of the present invention are: (1) The catalytic preparation method of D-mannose provided in the present invention can effectively reduce the amount of by-product glucose and greatly improve the conversion rate of D-mannose. Specifically: using an engineered bacterium expressing an isoamylase gene and an engineered bacterium expressing an α-glucan phosphorylase gene, a glucose phosphomutase gene, a bifunctional enzyme glucose phosphoisomerase / mannose 6-phosphate isomerase gene and a mannose 6-phosphate phosphatase gene, starch or a starch derivative is used as a substrate, adding a phosphate buffer solution and Mg 2+ , a preliminary catalytic system was constructed. After the reaction was completed, there were unreacted substrates and maltobiose in the reaction system. The saccharifying enzyme could hydrolyze the unreacted substrates and maltobiose into glucose. The polyphosphate glucose kinase transferred the phosphate group in sodium hexametaphosphate to glucose, converting all the glucose into phosphorylated glucose, and then generating D-mannose, thereby increasing the yield and removing by-products in the system.

[0026] Polyphosphoglucokinase (PPGK) plays a crucial role in the mannose synthesis pathway. It specifically transfers the terminal phosphate group of sodium hexametaphosphate to a glucose molecule, generating glucose-6-phosphate, which then reenters the D-mannose synthesis pathway. During this process, PPGK significantly reduces the byproduct glucose content in the reaction, correspondingly increasing mannose yield. This effective reduction in glucose content is also crucial for the subsequent D-mannose separation and purification steps, simplifying the process and improving the purity of the final product. However, during research, the inventors discovered that, in addition to its glucose phosphorylation function, polyphosphoglucokinase also has a side effect: it can rephosphorylate some of the already generated D-mannose, thereby limiting the D-mannose conversion rate. Therefore, to improve the D-mannose conversion efficiency, they screened for polyphosphoglucokinases with higher D-mannose conversion efficiency.

[0027] (2) The method of the present invention for catalytically preparing D-mannose has the advantages of low cost, low pollution, high yield, and few by-products, and is suitable for industrial production of D-mannose. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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.

[0029] Figure 1 The liquid phase diagrams are of the mannose reaction solution after saccharification enzyme treatment without the addition of polyphosphoglucokinase and sodium hexametaphosphate, and the final product after treatment under optimal conditions. DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are exemplary and 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 meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0031] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0032] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0033] Glucoamylase was purchased from Aladdin, product number A107823; The solutes in phosphate buffer solution are potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4), and the pH is 7.0.

[0034] Example 1 Construction of engineered bacteria expressing α-glucan phosphorylase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphomonomerase / mannose 6-phosphate isomerase gene, and mannose 6-phosphate phosphatase gene: In this embodiment, α-glucan phosphorylase aGP is derived from Arabidopsis thaliana The gene number on Uniprot is Q9SD76; PGM-glucose phosphoglucomutase is derived from Thermococcus kodakarensis The gene number on Uniprot is Q68BJ6; the bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase PGI is derived from Dictyoglomus turgidum The gene number on Uniprot is B8E0A4; mannose 6-phosphate phosphatase M6PP is derived from Pseudothermogo hypogea The gene is numbered A0A0X1KRU1 on Uniprot. These genomic DNAs can be obtained from the official website of ATCC (www.atcc.org).

[0035] After codon optimization, the amino acid sequences were sent to Jiangsu Jinweizhi Biotechnology Co., Ltd. for gene synthesis and constructed into the expression vector pET-32a. The resulting recombinant expression plasmids, pET-aGP, pET-PGM, pET-PGI, and pET-M6PP, were then transformed into four competent E. coli BL21-Trxb(DE3) cells. The transformants were plated onto ampicillin-resistant LB solid medium plates and incubated overnight at 37°C (approximately 16 hours). Transformants were selected from the LB solid medium to obtain recombinant E. coli containing the recombinant plasmids. These recombinant E. coli strains express the genes for α-glucan phosphorylase, phosphoglucomutase, the bifunctional enzymes phosphogluconate isomerase / mannose 6-phosphate isomerase, and mannose 6-phosphate phosphatase, respectively.

