A dual-enzyme immobilized enzyme for fructose synthesis, its preparation method, and its application.

Abstract

This invention discloses a dual-enzyme immobilized enzyme for fructose synthesis, obtained by immobilizing glucose-6-phosphate isomerase and a fructose-6-phosphate phosphatase mutant, TcPase_V237R, on resin. The invention also discloses its preparation method and applications. By rationally designing and site-directed mutagenesis of key sites in fructose-6-phosphate phosphatases from different sources, this invention obtained a fructose-6-phosphate phosphatase mutant, TcPase_V237R, with significantly enhanced substrate specificity for fructose-6-phosphate, effectively reducing non-specific hydrolysis of glucose-6-phosphate. When combined with glucose-6-phosphate isomerase TaPGI, a dual-enzyme immobilized enzyme is constructed using glucose-6-phosphate as a substrate, enabling highly efficient fructose synthesis. After 20 cycles of use, it retains over 90% of its residual activity, demonstrating promising prospects for industrial application.

Description

Technical Field

[0001] This invention relates to the field of enzymatic catalytic synthesis technology, specifically to a dual-enzyme immobilized enzyme for fructose synthesis, its preparation method, and its application. Background Technology

[0002] Fructose is an important sweetener, with a sweetness approximately 1.2-1.8 times that of sucrose, and is widely used in the food, beverage, and pharmaceutical industries. Currently, the main industrial process uses corn starch as a raw material, which is enzymatically hydrolyzed to produce glucose, and then catalyzed by glucose isomerase to produce high-fructose corn syrup. However, the glucose isomerase-catalyzed reaction is a reversible process, limited by thermodynamic equilibrium, resulting in limitations on product purity and yield.

[0003] The dephosphorylation cascade strategy provides a new pathway to circumvent the equilibrium limitations of traditional isomerization reactions. This route first uses glucose-6-phosphate isomerase to catalyze the isomerization of glucose-6-phosphate (G6P) to fructose-6-phosphate (F6P), followed by fructose-6-phosphate phosphatase to catalyze the hydrolysis and dephosphorylation of F6P, producing fructose and releasing inorganic phosphate. Since the dephosphorylation reaction catalyzed by fructose-6-phosphate phosphatase is thermodynamically irreversible, it effectively drives the first step of the isomerization reaction.

[0004] However, existing wild-type fructose-6-phosphate phosphatases suffer from poor substrate specificity and are prone to non-specific hydrolysis of structural analogs such as G6P during catalysis, leading to byproduct formation and affecting product purity. Therefore, rationally designing and mutating wild-type fructose-6-phosphate phosphatases using enzyme molecular engineering techniques to enhance substrate specificity for F6P and reduce side reactions has become crucial for improving the catalytic performance of dual-enzyme composite catalytic systems. Summary of the Invention

[0005] The purpose of this invention is to provide a dual-enzyme immobilized enzyme for fructose synthesis, its preparation method, and its application, in order to overcome the shortcomings of the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a dual-enzyme immobilized enzyme for fructose synthesis, which is obtained by immobilizing glucose-6-phosphate isomerase and a fructose-6-phosphate phosphatase mutant TcPase_V237R on a resin; wherein the fructose-6-phosphate phosphatase mutant TcPase_V237R is obtained by mutating valine at position 237 of fructose-6-phosphate phosphatase TcPase to arginine, i.e., V237R, and the amino acid sequence of the fructose-6-phosphate phosphatase TcPase is shown in SEQ ID NO: 5.

[0007] Furthermore, the glucose-6-phosphate isomerase is glucose-6-phosphate isomerase TaPGI, whose amino acid sequence is shown in SEQ ID NO: 1.

[0008] Furthermore, the resin is an amino resin.

[0009] Furthermore, the amino resin includes LX-1000NH amino resin or ESR-1 amino resin.

[0010] The second aspect of the present invention provides a method for preparing the above-mentioned dual-enzyme immobilized enzyme for fructose synthesis.

