A multi-enzyme immobilized enzyme for glucose-6-phosphate synthesis and a preparation method and application thereof

Abstract

The application discloses a multi-enzyme immobilized enzyme for glucose-6-phosphate synthesis, which is obtained by immobilizing three enzymes, starch debranching enzyme mutant AcDBE_E131R, maltodextrin phosphorylase mutant AtMalP_N188D and glucose phosphomutase mutant TkPGM_F360P, on a resin. The application also discloses a preparation method and application of the multi-enzyme immobilized enzyme. The application screens enzyme mutants with good thermal stability and high catalytic activity by site-directed mutagenesis of starch debranching enzymes, maltodextrin phosphorylases and glucose phosphomutases from different sources, and prepares the multi-enzyme immobilized enzyme AcDBE_E131R+AtMalP_N188D+TkPGM_F360P. The multi-enzyme immobilized enzyme is used as a catalyst, maltodextrin is used as a substrate, and glucose-6-phosphate is efficiently synthesized by one-pot method, and the conversion rate reaches 71.2%. After 50 times of reuse, the relative enzyme activity of the multi-enzyme immobilized enzyme still remains 85.1%.

Description

Technical Field

[0001] This invention relates to the field of enzymatic catalytic synthesis technology, specifically to a multi-enzyme immobilized enzyme for glucose-6-phosphate synthesis, its preparation method, and its application. Background Technology

[0002] Glucose-6-phosphate can be metabolized into various high-value compounds such as nucleotides, amino sugars, and glycosaminoglycans, which have wide applications in the pharmaceutical, nutritional, and biomanufacturing fields. Traditional chemical synthesis methods suffer from drawbacks such as heavy pollution and poor specificity, making it difficult to meet industrialization needs. Biosynthesis is mainly divided into fermentation and enzymatic synthesis. However, because glucose-6-phosphate is a key intermediate in several core metabolic pathways, it is easily and rapidly consumed and metabolized during fermentation, leading to difficulties in product accumulation. In contrast, enzymatic synthesis, with its advantages of being green, environmentally friendly, and allowing for controllable reactions, is gradually becoming a research hotspot.

[0003] Enzymatic synthesis uses maltodextrin as a substrate. Starch debranching enzyme hydrolyzes the α-1,6 glycosidic bonds in maltodextrin, eliminating the steric hindrance of branched chains to phosphorylase. Maltodextrin phosphorylase then catalyzes the cleavage of the α-1,4 glycosidic bonds and introduces a phosphate group, generating glucose-1-phosphate. This is then isomerized to glucose-6-phosphate by glucose phosphate mutase. However, existing enzymatic synthesis technologies still face many bottlenecks. Free enzyme systems are costly and difficult to recycle, and wild-type enzymes from different sources generally have low catalytic activity, leading to low conversion rates and increased production costs. Therefore, rational modification and optimization of key enzymes through enzyme engineering are needed to improve the economic feasibility of glucose-6-phosphate enzymatic synthesis. Summary of the Invention

[0004] The purpose of this invention is to provide a multi-enzyme immobilized enzyme for glucose-6-phosphate synthesis, its preparation method, and its application, in order to overcome the shortcomings of the prior art.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a multi-enzyme immobilized enzyme for the synthesis of glucose-6-phosphate, which is obtained by immobilizing three enzymes—a starch debranching enzyme mutant AcDBE_E131R, a maltodextrin phosphorylase mutant AtMalP_N188D, and a glucose phosphate mutase mutant TkPGM_F360P—on a resin.

[0007] The starch debranching enzyme mutant AcDBE_E131R is formed by mutating glutamic acid at position 131 of the wild-type starch debranching enzyme AcDBE to arginine, i.e., E131R. The amino acid sequence of the wild-type starch debranching enzyme AcDBE is shown in SEQ ID NO: 3.

[0008] The maltodextrin phosphorylase mutant AtMalP_N188D is formed by the mutation of asparagine at position 188 of the wild-type maltodextrin phosphorylase AtMalP to aspartic acid, i.e., N188D. The amino acid sequence of the wild-type maltodextrin phosphorylase AtMalP is shown in SEQ ID NO: 5.

[0009] The glucose phosphate mutase mutant TkPGM_F360P is formed by mutating phenylalanine at position 360 of the wild-type glucose phosphate mutase TkPGM to proline, i.e., F360P. The amino acid sequence of the wild-type glucose phosphate mutase TkPGM is shown in SEQ ID NO: 11.

