Method for preparing D-mannose through whole-cell catalysis

Abstract

Engineering bacteria for expressing an alpha-glucose phosphate mutase gene, a glucose phosphate mutase gene, a difunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene and a mannose 6-phosphate phosphatase gene are used as a whole-cell catalyst, starch or a starch derivative is used as a substrate, phosphate and Mg < 2 + > are added, and the whole-cell engineering bacteria are obtained. A whole-cell catalysis system is constructed, and starch or starch derivatives can be efficiently catalyzed to synthesize D-mannose. According to the method, the D-mannose is prepared through whole-cell catalysis, and the method has the advantages of being low in cost, low in pollution, high in yield and the like and is suitable for industrial production of the D-mannose.

Description

Technical Field

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

[0002] The information disclosed in this background technology section is only intended to enhance the 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 a person skilled in the art.

[0003] D-mannose is a common six-carbon monosaccharide, which is an isomer of glucose. It is in the form of white crystals or powder, tastes sweet with a bitter taste, is easily soluble in water, and is difficult to dissolve in ethanol. It rarely exists in a free state in nature, but mostly exists in the form of polysaccharides.

[0004] D-mannose is a functional rare sugar with important applications in biology and medicine. It not only regulates immunity, resists bacterial and viral infections, and promotes the normal synthesis of glycoproteins in the body, but can also be used as a raw material to synthesize some pharmaceutical and chemical raw materials and important carbohydrate drug precursors, such as immunostimulant drugs, anti-tumor drugs, vitamins and D-mannitol. In recent years, the demand for mannose in the fields of functional foods, feed additives and medicine has increased significantly, and its economic development value has become prominent.

[0005] At present, the preparation methods of D-mannose mainly include three approaches: plant extraction, chemical isomerization and bioenzyme. Plant extraction is mainly extracted directly from raw materials such as fruits and plants, and this method is widely used; chemical isomerization is to isomerize glucose into D-mannose under acidic, high temperature and high pressure with molybdic acid or molybdate as a catalyst. Due to the limitations of regional raw materials, harsh reaction conditions, complex processes, downstream purification difficulties, low conversion rates and other problems, the application of extraction and chemical methods has limitations and cannot meet the needs of industrial production. The preparation of D-mannose by bioenzyme has gradually become a research trend in the future. However, the current bioenzyme method for preparing D-mannose has the problems of low D-mannose conversion efficiency and many by-products, especially glucose. Glucose and mannose have significant similarities in chemical properties. In the process of producing mannose by biochemical means such as enzyme catalysis, glucose, as the main by-product, increases the technical challenges of subsequent separation and purification of the product. In particular, in the subsequent product separation process, in order to ensure that the purity and quality of the mannose product meet the predetermined standards, more precise and complex separation technology must be used. Therefore, how to effectively distinguish and remove glucose from mannose products has become one of the core problems that need to be solved urgently in the industrial production of mannose. Summary of the invention

[0006] In order to overcome the above problems, the present invention provides a method for preparing D-mannose by whole-cell catalysis.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a system for preparing D-mannose by whole-cell catalysis, comprising: phosphate, starch or starch derivatives, Mg 2+ And engineered bacteria expressing α-glucose phosphomutase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene and mannose 6-phosphate phosphatase gene.

[0009] In one or more embodiments, the phosphate concentration is 1-100 mM, preferably 10 mM.

[0010] In one or more embodiments, the starch derivative includes one or more of partially hydrolyzed starch, starch dextrin, maltodextrin, maltopolysaccharide or maltose.

[0011] In one or more embodiments, the concentration of the starch is 1 to 500 g / L, preferably 150 g / L.

[0012] In one or more embodiments, the Mg 2+ The concentration is 1-50 mM, preferably 5 mM.