[0011] Further, the process includes the following steps: using 50-200mM HEPES buffer (pH 6.0-8.0) as a solvent, glucose-6-phosphate isomerase, fructose-6-phosphate phosphatase mutant, and resin are added. The mass ratio of glucose-6-phosphate isomerase to resin is 5-50 mg:1 g, and the mass ratio of fructose-6-phosphate phosphatase mutant to resin is 50-200 mg:1 g. 1v / v%-5v / v% glutaraldehyde and 1v / v%-5v / v% polyethyleneimine are added. The immobilization temperature is 4-10℃, the stirring speed is 20-100 rpm, and the immobilization time is 2-5 h. After filtration and washing, the dual-enzyme immobilized enzyme is obtained.

[0012] The third aspect of the present invention provides the application of the above-mentioned dual-enzyme immobilized enzyme for fructose synthesis in fructose synthesis.

[0013] Furthermore, in application, the dual-enzyme immobilized enzyme is used as a catalyst, glucose-6-phosphate is used as a substrate, and in the presence of MgCl2, the temperature and stirring speed are controlled to carry out enzyme catalysis to produce fructose in the reaction medium.

[0014] Furthermore, the reaction medium is 50-200mM HEPES buffer with pH 6.0-8.0, the amount of enzyme used for dual-enzyme immobilization is 5-20g / L, the concentration of glucose-6-phosphate is 5-30g / L, the concentration of MgCl2 is 5-20mM, the temperature is controlled at 60-80℃, the stirring speed is 70-300rpm, and the reaction time is 4-10h.

[0015] The fourth aspect of the present invention provides a fructose-6-phosphate phosphatase mutant TcPase_V237R, wherein the fructose-6-phosphate phosphatase mutant TcPase_V237R is obtained by mutating valine at position 237 of fructose-6-phosphate phosphatase TcPase to arginine, i.e., V237R, and the amino acid sequence of the fructose-6-phosphate phosphatase TcPase is shown in SEQ ID NO: 5.

[0016] The beneficial effects of this invention are: This invention, through rational design and site-directed mutagenesis of key sites in fructose-6-phosphate phosphatases from different sources, yielded a fructose-6-phosphate phosphatase mutant, TcPase_V237R, with significantly enhanced substrate specificity for fructose-6-phosphate, effectively reducing non-specific hydrolysis of glucose-6-phosphate. When combined with glucose-6-phosphate isomerase TaPGI, a dual-enzyme immobilized enzyme was constructed. Using glucose-6-phosphate as a substrate, this enzyme achieved highly efficient fructose synthesis (the fructose conversion rate of the TaPGI+TcPase_V237R dual-enzyme immobilized enzyme was 94.2%, while that of the TaPGI+TcPase dual-enzyme immobilized enzyme was only 75.4%). After 20 cycles of application, over 90% of the residual activity was retained (compared to only 65.0% of the residual activity of the TaPGI+TcPase dual-enzyme immobilized enzyme after 20 cycles), demonstrating promising prospects for industrial application. Attached Figure Description

[0017] Figure 1 The results of SDS-PAGE analysis of TaPGI in Example 3 are shown.

[0018] Figure 2 The results of SDS-PAGE detection of PmPase and its mutants in Example 3 are shown.

[0019] Figure 3 The results of SDS-PAGE detection of TcPase and its mutants in Example 3 are shown.

[0020] Figure 4 The results show the substrate selectivity of fructose-6-phosphate phosphatase and its mutants.

[0021] Figure 5 The results show the determination of fructose synthesis catalyzed by glucose-6-phosphate isomerase, fructose-6-phosphate phosphatase and their mutant dual enzymes.

[0022] Figure 6 The results of 20 relative enzyme activity measurements were obtained for each dual-enzyme immobilized enzyme. Detailed Implementation

[0023] The present invention will be further explained below with reference to embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0024] Unless otherwise specified, the test conditions in the following examples are generally based on standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0025] The culture medium formulations involved in the following examples are as follows: SOC medium (1L): 20g tryptone, 5g yeast extract, 0.5g sodium chloride, 10mL 250mmol / L potassium chloride, 10mL 1M magnesium chloride, 10mL 1M magnesium sulfate, 5mL 1M D-glucose (added separately before use after sterilization by filtration through a 0.22μm filter membrane); pH 7.2.

