A process for the catalytic preparation of D-mannose

Abstract

The method for catalytically preparing D-mannose provided in the application can effectively reduce the amount of by-products glucose and fructose, and greatly improve the conversion rate of D-mannose. Specifically, the engineering bacteria expressing isoamylase gene and the engineering bacteria expressing alpha-glucan phosphorylase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene and mannose 6-phosphate phosphatase gene are used, starch or starch derivatives are used as substrates, phosphate buffer solution and Mg 2+ A preliminary catalytic system is constructed, and after the reaction is completed, there are unreacted substrates and maltodextrin in the reaction system, the saccharifying enzyme can hydrolyze the unreacted substrates and maltodextrin into glucose, the polyphosphate glucose kinase can convert all the glucose into phosphorylated glucose with the assistance of sodium hexametaphosphate, and then D-mannose is generated, thereby improving the yield and removing the by-products in the system.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for the catalytic preparation of D-mannose. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] D-Mannose is a common six-carbon monosaccharide, an isomer of glucose, and is a white crystalline or powdery substance with a sweet and slightly bitter taste. It is readily soluble in water but sparingly soluble in ethanol. It rarely exists in nature in its free state, mostly existing as a polysaccharide. D-Mannose is a functional rare sugar with important applications in biology and medicine. It not only plays a role in regulating immunity, resisting bacterial and viral invasion, and promoting the normal synthesis of glycoproteins in the body, but it can also be used as a raw material to synthesize some pharmaceutical and chemical raw materials and important precursors for sugar-based drugs, such as immunostimulants, anti-tumor drugs, vitamins, and D-mannitol. In recent years, the demand for mannose in functional foods, feed additives, and pharmaceuticals has increased significantly, highlighting its economic development value.

[0004] Currently, the main methods for preparing D-mannose include three approaches: plant extraction, chemical isomerization, and enzymatic methods. Plant extraction, primarily using direct extraction from fruits and plants, is widely applied. Chemical isomerization uses molybdic acid or molybdate as a catalyst to isomerize glucose into D-mannose under acidic, high-temperature, and high-pressure conditions. However, due to limitations in raw materials, harsh reaction conditions, complex processes, difficulties in downstream purification, and low conversion rates, both extraction and chemical methods have limitations and cannot meet the demands of industrial production. Enzymatic methods for preparing D-mannose are gradually becoming a future research trend. In the process of producing mannose using enzyme catalysis and other biochemical methods, glucose and fructose are the main byproducts, increasing the technical challenges of subsequent product separation and purification. Especially in the subsequent product separation process, to ensure that the purity and quality of the mannose product meet predetermined standards, more precise and complex separation techniques must be employed. Therefore, reducing the amount of byproducts glucose and fructose has become one of the core problems that urgently needs to be solved in the industrial production of mannose. Summary of the Invention

[0005] To overcome the above problems, the present invention provides a method for catalytic preparation of D-mannose.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention provides a system for the catalytic preparation of D-mannose, comprising: a phosphate buffer solution, starch or a starch derivative, and Mg 2+ Engineered bacteria expressing the α-glucan phosphorylase (aGP) gene, glucose phosphate mutase (PGM) gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase (PGI) gene, and mannose 6-phosphate phosphatase (M6PP) gene; engineered bacteria expressing the isoamylase (IA) gene, saccharifying enzyme, engineered bacteria expressing the limited polyphosphoglucose kinase (PPGK) gene, and sodium hexametaphosphate.

[0008] In one or more embodiments, the solute in the phosphate buffer solution is 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 1~100mM, preferably 10 mM.

[0009] In one or more embodiments, the starch derivative includes one or more of partially hydrolyzed starch, starch dextrin, maltodextrin, malt polysaccharide, or maltose.

[0010] In one or more embodiments, the concentration of the starch is 1 to 500 g / L, preferably 150 g / L.

[0011] In one or more embodiments, the Mg 2+ The concentration is 1~50 mM, preferably 5 mM.

[0012] In one or more embodiments, the method for preparing engineered bacteria expressing the α-glucan phosphorylase gene, glucose phosphate mutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene, and mannose 6-phosphate phosphatase gene is any one of the following A1)-A5):

[0013] A1) The nucleotides of the α-glucan phosphorylase gene, glucose phosphate mutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene, and mannose 6-phosphate phosphatase gene were respectively transferred into Escherichia coli to obtain 4 recombinant bacteria; the recombinant bacteria were induced to express and then permeabilized to obtain engineered bacteria.

[0014] A2) The nucleotides of the α-glucan phosphorylase gene, glucose phosphate mutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene, and mannose 6-phosphate phosphatase gene were co-transformed into the same strain of Escherichia coli in any arbitrary combination to obtain a recombinant strain; the recombinant strain was induced to express and then permeabilized to obtain engineered bacteria.