[0013] In one or more embodiments, the preparation method of the engineered bacteria expressing the α-glucose phosphomutase gene, the glucose phosphomutase gene, the bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene and the mannose 6-phosphate phosphatase gene is as shown in any one of the following A1)-A5):

[0014] A1) respectively transferring the nucleotides of α-glucose phosphomutase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene and mannose 6-phosphate phosphatase gene into Escherichia coli to obtain 4 recombinant bacteria; respectively inducing expression of the recombinant bacteria, and then permeabilizing the recombinant bacteria to obtain engineered bacteria;

[0015] A2) co-transforming the nucleotides of α-glucose phosphomutase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene and mannose 6-phosphate phosphatase gene into the same strain of Escherichia coli in any arrangement and combination to obtain a recombinant bacterium; inducing expression in the recombinant bacterium, and then permeabilizing it to obtain an engineered bacterium;

[0016] A3) any two nucleotides from the nucleotides of the α-glucose phosphomutase gene, the glucose phosphomutase gene, the bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene and the mannose 6-phosphate phosphatase gene are co-transformed into one strain of Escherichia coli in any arrangement and combination, and the remaining two nucleotides are co-transformed into another strain of Escherichia coli in any arrangement and combination to obtain two recombinant bacteria; inducing expression in each of the recombinant bacteria, and then permeabilizing the recombinant bacteria to obtain engineered bacteria;

[0017] A4) any two nucleotides from the nucleotides of the α-glucose phosphomutase gene, the glucose phosphomutase gene, the bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene and the mannose 6-phosphate phosphatase gene are co-transformed into one strain of Escherichia coli in any arrangement and combination, and the remaining two nucleotides are respectively transferred into two other strains of Escherichia coli to obtain three recombinant bacteria; the recombinant bacteria are respectively induced to express, and then permeabilized to obtain engineered bacteria;

[0018] A5) any three nucleotides from the nucleotides of the α-glucose phosphomutase gene, the glucose phosphomutase gene, the bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene and the mannose 6-phosphate phosphatase gene are co-transformed into one strain of Escherichia coli in any arrangement and combination, and the remaining one nucleotide is respectively transferred into another strain of Escherichia coli to obtain two recombinant bacteria; the recombinant bacteria are respectively induced to express, and then permeabilized to obtain engineered bacteria.

[0019] In one or more embodiments, in the system for whole-cell catalytic preparation of D-mannose, the concentration of α-glucan phosphorylase is 0.5%-3%, preferably 1%; the concentration of glucose phosphomutase is 0.5%-3%, preferably 1%; the concentration of the bifunctional enzyme glucose phosphomonomerase / mannose 6-phosphate isomerase is 0.5%-3%, preferably 2%; the concentration of mannose 6-phosphate phosphatase is 1%-8%, preferably 4%; wherein % is mass percentage.

[0020] In one or more embodiments, α-glucan phosphorylase is derived from Arabidopsis thaliana, and the gene number on Uniprot is Q9SD76; glucose phosphotyrosinase is derived from Thermococcus kodakarensis, and the gene number on Uniprot is Q68BJ6; the bifunctional enzyme glucose phosphotyrosinase / mannose 6-phosphate isomerase is derived from Thermococcus kodakarensis, and the gene number on Uniprot is Q5JE38; mannose 6-phosphate phosphatase is derived from Methanocaldococcus jannaschii, and the gene number on Uniprot is Q59000.

[0021] In one or more embodiments, the whole-cell catalytic system for producing D-mannose further comprises an engineered bacterium expressing an isoamylase gene, a 4-glucanotransferase gene, and a polyphosphoglucokinase gene.

[0022] Preferably, the method for preparing the engineered bacteria expressing the isoamylase gene, the 4-glucanotransferase gene and the polyphosphate glucokinase gene comprises:

[0023] The nucleotides of the isoamylase gene, the 4-glucanotransferase gene and the polyphosphate glucokinase gene were respectively transferred into Escherichia coli to obtain three recombinant bacteria; the recombinant bacteria were induced to express and then permeabilized to obtain engineered bacteria expressing the isoamylase gene, the 4-glucanotransferase gene and the polyphosphate glucokinase gene.

[0024] Further preferably, the isoamylase is derived from Pseudomonas amyloderamosa, and the gene number on Uniprot is P10342;

[0025] 4-Glucan transferase is derived from Streptococcus mutans, and the gene number on Uniprot is P49331;

[0026] Polyphosphokinase is derived from:

[0027] Mycolicibacterium paratuberculosis, the gene number on Uniprot is Q73W41;

[0028] Mycolicibacter sinensis , the gene number on Uniprot is F5YW32;

[0029] Mycobacterium bovis, the gene number on Uniprot is A0A0H3MD65;

[0030] Gordonia polyisoprenivorans, the gene number on Uniprot is H6MZT0;

[0031] Mycolicibacterium smegmatis, the gene number on Uniprot is I7G9D5;

[0032] Pseudomonas amyloderamosa, the gene number on Uniprot is Q49988;

[0033] Preferably, the polyphosphate glucokinase is derived from Pseudomonas amyloderamosa, and the gene number on Uniprot is Q49988.