[0026] LB liquid medium (1L): 10g tryptone, 5g yeast extract, 10g sodium chloride; pH 7.0.

[0027] LB solid medium is made by adding 20g of agar powder to 1L of LB liquid medium.

[0028] All of the above culture media need to be autoclaved at 121°C for 20 minutes.

[0029] Example 1: Construction of recombinant Escherichia coli containing glucose-6-phosphate isomerase and fructose-6-phosphate phosphatase I. Construction of Recombinant Plasmids Acidophilus pyrogen ( Thermoplasma acidophilum The gene fragment encoding the wild-type glucose-6-phosphate isomerase TaPGI in the genome (gene sequence as shown in SEQ ID NO: 2, amino acid sequence as shown in SEQ ID NO: 1); *Lithops microthermos* ( Petrotoga miotherma The gene fragment encoding wild-type fructose-6-phosphate phosphatase PmPase in the genome (gene sequence as shown in SEQ ID NO: 4, amino acid sequence as shown in SEQ ID NO: 3); Thermococcus ( Thermococcus camini The gene fragment encoding wild-type fructose-6-phosphate phosphatase TcPase in the genome (gene sequence as shown in SEQ ID NO: 6, amino acid sequence as shown in SEQ ID NO: 5) was synthesized by Sangon Biotech (Shanghai) Co., Ltd. after codon optimization of E. coli. The optimized gene fragment was constructed into the vector pET28a(+) to obtain the recombinant plasmid.

[0030] II. Transformation of Escherichia coli with recombinant plasmids E. coli BL21(DE3) The recombinant plasmid obtained in step one of Example 1 was transformed into the strain using a heat shock method. E. coli In BL21(DE3). The specific steps are as follows: 100 μL in each tube E. coliAdd 10 μL of 1 ng / μL recombinant plasmid to a suspension of BL21(DE3) competent cells (OD approx. 0.4-0.6), mix gently, and incubate on ice for 30 min. Transfer to a 42°C water bath and heat shock for 90 s. Quickly transfer to an ice bath and cool for 3 min. Add 700 μL of antibiotic-free SOC liquid medium to each tube and incubate at 37°C and 100 rpm on a shaker for 40 min. After incubation, centrifuge the bacterial culture at 4°C and 12000 rpm for 10 min, discard 600 μL of supernatant, and spread the remaining bacterial culture onto LB agar plates containing 50 μg / mL kanamycin sulfate. Incubate overnight at 37°C with the plates inverted.

[0031] Selection of positive clones: Four clones were selected and transferred to 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate. The cultures were incubated at 37°C and 200 rpm for 8 h. Plasmids were extracted using the Mini-Plasmid Rapid Isolation Kit (Beijing Bodatech Biotechnology Co., Ltd.). 20 μL of plasmid was taken and sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing to confirm successful construction.

[0032] Example 2 Construction of recombinant Escherichia coli with fructose-6-phosphate phosphatase mutant I. Construction of Recombinant Plasmids The sequences of fructose-6-phosphate phosphatase PmPase and fructose-6-phosphate phosphatase TcPase in step one of Example 1 were analyzed, and multiple single-point mutants were designed using bioinformatics methods, as shown in Table 1.

[0033] Table 1

[0034] PmPase_S47H is a mutation of serine at position 47 of wild-type fructose-6-phosphate phosphatase PmPase to histidine, i.e., S47H.

[0035] PmPase_F123R is a wild-type fructose-6-phosphate phosphatase PmPase with a phenylalanine mutation at position 123 replaced by arginine, hence F123R.

[0036] PmPase_L191K is the wild-type fructose-6-phosphate phosphatase PmPase with a leucine mutation at position 191, i.e., L191K.