[0010] Furthermore, the resin is an amino resin.

[0011] Furthermore, the amino resin includes LX-1000NH amino resin or ESR-1 amino resin.

[0012] The second aspect of this invention provides a method for preparing the above-mentioned multi-enzyme immobilized enzyme for glucose-6-phosphate synthesis, comprising the following steps: using 50-200 mM HEPES buffer, pH 6.0-8.0 as a solvent, adding starch debranching enzyme mutant AcDBE_E131R, maltodextrin phosphorylase mutant AtMalP_N188D, glucose phosphate mutase mutant TkPGM_F360P, and resin; the mass ratio of starch debranching enzyme mutant AcDBE_E131R to resin is 10-100. The mass ratio of maltodextrin phosphorylase mutant AtMalP_N188D to resin was 20-200 mg:1g, and the mass ratio of glucose phosphate mutase mutant TkPGM_F360P to resin was 10-100 mg:1g. 1v / v%-5v / v% glutaraldehyde and 1v / v%-5v / v% polyethyleneimine were added. The immobilization temperature was 4-10℃, the stirring speed was 20-100 rpm, and the immobilization time was 2-5 h. After filtration and washing, the multi-enzyme immobilized enzyme was obtained.

[0013] The third aspect of the present invention provides the application of the above-mentioned multi-enzyme immobilized enzyme for glucose-6-phosphate synthesis in the synthesis of glucose-6-phosphate. In the application, the multi-enzyme immobilized enzyme is used as a catalyst, maltodextrin is used as a substrate, and in the presence of phosphate and MgCl2, the reaction temperature and stirring speed are controlled to carry out enzyme catalysis to generate glucose-6-phosphate in the reaction medium.

[0014] Furthermore, the phosphate includes KH2PO4.

[0015] Furthermore, the reaction medium is 50-200mM HEPES buffer with pH 6.0-8.0, the amount of multi-enzyme immobilized enzyme is 5-20g / L, the concentration of maltodextrin is 10-100mg / mL, the concentration of phosphate is 20-200mM, the concentration of MgCl2 is 2-20mM, the reaction temperature is 60-80℃, the stirring speed is 70-300rpm, and the reaction time is 6-20h.

[0016] The fourth aspect of the present invention provides a starch debranching enzyme mutant AcDBE_E131R, which is a mutation of glutamic acid at position 131 of wild-type starch debranching enzyme AcDBE to arginine, namely E131R, and the amino acid sequence of the wild-type starch debranching enzyme AcDBE is shown in SEQ ID NO: 3.

[0017] The fifth aspect of the present invention provides a maltodextrin phosphorylase mutant AtMalP_N188D, which is a mutation of asparagine at position 188 of wild-type maltodextrin phosphorylase AtMalP to aspartic acid, i.e., N188D. The amino acid sequence of the wild-type maltodextrin phosphorylase AtMalP is shown in SEQ ID NO: 5.

[0018] The sixth aspect of the present invention provides a glucose phosphate mutase mutant TkPGM_F360P, which is a mutation of phenylalanine at position 360 of wild-type glucose phosphate mutase TkPGM to proline, i.e., F360P. The amino acid sequence of the wild-type glucose phosphate mutase TkPGM is shown in SEQ ID NO: 11.

[0019] The beneficial effects of this invention are:

[0020] This invention screens out enzyme mutants with good thermal stability and high catalytic activity by performing site-directed mutagenesis on starch debranching enzyme, maltodextrin phosphorylase, and glucose phosphate mutase from different sources. The obtained starch debranching enzyme mutant AcDBE_E131R, maltodextrin phosphorylase mutant AtMalP_N188D, and glucose phosphate mutase mutant TkPGM_F360P all maintained relative enzyme activities of over 85% after heat treatment at 60℃ for 60 h. Using the three enzyme mutants mentioned above, a multi-enzyme immobilized enzyme AcDBE_E131R+AtMalP_N188D+TkPGM_F360P was prepared. Using this enzyme as a catalyst and maltodextrin as a substrate, glucose-6-phosphate was synthesized efficiently in a one-pot process with a conversion rate of 71.2%. After 50 cycles of use, it still retained 85.1% of the relative enzyme activity (compared to only 30.4% for the glucose-6-phosphate conversion rate of the multi-enzyme immobilized enzyme AcDBE+AtMalP+TkPGM, which retained only 23.4% of the relative enzyme activity after 80 cycles), demonstrating excellent prospects for industrial application. Attached Figure Description

[0021] Figure 1 The results of SDS-PAGE analysis of starch debranching enzyme SsDBE and its mutants in Example 3 are shown.