[0034] Preferably, in the system for preparing D-mannose by whole cell catalysis, the concentration of isoamylase is 0.1% to 2%, preferably 0.4%; the concentration of 4-glucanotransferase is 0.1% to 2%, preferably 0.8%; and the concentration of polyphosphate glucokinase is 0.1% to 2%, preferably 1%.

[0035] The second aspect of the present invention provides a method for preparing D-mannose by whole-cell catalysis, using starch or starch derivatives as substrates, adding phosphate and Mg 2+ D-mannose is prepared by whole-cell catalysis using engineered bacteria expressing α-glucose phosphomutase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphomitomerase / mannose 6-phosphate isomerase gene and mannose 6-phosphate phosphatase gene.

[0036] In one or more embodiments, in the process of catalytic preparation of D-mannose, starch or starch derivatives are treated with engineered bacteria expressing isoamylase genes, and then D-mannose is prepared by whole-cell catalysis using engineered bacteria expressing α-glucose phosphomutase genes, glucose phosphomutase genes, bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase genes and mannose 6-phosphate phosphatase genes, engineered bacteria expressing 4-glucan transferase genes, and engineered bacteria expressing polyphosphate glucokinase genes.

[0037] Preferably, the temperature for treating starch or starch derivatives with the engineered bacteria expressing the isoamylase gene is 70-85° C., preferably 80° C.; and the treatment time is 8-16 h, preferably 12 h.

[0038] In one or more embodiments, the whole-cell catalytic reaction temperature is 40 to 80° C., preferably 65° C.; the reaction time is 10 to 100 hours, preferably 16 to 30 hours.

[0039] The beneficial effects of the present invention are:

[0040] (1) In the present invention, an engineered bacterium expressing an α-glucose phosphomutase gene, a glucose phosphomutase gene, a bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene, and a mannose 6-phosphate phosphatase gene is used as a whole-cell catalyst, starch or a starch derivative is used as a substrate, and phosphate and Mg are added. 2+ , a whole-cell catalytic system was constructed that can efficiently catalyze the synthesis of D-mannose from starch or starch derivatives.

[0041] (2) Isoamylase is an endo-amylase that can specifically cleave the α-1,6-glycosidic bond at the branch point of amylopectin, cutting off the entire side chain to form straight-chain starch, which helps glucan phosphorylase to degrade starch more completely. Therefore, using engineered bacteria expressing the isoamylase gene to treat starch or starch derivatives can improve the efficiency of D-mannose synthesis.

[0042] (3) Polyphosphoglucokinase (PPGK) plays a vital role in the mannose synthesis pathway. It can specifically transfer the terminal phosphate group of polyphosphate to the glucose molecule to generate 6-phosphate glucose, which then re-enters the mannose synthesis pathway. In this process, PPGK significantly reduces the content of glucose as a byproduct in the reaction, and correspondingly increases the yield of mannose. The effective reduction of glucose content is also crucial for the subsequent mannose separation and purification steps, because it can simplify the operation process and improve the purity of the final product. However, the inventors found in the research process that in addition to the phosphorylation function of glucose, polyphosphoglucokinase also has a side effect: it can re-phosphorylate part of the generated D-mannose, thereby limiting the increase in the conversion rate of D-mannose. Therefore, in order to improve the conversion efficiency of D-mannose, polyphosphoglucokinase with higher D-mannose conversion efficiency was obtained through screening.

[0043] (4) The method of the present invention utilizes whole-cell catalysis to prepare D-mannose, which 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

[0044] The accompanying drawings in the specification, 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.

[0045] Figure 1In figure a, mannose was detected by HPLC in Example 8 and Example 6, in which the horizontal coordinates are 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 10.0, 12.0, 13.0, 14.0, 15.0, 16.0 and 17.0 from left to right; the vertical coordinates are -10.0, -5.0, 0.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0 and 60.0 from bottom to top; and b is an enlarged view of part of the data in figure a. DETAILED DESCRIPTION

[0046] It should be noted that the following detailed descriptions are exemplary and are 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.