[0036] (1) Pick a single clone and inoculate (pipette tip directly into the test tube) into a test tube containing 5 mL of LB liquid medium with resistance, and culture at 37°C, 200 rpm overnight (about 16 hours). Take 5 mL of the overnight culture seed liquid and transfer it to a shake flask containing 500 mL of LB liquid medium with resistance, and culture at 37°C, 200 rpm until the OD 600When the concentration reaches about 0.4-0.6 (about 3 h), cool down to 16 °C, add IPTG to a final concentration of 0.1 mM, and induce expression for 16 h.

[0037] (2) Centrifuge the overnight cultured cells at 5500 r / min for 15 min to collect the cells.

[0038] (3) Weigh and resuspend the bacterial sludge in a ratio of 0.2 g of bacterial cells to 1 mL of reaction buffer to obtain the engineered bacterial solution.

[0039] Example 2 Optimization of reaction temperature for the initial catalytic synthesis of D-mannose using engineered bacteria expressing α-glucan phosphorylase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphomonomerase / mannose 6-phosphate isomerase gene, and mannose 6-phosphate phosphatase gene: Thanks to the positive effect of high temperature on substrate solubility, the reaction proceeds in a positive direction as the reaction temperature gradually increases. However, excessively high temperatures can disrupt the enzyme's spatial structure and lead to enzyme inactivation. Therefore, it is necessary to find and maintain an optimal reaction temperature range.

[0040] The 1 mL reaction system involved in this example is as follows: starch concentration 150 g / L, phosphate buffer final concentration 10 mM, Mg 2+ The final concentration of aGP was 5 mM. aGP, PGM, PGI, and M6PP were the engineered bacteria prepared in Example 1. 100 μL of each was added and the mixture was made up to 1 mL with water.

[0041] The reaction system described in the above example was placed in a thermostatic reactor at 55°C, 60°C, 65°C, and 70°C for reaction. The rotation speed was set to 700 rpm. After 16 hours of reaction, samples were taken and analyzed by HPLC. The results are shown in Table 1. It was found that when the reaction temperature was 65°C, the D-mannose conversion rate was the highest, reaching 49.38%.

[0042] Table 1 D-mannose conversion rate at different temperatures

[0043] Example 3 Optimization of phosphate buffer concentration for the initial catalytic synthesis of D-mannose using engineered bacteria expressing α-glucan phosphorylase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphomonomerase / mannose 6-phosphate isomerase gene, and mannose 6-phosphate phosphatase gene: The 1 mL reaction system involved in this example is: starch concentration 150 g / L, Mg 2+The final concentration of was 5 mM, the final concentrations of phosphate buffer were 0, 5, 10, 20, 30, 50 and 100 mM, respectively. aGP, PGM, PGI and M6PP were the engineered bacterial solutions prepared in Example 1 above, 100 μL of each was added, and water was added to make up to 1 mL.

[0044] The reaction system described in the above example was placed in a thermostated reactor at 65°C and 200 rpm for 16 hours. Samples were then collected and analyzed by HPLC. The results are shown in Table 2. The yield of D-mannose initially increased and then decreased with increasing phosphate buffer concentration, with the optimal phosphate concentration being between 10 and 30 mM.

[0045] Table 2 Reaction results of different phosphate buffer concentrations

[0046] Example 4 The Mg2+ catalyzed synthesis of D-mannose by an engineered bacterium expressing α-glucan phosphorylase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphoisomerase / mannose 6-phosphate isomerase gene and mannose 6-phosphate phosphatase gene was performed. 2+ Concentration optimization: The 1 mL reaction system involved in this example is as follows: starch concentration 150 g / L, phosphate buffer final concentration 10 mM, Mg 2+ The final concentrations of aGP, PGM, PGI, and M6PP were 2, 5, 10, 20, 30, and 50 mM, respectively. 100 μL of each of aGP, PGM, PGI, and M6PP were the engineered bacterial solutions prepared in Example 1 above, and the mixture was filled to 1 mL with water.

[0047] The reaction system described in the above example was placed in a constant temperature reactor at 65°C for reaction, with the speed set at 200 rpm. After 16 hours of reaction, samples were taken and HPLC analysis was performed. The results are shown in Table 3. 2+ When the final concentration was 5 mM, the D-mannose conversion rate was the highest at 51.50%.