[0015] A3) Two nucleotides from the α-glucan phosphorylase gene, glucose phosphate mutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene, and mannose 6-phosphate phosphatase gene were co-transformed into one strain of *E. coli* in any combination, and the remaining two nucleotides were co-transformed into another strain of *E. coli* in any combination to obtain two recombinant bacteria; the recombinant bacteria were induced to express and then permeabilized to obtain engineered bacteria;

[0016] A4) Any two nucleotides from the α-glucan phosphorylase gene, glucose phosphate mutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene, and mannose 6-phosphate phosphatase gene were co-transformed into one strain of *E. coli* in any combination. The remaining two nucleotides were transformed into two other strains of *E. coli*, respectively, to obtain three recombinant bacteria. The recombinant bacteria were induced to express their contents and then permeabilized to obtain engineered bacteria.

[0017] A5) Any three nucleotides from the α-glucan phosphorylase gene, glucose phosphate mutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene, and mannose 6-phosphate phosphatase gene were co-transformed into one strain of Escherichia coli in any combination, and the remaining nucleotide was transformed into another strain of Escherichia coli to obtain two recombinant bacteria; the recombinant bacteria were induced to express and then permeabilized to obtain engineered bacteria.

[0018] Preferably, the following method is used: A1) The nucleotides of the α-glucan phosphorylase gene, glucose phosphate mutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene and mannose 6-phosphate phosphatase gene are respectively transferred into Escherichia coli to obtain 4 recombinant bacteria; the recombinant bacteria are induced to express and then permeabilized to obtain engineered bacteria.

[0019] Preferably, in the system for catalytic preparation of D-mannose, the concentration of α-glucan phosphorylase is 0.5%~3%, preferably 1%; the concentration of glucose phosphate mutase is 0.5%~3%, preferably 1%; the concentration of the bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase is 0.5%~3%, preferably 2%; and the concentration of mannose 6-phosphate phosphatase is 1%~8%, preferably 4%; wherein % cell count is a percentage by mass.

[0020] In one or more embodiments, α-glucan phosphorylase is derived from... Arabidopsis thaliana The gene is numbered Q9SD76 on Uniprot; glucose-phosphoryltransferase originates from Thermococcus kodakarensisThe gene's Uniprot number is Q68BJ6; the bifunctional enzyme glucose-6-phosphate isomerase / mannose-6-phosphate isomerase originates from... Dictyoglomus turgidum The gene's serial number on Uniprot is B8E0A4; mannose-6-phosphate phosphatase originates from... Pseudothermogo hypogea The gene is numbered A0A0X1KRU1 on Uniprot.

[0021] In one or more embodiments, the method for preparing engineered bacteria expressing the isoamylase gene includes:

[0022] The nucleotides of the isoamylase gene were transferred into Escherichia coli to obtain recombinant bacteria; the recombinant bacteria were induced to express the gene and then permeabilized to obtain engineered bacteria expressing the isoamylase gene.

[0023] In one or more embodiments, isoamylase is derived from Pseudomonas amyloderamos The gene is numbered P10342 on Uniprot.

[0024] In one or more embodiments, the method for preparing the engineered bacteria defining the polyphosphoglucosamine gene includes:

[0025] The nucleotides defining the polyphosphoglucosamine gene were transferred into Escherichia coli to obtain recombinant bacteria. The recombinant bacteria were then induced to express the gene and then permeabilized to obtain engineered bacteria expressing the polyphosphoglucosamine gene.

[0026] In one or more embodiments, polyphosphoglucoskinase is derived from:

[0027] Mycobacterium paratuberculosis The gene is numbered Q73W41 on Uniprot.

[0028] Mycobacterium sinensis The gene is numbered F5YW32 on Uniprot.

[0029] Mycobacterium bovis The gene's serial number on Uniprot is A0A0H3MD65;

[0030] Gordonia polyisoprenivorans The gene is numbered H6MZT0 on Uniprot.

[0031] Mycobacterium smegmatis The gene is numbered I7G9D5 on Uniprot.

[0032] Thermobifida halotolerans The gene is numbered NI17_011080 on KEGG.

[0033] Preferably, the polyphosphoglucoskinase is derived from Thermobifida halotolerans The gene is numbered NI17_011080 on KEGG.

[0034] In one or more embodiments, the concentration of the saccharifying enzyme is 0.8~1.2 U / mL, preferably 1 U / mL.

[0035] In one or more embodiments, the concentration of isoamylase is 0.1% to 2%, preferably 0.4%; the concentration of polyphosphoglucosidase is 0.1% to 2%, preferably 1%; wherein % bacterial cells is a mass percentage.