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

[0048] Example 1

[0049] 1.1 Construction of engineered bacteria expressing α-glucose phosphomutase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphomitomerase / mannose 6-phosphate isomerase gene and mannose 6-phosphate phosphatase gene:

[0050] In this embodiment, α-glucan phosphorylase aGP is derived from Arabidopsis thaliana, and the gene number on Uniprot is Q9SD76; phosphoglucomutase PGM is derived from Thermococcus kodakarensis, and the gene number on Uniprot is Q68BJ6; bifunctional enzyme phosphoglucoside isomerase / mannose 6-phosphate isomerase PGI is derived from Thermococcus kodakarensis, and the gene number on Uniprot is Q5JE38; mannose 6-phosphate phosphatase M6PP is derived from Methanocaldococcus jannaschii, and the gene number on Uniprot is Q59000. These genomic DNAs can be obtained from the official website of ATCC (www.atcc.org).

[0051] After the codons were optimized, the amino acid sequences were sent to Jiangsu Jinweizhi Biotechnology Co., Ltd. for gene synthesis and constructed into the expression vector pET-32a to obtain the expression recombinant plasmids pET-aGP, pET-PGM, pET-PGI, and pET-M6PP, respectively, and then transformed into Escherichia coli BL21-Trxb (DE3) competent cells, and the transformation products were spread on ampicillin-resistant LB solid medium plates and cultured overnight at 37°C (about 16 hours). The transformants were picked on the LB solid medium to obtain the recombinant Escherichia coli containing the recombinant plasmid.

[0052] 1.2 Obtaining enzymes for whole-cell catalytic production of D-mannose using engineered bacteria expressing α-glucose phosphomutase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene and mannose 6-phosphate phosphatase gene:

[0053] (1) Pick a single clone and inoculate (directly insert the pipette tip 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 cultured seed culture 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 600 When the pH 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.

[0054] (2) Centrifuge the overnight culture at 5500 rpm for 15 min to collect the cells.

[0055] (3) Weigh the bacterial sludge in a ratio of 0.2 g of bacterial cells to 1 mL of reaction buffer and resuspend.

[0056] Example 2 Optimization of reaction temperature

[0057] Thanks to the positive effect of high temperature on substrate solubilization, as the reaction temperature gradually increases, the reaction is conducive to the forward progress. However, too high a temperature may destroy the spatial structure of the enzyme and cause enzyme inactivation. Therefore, it is necessary to find and maintain the most suitable reaction temperature range.

[0058] The 1 mL reaction system involved in this example is: starch concentration 150 g / L, phosphate buffer final concentration 10 mM, Mg 2+ The final concentration is 10 mM. aGP, PGM, PGI, and M6PP are crude enzyme solutions prepared in Example 1. 100 μL of each is added and water is added to make up to 1 mL.

[0059] The reaction system described in the above embodiment was placed in a constant temperature reactor at 55°C, 60°C, 65°C and 70°C for reaction, and the rotation speed was set to 700 rpm. After 16 hours of reaction, samples were taken and HPLC detection was performed. The results are shown in Table 1. It was found that when the reaction temperature was 65°C, the D-mannose conversion rate was as high as 49.38%.

[0060] Table 1 D-mannose conversion rate at different temperatures

[0061] Reaction temperature℃ Mannose production rate % 55 32.55 60 45.62 65 49.38 70 41.70

[0062] Example 3 Optimization of phosphate concentration

[0063] The 1 mL reaction system involved in this example is: starch concentration 150 g / L, Mg 2+ The final concentration of is 10 mM, the final concentrations of phosphate buffer are 0, 5, 10, 20, 30, 50 and 100 mM, respectively. aGP, PGM, PGI and M6PP are the crude enzyme solutions prepared in Example 1 above, 100 μL of each are added, and water is added to make up to 1 mL.

[0064] The reaction system described in the above embodiment was placed in a 65°C constant temperature reactor for reaction, the speed was set to 200 rpm, and after 16 hours of reaction, samples were taken and HPLC detection was performed. The results are shown in Table 2, and it was found that the generation rate of D-mannose first increased and then decreased with the increase of phosphate concentration, and 10-30 μm was the optimal phosphate concentration.

[0065] Table 2 Reaction results of different phosphate concentrations

[0066] Final phosphate concentration mM Mannose production rate % 0 44.88 5 49.64 10 52.35 20 52.22 30 52.09 50 49.20 100 45.46

[0067] Example 4Mg 2+ Concentration Optimization

[0068] The 1 mL reaction system involved in this example is: 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 crude enzyme solutions prepared in Example 1. 100 μL of each was added and the mixture was made up to 1 mL with water.