[0048] Table 3 Mg 2+ The reaction results corresponding to different concentrations

[0049] Example 5 The ratio of each enzyme when initially catalyzing the synthesis of D-mannose using an engineered bacterium expressing the α-glucan phosphorylase gene, the glucose phosphomutase gene, the bifunctional enzyme glucose phosphoisomerase / mannose 6-phosphate isomerase gene, and the mannose 6-phosphate phosphatase gene is as follows: The 1 mL reaction system involved in this example is as follows: starch concentration 150 g / L, final concentration of phosphate buffer 10 mM, Mg 2+ The final concentration of aGP is 5 mM, aGP, PGM, PGI, and M6PP are the engineered bacterial solutions prepared in Example 1, and water is added to make up to 1 mL.

[0050] The reaction system described in the above example was placed in a thermostated reactor at 65°C and 200 rpm for 16 hours. Samples were then collected and analyzed by HPLC. The results are shown in Table 4. When the addition levels of aGP were 1%, PGM was 1%, PGI was 2%, and M6PP was 4%, the highest D-mannose production rate was 54.84%, where % bacterial cells is percentage by mass.

[0051] Table 4 Reaction results of different enzyme ratios

[0052] Example 6 Construction of engineered bacteria expressing isoamylase gene, and optimization of substrate treatment temperature and time: (1) Isoamylase is an endo-amylase that can specifically cut the α-1,6-glycosidic bond at the branch point of amylose, cutting off the entire side chain to form amylose, which helps glucan phosphorylase to degrade starch more completely.

[0053] In this embodiment, isoamylase IA is derived from Pseudomonas amyloderamos The gene is registered on Uniprot as P10342. After codon optimization, the amino acid sequence was sent to Jiangsu Jinweizhi Biotechnology Co., Ltd. for gene synthesis and integration into the expression vector pET-32a, creating the recombinant expression vector pET32a-IA. The plasmid was transformed into Escherichia coli BL21-Trxb(DE3). The transformation product was plated onto ampicillin-resistant LB solid medium plates and incubated overnight at 37°C (approximately 16 hours). Transformants were harvested from the LB solid medium to obtain recombinant E. coli containing the recombinant plasmid, expressing the isoamylase gene.

[0054] Pick a single colony and inoculate (pipette tip directly into the test tube) into a test tube containing 5 mL of LB liquid medium with resistance. Culture overnight at 37°C and 200 rpm (about 16 hours). Take 5 mL of the overnight culture seed solution and transfer it to a shake flask containing 500 mL of LB liquid medium with resistance. Culture at 37°C and 200 rpm until the OD 600 When the concentration reaches about 0.4-0.6 (about 3 h), cool down to 16 °C, add IPTG to a final concentration of 0.1 mM, and induce expression for 16 h.

[0055] The overnight cultured cells were centrifuged at 5500 r / min for 15 min to collect the cells.

[0056] Weigh the bacterial slurry according to the ratio of 0.2 g of bacterial cells to 1 mL of reaction buffer and resuspend it.

[0057] (2) Substrate pretreatment system: starch concentration 400 g / L, isoamylase IA addition amount 4%, % is the mass fraction of the bacteria, substrates were treated at different temperatures for different time periods, and catalytic reactions were carried out respectively.

[0058] The reaction system for 1 mL was as follows: starch concentration treated with isoamylase IA was 150 g / L, phosphate buffer solution had a final concentration of 10 mM, Mg 2+ The final concentration of aGP was 5 mM. aGP, PGM, PGI, and M6PP were obtained from the engineered bacterial solution prepared in Example 1. The addition levels of aGP, PGM, PGI, and M6PP were 1%, 1%, 2%, and 4%, respectively, where % represents the percentage of bacterial mass. The suspension was then made up to 1 mL with water. The reaction was carried out in a thermostatic reactor at 65°C at 200 rpm. After 16 hours of reaction, samples were collected and analyzed by HPLC. The results are shown in Table 5. The substrate, treated with isoamylase IA at 80°C for 12 hours, achieved the highest D-mannose conversion rate of 72.36%.