[0037] TcPase_D46Q is a wild-type fructose-6-phosphate phosphatase TcPase with an aspartic acid mutation at position 46 replaced by glutamine, i.e., D46Q.

[0038] TcPase_Y157F is a wild-type fructose-6-phosphate phosphatase TcPase with a tyrosine mutation at position 157 to phenylalanine, i.e., Y157F.

[0039] TcPase_V237R is a wild-type fructose-6-phosphate phosphatase TcPase with a valine mutation at position 237 to arginine, hence V237R.

[0040] All the mutants were codon optimized by Sangon Biotech (Shanghai) Co., Ltd., and the optimized gene fragments were synthesized and constructed into the vector pET-28a(+) to obtain recombinant plasmids.

[0041] II. Transformation of Escherichia coli with recombinant plasmids E. coli BL21(DE3) The recombinant plasmid obtained in step one of Example 2 was transformed into the strain using a heat shock method. E. coli In BL21(DE3). The specific steps are as follows: 100 μL in each tube E. coli Add 10 μL of 1 ng / μL recombinant plasmid to a suspension of BL21(DE3) competent cells (OD approx. 0.4-0.6), mix gently, and incubate on ice for 30 min. Transfer to a 42°C water bath and heat shock for 90 s. Quickly transfer to an ice bath and cool for 3 min. Add 700 μL of antibiotic-free SOC liquid medium to each tube and incubate at 37°C and 100 rpm on a shaker for 40 min. After incubation, centrifuge the bacterial culture at 4°C and 12000 rpm for 10 min, discard 600 μL of supernatant, and spread the remaining bacterial culture onto LB agar plates containing 50 μg / mL kanamycin sulfate. Incubate overnight at 37°C with the plates inverted.

[0042] Selection of positive clones: Four clones were selected and transferred to 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate. The cultures were incubated at 37°C and 200 rpm for 8 h. Plasmids were extracted using the Mini-Plasmid Rapid Isolation Kit (Beijing Bodatech Biotechnology Co., Ltd.). 20 μL of plasmid was taken and sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing to confirm successful construction.

[0043] Example 3: Induced expression culture of recombinant Escherichia coli The recombinant *E. coli* strains constructed in Examples 1 and 2, as well as the recombinant *E. coli* strain from the control group (pET-28a(+) empty vector transformant, as a negative control), were inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured overnight at 37°C with shaking at 200 rpm. 10 mL of the culture was then transferred to 1 L of LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured at 37°C with shaking at 200 rpm until OD (dose retardation). 600 The concentration was approximately 0.6-0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to the culture to a final concentration of 0.5 mM, and the culture was induced at 28°C and 200 rpm for 12 h. The culture medium was centrifuged at 4°C and 12000 rpm for 10 min, the supernatant was discarded, and the precipitate (bacterial cells) was collected. The bacterial cells were dispersed in 14 mL of 50 mM HEPES buffer (pH 7.5) and sonicated in an ice bath. The sonication parameters were: ultrasonic power of the ultrasonic cell disruptor (purchased from Ningbo Xinzhi Biotechnology Co., Ltd., model JY92-IIN) was set to 10%, and the sonication time was 30 min (2 s working time, 3 s interval). A portion of the sonicated bacterial cells (i.e., whole cell lysate) was directly used for SDS-PAGE detection, and the other portion was centrifuged at 4°C and 12000 rpm for 10 min. The supernatant and precipitate were then used for SDS-PAGE detection separately. Figures 1 to 3 As shown, the SDS-PAGE results indicate that the target protein has a high soluble expression level and the correct molecular weight.

[0044] Example 4 Purification of recombinant protein The recombinant *E. coli* strains constructed in Examples 1 and 2 were inoculated into 50 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured overnight at 37°C with shaking at 200 rpm. 50 mL of the culture was then transferred to 5 L of LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured at 37°C with shaking at 200 rpm until OD (dose eluent) was reached. 600 Approximately 0.6–0.8. Add IPTG to the culture to a final concentration of 0.5 mM and induce culture at 28°C and 200 rpm for 12 h. Centrifuge the culture medium at 4°C and 12000 rpm for 10 min, discard the supernatant, collect the precipitate (bacterial cells), and wash the bacterial cells three times with physiological saline to obtain wet bacterial cells.

