Method for regulating synthesis of oligogalactose by using a polysucrose macromolecular system and application thereof

Abstract

This invention discloses a method and its application for regulating the synthesis of galactooligosaccharides (GOS) using a polysucrose macromolecular system, belonging to the fields of food biotechnology and enzyme engineering technology. The invention demonstrates the high efficiency of GOS synthesis by regulating β-galactosidase using polysucrose. The synthesis efficiency shows that as the polysucrose concentration increases, the GOS synthesis rate initially increases and then decreases. The corresponding trends for glucose and GOS are the same, while those for galactose and lactose are opposite. The increased GOS synthesis rate is related to enhanced enzyme hydrolysis activity. Hydrolysis of lactose produces large amounts of glucose and galactose. Galactose participates in the transglycosylation reaction to synthesize GOS, and a high galactose concentration promotes the transglycosylation reaction, thus increasing the GOS synthesis rate. As the polysucrose concentration increases, hydrolysis activity decreases, and the amount of galactose produced also decreases, limiting the transglycosylation reaction and reducing the GOS synthesis rate. This invention achieves synergistic optimization of enzyme hydrolysis and transglycosylation activity by precisely controlling the polysucrose concentration, significantly improving the GOS synthesis rate.

Description

Technical Field

[0001] This invention relates to the fields of food biotechnology and enzyme engineering technology, specifically to a method and application of regulating the synthesis of galactooligosaccharides using a polysucrose macromolecular system. Background Technology

[0002] Galacto-oligosaccharides (GOS) are natural prebiotics that can efficiently promote the growth of beneficial gut bacteria and are widely used in the food and health product industries. Industrially, GOS is mainly synthesized by catalyzing the transglycosylation of lactose using β-galactosidase. However, in traditional aqueous solutions, the enzyme's hydrolytic activity is too high while the transglycosylation activity is too low, resulting in GOS yields generally below 30%, which is insufficient to meet the demands of efficient industrial production.

[0003] Macromolecular crowding systems can mimic intracellular crowding environments, altering enzyme catalytic preferences through size exclusion, conformational regulation, and water activity modulation. Ficoll, a food-grade macromolecular crowding agent, is non-toxic, non-immunogenic, and possesses moderate water solubility and viscosity, making it suitable for constructing food-grade enzyme catalytic systems. Currently, there are no reports on using Ficoll concentration gradients to construct regulatory systems and directionally enhance GOS synthesis efficiency.

[0004] Currently, the green preparation of functional oligosaccharides has become a key development direction in the field of food biotechnology. Galacto-oligosaccharides, with their stable physicochemical properties and excellent probiotic functions, are seeing their application scenarios continuously expand in areas such as infant formula, health foods, and special diets, leading to a steady increase in industry scale. High-efficiency synthesis technology has become the focus of industry competition. Traditional aqueous solution enzyme catalysis systems, due to their limited microenvironment and uncontrolled enzyme conformation, struggle to maximize transglycosylation activity, resulting in low product yields, difficulties in separation and purification, and insufficient economic efficiency, thus hindering the high-quality development of the industry.

[0005] Macromolecular crowding regulation technology optimizes the enzyme catalytic microenvironment in a gentle, non-genetically modified, and non-chemically modified manner, meeting food processing safety requirements and representing an important pathway to improve enzyme catalytic efficiency and selectivity. Sucrose, as a safe and stable food-grade macromolecule, possesses excellent water solubility, biocompatibility, and processing adaptability. It can regulate the spatial conformation and catalytic behavior of enzyme proteins through intermolecular interactions without affecting food quality and product safety, thus meeting the needs of continuous industrial food production. Summary of the Invention