[0059] Table 5 Effects of isoamylase treatment at different temperatures and for different durations

[0060] Example 7 α-1,4-Glucoamylase, also known as glucoamylase, is scientifically known as α-1,4-glucanglucohydrolase. Add 1 U / mL of saccharifying enzyme to the catalytic reaction solution in Example 6 to hydrolyze the unreacted substrate and maltobiose into glucose. Polyphosphate glucokinase converts all glucose into phosphorylated glucose, which in turn produces D-mannose. This improves yield while removing byproducts from the system.

[0061] Screening of polyphosphoglucokinase PPGK: In this embodiment, the polyphosphate glucose kinase is derived from: Mycobacterium paratuberculosis , the gene number on Uniprot is Q73W41; Mycobacterium sinensis , the gene number on Uniprot is F5YW32; Mycobacterium bovis , the gene number on Uniprot is A0A0H3MD65; Gordonia polyisoprenivorans , the gene number on Uniprot is H6MZT0; Mycobacterium smegmatis , the gene number on Uniprot is I7G9D5; Thermobifida halotolerans , the gene number in KEGG is NI17_011080; The above genes were used to construct recombinant expression vectors pET32a-PPGK (Q73W41 / F5YW32 / A0A0H3MD65 / H6MZT0 / I7G9D5 / NI17_011080). Each plasmid was transformed into Escherichia coli BL21-Trxb(DE3), and fermented to obtain engineered bacteria expressing the corresponding enzymes. The engineered bacterial culture was prepared as described in Example 1.

[0062] The reaction system for 1 mL was as follows: 500 μL of mannose reaction solution after glucoamylase treatment, phosphate buffer solution with a final concentration of 10 mM, Mg 2+ The final concentration of 5 mM sodium hexametaphosphate, 60 mM sodium hexametaphosphate, and 1% PPGK were added, where % represents the percentage of bacterial cells by mass. The mixture was made up to 1 mL with water. The reaction was carried out in a thermostated reactor at 65°C and 200 rpm. After 24 hours of reaction, samples were collected and analyzed by HPLC. The results are shown in Table 6. KEGG: NI17_011080 enzyme effectively reduced glucose conversion and increased mannose conversion.

[0063] Table 6 Results of reactions catalyzed by different polyphosphoglucokinases (PPGK)

[0064] Example 8 Optimization of polyphosphoglucokinase (PPGK) enzyme dosage If the amount of PPGK added is insufficient, glucose cannot be completely removed; if the amount of PPGK added is too high, the D-mannose yield will be reduced.

[0065] The reaction system for 1 mL was as follows: 500 μL of mannose reaction solution after glucoamylase treatment, phosphate buffer solution with a final concentration of 10 mM, Mg 2+ The final concentration of 5 mM sodium hexametaphosphate was 60 mM. PPGK was added at 0.5, 1, 1.5, 2, 2.5, and 3% concentrations, respectively, where % represents mass percentage. The mixture was made up to 1 mL with water. The reaction was carried out in a thermostated reactor at 65°C and 200 rpm. After 8 hours of reaction, samples were collected and analyzed by HPLC. The results are shown in Table 7. A 1.5% PPGK addition achieved complete glucose removal and achieved the highest mannose conversion rate of 91.75%.

[0066] Table 7 Reaction results of different addition amounts of PPGK

[0067] Example 9 Optimization of the amount of sodium hexametaphosphate added If the amount of sodium hexametaphosphate added is insufficient, glucose cannot be completely removed; if the amount of sodium hexametaphosphate added is too high, the D-mannose yield will be reduced.

[0068] 1 mL reaction system: 500 μl of mannose reaction solution after glucoamylase treatment, phosphate buffer solution with a final concentration of 10 mM, Mg 2+ The final concentration of sodium hexametaphosphate was 5 mM, the amount of PPGK added was 1.5%, where % represents mass percentage. The final concentrations of sodium hexametaphosphate were 30, 40, 50, 60, and 70 mM, respectively. Water was added to 1 mL. The reaction was carried out in a thermostated reactor at 65°C and 200 rpm. After 8 hours of reaction, samples were collected and analyzed by HPLC. The results are shown in Table 8. A final sodium hexametaphosphate concentration of 40 mM completely removed glucose and achieved the highest mannose conversion rate.