[0045] Weigh 20g of wet bacterial cells and suspend them in 70mL of 50mM HEPES buffer (pH 7.5). Sonicate the cells in an ice bath. The sonication parameters were: 10% ultrasonic power and 30 min sonication time (2s working time, 3s interval). Centrifuge the sonicated cells at 4℃ and 12000rpm for 10 min, and collect the supernatant as the crude enzyme solution. Purify the crude enzyme solution using a His-Trap HPaffinity column. After ultrafiltration and desalting, obtain pure enzyme solutions. Concentrate each pure enzyme solution to 5mg / mL using a 30KD ultrafiltration tube at 4℃ and 6000rpm for 30 min, and store at 4℃ for later use.

[0046] Example 5: HPLC determination of glucose and fructose content Sample preparation method: Dissolve and dilute the sample with ultrapure water until the glucose and fructose contents are approximately 0.2-5 mg / mL, filter with a 0.22 μm aqueous filter membrane, and then inject for detection.

[0047] HPLC detection method: An Agilent 1260 Infinity HPLC system with a differential refractive index detector was used. The chromatographic column was Sepax Carbomix Ca-NP5 with 8% crosslinking degree (7.8×300mm, 5μm). The mobile phase was pure aqueous solution. The column temperature was 80℃, the flow rate was 0.6mL / min, the detector temperature was 35℃, and the run time was 16min.

[0048] Example 6 Enzyme activity assay of fructose-6-phosphate phosphatase and its mutants The activity of fructose-6-phosphate phosphatase and its mutants was characterized by determining the fructose content using HPLC. Enzyme activity assay conditions: Total reaction volume 10 mL, solvent 50 mM HEPES buffer (pH 7.5), including 18 g / L fructose-6-phosphate, 5 mM MgCl2, and 100 μL of 5 mg / mL fructose-6-phosphate phosphatase or its mutant enzyme solution (enzyme solution obtained in Example 4). The reaction was carried out at 70 °C and 70 rpm for 30 min, followed by the addition of 0.5 mL of 10 v / v% sulfuric acid solution to terminate the reaction. The fructose content of the product was determined according to the method in Example 5. Enzyme activity was defined as the amount of enzyme required to catalyze the formation of 1 μmol of fructose per minute (1 U). The enzyme activity test results of fructose-6-phosphate phosphatase and its mutants are shown in Table 2.

[0049] Table 2

[0050] The results above show that the fructose-6-phosphate phosphatase mutant constructed in Example 2 has higher enzyme activity compared to wild-type fructose-6-phosphate phosphatase Impasse and TcPase. Specifically, the enzyme activity of TcPase_V237R is approximately 3 times higher than that of TcPase.

[0051] Example 7: Substrate selectivity of fructose-6-phosphate phosphatase and its mutants The substrate selectivity of fructose-6-phosphate phosphatase and its mutants to fructose-6-phosphate (F6P) and glucose-6-phosphate (G6P) was determined. The total reaction volume was 10 mL, and the solvent was 50 mM HEPES buffer (pH 7.5), including 10 g / L fructose-6-phosphate or glucose-6-phosphate, 5 mM MgCl2, and 300 μL of 5 mg / mL fructose-6-phosphate phosphatase or its mutant enzyme solution (the enzyme solution obtained in Example 4). The reaction was carried out at 70 °C and 70 rpm for 6 h, and then 0.5 mL of 10 v / v% sulfuric acid solution was added to terminate the reaction. The product content was determined according to the method of Example 5. The relative activities of each enzyme to glucose-6-phosphate were calculated with the activity of each enzyme to fructose-6-phosphate as 100%. The experimental results showed that ( Figure 4 The above-mentioned fructose-6-phosphate phosphatase mutants showed significantly improved selectivity for fructose-6-phosphate compared to wild-type fructose-6-phosphate phosphatase, especially PmPase_F123R and TcPase_V237R.