[0069] The reaction system described in the above embodiment was placed in a 65°C constant temperature reactor for reaction, the speed was set to 200 rpm, and after 16 hours of reaction, samples were taken and HPLC detection was performed. The results are shown in Table 3. It was found that Mg 2+ When the final concentration was 5 mM, the D-mannose conversion rate was the highest at 51.50%.

[0070] Table 3Mg 2+ The reaction results corresponding to different concentrations

[0071]

[0072]

[0073] Example 5 Determination of the ratio of each enzyme in the whole cell catalytic system

[0074] 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 is 10 mM. aGP, PGM, PGI, and M6PP are crude enzyme solutions prepared in Example 1. 100 μL of each is added and water is added to make up to 1 mL.

[0075] The reaction system described in the above embodiment was placed in a 65°C constant temperature reactor for reaction, the speed was set to 200 rpm, and after 16 hours of reaction, samples were taken and HPLC detection was performed. The results are shown in Table 4, and it was found that when the addition amount of Agp was 1%, the addition amount of PGM was 1%, the addition amount of PGI was 2%, and the addition amount of M6PP was 4%, % being the mass percentage, the highest generation rate of D-mannose was 54.84%.

[0076] Table 4 Reaction results of different enzyme ratios

[0077]

[0078] Example 6 Optimization of temperature and time for isoamylase treatment of substrate

[0079] Isoamylase is an endo-amylase that can specifically cut the α-1,6-glycosidic bond at the branch point of amylose, cut off the entire side chain to form amylose, and help glucan phosphorylase degrade starch more completely.

[0080] In this embodiment, isoamylase IA is derived from Pseudomonas amyloderamosa, and the gene number on Uniprot is P10342. The gene constructs the recombinant expression vector pET32a-IA. The plasmids are transformed into Escherichia coli BL21-Trxb (DE3), and fermented to obtain whole cells expressing the corresponding enzymes, and the crude enzyme solution is prepared in the manner of the above embodiment 1. Substrate pretreatment system: starch concentration 400g / L, isoamylase IA addition amount 4%, % is mass fraction, and substrates of different lengths are treated at different temperatures for different times, and catalytic reactions are performed respectively.

[0081] The reaction system for 1 mL was as follows: the concentration of starch treated with IA was 150 g / L, the final concentration of phosphate buffer was 10 mM, and the Mg2+ The final concentration is 5mM, aGP, PGM, PGI, M6PP are the crude enzyme solutions prepared in Example 1, and the addition amounts are 1%, 1%, 2%, 4%, % is the mass percentage, and water is added to 1mL. Place in a 65℃ constant temperature reactor for reaction, the speed is set to 200rpm, after 16h of reaction, sample and perform HPLC detection. The results are shown in Table 5. The substrate treated with isoamylase IA at 80℃ for 12h is catalyzed, and the D-mannose conversion rate is as high as 72.36%.

[0082] Table 5 Effects of isoamylase treatment at different temperatures and for different durations

[0083]

[0084]

[0085] Example 7 Screening of polyphosphoglucokinase PPGK

[0086] In this example, the glucanotransferase 4GT is derived from the Streptococcus mutans gene and is numbered P49331 on Uniprot.

[0087] Polyphosphokinase is derived from:

[0088] Mycolicibacterium paratuberculosis, the gene number on Uniprot is Q73W41;

[0089] Mycolicibacter sinensis , the gene number on Uniprot is F5YW32;

[0090] Mycobacterium bovis, the gene number on Uniprot is A0A0H3MD65;

[0091] Gordonia polyisoprenivorans, the gene number on Uniprot is H6MZT0;

[0092] Mycolicibacterium smegmatis, the gene number on Uniprot is I7G9D5;

[0093] Pseudomonas amyloderamosa, the gene number on Uniprot is Q49988;

[0094] The above genes were used to construct recombinant expression vectors pET32a-4GT and pET32a-PPGK (Q73W41 / F5YW32 / A0A0H3MD65 / H6MZT0 / I7G9D5 / Q49988), respectively. The plasmids were transformed into Escherichia coli BL21-Trxb (DE3), and fermented to obtain whole cells expressing the corresponding enzymes, and crude enzyme solution was prepared according to the method in Example 2 above.