[0006] The purpose of this invention is to provide a method and application for the controlled synthesis of galactooligosaccharides (GOS) using a polysucrose macromolecular system. By precisely controlling the concentration of polysucrose, the method aims to achieve synergistic optimization of enzyme hydrolysis and transglycosidase activity, thereby significantly improving the GOS synthesis rate.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for the synthesis of galactooligosaccharides by regulating a polysucrose macromolecular system. The method involves changing the aqueous solution reaction system to a polysucrose macromolecular reaction system, and obtaining the results by detecting the enzyme activity, RMSD and Rg values ​​of β-galactosidase, and the hydrogen bonds, α-helices and β-sheets in the secondary structure of β-galactosidase under different concentrations of polysucrose macromolecular reaction system. The materials used for testing are: sucrose with a molecular weight of 70 kDa, silica gel plate, lactose, oNPG, o-nitrophenol, dipotassium hydrogen phosphate trihydrate, citric acid monohydrate, EDTA dihydrate, sodium carbonate, n-butanol, n-propanol, ethanol, diphenylamine, aniline, acetone, and phosphoric acid.

[0008] As a preferred technical solution of the present invention: preparing polysucrose macromolecular solutions of different concentrations, the preparation method includes the following steps. A. Prepare pH 4.5 phosphate buffer: Weigh 22.5 g of dipotassium hydrogen phosphate trihydrate and 11.2 g of citric acid monohydrate, dissolve and dilute to 1000 mL to obtain pH 4.5 phosphate buffer; B. Preparation of saturated sucrose solution: Weigh 840 mg of sucrose with a molecular weight of 70 kDa and dissolve it in 1 mL of the phosphate buffer obtained in step A to obtain a saturated sucrose macromolecular solution with a concentration of 12 mM. C. Construction of the polysucrose macromolecular enzyme activity modification system: The saturated polysucrose solution obtained in step B was serially diluted with pH 4.5 phosphate buffer to prepare polysucrose macromolecular solutions with concentrations of 2, 4, 6, 8, 10, and 12 mM, thus constructing the polysucrose macromolecular enzyme activity modification system.

[0009] As a preferred technical solution of the present invention, the catalytic reaction conditions of the β-galactosidase in the polysucrose macromolecular enzyme activity modification system are: reaction temperature 55℃, transglycosylation reaction time 60 min, and hydrolysis activity determination reaction time 10 min.

[0010] As a preferred embodiment of the present invention, the β-galactosidase activity detection steps are as follows: A. Substrate preparation: Dissolve 10 mg oNPG in 1 mL of pH 4.5 phosphate buffer to prepare the oNPG substrate solution; B. Construction of the reaction system: Add 10 μL of substrate oNPG solution and 1 μL of 2 U / mL β-galactosidase solution to a centrifuge tube, add different volumes of saturated polysucrose macromolecule solution and pH 4.5 phosphate buffer, control the polysucrose concentration and make up the system volume to 100 μL; C. Enzymatic reaction: The reaction system was placed at 55 ℃ for 10 min, and the absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) reader. D. Standard curve plotting: Measure the absorbance of NPG standards with concentrations of 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, and 0.14 mM and plot the standard curve. E. Enzyme activity calculation: Convert the absorbance value into hydrolysis activity according to the standard curve. The calculation formula is: Enzyme activity (U / g) = (C×Vtotal×D) / (Venzyme×t×m); In the formula: C is the concentration of o-nitrophenol in the sample solution, in mM; Vtotal is the total volume of the reaction reagents, in μL; D is the dilution factor; Venzyme is the volume of the enzyme solution involved in the reaction, in μL; t is the reaction time, in min; and m is the mass of the enzyme powder weighed, in g.

[0011] As a preferred technical solution of the present invention, the concentration setting of the polysucrose macromolecular system enzyme activity modification system follows the following principles: the polysucrose concentration gradient is 2, 4, 6, 8, 10, 12 mM, with a pure aqueous solution reaction system as a control; the substrate oNPG concentration gradient in the hydrolysis activity assay is 0.02-0.14 mM, the lactose substrate concentration in the transglycosylation reaction is 190 mg / mL; the enzyme solution concentration is 2 U / mL, and the enzyme addition amount in the transglycosylation reaction is 2 U / μL.