[0052] Example 8: Synthesis of fructose by dual enzyme catalysis of glucose-6-phosphate isomerase, fructose-6-phosphate phosphatase and their mutants. The fructose-6-phosphate phosphatase mutants PmPase_F123R or TcPase_V237R, which exhibited strong F6P preference and were screened in Example 7, were coupled with glucose-6-phosphate isomerase TaPGI to catalyze the synthesis of fructose using glucose-6-phosphate as a substrate. The total reaction volume was 10 mL, and the solvent was 50 mM HEPES buffer (pH 7.5), including 10 g / L glucose-6-phosphate, 5 mM MgCl2, 20 μL of 5 mg / mL glucose-6-phosphate isomerase enzyme solution, and 300 μL of 5 mg / mL fructose-6-phosphate phosphatase enzyme solution (the enzyme solution obtained in Example 4). The reaction was carried out at 70 °C and 70 rpm for 6 h, and then 0.5 mL of 10 v / v% sulfuric acid solution was added to terminate the reaction. The glucose and fructose contents were determined according to the method in Example 5, and their conversion rates were calculated. Figure 5As shown in the figure, the fructose-6-phosphate phosphatase mutant TcPase_V237R exhibited good catalytic performance. When used in combination with glucose-6-phosphate isomerase TaPGI, it could completely convert the substrate into fructose and glucose, with a fructose conversion rate of 94.1%, which is higher than the 79.4% fructose conversion rate when wild-type fructose-6-phosphate phosphatase TcPase was used in combination with glucose-6-phosphate isomerase TaPGI.

[0053] Glucose conversion rate % = (mass concentration of glucose / molar mass of glucose) / (mass concentration of glucose-6-phosphate / molar mass of glucose-6-phosphate) Fructose conversion rate % = (mass concentration of fructose / molar mass of fructose) / (mass concentration of glucose-6-phosphate / molar mass of glucose-6-phosphate) Example 9 Preparation of dual-enzyme immobilized enzyme and its catalytic reaction Preparation of the two-enzyme immobilized enzyme: The total volume of the reaction system was 20 mL, and the solvent was 50 mM HEPES buffer (pH 7.5), including 1.5 mL of glucose-6-phosphate isomerase TaPGI enzyme solution (5 mg / mL) obtained in Example 4, 15 mL of fructose-6-phosphate phosphatase enzyme solution TcPase (5 mg / mL) or fructose-6-phosphate phosphatase mutant enzyme solution TcPase_V237R (5 mg / mL) obtained in Example 4, 1 g LX-1000NH amino resin (Xi'an Lanxiao Technology New Material Co., Ltd.) (amino resin is not included in the total volume of the system), 0.5 mL glutaraldehyde, and 0.2 mL polyethyleneimine. The mixture was stirred at 10 °C and 50 rpm for 5 h, and then filtered to obtain a solid. The solid was washed with 100 mL of deionized water and filtered again to obtain the two-enzyme immobilized enzyme.

[0054] The dual-enzyme immobilized enzyme-catalyzed reaction: The total reaction volume was 10 mL, and the solvent was 50 mM HEPES buffer (pH 7.5), including 20 mM MgCl2, 10 g / L glucose-6-phosphate, and 10 g / L dual-enzyme immobilized enzyme (the dual-enzyme immobilized enzyme was not included in the total reaction volume). The reaction was carried out at 70 °C and 70 rpm for 6 h, and the dual-enzyme immobilized enzyme in the reaction solution was filtered off. The amount of fructose generated in the reaction was detected according to the method in Example 5. The fructose conversion rate was calculated as follows: fructose conversion rate % = (mass concentration of fructose / molar mass of fructose) / (mass concentration of glucose-6-phosphate / molar mass of glucose-6-phosphate). The calculated fructose conversion rate of the TaPGI+TcPase_V237R dual-enzyme immobilized enzyme was 94.2%, while that of the TaPGI+TcPase dual-enzyme immobilized enzyme was only 75.4%.