[0095] The reaction system for 1 mL was as follows: the concentration of starch treated with IA was 150 g / L, the final concentration of phosphate buffer was 10 mM, and the Mg 2+ The final concentration is 5mM, aGP, PGM, PGI, M6PP are the crude enzyme solutions prepared in Example 1, and the addition amounts are 1%, 1%, 2%, 4%, 4GT addition amount is 2%, PPGK addition amount is 1%, % is mass percentage, and water is added to 1mL. Place in a 65℃ constant temperature reactor for reaction, the speed is set to 200rpm, after 24h of reaction, take a sample, and perform HPLC detection. The results are shown in Table 6. Uniprot: Q49988 enzyme can effectively reduce the glucose conversion rate and improve the mannose conversion rate.

[0096] Table 6 Results of reactions catalyzed by different polyphosphate glucose kinases PPGK

[0097]

[0098] Example 8 Optimization of polyphosphoglucokinase PPGK reaction system

[0099] 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.

[0100] The reaction system for 1 mL was as follows: the concentration of starch treated with IA was 150 g / L, the final concentration of phosphate buffer was 10 mM, and the Mg 2+ The final concentration is 5mM, aGP, PGM, PGI, M6PP are the crude enzyme solutions prepared in Example 1, and the addition amounts are 1%, 1%, 2%, 4%, 4GT is 2%, and PPGK is 0.5 / 1 / 1.5 / 2 / 2.5 / 3%, % is the mass percentage, and water is added to 1mL. Place in a 65℃ constant temperature reactor for reaction, the speed is set to 200rpm, after 24h of reaction, take samples, and perform HPLC detection. The results are shown in Table 7. When the addition amount of PPGK is 2%, glucose can be completely removed, and the mannose conversion rate is the highest.

[0101] Table 7 Reaction results of different addition amounts of PPGK

[0102]

[0103] Figure 1 The liquid phase diagram of mannose detected by HPLC in Example 8 and Example 6.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A system for preparing D-mannose by whole-cell catalysis, characterized in that: include: Phosphate, starch or starch derivatives, Mg 2+ And engineered bacteria expressing α-glucose phosphomutase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene and mannose 6-phosphate phosphatase gene.

2. The whole-cell catalytic system for preparing D-mannose according to claim 1, characterized in that: The phosphate concentration is 1 to 100 mM; The starch derivatives include one or more of partially hydrolyzed starch, starch dextrin, maltodextrin, maltopolysaccharide or maltose; The concentration of the starch is 1 to 500 g / L; The Mg 2+ The concentration is 1~50mM.

3. The whole-cell catalytic system for preparing D-mannose according to claim 1, characterized in that: The preparation method of the engineered bacteria expressing the α-glucose phosphomutase gene, the glucose phosphomutase gene, the bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene and the mannose 6-phosphate phosphatase gene is as shown in any one of A1) to A5) below: A1) respectively transferring the nucleotides of α-glucose phosphomutase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene and mannose 6-phosphate phosphatase gene into Escherichia coli to obtain 4 recombinant bacteria; respectively inducing expression of the recombinant bacteria, and then permeabilizing the recombinant bacteria to obtain engineered bacteria; A2) co-transforming the nucleotides of α-glucose phosphomutase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene and mannose 6-phosphate phosphatase gene into the same strain of Escherichia coli in any arrangement and combination to obtain a recombinant bacterium; inducing expression in the recombinant bacterium, and then permeabilizing it to obtain an engineered bacterium; A3) any two nucleotides from the nucleotides of the α-glucose phosphomutase gene, the glucose phosphomutase gene, the bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene and the mannose 6-phosphate phosphatase gene are co-transformed into one strain of Escherichia coli in any arrangement and combination, and the remaining two nucleotides are co-transformed into another strain of Escherichia coli in any arrangement and combination to obtain two recombinant bacteria; inducing expression in each of the recombinant bacteria, and then permeabilizing the recombinant bacteria to obtain engineered bacteria; A4) any two nucleotides from the nucleotides of the α-glucose phosphomutase gene, the glucose phosphomutase gene, the bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene and the mannose 6-phosphate phosphatase gene are co-transformed into one strain of Escherichia coli in any arrangement and combination, and the remaining two nucleotides are respectively transferred into two other strains of Escherichia coli to obtain three recombinant bacteria; the recombinant bacteria are respectively induced to express, and then permeabilized to obtain engineered bacteria; A5) any three nucleotides from the nucleotides of the α-glucose phosphomutase gene, the glucose phosphomutase gene, the bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase gene and the mannose 6-phosphate phosphatase gene are co-transformed into one strain of Escherichia coli in any arrangement and combination, and the remaining one nucleotide is respectively transferred into another strain of Escherichia coli to obtain two recombinant bacteria; the recombinant bacteria are respectively induced to express, and then permeabilized to obtain engineered bacteria.