[0012] As a preferred technical solution of the present invention: the β-galactosidase is placed in a 4 mM polysucrose macromolecular enzyme activity modification system, and oligogalactose is synthesized by transglycosylation catalysis of the enzyme under the conditions of lactose substrate concentration of 190 mg / mL, reaction temperature of 55 ℃ and reaction time of 60 min.

[0013] As a preferred technical solution of the present invention: the activity of β-galactosidase in the efficient synthesis of oligogalactosidase regulated by sucrose at different reaction temperatures is measured. A pH 4.5 phosphate buffer is prepared, and the experimental temperature for hydrolysis activity determination is modified to 35, 45, 55, 65, 75, and 85°C. The hydrolysis activity of β-galactosidase in the sucrose macromolecular reaction system at different reaction temperatures is measured.

[0014] As a preferred technical solution of the present invention: the activity of polysucrose-regulated β-galactosidase for efficient synthesis of oligogalactosidase is determined at different reaction times. The water bath temperature is set to 55°C. The enzyme mother liquor is mixed with polysucrose macromolecular reagent. The control group is the enzyme mixed with buffer. One sample is placed in the water bath every 10 min until the first sample is in the water bath for 30 min. All samples are then taken out. Through this operation, four samples are obtained for 0, 10, 20 and 30 min of water bath. The hydrolysis activity of each group of four samples is measured according to the enzyme activity assay method.

[0015] As a preferred technical solution of the present invention: molecular dynamics simulation was performed using Gromacs software. The crystal structure of β-galactosidase was obtained from Protein Data Bank, PDB number 4IUG, after removing other molecules and water. The simulation experiment was conducted under the following conditions: an AMBER99SB-ILDN force field was added; the model was a rectangular box; solvation was performed using the SPC216 water model; the temperature was set to 300 K; the pressure was 1 bar to simulate atmospheric pressure; and the ion concentration was determined by adding Na+. + or Cl - Ions are used to balance the charge; the total number of steps for temperature equilibrium is 50,000, with a step size of 0.002 ns; the total number of steps for pressure equilibrium is 100,000, with a step size of 0.002 ns; for the equilibrium system, molecular dynamics simulations are performed for 20 ns with a step size of 0.002 ps.

[0016] As a preferred technical solution of the present invention: by utilizing the size exclusion effect and enzyme conformation regulation of the polysucrose macromolecular enzyme activity modification system, the hydrolytic side reaction of β-galactosidase is inhibited and the main reaction of transglycosylation is promoted, so as to achieve the efficient synthesis of galactooligosaccharides. Moreover, the polysucrose is non-toxic and non-immunogenic, meets the food-grade safety requirements, and can be directly applied to the industrial production of functional food additive galactooligosaccharides.

[0017] By adopting the above technical solution, the beneficial effects of the present invention are: The β-galactosidase hydrolysis activity in the 1.4 mM polysucrose macromolecular reaction system was 2.05 U / mL, which was 1.1 times that of the pure aqueous solution reaction system. Under the same concentration conditions, the GOS synthesis rate reached a maximum of 38.6%, which was 1.23 times that of the pure aqueous solution reaction.

[0018] 2. The results of enzyme reaction kinetics and stability analysis showed that the Km value was the smallest (2.42 mM) under the reaction conditions, and the enzyme molecule had the strongest affinity for the substrate molecule; at the same time, the thermal stability of the enzyme molecule was 2.95 times that of the pure aqueous solution reaction system.