[0036] In one or more embodiments, the final concentration of sodium hexametaphosphate is 30-70 mM, preferably 40 mM.

[0037] A second aspect of the present invention provides a method for the catalytic preparation of D-mannose, using starch or a starch derivative as a substrate, adding a phosphate buffer solution and Mg 2+ Starch or starch derivatives were treated with engineered bacteria expressing isoamylase genes; D-mannose was prepared by preliminary catalysis using engineered bacteria expressing α-glucan phosphorylase genes, glucose phosphate mutase genes, bifunctional enzymes glucose phosphate isomerase / mannose 6-phosphate isomerase genes, and mannose 6-phosphate phosphatase genes; then, saccharifying enzymes, engineered bacteria expressing limited polyphosphoglucose kinase genes, and sodium hexametaphosphate were added for further catalysis.

[0038] In one or more embodiments, the temperature for treating starch or starch derivatives with engineered bacteria expressing the isoamylase gene is 70-85 °C, preferably 80 °C; and the treatment time is 8-16 h, preferably 12 h.

[0039] In one or more embodiments, the catalytic reaction temperature is 40~80 ℃, preferably 65 ℃; the reaction time is 10~100 h, preferably 16~30 h.

[0040] The beneficial effects of this invention are as follows:

[0041] (1) The method for catalytically preparing D-mannose provided in this invention can effectively reduce the amount of glucose by-product and greatly improve the conversion rate of D-mannose. Specifically: using engineered bacteria expressing isoamylase genes and engineered bacteria expressing α-glucan phosphorylase genes, glucose phosphate mutase genes, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase genes, and mannose 6-phosphate phosphatase genes, starch or starch derivatives are used as substrates, and phosphate buffer solution and Mg2+ are added. 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. Polyphosphoglucokinase converted all glucose into phosphorylated glucose by transferring the phosphate group in sodium hexametaphosphate to glucose, thereby generating D-mannose, which improved the yield and removed byproducts in the system.

[0042] Polyphosphoglucokinase (PPGK) plays a crucial role in the mannose synthesis pathway. It specifically transfers the terminal phosphate group of sodium hexametaphosphate to glucose molecules, generating glucose-6-phosphate, which then re-enters the D-mannose synthesis pathway. In this process, PPGK significantly reduces the content of glucose as a byproduct, thereby increasing mannose yield. This effective reduction in glucose content is also essential for subsequent D-mannose separation and purification steps, as it simplifies the process and improves the purity of the final product. However, the inventors discovered a side effect during their research: PPGK, in addition to its glucose phosphorylation function, can re-phosphorylate some of the already generated D-mannose, thus limiting the conversion rate. Therefore, to improve D-mannose conversion efficiency, PPGK with higher conversion efficiency was screened for.

[0043] (2) The method of preparing D-mannose by catalysis in this invention has the advantages of low cost, low pollution, high yield and few by-products, and is suitable for industrial production of D-mannose. Attached Figure Description

[0044] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0045] Figure 1 The images show the liquid phase diagrams of the mannose reaction solution after treatment with glucokinase without the addition of polyphosphoglucosamine and sodium hexametaphosphate, and the final product after treatment under optimal conditions. Detailed Implementation

[0046] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0047] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0048] To enable those skilled in the art to better 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.

[0049] The saccharifying enzyme was purchased from Aladdin, product code A107823;

[0050] The solutes in the phosphate buffer solution are potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4), and the pH is 7.0.

[0051] Example 1

[0052] Construction of engineered bacteria expressing α-glucan phosphorylase gene, glucose phosphate mutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene, and mannose 6-phosphate phosphatase gene:

[0053] In this embodiment, α-glucan phosphorylase aGP is derived from... Arabidopsis thaliana The gene is numbered Q9SD76 on Uniprot; glucose-phosphoryltransferase PGM originates from... Thermococcus kodakarensis The gene's Uniprot number is Q68BJ6; the bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase PGI is derived from... Dictyoglomus turgidum The gene's serial number on Uniprot is B8E0A4; mannose-6-phosphate phosphatase M6PP originates from... Pseudothermogo hypogea The gene is designated A0A0X1KRU1 on Uniprot. These genomic DNA sequences are available from the ATCC website (www.atcc.org).