4. The whole-cell catalytic system for preparing D-mannose according to claim 1, characterized in that: In the system for preparing D-mannose by whole cell catalysis, the concentration of α-glucan phosphorylase is 0.5%-3%; the concentration of glucose phosphomutase is 0.5%-3%; the concentration of bifunctional enzyme glucose phosphomonomerase / mannose 6-phosphate isomerase is 2%; and the concentration of mannose 6-phosphate phosphatase is 1%-8%.

5. The whole-cell catalytic system for preparing D-mannose according to claim 1, characterized in that: α-Glucan phosphorylase is derived from Arabidopsis thaliana, and the gene number on Uniprot is Q9SD76; glucose phosphomutase is derived from Thermococcus kodakarensis, and the gene number on Uniprot is Q68BJ6; the bifunctional enzyme glucose phosphomerase / mannose 6-phosphate isomerase is derived from Thermococcus kodakarensis, and the gene number on Uniprot is Q5JE38; mannose 6-phosphate phosphatase is derived from Methanocaldococcus jannaschii, and the gene number on Uniprot is Q59000.

6. The whole-cell catalytic system for preparing D-mannose according to claim 1, characterized in that: The whole-cell catalytic system for preparing D-mannose also includes an engineered bacterium expressing an isoamylase gene, a 4-glucanotransferase gene, and a polyphosphate glucokinase gene; The method for preparing the engineered bacteria expressing the isoamylase gene, the 4-glucanotransferase gene and the polyphosphate glucokinase gene comprises: The nucleotides of the isoamylase gene, the 4-glucanotransferase gene and the polyphosphate glucokinase gene were respectively transferred into Escherichia coli to obtain three recombinant bacteria; the recombinant bacteria were induced to express and then permeabilized to obtain engineered bacteria expressing the isoamylase gene, the 4-glucanotransferase gene and the polyphosphate glucokinase gene.

7. The whole-cell catalytic system for preparing D-mannose according to claim 1, characterized in that: Isoamylase is derived from Pseudomonas amyloderamosa, and the gene number on Uniprot is P10342; 4-Glucan transferase is derived from Streptococcus mutans, and the gene number on Uniprot is P49331; Polyphosphokinase is derived 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; Pseudomonas amyloderamosa, the gene number on Uniprot is Q49988; In the system for preparing D-mannose by whole cell catalysis, the concentration of isoamylase is 0.1% to 2%; the concentration of 4-glucanotransferase is 0.1% to 2%; and the concentration of polyphosphate glucokinase is 0.1% to 2%.

8. A method for preparing D-mannose by whole-cell catalysis, characterized in that: Starch or starch derivatives are used as substrates, and phosphate and Mg are added 2+ D-mannose is prepared by whole-cell catalysis using engineered bacteria expressing α-glucose phosphomutase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphomitomerase / mannose 6-phosphate isomerase gene and mannose 6-phosphate phosphatase gene.

9. The method according to claim 8, characterized in that In the process of catalytic preparation of D-mannose, starch or starch derivatives are treated with engineered bacteria expressing isoamylase genes, and then D-mannose is prepared by whole-cell catalysis using engineered bacteria expressing α-glucose phosphomutase genes, glucose phosphomutase genes, bifunctional enzyme glucose phosphomutase / mannose 6-phosphate isomerase genes and mannose 6-phosphate phosphatase genes, engineered bacteria expressing 4-glucanotransferase genes, and engineered bacteria expressing polyphosphate glucokinase genes; The temperature for treating starch or starch derivatives with the engineered bacteria expressing the isoamylase gene is 70 to 85° C., and the treatment time is 8 to 16 hours.

10. The method according to claim 8, characterized in that The whole-cell catalytic reaction temperature is 40 to 80° C., preferably 65° C.; the reaction time is 10 to 100 hours, preferably 16 to 30 hours.

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