[0019] 3. Molecular dynamics simulations show that, compared with the pure aqueous solution reaction system, the RMSD and Rg values ​​of β-galactosidase are reduced in the polysucrose macromolecular reaction system, indicating increased enzyme molecule rigidity. Changes in hydrogen bonds, α-helices, and β-sheets in the secondary structure of β-galactosidase lead to alterations in the TIM barrel-shaped catalytic domain, affecting catalytic activity. The reduced hydrophilic accessible surface area of ​​the enzyme molecule decreases, reducing enzyme-water contact, which is beneficial for transglycosylation reactions. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the effect of sucrose concentration on GOS synthesis rate and hydrolysis activity in this invention. Figure 2 This is a schematic diagram illustrating the effect of temperature / reaction time on the GOS synthesis rate in the polysucrose macromolecular system of this invention; Figure 3 This is a schematic diagram of β-galactosidase RMSD and Rg in the polysucrose macromolecular system of this invention; Figure 4 This is a schematic diagram comparing the three-dimensional structures of β-galactosidase in the aqueous solution system and the 4 mM polysucrose biphase system of the present invention. Figure 5 This is a schematic diagram illustrating the effect of sucrose concentration on the water activity and enzyme hydrolysis activity of the reaction system in this invention. Figure 6 This is a schematic diagram of the simulated three-dimensional structure of β-galactosidase in the 4 mM polysucrose macromolecular system of the present invention; Figure 7 This is a schematic diagram of the β-galactosidase reaction kinetics in the polysucrose macromolecular reaction system of the present invention. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to specific embodiments. The scope of protection of the present invention is not limited to the following embodiments, and all equivalent transformations based on the technical solutions of the present invention are within the scope of protection of the present invention.

[0022] The experimental materials used in this invention were as follows: β-galactosidase (from Aspergillus oryzae) and polysucrose (molecular weight 70 kDa) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; silica gel plates were purchased from Yantai Jiangyou Silica Gel Development Co., Ltd.; lactose, oNPG, o-nitrophenol, dipotassium hydrogen phosphate trihydrate, and citric acid monohydrate were all of analytical grade; SpectraMax Plus 384 microplate reader, P901 pH meter, and HD-3A water activity meter were all standard experimental instruments.

[0023] Figure 1 In the figure, the horizontal axis represents the concentration of sucrose (mM), the vertical axis represents the GOS synthesis rate (%) on the left and the hydrolysis activity (U / mL) on the right. Figure 2 In the figure, the horizontal axis represents the reaction temperature (°C), and the vertical axis represents the GOS synthesis rate (%). Figure 3 In the figure, the horizontal axis represents the simulation time (ns), and the vertical axes are RMSD (nm) and Rg (nm), respectively. Figure 4 In the diagram, gray represents the enzyme structure in an aqueous solution system, and green represents the enzyme structure in a 4 mM polysucrose biphasic system. Figure 5 In the figure, the horizontal axis represents the concentration of sucrose (mM), the vertical axis represents the hydrolytic activity (U / mL) on the left and the water activity on the right; Figure 6In the diagram, red represents the TIM barrel-shaped catalytic domain (Asp40-Thr397), while green, blue, yellow, orange, and cyan represent the other domains. Figure 7 In the table, the column headers are sucrose concentration (mM), Vmax (mM / min), Km (mM), and Kcat (min-1), respectively.

[0024] Example 1 1. Preparation of polysucrose macromolecular system (1) Prepare pH 4.5 dipotassium hydrogen phosphate-citric acid buffer: Weigh 22.5 g dipotassium hydrogen phosphate trihydrate and 11.2 g citric acid monohydrate, place them in a 1000 mL volumetric flask, add deionized water to dissolve and dilute to the mark, shake well and set aside. (2) Preparation of saturated polysucrose solution: Weigh 840 mg of polysucrose with a molecular weight of 70 kDa, dissolve it in 1 mL of the above buffer solution to prepare a 12 mM saturated polysucrose macromolecular solution; (3) Preparation of polysucrose solutions of various concentrations: The 12 mM saturated polysucrose solution was serially diluted with pH 4.5 buffer to obtain 4, 6, 8 and 10 mM polysucrose solutions; (4) Preparation of polysucrose macromolecular system: Add lactose to the above 4 mM polysucrose solution and stir thoroughly until the final lactose concentration is 190 mg / mL to obtain polysucrose macromolecular reaction system of different concentrations. Seal and store for later use.