[0022] Figure 2 The results of SDS-PAGE analysis of starch debranching enzyme AcDBE and its mutants in Example 3 are shown.

[0023] Figure 3 The results of SDS-PAGE analysis of maltodextrin phosphorylase AtMalP and its mutants in Example 3 are shown.

[0024] Figure 4 The results of SDS-PAGE analysis of maltodextrin phosphorylase TpMalP and its mutants in Example 3 are shown.

[0025] Figure 5 The results of SDS-PAGE analysis of glucose phosphate mutase TtPGM and its mutants in Example 3 are shown.

[0026] Figure 6 The results of SDS-PAGE analysis of glucose phosphate mutase TkPGM and its mutants in Example 3 are shown.

[0027] Figure 7 The results show the thermostability of starch debranching enzymes SsDBE and AcDBE and their mutants.

[0028] Figure 8 Results of thermostability determination of maltodextrin phosphorylases AtMalP and TpMalP and their mutants.

[0029] Figure 9 The results show the thermostability of glucose phosphate mutases TtPGM and TkPGM and their mutants.

[0030] Figure 10 The results of 50 relative enzyme activity assays were performed on each immobilized enzyme. Detailed Implementation

[0031] 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.

[0032] 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.

[0033] The culture medium and reagent formulations involved in the following examples are as follows:

[0034] SOC medium (1L): 20g tryptone, 5g yeast extract, 0.5g sodium chloride, 10mL 250mM 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.

[0035] LB liquid medium (1L): 10g tryptone, 5g yeast extract, 10g sodium chloride; pH 7.0.

[0036] LB solid medium is made by adding 20g of agar powder to 1L of LB liquid medium.

[0037] All of the above culture media need to be autoclaved at 121°C for 20 minutes.

[0038] 3,5-Dinitrosalicylic acid (DNS) solution (1L): Add 10g NaOH, 10g 3,5-dinitrosalicylic acid, 200g potassium sodium tartrate, 2g phenol, and 5g anhydrous sodium sulfite in that order; let stand for one week after preparation before use, and store away from light.

[0039] Example 1: Construction of recombinant Escherichia coli containing starch debranching enzyme, maltodextrin phosphorylase, and glucose phosphate mutase

[0040] I. Construction of Recombinant Plasmids

[0041] Sulfide leaf fungi ( Saccharolobus solfataricus The gene fragment encoding the wild-type starch debranching enzyme SsDBE in the genome (gene sequence shown in SEQ ID NO: 2, amino acid sequence shown in SEQ ID NO: 1), a heat-soluble cellulose-decomposing bacterium ( Acidothermus cellulolyticus The gene segment encoding wild-type starch debranching enzyme in the AcDBE genome (gene sequence as shown in SEQ ID NO: 4, amino acid sequence as shown in SEQ ID NO: 3);

[0042] Clostridium thermophilum ( Acetivibrio thermocellus The gene fragment encoding the wild-type maltodextrin phosphorylase AtMalP in the genome (gene sequence as shown in SEQ ID NO: 6, amino acid sequence as shown in SEQ ID NO: 5); *Thermophilus* ( Thermotoga petrophila The gene fragment encoding wild-type maltodextrin phosphorylase TpMalP in the genome (gene sequence as shown in SEQ ID NO: 8, amino acid sequence as shown in SEQ ID NO: 7);

[0043] Thermophilic bacteria ( Thermus thermophilusThe gene fragment encoding glucose-phosphate mutase TtPGM in the genome of *Thermococcus thermophilus* (gene sequence as shown in SEQ ID NO: 10, amino acid sequence as shown in SEQ ID NO: 9) Thermococcus kodakarensis The gene fragment encoding glucose phosphate mutase TkPGM in the genome (gene sequence as shown in SEQ ID NO: 12, amino acid sequence as shown in SEQ ID NO: 11) 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.

[0044] II. Transformation of Escherichia coli with recombinant plasmids E. coli BL21(DE3)

[0045] 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. 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.

[0046] 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.