[0055] The immobilized enzymes were washed with 10 mL of deionized water and reused in the reaction, for a total of 20 reuses. The relative enzyme activities of the immobilized enzymes were calculated at different reuse counts (with the enzyme activity measured at 0 reuses for each immobilized enzyme as 100%). Figure 6 As shown, the TaPGI+TcPase_V237R dual-enzyme immobilized enzyme retained 90.2% of its relative enzyme activity after 20 applications, while the TaPGI+TcPase dual-enzyme immobilized enzyme retained only 65.0% of its relative enzyme activity after 20 applications.

Claims

1. A dual-enzyme immobilized enzyme for fructose synthesis, characterized in that, It is obtained by immobilizing glucose-6-phosphate isomerase and fructose-6-phosphate phosphatase mutant TcPase_V237R on resin; the fructose-6-phosphate phosphatase mutant TcPase_V237R is obtained by mutating valine at position 237 of fructose-6-phosphate phosphatase TcPase to arginine, i.e., V237R, and the amino acid sequence of fructose-6-phosphate phosphatase TcPase is shown in SEQ ID NO:

5.

2. The dual-enzyme immobilized enzyme for fructose synthesis according to claim 1, characterized in that, The glucose-6-phosphate isomerase is glucose-6-phosphate isomerase TaPGI, and its amino acid sequence is shown in SEQ ID NO:

1.

3. A dual-enzyme immobilized enzyme for fructose synthesis according to claim 1 or 2, characterized in that, The resin is an amino resin.

4. The dual-enzyme immobilized enzyme for fructose synthesis according to claim 3, characterized in that, The amino resin includes LX-1000NH amino resin or ESR-1 amino resin.

5. A method for preparing a dual-enzyme immobilized enzyme for fructose synthesis as described in any one of claims 1-4.

6. The preparation method according to claim 5, characterized in that, The process includes the following steps: using 50-200mM HEPES buffer (pH 6.0-8.0) as solvent, glucose-6-phosphate isomerase, fructose-6-phosphate phosphatase mutant, and resin are added. The mass ratio of glucose-6-phosphate isomerase to resin is 5-50 mg:1 g, and the mass ratio of fructose-6-phosphate phosphatase mutant to resin is 50-200 mg:1 g. 1v / v%-5v / v% glutaraldehyde and 1v / v%-5v / v% polyethyleneimine are added. The immobilization temperature is 4-10℃, the stirring speed is 20-100 rpm, and the immobilization time is 2-5 h. After filtration and washing, the dual-enzyme immobilized enzyme is obtained.

7. The application of the dual-enzyme immobilized enzyme for fructose synthesis as described in any one of claims 1-4 in fructose synthesis.

8. The application according to claim 7, characterized in that, In application, the dual-enzyme immobilized enzyme is used as a catalyst, glucose-6-phosphate is used as a substrate, and in the presence of MgCl2, the temperature and stirring speed are controlled to carry out enzyme catalysis to produce fructose in the reaction medium.

9. The application according to claim 8, characterized in that, The reaction medium is 50-200mM HEPES buffer with pH 6.0-8.

0. The amount of enzyme used for dual-enzyme immobilization is 5-20g / L, the concentration of glucose-6-phosphate is 5-30g / L, the concentration of MgCl2 is 5-20mM, the temperature is controlled at 60-80℃, the stirring speed is 70-300rpm, and the reaction time is 4-10h.

10. A fructose-6-phosphate phosphatase mutant, TcPase_V237R, characterized in that, The fructose-6-phosphate phosphatase mutant TcPase_V237R is obtained by mutating valine at position 237 of fructose-6-phosphate phosphatase TcPase to arginine, i.e., V237R. The amino acid sequence of fructose-6-phosphate phosphatase TcPase is shown in SEQ ID NO: 5.

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