[0025] 2. Determination of water activity in the polysucrose macromolecular reaction system Weigh 22.5 g of dipotassium hydrogen phosphate trihydrate and 11.2 g of citric acid monohydrate, dissolve and dilute to 1000 mL to obtain a pH 4.5 phosphate buffer. Weigh 840 mg of sucrose and dissolve it in 1 mL of phosphate buffer to obtain a saturated sucrose macromolecular solution with a concentration of 12 mM. Dilute the saturated sucrose macromolecular solution to obtain sucrose solutions with concentrations of 2, 4, 6, 8, 10, and 12 mM. Measure the water activity of the above samples using a water activity meter, with the phosphate buffer as the control group, to obtain the water activity of the reaction system of different concentrations of sucrose macromolecular solution.

[0026] 3. Determination of β-galactosidase activity in polysucrose macromolecular systems (1) Hydrolysis activity determination Take the polysaccharide systems prepared in step 1 at various concentrations, dissolve 10 mg of substrate oNPG in 1 mL of phosphate buffer, add 10 μL of substrate oNPG solution and 1 μL of 2 U / mL enzyme solution to a centrifuge tube, and control the concentration of polysaccharide macromolecules in the system during the reaction by adding different volumes of saturated polysaccharide macromolecule solution and buffer, and make up the volume to 100 μL. Measure the absorbance after 10 min at 55 ℃ and pH 4.5 using a microplate reader. Plot a standard curve by measuring the absorbance of substrate ONPG standards at concentrations of 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, and 0.14 mM, thus converting the absorbance values ​​into hydrolytic activity. The results show that the hydrolytic activity in the 4 mM polysaccharide system is 2.05 U / mL (1.10 times that of the aqueous solution). The formula for calculating the hydrolytic activity is as follows:

[0027] In the formula: C represents the concentration of o-nitrophenol in the sample solution, obtained from the standard curve, in mM; Vtotal represents the total volume of the reaction reagents, in μL; D represents the dilution factor; Venzyme represents the volume of enzyme solution involved in the reaction, in μL; t represents the reaction time, in min; and m represents the mass of enzyme powder weighed, in g.

[0028] The experimental temperatures for hydrolysis activity determination were modified to 35, 45, 55, 65, 75, and 85℃, while the rest were performed under the same conditions. The hydrolysis activity of β-galactosidase in the polysucrose macromolecular reaction system at different reaction temperatures was measured.

[0029] (2) Determination of GOS synthesis rate Take the polysaccharide systems prepared in step 1 at various concentrations, prepare 190 mg / mL saturated lactose solutions, and then dissolve 140, 280, 420, 560, 700, and 840 mg of polysaccharide macromolecules respectively to obtain saturated lactose solutions containing 2, 4, 6, 8, 10, and 12 mM polysaccharide macromolecule lactose solutions in centrifuge tubes. Add 100 μL of the polysaccharide macromolecule lactose solution to a centrifuge tube, and add β-galactosidase solution at a concentration of 2 U / μL. React at 55 ℃ and pH 5.5 for 30 min, and then inactivate the enzyme by boiling in a water bath for 10 min. Perform thin-layer chromatography analysis on the transglycosylation reaction products, scan the TLC plate, and use ImageJ software to determine the gray values ​​of monosaccharides (glucose, galactose), disaccharides (lactose), and GOS. Calculate the percentage of each sugar in the total sugar content based on the gray value. The results showed that the GOS synthesis rate in the 4 mM sucrose system reached 38.6%, which was 1.23 times that of the aqueous solution system (31.2%). The formula for calculating the GOS synthesis rate is as follows:

[0030] In the formula: the amount of GOS synthesized is the corresponding gray value of the GOS blot in thin-layer chromatography; the total sugar is the sum of the corresponding gray values ​​of monosaccharides, disaccharides, and GOS blots in thin-layer chromatography.

[0031] The reaction time for the GOS synthesis rate determination experiment was modified to 10, 30, 60, 90, 120, and 150 min, while the rest were carried out under the same conditions. The β-galactosidase GOS synthesis rate was measured in the polysucrose macromolecular reaction system at different times.