[0069] Table 8 Reaction results of different addition amounts of sodium hexametaphosphate

[0070] Based on the optimal type of polyphosphate glucokinase, optimal amount of polyphosphate glucokinase added, and optimal amount of sodium hexametaphosphate added defined in Examples 7, 8, and 9, HPLC detection was performed on the mannose reaction solution after glucoamylase treatment without the addition of polyphosphate glucokinase and sodium hexametaphosphate, and the final product after treatment under the optimal conditions. The results were as follows: Figure 1 As shown, from Figure 1 It can be seen that polyphosphate glucose kinase converts all glucose into phosphorylated glucose by transferring the phosphate group in sodium hexametaphosphate to glucose, thereby generating D-mannose, thereby increasing the yield and removing the by-product glucose in the system.

[0071] 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 system for catalytically preparing D-mannose, characterized in that: include: Phosphate buffer solution, starch or starch derivatives, Mg 2+ As well as engineered bacteria expressing α-glucan phosphorylase (aGP) gene, glucose phosphomutase (PGM) gene, bifunctional enzyme glucose phosphomonomerase / mannose 6-phosphate isomerase (PGI) gene and mannose 6-phosphate phosphatase (M6PP) gene; engineered bacteria expressing isoamylase (IA) gene, saccharifying enzyme, engineered bacteria expressing defined polyphosphate glucokinase (PPGK) gene and sodium hexametaphosphate.

2. The system according to claim 1, wherein The solutes in the phosphate buffer solution are potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4), with a pH of 6.8 to 7.2, preferably 7.0; the concentration of the solutes in the phosphate buffer solution is 1 to 100 mM, preferably 10 mM; Alternatively, the starch derivative comprises one or more of partially hydrolyzed starch, starch dextrin, maltodextrin, maltopolysaccharide or maltose.

3. The system according to claim 1, wherein: The concentration of the starch is 1-500 g / L, preferably 150 g / L; Or, the Mg 2+ The concentration is 1~50 mM, preferably 5 mM.

4. The system according to claim 1, wherein: The method for preparing an engineered bacterium expressing an α-glucan phosphorylase gene, a phosphoglucomutase gene, a bifunctional enzyme phosphoglucoisomerase / mannose 6-phosphate isomerase gene, and a mannose 6-phosphate phosphatase gene is as shown in any one of A1) to A5) below: A1) nucleotide sequences of the α-glucan phosphorylase gene, the phosphoglucomutase gene, the bifunctional enzyme phosphoglucoisomerase / mannose 6-phosphate isomerase gene, and the mannose 6-phosphate phosphatase gene were separately transferred into Escherichia coli to obtain four recombinant strains; the recombinant strains were induced for expression and then permeabilized to obtain engineered strains; A2) co-transforming the nucleotide sequences of the α-glucan phosphorylase gene, the phosphoglucomutase gene, the bifunctional enzyme phosphoglucoisomerase / mannose 6-phosphate isomerase gene, and the mannose 6-phosphate phosphatase gene into the same Escherichia coli strain in any permutation and combination to obtain a recombinant strain; inducing expression in the recombinant strain, and then permeabilizing the strain to obtain an engineered strain; A3) co-transforming any two nucleotides from the α-glucan phosphorylase gene, the phosphoglucomutase gene, the bifunctional enzyme phosphogluconate isomerase / mannose 6-phosphate isomerase gene, and the mannose 6-phosphate phosphatase gene into one strain of Escherichia coli in any permutation and combination, and co-transforming the remaining two nucleotides into another strain of Escherichia coli in any permutation and combination, to obtain two recombinant strains; inducing expression in each recombinant strain, and then permeabilizing the recombinant strains to obtain engineered strains; A4) co-transferring any two nucleotides from the α-glucan phosphorylase gene, the phosphoglucomutase gene, the bifunctional enzyme phosphoglucoisomerase / mannose 6-phosphate isomerase gene, and the mannose 6-phosphate phosphatase gene into one strain of Escherichia coli in any permutation and combination, and transferring the remaining two nucleotides into two other strains of Escherichia coli to obtain three recombinant strains; inducing expression in each of the recombinant strains, and then permeabilizing them to obtain engineered strains; A5) transferring any three nucleotides from the α-glucan phosphorylase gene, the phosphoglucomutase gene, the bifunctional enzyme phosphoglucoisomerase / mannose 6-phosphate isomerase gene, and the mannose 6-phosphate phosphatase gene into one strain of Escherichia coli in any permutation and combination, and transferring the remaining nucleotide into another strain of Escherichia coli to obtain two recombinant strains; inducing expression in each recombinant strain, and then permeabilizing the recombinant strain to obtain an engineered strain; Preferably: A1) the nucleotides of the α-glucan phosphorylase gene, the glucose phosphomutase gene, the bifunctional enzyme glucose phosphomonomerase / mannose 6-phosphate isomerase gene, and the mannose 6-phosphate phosphatase gene are separately transferred into Escherichia coli to obtain four recombinant bacteria; the recombinant bacteria are induced to express respectively, and then permeabilized to obtain engineered bacteria.