[0054] After codon optimization, the above amino acid sequences were sent to Jiangsu Genewise Biotechnology Co., Ltd. for gene synthesis and constructed into the expression vector pET-32a, yielding the recombinant expression plasmids pET-aGP, pET-PGM, pET-PGI, and pET-M6PP. These plasmids were then transformed into four E. coli BL21-Trxb(DE3) competent cells. The transformation products were plated on ampicillin-resistant LB agar plates and cultured overnight at 37 °C (approximately 16 h). Transformants were picked from the LB agar plates to obtain recombinant E. coli containing the recombinant plasmids, namely, engineered bacteria expressing α-glucan phosphorylase gene, engineered bacteria expressing glucose phosphate mutase gene, engineered bacteria expressing the bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene, and engineered bacteria expressing mannose 6-phosphate phosphatase gene.

[0055] (1) Pick a single colony and inoculate it (directly inject the pipette tip into the test tube) into a test tube containing 5 mL of LB liquid medium with antibiotic resistance. Incubate overnight at 37 °C and 200 rpm (approximately 16 h). Take 5 mL of the overnight cultured seed culture and transfer it to a shake flask containing 500 mL of LB liquid medium with antibiotic resistance. Incubate at 37 °C and 200 rpm until OD (outlet count) is reached. 600 Once the concentration reaches approximately 0.4–0.6 (about 3 h), cool to 16 °C, add IPTG to a final concentration of 0.1 mM, and induce expression for 16 h.

[0056] (2) Centrifuge the overnight cultured cells at 5500 r / min for 15 min and collect the cells.

[0057] (3) Weigh the bacterial sludge according to the ratio of 0.2 g of bacterial cells to 1 mL of reaction buffer and resuspend it to obtain the engineered bacterial solution.

[0058] Example 2

[0059] Optimization of reaction temperature for the preliminary catalytic synthesis of D-mannose using engineered bacteria expressing α-glucan phosphorylase gene, glucose phosphate mutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene, and mannose 6-phosphate phosphatase gene:

[0060] Thanks to the positive effect of high temperature on substrate solubilization, gradually increasing the reaction temperature favors the forward reaction. However, excessively high temperatures may damage the enzyme's spatial structure, leading to enzyme inactivation. Therefore, it is necessary to find and maintain an optimal reaction temperature range.

[0061] The 1 mL reaction system involved in this embodiment is as follows: starch concentration 150 g / L, phosphate buffer final concentration 10 mM, Mg 2+The final concentration was 5 mM. aGP, PGM, PGI, and M6PP were engineered bacteria prepared in Example 1 above. 100 μL of each was added, and water was added to make up to 1 mL.

[0062] The reaction system described in the above embodiments was placed in constant temperature reactors at 55 ℃, 60 ℃, 65 ℃ and 70 ℃ for reaction. The rotation speed was set to 700 rpm. After 16 h of reaction, samples were taken and analyzed by HPLC. The results are shown in Table 1. It was found that the D-mannose conversion rate was the highest at 65 ℃, which was 49.38%.

[0063] Table 1. D-mannose conversion rate at different temperatures

[0064]

[0065] Example 3

[0066] Optimization of phosphate buffer concentration during the initial catalytic synthesis of D-mannose using engineered bacteria expressing α-glucan phosphorylase gene, glucose phosphate mutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene, and mannose 6-phosphate phosphatase gene:

[0067] The 1 mL reaction system involved in this embodiment is: starch concentration 150 g / L, Mg 2+ The final concentration of the phosphate buffer was 5 mM, and the final concentrations of the phosphate buffer were 0, 5, 10, 20, 30, 50 and 100 mM, respectively. aGP, PGM, PGI and M6PP were the engineered bacterial cultures prepared in Example 1 above. 100 μL of each was added and water was added to make up to 1 mL.

[0068] The reaction system described in the above examples was placed in a constant-temperature reactor at 65 ℃ and the rotation speed was set to 200 rpm. After 16 h of reaction, samples were taken and analyzed by HPLC. The results are shown in Table 2. It was found that the formation rate of D-mannose first increased and then decreased with the increase of phosphate buffer concentration, with 10~30 mM being the optimal phosphate concentration.

[0069] Table 2. Reaction results at different phosphate buffer concentrations

[0070]

[0071] Example 4

[0072] When engineered bacteria expressing α-glucan phosphorylase gene, glucose phosphate mutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene, and mannose 6-phosphate phosphatase gene were used to initially catalyze the synthesis of D-mannose, Mg 2+ Concentration optimization:

[0073] The 1 mL reaction system involved in this embodiment is as follows: starch concentration 150 g / L, phosphate buffer final concentration 10 mM, Mg 2+ The final concentrations were 2, 5, 10, 20, 30 and 50 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.

[0074] The reaction system described in the above embodiments was placed in a constant-temperature reactor at 65 ℃ and the rotation speed was set to 200 rpm. After 16 h of reaction, samples were taken and analyzed by HPLC. The results are shown in Table 3, and Mg was found to be present. 2+ At a final concentration of 5 mM, the D-mannose conversion rate was the highest at 51.50%.