[0032] 4. Structural verification of β-galactosidase in a polysucrose macromolecular system (molecular dynamics simulation) (1) Construction of simulation system: The crystal structure of Aspergillus oryzae β-galactosidase (PDB:4IUG) was obtained and impurities and water were removed; a polysucrose model with a degree of polymerization of 50 was constructed using Glycam, and 10 polysucrose molecules and 1 enzyme protein were inserted into a 30×30×36 nm virtual box. A 4 mM polysucrose reaction system was constructed by limiting the number of water molecules, with a polysucrose-free aqueous solution system as a control. (2) Simulation conditions: Gromacs software, AMBER99SB-ILDN force field, SPC216 water model, temperature 300 K, pressure 1 bar, Na added + / Cl - Balance the charge; perform 50,000 temperature equilibration steps and 100,000 pressure equilibration steps, and conduct 20 ns molecular dynamics simulations on the equilibrium system with a step size of 0.002 ps. (3) Structural analysis: The three-dimensional structure of the enzyme was analyzed using PyMOL 2.5.4 software, and parameters such as RMSD, Rg, number of hydrogen bonds, proportion of secondary structures, and hydrophilic accessible surface area were calculated. The results showed that in the 4 mM sucrose system, the enzyme RMSD = 0.174 nm and Rg = 3.093 nm, which were lower than those in the aqueous solution system; the number of hydrogen bonds increased to 765; α-helices were 101.11%, β-sheets were 103.27%, and random coils were 97.84%; the hydrophilic accessible surface area decreased to 170.79 nm. 2 The hydrophilic / hydrophobic accessible surface area is 1.03, demonstrating that the polysucrose macromolecular system can directionally regulate the conformation of enzymes.

[0033] 5. Determination of the thermostability and kinetics of β-galactosidase in polysucrose macromolecular systems (1) Determination of thermal stability A mixture of 2 U / μL enzyme solution and 4 mM sucrose macromolecules was prepared, with an enzyme solution-buffer mixture as a control. The mixture was placed in a 55 °C water bath for 0, 10, 20, and 30 min, and the hydrolytic activity was measured. The results showed that after 30 min of treatment, the enzyme hydrolytic activity in the 4 mM sucrose system was 0.53 U / mL, which was 2.95 times that of the aqueous solution system (0.18 U / mL), demonstrating that the biphasic system significantly improved the enzyme's thermostability.

[0034] (2) Determination of enzyme-catalyzed reaction kinetics In a 4 mM sucrose system and an aqueous control, the reaction was carried out at 55 ℃ and pH 4.5 for 5 min. The reaction rate was measured, and Km, Vmax, and Kcat were calculated by plotting 1 / V against 1 / [S]. The results are shown in Table 1. In the 4 mM sucrose system, Km = 2.42 mM (minimum), Vmax = 2.62 mM / min, and Kcat = 78.60 min. -1 This demonstrates that at this concentration, the enzyme has the strongest affinity for the substrate and the highest catalytic efficiency.

[0035] Example 2 1. Preparation of polysucrose macromolecular system (1) Prepare pH 4.5 dipotassium hydrogen phosphate-citric acid buffer: Weigh 22.5 g dipotassium hydrogen phosphate trihydrate and 11.2 g citric acid monohydrate, place them in a 1000 mL volumetric flask, add deionized water to dissolve and dilute to the mark, shake well and set aside. (2) Preparation of saturated polysucrose solution: Weigh 840 mg of polysucrose with a molecular weight of 70 kDa, dissolve it in 1 mL of the above buffer solution to prepare a 12 mM saturated polysucrose macromolecular solution; (3) Preparation of polysucrose solutions of various concentrations: The 12 mM saturated polysucrose solution was serially diluted with pH 4.5 buffer to obtain 4, 6, 8 and 10 mM polysucrose solutions; (4) Preparation of polysucrose macromolecular system: Add lactose to the above 8 mM polysucrose solution and stir thoroughly until the final lactose concentration is 190 mg / mL to obtain polysucrose macromolecular reaction system of different concentrations. Seal and store for later use.