[0047] Example 2: Construction of recombinant *E. coli* with starch debranching enzyme mutant, maltodextrin phosphorylase mutant, and glucose phosphate mutase mutant

[0048] I. Construction of Recombinant Plasmids

[0049] The sequences of wild-type starch debranching enzymes (SsDBE, AcDBE), maltodextrin phosphorylases (AtMalP, TpMalP), and wild-type glucose phosphate mutases (TtPGM, TkPGM) in step one of Example 1 were analyzed. Using bioinformatics methods, several single-point mutants were designed, as shown in Table 1.

[0050] Table 1

[0051] enzyme name mutation site SsDBE M222L or T353S or L378D AcDBE E131R or A425L or G622P AtMalP N188D or Q301R or Y422H TpMalP D167K, Y281L, or R751P TtPGM T145N or L192P or E345K TkPGM I145L or V178G or F360P

[0052] SsDBE_M222L is a wild-type starch debranching enzyme SsDBE with a methionine mutation at position 222 replaced by leucine.

[0053] SsDBE_T353S is a mutation where the threonine at position 353 of the wild-type starch debranching enzyme SsDBE is replaced with serine.

[0054] SsDBE_L378D is a wild-type starch debranching enzyme SsDBE with a leucine mutation at position 378, replacing it with aspartic acid.

[0055] AcDBE_E131R is a wild-type starch debranching enzyme AcDBE with a glutamic acid mutation at position 131 to arginine.

[0056] AcDBE_A425L is a wild-type starch debranching enzyme AcDBE with a mutation at position 425 (alanine) replaced by leucine.

[0057] AcDBE_G622P is a wild-type starch debranching enzyme AcDBE with a glycine mutation at position 622 to proline.

[0058] AtMalP_N188D is a mutation at position 188 of the wild-type maltodextrin phosphorylase AtMalP, where asparagine is replaced by aspartic acid.

[0059] AtMalP_Q301R is a wild-type maltodextrin phosphorylase AtMalP with a glutamine-arginine mutation at position 301.

[0060] AtMalP_Y422H is a mutation at position 422 of the wild-type maltodextrin phosphorylase AtMalP, replacing tyrosine with histidine.

[0061] TpMalP_D167K is a mutation at position 167 of the wild-type maltodextrin phosphorylase TpMalP, where aspartic acid is replaced with lysine.

[0062] TpMalP_Y281L is a wild-type maltodextrin phosphorylase TpMalP with a tyrosine mutation at position 281 to leucine.

[0063] TpMalP_R751P is a mutation at position 751 of the wild-type maltodextrin phosphorylase TpMalP, where arginine is replaced with proline.

[0064] TtPGM_T145N is a mutation of threonine at position 145 of the wild-type glucose phosphate mutase TtPGM to asparagine.

[0065] TtPGM_L192P is a mutation of leucine at position 192 of the wild-type glucose phosphate mutase TtPGM, which is replaced with proline.

[0066] TtPGM_E345K is a mutation of glutamic acid at position 345 of the wild-type glucose phosphate mutase TtPGM, replacing it with lysine.

[0067] TkPGM_I145L is a wild-type glucose phosphate mutase TkPGM with a mutation of isoleucine at position 145 to leucine.

[0068] TkPGM_V178G is a wild-type glucose phosphate mutase TkPGM with a valine mutation at position 178 to glycine.

[0069] TkPGM_F360P is a wild-type glucose phosphate mutase TkPGM with a phenylalanine mutation at position 360 replaced by proline.

[0070] 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.

[0071] II. Transformation of Escherichia coli with recombinant plasmids E. coli BL21(DE3)

[0072] 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.

[0073] 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.

[0074] Example 3: Induction and expression culture of recombinant Escherichia coli

[0075] 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 (cells) was collected. The starch debranching enzyme cells were resuspended in 14 mL of 50 mM MES buffer (pH 6.0), and the maltodextrin phosphorylase or glucose phosphate mutase cells were resuspended in 14 mL of 50 mM HEPES buffer (pH 7.0). The cells were then subjected to ultrasonic disruption in an ice bath. The ultrasonic disruption parameters were as follows: the ultrasonic power of the ultrasonic cell disruptor (purchased from Ningbo Xinzhi Biotechnology Co., Ltd., model JY92-IIN) was set to 10%, and the ultrasonic disruption time was 30 min (2 s working time, 3 s interval). A portion of the ultrasonically disrupted bacterial cells (i.e., whole-cell lysate) was directly used for SDS-PAGE analysis, while the other portion was centrifuged at 4°C and 12,000 rpm for 10 min. The supernatant and precipitate were then separately used for SDS-PAGE analysis. Figure 1-6 As shown, the SDS-PAGE results indicate that the soluble expression levels of each target protein are high and the molecular weights are correct.