[0075] Table 3 Mg 2+ Reaction results corresponding to different concentrations

[0076]

[0077] Example 5

[0078] The enzyme ratio for the initial catalytic synthesis of D-mannose using engineered bacteria expressing α-glucan phosphorylase gene, glucose phosphate mutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene, and mannose 6-phosphate phosphatase gene was as follows:

[0079] The 1 mL reaction system involved in this embodiment is as follows: starch concentration 150 g / L, phosphate buffer final concentration 10 mM, Mg 2+ The final concentration was 5 mM. aGP, PGM, PGI, and M6PP were the engineered bacterial solutions prepared in Example 1 above. Water was added to make up to 1 mL.

[0080] The reaction system described in the above examples was placed in a constant temperature reactor at 65 ℃ and the rotation speed was set to 200 rpm. After 16 h of reaction, samples were taken and analyzed by HPLC. The results are shown in Table 4. It was found that when the aGP addition amount was 1%, the PGM addition amount was 1%, the PGI addition amount was 2%, and the M6PP addition amount was 4%, the D-mannose production rate was the highest at 54.84% (% cell count by mass).

[0081] Table 4. Reaction results with different enzyme ratios

[0082]

[0083] Example 6

[0084] Construction of engineered bacteria expressing isoamylase genes, and optimization of substrate treatment temperature and time:

[0085] (1) Isoamylase is an endoamylase that can specifically cleave the α-1,6-glycosidic bonds at the branch points of branched starch, cutting off the entire side chain to form amylose, which helps glucan phosphorylase to degrade starch more completely.

[0086] In this embodiment, isoamylase IA is derived from Pseudomonas amyloderamos The gene, numbered P10342 on Uniprot, was sent to Jiangsu Genewise Biotechnology Co., Ltd. for gene synthesis after codon optimization. The gene was then constructed into the expression vector pET-32a, forming the recombinant expression vector pET32a-IA. The plasmid was transformed into *E. coli* BL21-Trxb(DE3), and the transformation product was plated on ampicillin-resistant LB agar plates and incubated overnight at 37 °C (approximately 16 h). Transformants were picked from the LB agar plates to obtain recombinant *E. coli* containing the recombinant plasmid, which is an engineered bacterium expressing the isoamylase gene.

[0087] Select a single colony and inoculate it (directly inject the colony into a test tube) into a test tube containing 5 mL of LB liquid medium with antibiotic resistance. Incubate overnight (approximately 16 h) at 37 °C and 200 rpm. Take 5 mL of the overnight culture and transfer it to a shake flask containing 500 mL of LB liquid medium with antibiotic resistance. Incubate at 37 °C and 200 rpm until OD (outlet count) reaches 100%. 600 Once the concentration reaches approximately 0.4–0.6 (about 3 h), cool to 16 °C, add IPTG to a final concentration of 0.1 mM, and induce expression for 16 h.

[0088] Collect the bacterial cells after overnight culture by centrifuging at 5500 r / min for 15 min.

[0089] Weigh out the bacterial sludge according to the ratio of 0.2 g of bacterial cells to 1 mL of reaction buffer and resuspend it.

[0090] (2) Substrate pretreatment system: starch concentration 400 g / L, isoamylase IA added 4%, % is the mass fraction of the cells, substrates were treated at different temperatures for different durations and catalytic reactions were carried out respectively.

[0091] The 1 mL reaction system consisted of: starch treated with isoamylase IA at a concentration of 150 g / L, phosphate buffer at a final concentration of 10 mM, and Mg... 2+The final concentration was 5 mM. aGP, PGM, PGI, and M6PP were the engineered bacterial cultures prepared in Example 1 above. When the addition amounts of aGP were 1%, PGM were 1%, PGI were 2%, and M6PP were 4%, the percentages (% represents the percentage of bacterial mass) were added, and water was added to make up to 1 mL. The reaction was carried out in a 65 ℃ constant temperature reactor with a rotation speed of 200 rpm. After 16 h of reaction, samples were taken and analyzed by HPLC. The results are shown in Table 5. The highest D-mannose conversion rate was 72.36% when the substrate was treated with isoamylase IA at 80 ℃ for 12 h for catalytic reaction.

[0092] Table 5. Effects of isoamylase treatment at different temperatures and for different durations.

[0093]

[0094] Example 7

[0095] Glycoamylase, also known as glucoamylase, is scientifically named α-1,4-glucanglucohydrolase. In the catalytic reaction solution of Example 6, 1 U / mL was added to hydrolyze the unreacted substrate and maltobiose into glucose. Polyphosphoglucokinase then converted all the glucose into phosphorylated glucose, thereby generating D-mannose, increasing the yield while removing byproducts from the system.