[0036] 2. Determination of water activity in the polysucrose macromolecular reaction system Weigh 22.5 g of dipotassium hydrogen phosphate trihydrate and 11.2 g of citric acid monohydrate, dissolve and dilute to 1000 mL to obtain a pH 4.5 phosphate buffer. Weigh 840 mg of sucrose and dissolve it in 1 mL of phosphate buffer to obtain a saturated sucrose macromolecular solution with a concentration of 12 mM. Dilute the saturated sucrose macromolecular solution to obtain sucrose solutions with concentrations of 2, 4, 6, 8, 10, and 12 mM. Measure the water activity of the above samples using a water activity meter, with the phosphate buffer as the control group, to obtain the water activity of the reaction system of different concentrations of sucrose macromolecular solution.

[0037] 3. Determination of β-galactosidase activity in polysucrose macromolecular systems (1) Hydrolysis activity determination Take the polysaccharide systems prepared in step 1 at various concentrations, dissolve 10 mg of substrate oNPG in 1 mL of phosphate buffer, add 10 μL of substrate oNPG solution and 1 μL of enzyme solution with a concentration of 2 U / mL to a centrifuge tube, and control the concentration of polysaccharide macromolecules in the system during the reaction by adding different volumes of saturated polysaccharide macromolecule solution and buffer, and make up the volume to 100 μL. Measure the absorbance value after 10 min of reaction at 55 ℃ and pH 4.5 using a microplate reader. Plot a standard curve by measuring the absorbance values ​​of substrate ONPG standards at concentrations of 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, and 0.14 mM, thereby converting the absorbance value into hydrolytic activity. The results show that the hydrolytic activity gradually decreases in the 8 mM system, and the hydrolytic activity of the aqueous solution system is 1.86 U / mL. The formula for calculating the hydrolytic activity is as follows:

[0038] In the formula: C represents the concentration of o-nitrophenol in the sample solution, obtained from the standard curve, in mM; Vtotal represents the total volume of the reaction reagents, in μL; D represents the dilution factor; Venzyme represents the volume of enzyme solution involved in the reaction, in μL; t represents the reaction time, in min; and m represents the mass of enzyme powder weighed, in g.

[0039] The experimental temperatures for hydrolysis activity determination were modified to 35, 45, 55, 65, 75, and 85℃, while the rest were performed under the same conditions. The hydrolysis activity of β-galactosidase in the polysucrose macromolecular reaction system at different reaction temperatures was measured.

[0040] (2) Determination of GOS synthesis rate Take the polysaccharide systems prepared in step 1 at various concentrations, prepare 190 mg / mL saturated lactose solutions, and then dissolve 140, 280, 420, 560, 700, and 840 mg of polysaccharide macromolecules respectively to obtain saturated lactose solutions containing 2, 4, 6, 8, 10, and 12 mM polysaccharide macromolecule lactose solutions in centrifuge tubes. Add 100 μL of polysaccharide macromolecule lactose solution to centrifuge tubes, and add β-galactosidase solution at a concentration of 2 U / μL. React at 55 ℃ and pH 5.5 for 30 min, and then inactivate the enzyme by boiling in a water bath for 10 min. Perform thin-layer chromatography analysis on the transglycosylation reaction products, scan the TLC plate, and use ImageJ software to determine the gray values ​​of monosaccharides (glucose, galactose), disaccharides (lactose), and GOS. Calculate the percentage of each sugar in the total sugar content based on the gray value. The results show that the GOS synthesis rate gradually decreases in the 8 mM system. The formula for calculating the GOS synthesis rate is as follows:

[0041] In the formula: the amount of GOS synthesized is the corresponding gray value of the GOS blot in thin-layer chromatography; the total sugar is the sum of the corresponding gray values ​​of monosaccharides, disaccharides, and GOS blots in thin-layer chromatography.

[0042] The reaction time for the GOS synthesis rate determination experiment was modified to 10, 30, 60, 90, 120, and 150 min, while the rest were carried out under the same conditions. The β-galactosidase GOS synthesis rate was measured in the polysucrose macromolecular reaction system at different times.