[0076] Example 4 Purification of recombinant protein

[0077] 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.

[0078] Weigh 20g of wet bacterial cells. Suspend starch-debranched cells in 70mL of 50mM MES buffer (pH 6.0). Suspend maltodextrin phosphorylase cells or glucose phosphate mutase cells in 70mL of 50mM HEPES buffer (pH 7.0). Perform ultrasonic disruption in an ice bath. The ultrasonic disruption parameters are: ultrasonic power of the ultrasonic cell disruptor (purchased from Ningbo Xinzhi Biotechnology Co., Ltd., model JY92-IIN) at 10%, and ultrasonic disruption time at 30min (2s working time, 3s interval). Centrifuge the disrupted bacterial cells at 4℃ and 12000rpm for 10min, and collect the supernatant as the crude enzyme solution. Purify the crude enzyme solution using a His-Trap HP affinity column. After ultrafiltration and desalting, obtain a pure enzyme solution. Concentrate the pure enzyme solution to 5mg / mL using a 30KD ultrafiltration tube at 4℃ and 6000rpm for 30min. The obtained starch debranching enzyme, maltodextrin phosphorylase, glucose phosphate mutase, and their mutant enzyme solutions (5 mg / mL) were stored at 4°C for later use. The enzyme solutions required for the following examples were provided in this example; if some enzyme solutions are insufficient, they can be prepared by scaling up the process.

[0079] Example 5: Determination of glucose-1-phosphate and glucose-6-phosphate content by HPLC

[0080] Sample preparation method: Dissolve and dilute the sample with ultrapure water to a glucose-1-phosphate or glucose-6-phosphate content of approximately 0.2-10 mg / mL. Filter the sample through a 0.22 μm aqueous filter membrane before injection. If the sample concentration is below 0.2 mg / mL, it needs to be concentrated before measurement.

[0081] HPLC detection method: An Agilent 1260 Infinity HPLC system with a differential refractive index detector was used. The chromatographic column was a Welch Xtimate Sugar-H column (7.8×300mm, 5μm). The mobile phase was sulfuric acid solution (pH 1.15). The column temperature was 80℃, the flow rate was 0.8mL / min, the detector temperature was 35℃, and the run time was 10min.

[0082] Example 6: Determination of maltodextrin content by HPLC

[0083] Sample preparation method: Dissolve and dilute the sample with ultrapure water until the maltodextrin content is about 1-10 mg / mL, filter it through a 0.22 μm aqueous filter membrane and then inject it for detection.

[0084] HPLC detection method: An Agilent 1260 Infinity HPLC system with an evaporative light scattering detector was used. The chromatographic column was a Welch Xtimate Sugar-H column (7.8×300mm, 5μm). The mobile phase was formic acid solution (pH 1.15). The column temperature was 80℃, the flow rate was 0.8mL / min, the run time was 10min, and the detector parameters were: evaporator temperature 80℃, nebulizer temperature 80℃, and gas flow rate 1.0L / min.

[0085] Example 7 Enzyme activity assay of starch debranching enzyme and its mutant

[0086] The enzyme activity of starch debranching enzyme and its mutants was determined by the 3,5-dinitrosalicylic acid (DNS) method. Enzyme activity assay conditions: Total reaction volume 1 mL, solvent 50 mM MES buffer (pH 6.0), including 0.5% (w / v) limiting dextrin solution, 1 μg of the starch debranching enzyme or its mutant (10 μL of the 5 mg / mL enzyme solution obtained in Example 4 was diluted to 0.1 mg / mL with 50 mM MES buffer (pH 6.0) and added), reacted at 60 °C and 70 rpm for 3 min. After adding 1 mL of DNS solution and mixing, the mixture was boiled in a water bath for 7 min for color development, then rapidly cooled to room temperature in water, and the absorbance was measured at 540 nm. Enzyme activity was defined as the amount of enzyme required to catalyze the production of 1 μmol of reducing sugar (a mixture of maltodextrins, mainly maltotriose) per minute under the above assay conditions.

[0087] The enzyme activity test results of starch debranching enzyme and its mutants are shown in Table 2.