[0096] Screening for polyphosphoglucosinolate kinase (PPGK):

[0097] In this embodiment, the polyphosphoglucoskinase is derived from:

[0098] Mycobacterium paratuberculosis The gene is numbered Q73W41 on Uniprot.

[0099] Mycobacterium sinensis The gene is numbered F5YW32 on Uniprot.

[0100] Mycobacterium bovis The gene's serial number on Uniprot is A0A0H3MD65;

[0101] Gordonia polyisoprenivorans The gene is numbered H6MZT0 on Uniprot.

[0102] Mycobacterium smegmatis The gene is numbered I7G9D5 on Uniprot.

[0103] Thermobifida halotolerans The gene is numbered NI17_011080 on KEGG.

[0104] The above genes were used to construct recombinant expression vectors pET32a-PPGK (Q73W41 / F5YW32 / A0A0H3MD65 / H6MZT0 / I7G9D5 / NI17_011080). All plasmids were transformed into *E. coli* BL21-Trxb(DE3) and fermented to obtain engineered bacteria expressing the corresponding enzymes. The engineered bacterial culture was prepared according to the method described in Example 1 above.

[0105] The 1 mL reaction system consisted of: 500 μL of mannose reaction solution treated with saccharifying enzyme, phosphate buffer with a final concentration of 10 mM, and Mg... 2+ The final concentration of the enzyme was 5 mM, sodium hexametaphosphate was 60 mM, and PPGK was added at 1% (% represents the percentage of bacterial cell mass). Water was added to bring the volume to 1 mL. The reaction was carried out in a 65 ℃ constant temperature reactor at 200 rpm for 24 h. Samples were then taken for HPLC analysis. The results are shown in Table 6. KEGG: NI17_011080 enzyme can effectively reduce glucose conversion and increase mannose conversion.

[0106] Table 6 Results of PPGK-catalyzed reactions with different polyphosphoglucosamine kinases

[0107]

[0108] Example 8: Optimization of PPGK enzyme dosage

[0109] Insufficient PPGK addition will fail to remove all glucose; excessive PPGK addition will reduce D-mannose yield.

[0110] The 1 mL reaction system consisted of: 500 μL of mannose reaction solution treated with saccharifying enzyme, phosphate buffer with a final concentration of 10 mM, and Mg... 2+ The final concentration of the reagent was 5 mM, and the sodium hexametaphosphate was 60 mM. The PPGK addition amounts were 0.5%, 1%, 1.5%, 2%, 2.5%, and 3%, respectively (% is by mass percentage). Water was added to bring the volume to 1 mL. The reaction was carried out in a 65 ℃ constant temperature reactor at a rotation speed of 200 rpm. After 8 h of reaction, samples were taken for HPLC analysis. The results are shown in Table 7. When the PPGK addition amount was 1.5%, glucose could be completely removed, and the mannose conversion rate was the highest at 91.75%.

[0111] Table 7. Results of reactions with different amounts of PPGK added.

[0112]

[0113] Example 9: Optimization of Sodium Hexametaphosphate Addition Amount

[0114] Insufficient sodium hexametaphosphate will not remove all glucose; excessive sodium hexametaphosphate will reduce the yield of D-mannose.

[0115] The 1 mL reaction system consisted of: 500 μL of mannose reaction solution treated with saccharifying enzyme, phosphate buffer solution with a final concentration of 10 mM, and Mg... 2+ The final concentration of sodium hexametaphosphate was 5 mM, and the amount of PPGK added was 1.5% (% is by mass percentage). The final concentrations of sodium hexametaphosphate were 30, 40, 50, 60, and 70 mM, respectively, and water was added to bring the volume to 1 mL. The reaction was carried out in a 65 ℃ constant-temperature reactor at a rotation speed of 200 rpm for 8 h. Samples were then taken for HPLC analysis. The results are shown in Table 8. A final sodium hexametaphosphate concentration of 40 mM was sufficient to completely remove glucose, and the mannose conversion rate was the highest.

[0116] Table 8. Reaction results with different amounts of sodium hexametaphosphate.

[0117]

[0118] Based on the optimal types, amounts, and amounts of polyphosphoglucosamine defined in Examples 7, 8, and 9, HPLC analysis was performed on the mannose reaction solution treated with saccharifying enzymes without the addition of polyphosphoglucosamine and sodium hexametaphosphate, as well as the final product treated under optimal conditions. The results... Figure 1 As shown, from Figure 1 It can be seen that polyphosphoglucosidase converts all glucose into phosphorylated glucose by transferring phosphate groups from sodium hexametaphosphate to glucose, thereby generating D-mannose, which increases the yield while removing glucose as a byproduct in the system.