5. The system according to claim 4, wherein: The concentration of α-glucan phosphorylase is 0.5% to 3%, preferably 1%; the concentration of phosphoglucomutase is 0.5% to 3%, preferably 1%; the concentration of the bifunctional enzyme phosphoglucoisomerase / mannose 6-phosphate isomerase is 0.5% to 3%, preferably 2%; the concentration of mannose 6-phosphate phosphatase is 1% to 8%, preferably 4%; wherein the % bacterial cell is a mass percentage.

6. The system according to claim 1, wherein: 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.

7. The system according to claim 1, wherein: The preparation method of the engineered bacteria expressing the isoamylase gene comprises: The nucleotides of the isoamylase gene are respectively transferred into Escherichia coli to obtain recombinant bacteria; the recombinant bacteria are induced to express, and then permeabilized to obtain engineered bacteria expressing the isoamylase gene; Or, isoamylase is derived from Pseudomonas amyloderamosa , the gene number on Uniprot is P10342; Alternatively, the method for preparing the engineered bacteria containing the polyphosphate glucokinase gene comprises: The nucleotides of the polyphosphate glucokinase gene are respectively transferred into Escherichia coli to obtain recombinant bacteria; the recombinant bacteria are induced to express, and then permeabilized to obtain engineered bacteria expressing the polyphosphate glucokinase gene; or, polyphosphoglucokinase, respectively, from: Mycolicibacterium paratuberculosis , the gene number on Uniprot is Q73W41; Mycolicibacter sinensis , the gene number on Uniprot is F5YW32; Mycobacterium bovis , the gene number on Uniprot is A0A0H3MD65; Gordonia polyisoprenivorans , the gene number on Uniprot is H6MZT0; Mycolicibacterium smegmatis , the gene number on Uniprot is I7G9D5; Thermobifida halotolerans , the gene number in KEGG is NI17_011080; Preferably, the polyphosphoglucokinase is derived from Thermobifida halotolerans The KEGG number of this gene is NI17_011080.

8. The system according to claim 1, wherein: The concentration of saccharifying enzyme is 0.8~1.2 U / mL, preferably 1 U / mL; Alternatively, the concentration of isoamylase is 0.1% to 2%, preferably 0.4%; the concentration of polyphosphate glucokinase is 0.1% to 2%, preferably 1%; wherein % bacterial cells are percentage by mass; Alternatively, the final concentration of the sodium hexametaphosphate is 30-70 mM, preferably 40 mM.

9. A method for catalytically preparing D-mannose, characterized in that: Starch or starch derivatives were used as substrates, and phosphate buffer solution and Mg were added. 2+ , after treating starch or starch derivatives with engineered bacteria expressing isoamylase genes; using engineered bacteria expressing α-glucan phosphorylase genes, glucose phosphomutase genes, bifunctional enzyme glucose phosphomonomerase / mannose 6-phosphate isomerase genes and mannose 6-phosphate phosphatase genes for preliminary catalysis, then adding saccharifying enzymes and engineered bacteria expressing limited polyphosphate glucokinase genes and sodium hexametaphosphate for further catalysis to prepare D-mannose.

10. The method according to claim 9, wherein The temperature for treating starch or starch derivatives with the engineered bacteria expressing the isoamylase gene is 70-85°C, preferably 80°C; the treatment time is 8-16 hours, preferably 12 hours; Alternatively, the catalytic reaction temperature is 40-80°C, preferably 65°C; the reaction time is 10-100 h, preferably 16-30 h.

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