[0088] Table 2

[0089] enzymes Enzyme activity (U / mg) SsDBE 405±13 SsDBE_M222L 485±25 SsDBE_T353S 936±20 SsDBE_L378D 675±14 AcDBE 527±16 AcDBE_E131R 1230±27 AcDBE_A425L 925±15 AcDBE_G622P 872±13

[0090] The results above show that, compared with wild-type starch debranching enzymes SsDBE and AcDBE, the starch debranching enzyme mutants constructed in Example 2 all have higher enzyme activities. Among them, the starch debranching enzyme mutant AcDBE_E131R has an enzyme activity of 1230 U / mg.

[0091] Example 8 Enzyme activity assay of maltodextrin phosphorylase and its mutants

[0092] Enzyme activity assay conditions: Total reaction volume 5 mL, solvent 50 mM HEPES buffer (pH 7.0), including 10 mg / mL maltodextrin, 100 mM KH₂PO₄, 5 mM MgCl₂, 10 μg of the maltodextrin phosphorylase or its mutant (2 μL of the 5 mg / mL enzyme solution obtained in Example 4), reacted at 60 °C and 70 rpm for 5 min, then boiled for 5 min to terminate the reaction. The glucose-1-phosphate concentration was determined according to the HPLC method of Example 5. Enzyme activity was defined as: 1 U is the amount of enzyme required to catalyze the formation of 1 μmol of glucose-1-phosphate per minute.

[0093] The enzyme activity test results of maltodextrin phosphorylase and its mutants are shown in Table 3.

[0094] Table 3

[0095] enzymes Enzyme activity (U / mg) AtMalP 115±4 AtMalP_N188D 345±12 AtMalP_Q301R 206±7 AtMalP_Y422H 190±8 TpMalP 75±2 TpMalP_D167K 272±10 TpMalP_Y281L 125±5 TpMalP_R751P 207±8

[0096] The results above show that the maltodextrin phosphorylase mutant constructed in Example 2 has higher enzyme activity compared to wild-type maltodextrin phosphorylases AtMalP and TpMalP. Specifically, the maltodextrin phosphorylase mutant AtMalP_N188D achieved an enzyme activity of 345 U / mg, which is three times higher than that of wild-type maltodextrin phosphorylase AtMalP, indicating a significant improvement in catalytic activity.

[0097] Example 9 Enzyme activity assay of glucose phosphate mutase and its mutants

[0098] Enzyme activity assay conditions: Total reaction volume 10 mL, solvent 50 mM HEPES buffer (pH 7.0), including 5 mM MgCl2, 10 mM glucose-1-phosphate, 200 μM glucose-1,6-bisphosphate, 2 μg of the glucose phosphate mutase or its mutant (20 μL of the 5 mg / mL enzyme solution obtained in Example 4 was diluted to 0.1 mg / mL with 50 mM HEPES buffer (pH 7.0)). The reaction was carried out at 60°C and 70 rpm for 5 min, then 0.3 mL of 10% (v / v) sulfuric acid was added to terminate the reaction. The glucose-6-phosphate concentration was determined according to the HPLC method of Example 5. Enzyme activity was defined as: 1 U is the amount of enzyme required to catalyze the formation of 1 μmol of glucose-6-phosphate per minute.

[0099] The enzyme activity test results of glucose phosphate mutase and its mutants are shown in Table 4.

[0100] Table 4

[0101] enzymes Enzyme activity (U / mg) TtPGM 385±9 TtPGM_T145N 790±14 TtPGM_L192P 505±21 TtPGM_E345K 820±13 TkPGM 425±14 TkPGM_I145L 620±8 TkPGM_V178G 775±10 TkPGM_F360P 980±19

[0102] The results above show that, compared with wild-type glucose phosphate mutases TtPGM and TkPGM, the glucose phosphate mutase mutants constructed in Example 2 have higher enzyme activity. Among them, the enzyme activity of glucose phosphate mutase mutant TkPGM_F360P reaches 980 U / mg, showing good application prospects.