[0119] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included 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+ And the expression of the α-glucan phosphorylase (aGP) gene, glucose phosphate mutase (PGM) gene, and the bifunctional enzyme glucose phosphate isomerase / mannose 6 Phosphoisomerase (PGI) gene and mannose-6 Engineered bacteria with the M6PP gene; Engineered bacteria expressing the isoamylase (IA) gene, saccharifying enzyme, engineered bacteria expressing the limited polyphosphoglucosidase (PPGK) gene, and sodium hexametaphosphate; Polyphosphoglucokinase originates from Thermobifida halotolerans The gene's KEGG identifier is NI17_011080; the concentration of polyphosphoglucosamine is 1.5%; where % of bacterial cells is by mass percentage; The concentration of the saccharifying enzyme is 1 U / mL; The final concentration of sodium hexametaphosphate is 40 mM.

2. The system as described in claim 1, characterized in that, The solutes in the phosphate buffer solution are potassium dihydrogen phosphate (KH₂PO₄) and dipotassium hydrogen phosphate (K₂HPO₄), with a pH of 6.8–7.2; the concentration of the solutes in the phosphate buffer solution is 1–100 mM. Alternatively, the starch derivative may include one or more of partially hydrolyzed starch, starch dextrin, maltodextrin, malt polysaccharide, or maltose.

3. The system as described in claim 2, characterized in that, The pH of the phosphate buffer solution is 7.

0.

4. The system as described in claim 2, characterized in that, The concentration of the solute in the phosphate buffer solution is 10 mM.

5. The system as described in claim 1, characterized in that, The concentration of the starch is 1~500 g / L; Or, the Mg 2+ The concentration ranges from 1 to 50 mM.

6. The system as described in claim 5, characterized in that, The concentration of the starch is 150 g / L.

7. The system as described in claim 5, characterized in that, The Mg 2+ The concentration was 5 mM.

8. The system as described in claim 1, characterized in that, α-glucan phosphorylase gene, glucose phosphate mutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6 Phosphoisomerase gene and mannose 6 The method for preparing engineered bacteria containing the phosphophosphatase gene is as follows (A1). A5) As shown in any of them: A1) Incorporate α-glucan phosphorylase gene, glucose phosphate mutase gene, and the bifunctional enzyme glucose phosphate isomerase / mannose 6 Phosphoisomerase gene and mannose 6 Nucleotides of the phosphophosphatase gene were transferred into Escherichia coli to obtain four recombinant bacteria; the recombinant bacteria were induced to express their contents and then permeabilized to obtain engineered bacteria. A2) Incorporate α-glucan phosphorylase gene, glucose phosphate mutase gene, and the bifunctional enzyme glucose phosphate isomerase / mannose 6 Phosphoisomerase gene and mannose 6 The nucleotides of the phosphophosphatase gene were co-transferred into the same strain of Escherichia coli in any arrangement to obtain a recombinant strain; the recombinant strain was induced to express and then permeabilized to obtain an engineered strain. A3) Incorporate α-glucan phosphorylase gene, glucose phosphate mutase gene, and the bifunctional enzyme glucose phosphate isomerase / mannose 6 Phosphoisomerase gene and mannose 6 Two nucleotides from the phosphophosphatase gene were co-transferred into one strain of Escherichia coli in any combination, and the remaining two nucleotides were co-transferred into another strain of Escherichia coli in any combination, resulting in two recombinant bacteria. The recombinant bacteria were then induced to express their contents and then permeabilized to obtain engineered bacteria. A4) Incorporate α-glucan phosphorylase gene, glucose phosphate mutase gene, and the bifunctional enzyme glucose phosphate isomerase / mannose 6 Phosphoisomerase gene and mannose 6 Two nucleotides from the phosphophosphatase gene were co-transferred into one strain of Escherichia coli in any combination, and the remaining two nucleotides were transferred into two other strains of Escherichia coli, respectively, to obtain three recombinant bacteria. The recombinant bacteria were induced to express their contents and then permeabilized to obtain engineered bacteria. A5) Incorporate α-glucan phosphorylase gene, glucose phosphate mutase gene, and the bifunctional enzyme glucose phosphate isomerase / mannose 6 Phosphoisomerase gene and mannose 6 Three nucleotides from the phosphophosphatase gene were co-transferred into one strain of Escherichia coli in any combination, and the remaining nucleotide was transferred into another strain of Escherichia coli to obtain two recombinant bacteria. The recombinant bacteria were then induced to express their contents and then permeabilized to obtain engineered bacteria.