[0103] Example 10: Determination of the thermostability of wild-type enzymes and their mutants

[0104] The enzyme solutions (5 mg / mL) of starch debranching enzyme, maltodextrin phosphorylase, and glucose phosphate mutase and their mutants obtained in Example 4 were each incubated in a 60°C water bath for 10 h, 20 h, 30 h, 40 h, 50 h, and 60 h, respectively. After incubation, the heat-treated samples were immediately transferred to an ice-water mixture to terminate the heat effect, resulting in heat-treated enzyme solutions. Enzyme activity was measured under the same reaction conditions according to the corresponding enzyme activity assay methods. The relative enzyme activities of starch debranching enzyme, maltodextrin phosphorylase, and glucose phosphate mutase and their mutants after heat treatment for different times were calculated (with the highest enzyme activity (i.e., enzyme activity at 0 h) in the thermal stability assay as 100%). The results are as follows: Figure 7-9 As shown in the results, the thermostability of the various starch debranching enzyme mutants, maltodextrin phosphorylase mutants, and glucose phosphate mutase mutants was significantly improved compared with the wild type. Specifically, the starch debranching enzyme mutant AcDBE_E131R, the maltodextrin phosphorylase mutant AtMalP_N188D, and the glucose phosphate mutase mutant TkPGM_F360P all maintained relative enzyme activities above 85% after heat treatment at 60℃ for 60 h.

[0105] Example 11 Preparation of multi-enzyme immobilized enzymes and their application in glucose-6-phosphate synthesis

[0106] Preparation of multi-enzyme immobilized enzyme: The total volume of the reaction system was 20 mL, and the solvent was 50 mM HEPES buffer (pH 7.0). The reaction system included 2 mL of starch debranching enzyme AcDBE or its mutant AcDBE_E131R obtained in Example 4 (5 mg / mL), 4 mL of maltodextrin phosphorylase AtMalP or its mutant AtMalP_N188D obtained in Example 4 (5 mg / mL), 2 mL of glucose phosphate mutase TkPGM or its mutant TkPGM_F360P obtained in Example 4 (5 mg / mL), 1 g of LX-1000NH amino resin (Xi'an Lanxiao Technology New Material Co., Ltd.) (the amino resin was not included in the total volume of the system), 0.5 mL of glutaraldehyde, and 0.2 mL of polyethyleneimine. The mixture was stirred at 10 °C and 50 rpm for 5 h. The solid was then filtered to obtain the multi-enzyme immobilized enzyme. The solid was washed with 100 mL of deionized water and filtered again to obtain the multi-enzyme immobilized enzyme.

[0107] Multi-enzyme immobilized enzyme-catalyzed reaction: The total volume of the reaction system was 10 mL, the solvent was 50 mM HEPES buffer (pH 7.0), including 50 mg / mL maltodextrin, 100 mM KH2PO4, 20 mM MgCl2, and 10 g / L of multi-enzyme immobilized enzyme (the multi-enzyme immobilized enzyme was not included in the total volume of the reaction system). The reaction temperature was 60 °C, the stirring speed was 70 rpm, and the reaction time was 6 h. After the reaction, the multi-enzyme immobilized enzyme in the reaction solution was immediately filtered off. The amounts of maltodextrin, glucose-1-phosphate, and glucose-6-phosphate were detected according to the methods described in Examples 5 and 6, and the conversion rate of glucose-6-phosphate was calculated. The formula for calculating the conversion rate of glucose-6-phosphate is: Conversion rate of glucose-6-phosphate (%) = actual molar concentration of glucose-6-phosphate generated / molar concentration of glucose-6-phosphate that can be generated from complete substrate conversion × 100%. Calculations showed that the glucose-6-phosphate conversion rate of the multi-enzyme immobilized enzyme AcDBE_E131R+AtMalP_N188D+TkPGM_F360P reached 71.2%, while the glucose-6-phosphate conversion rate of AcDBE+AtMalP+TkPGM was only 30.4%.

[0108] The filtered immobilized multi-enzyme was washed with 10 mL of deionized water and filtered again. The reaction mixture was reused 80 times. The relative enzyme activity of the immobilized multi-enzyme was calculated at different reuse counts (with the enzyme activity measured at 0 reuses as 100%). The experimental results are as follows: Figure 10As shown, the multi-enzyme immobilized enzyme AcDBE_E131R+AtMalP_N188D+TkPGM_F360P retained 85.1% of its relative enzyme activity after 80 applications, while the multi-enzyme immobilized enzyme AcDBE+AtMalP+TkPGM retained only 23.4% of its relative enzyme activity after 80 applications.