9. The system as described in claim 8, characterized in that, α-glucan phosphorylase gene, glucose phosphate mutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6 Phosphoisomerase gene and mannose 6 The method for preparing engineered bacteria containing phosphatase genes is as follows: A1) Introduce α-glucan phosphorylase gene, glucose phosphate mutase gene, and bifunctional enzyme glucose phosphate isomerase / mannose 6... Phosphoisomerase gene and mannose 6 Nucleotides of the phosphophosphatase gene were transferred into Escherichia coli to obtain four recombinant strains. The recombinant strains were then induced to express their contents and subjected to permeation treatment to obtain engineered strains.

10. The system as described in claim 8, characterized in that, The concentration of α-glucan phosphorylase was 0.5%–3%; the concentration of glucose phosphate mutase was 0.5%–3%; the bifunctional enzyme glucose phosphate isomerase / mannose 6 The concentration of phosphatidyl isomerase is 0.5%–3%; mannose 6 The concentration of phosphatase is 1% to 8%; where % of bacterial cells is a mass percentage.

11. The system as claimed in claim 10, characterized in that, The concentration of α-glucan phosphorylase was 1%; the concentration of glucose phosphate mutase was 1%; the bifunctional enzyme glucose phosphate isomerase / mannose 6 The concentration of phosphatidyl isomerase was 2%; mannose 6 The concentration of phosphatase was 4%.

12. The system as claimed in claim 1, characterized in that, α-glucan phosphorylase originates from Arabidopsis thaliana The gene is numbered Q9SD76 on Uniprot; glucose-phosphoryltransferase originates from Thermococcus kodakarensis The gene's Uniprot number is Q68BJ6; the bifunctional enzyme glucose phosphate isomerase / mannose 6 Phosphoisomerase originates from Dictyoglomus turgidum The gene's serial number on Uniprot is B8E0A4; mannose 6 Phosphophosphatase originates from Pseudothermotoga hypogea The gene is numbered A0A0X1KRU1 on Uniprot.

13. The system as claimed in claim 1, characterized in that, Methods for preparing engineered bacteria expressing isoamylase genes include: The nucleotides of the isoamylase gene were transferred into Escherichia coli to obtain recombinant bacteria; the recombinant bacteria were induced to express the gene and then permeabilized to obtain engineered bacteria expressing the isoamylase gene. Or, isoamylase originates from Pseudomonas amyloderamosa The gene is numbered P10342 on Uniprot. Alternatively, the method for preparing the engineered bacteria containing the defined polyphosphoglucosamine gene includes: The nucleotides defining the polyphosphoglucosamine gene were transferred into Escherichia coli to obtain recombinant bacteria. The recombinant bacteria were then induced to express the gene and then permeabilized to obtain engineered bacteria expressing the polyphosphoglucosamine gene.

14. The system as claimed in claim 1, characterized in that, The concentration of isoamylase is 0.1% to 2%; where % bacterial cells is a mass percentage.

15. The system as described in claim 14, characterized in that, The concentration of isoamylase was 0.4%; where % bacterial cells represent a mass percentage.

16. A method for catalytically preparing D-mannose, characterized in that, Using starch or starch derivatives as substrates, phosphate buffer solution and Mg2+ are added. 2+ Starch or starch derivatives were treated with engineered bacteria expressing isoamylase genes; then, the α-glucan phosphorylase gene, glucose phosphate mutase gene, and bifunctional enzyme glucose phosphate isomerase / mannose 6 were used. Phosphoisomerase gene and mannose 6 The engineered bacteria with the phosphatase gene were initially used for catalysis, and then saccharifying enzyme, engineered bacteria expressing the limited polyphosphoglucosinolate gene, and sodium hexametaphosphate were added to catalyze the preparation of D-mannose again. Polyphosphoglucokinase originates from Thermobifida halotolerans The gene's KEGG identifier is NI17_011080; the concentration of polyphosphoglucosamine is 1.5%; where % of bacterial cells is by mass percentage; The concentration of the saccharifying enzyme is 1 U / mL; The final concentration of sodium hexametaphosphate is 40 mM.

17. The method as described in claim 16, characterized in that, The temperature for treating starch or starch derivatives with engineered bacteria expressing the isoamylase gene is 70–85 °C; the treatment time is 8–16 h. Alternatively, the catalytic reaction temperature is 40~80 ℃; the reaction time is 10~100 h.

18. The method as described in claim 17, characterized in that, Starch or starch derivatives were treated with engineered bacteria expressing the isoamylase gene at a temperature of 80 °C for 12 h.

19. The method as described in claim 17, characterized in that, The catalytic reaction temperature was 65 °C; the reaction time was 16-30 h.

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