Claims

1. A multi-enzyme immobilized enzyme for glucose-6-phosphate synthesis, characterized in that, It was obtained by immobilizing three enzymes—starch debranching enzyme mutant AcDBE_E131R, maltodextrin phosphorylase mutant AtMalP_N188D, and glucose phosphate mutase mutant TkPGM_F360P—on resin. The starch debranching enzyme mutant AcDBE_E131R is formed by mutating glutamic acid at position 131 of the wild-type starch debranching enzyme AcDBE to arginine, i.e., E131R. The amino acid sequence of the wild-type starch debranching enzyme AcDBE is shown in SEQ ID NO:

3. The maltodextrin phosphorylase mutant AtMalP_N188D is formed by the mutation of asparagine at position 188 of the wild-type maltodextrin phosphorylase AtMalP to aspartic acid, i.e., N188D. The amino acid sequence of the wild-type maltodextrin phosphorylase AtMalP is shown in SEQ ID NO:

5. The glucose phosphate mutase mutant TkPGM_F360P is formed by mutating phenylalanine at position 360 of the wild-type glucose phosphate mutase TkPGM to proline, i.e., F360P. The amino acid sequence of the wild-type glucose phosphate mutase TkPGM is shown in SEQ ID NO:

11.

2. The multi-enzyme immobilized enzyme for glucose-6-phosphate synthesis according to claim 1, characterized in that, The resin is an amino resin.

3. The multi-enzyme immobilized enzyme for glucose-6-phosphate synthesis according to claim 2, characterized in that, The amino resin includes LX-1000NH amino resin or ESR-1 amino resin.

4. A method for preparing a multi-enzyme immobilized enzyme for glucose-6-phosphate synthesis according to any one of claims 1-3, characterized in that, The steps include: using 50-200mM HEPES buffer (pH 6.0-8.0) as solvent, adding starch debranching enzyme mutant AcDBE_E131R, maltodextrin phosphorylase mutant AtMalP_N188D, glucose phosphate mutase mutant TkPGM_F360P, and resin; the mass ratio of starch debranching enzyme mutant AcDBE_E131R to resin is 10-100mg:1g, and maltodextrin phosphorylase mutant A... The mass ratio of tMalP_N188D to resin was 20-200 mg:1 g, the mass ratio of glucose phosphate mutase mutant TkPGM_F360P to resin was 10-100 mg:1 g, and 1 v / v%-5 v / v% glutaraldehyde and 1 v / v%-5 v / v% polyethyleneimine were added. The immobilization temperature was 4-10℃, the stirring speed was 20-100 rpm, and the immobilization time was 2-5 h. After filtration and washing, the multi-enzyme immobilized enzyme was obtained.

5. The application of the multi-enzyme immobilized enzyme for glucose-6-phosphate synthesis according to any one of claims 1-3, characterized in that, In application, the multi-enzyme immobilized enzyme is used as a catalyst, maltodextrin is used as a substrate, and in the presence of phosphate and MgCl2, the reaction temperature and stirring speed are controlled to catalyze the production of glucose-6-phosphate in the reaction medium.

6. The application according to claim 5, characterized in that, The phosphate includes KH2PO4.

7. The application according to claim 5 or 6, characterized in that, The reaction medium is 50-200mM HEPES buffer with pH 6.0-8.

0. The amount of multi-enzyme immobilized enzyme is 5-20g / L, the concentration of maltodextrin is 10-100mg / mL, the concentration of phosphate is 20-200mM, the concentration of MgCl2 is 2-20mM, the reaction temperature is 60-80℃, the stirring speed is 70-300rpm, and the reaction time is 6-20h.

8. A starch debranching enzyme mutant AcDBE_E131R, characterized in that, The wild-type starch debranching enzyme AcDBE has a glutamic acid mutation at position 131, which is replaced by arginine, i.e., E131R. The amino acid sequence of the wild-type starch debranching enzyme AcDBE is shown in SEQ ID NO:

3.

9. A maltodextrin phosphorylase mutant AtMalP_N188D, characterized in that, The wild-type maltodextrin phosphorylase AtMalP has an asparagine mutation at position 188 to aspartic acid, namely N188D. The amino acid sequence of the wild-type maltodextrin phosphorylase AtMalP is shown in SEQ ID NO:

5.

10. A glucose-phosphoryltransferase mutant TkPGM_F360P, characterized in that, The wild-type glucose phosphate mutase TkPGM has a phenylalanine mutated to proline at position 360, i.e., F360P. The amino acid sequence of the wild-type glucose phosphate mutase TkPGM is shown in SEQ ID NO: 11.

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