[0043] 4. Structural verification of β-galactosidase in a polysucrose macromolecular system (molecular dynamics simulation) (1) Construction of simulation system: The crystal structure of Aspergillus oryzae β-galactosidase (PDB:4IUG) was obtained and impurities and water were removed; a polysucrose model with a degree of polymerization of 50 was constructed using Glycam, and 10 polysucrose molecules and 1 enzyme protein were inserted into a 30×30×36 nm virtual box. A 4 mM polysucrose reaction system was constructed by limiting the number of water molecules, with a polysucrose-free aqueous solution system as a control. (2) Simulation conditions: Gromacs software, AMBER99SB-ILDN force field, SPC216 water model, temperature 300 K, pressure 1 bar, Na added + / Cl - Balance the charge; perform 50,000 temperature equilibration steps and 100,000 pressure equilibration steps, and conduct 20 ns molecular dynamics simulations on the equilibrium system with a step size of 0.002 ps. (3) Structural analysis: The three-dimensional structure of the enzyme was analyzed using PyMOL 2.5.4 software, and parameters such as RMSD, Rg, number of hydrogen bonds, proportion of secondary structures, and hydrophilic accessible surface area were calculated. The results showed that in the 4 mM sucrose system, the enzyme RMSD = 0.174 nm and Rg = 3.093 nm, which were lower than those in the aqueous solution system; the number of hydrogen bonds increased to 765; α-helices were 101.11%, β-sheets were 103.27%, and random coils were 97.84%; the hydrophilic accessible surface area decreased to 170.79 nm. 2 The hydrophilic / hydrophobic accessible surface area is 1.03, demonstrating that the polysucrose macromolecular system can directionally regulate the conformation of enzymes.

[0044] 5. Determination of the thermostability and kinetics of β-galactosidase in polysucrose macromolecular systems (1) Determination of thermal stability A mixture of 2 U / μL enzyme solution and 4 mM sucrose macromolecular system was prepared, with an enzyme solution-buffer mixture as a control. The mixture was placed in a 55 ℃ water bath for 0, 10, 20, and 30 min, and the hydrolytic activity was measured. The results showed that after 30 min of treatment, the enzyme hydrolytic activity in the 4 mM sucrose system was 0.53 U / mL, which was 2.95 times that of the aqueous solution system (0.18 U / mL), demonstrating that the sucrose macromolecular system significantly improved the enzyme's thermostability.

[0045] (2) Determination of enzyme-catalyzed reaction kinetics In an 8 mM sucrose system and an aqueous control, different lactose substrate concentrations were set, and the reaction was carried out at 55 ℃ and pH 4.5 for 5 min. The reaction rate was measured, and Km, Vmax, and Kcat were calculated by plotting 1 / V against 1 / [S]. The results are shown in Table 1. In the 8 mM sucrose system, Km = 3.31 mM, Vmax = 1.34 mM / min, and Kcat = 40.20 min. -1 This demonstrates that at this concentration, the enzyme has the strongest affinity for the substrate and the highest catalytic efficiency.

[0046] Example 3 1. Preparation of polysucrose macromolecular system (1) Prepare pH 4.5 dipotassium hydrogen phosphate-citric acid buffer: Weigh 22.5 g dipotassium hydrogen phosphate trihydrate and 11.2 g citric acid monohydrate, place them in a 1000 mL volumetric flask, add deionized water to dissolve and dilute to the mark, shake well and set aside. (2) Preparation of saturated polysucrose solution: Weigh 840 mg of polysucrose with a molecular weight of 70 kDa, dissolve it in 1 mL of the above buffer solution to prepare a 12 mM saturated polysucrose macromolecular solution; (3) Preparation of polysucrose solutions of various concentrations: The 12 mM saturated polysucrose solution was serially diluted with pH 4.5 buffer to obtain 4, 6, 8 and 10 mM polysucrose solutions; (4) Preparation of polysucrose macromolecular system: Add lactose to the above 12 mM polysucrose solution and stir thoroughly until the final lactose concentration is 190 mg / mL to obtain polysucrose macromolecular reaction system of different concentrations. Seal and store for later use.

[0047] 2. Determination of water activity in the polysucrose macromolecular reaction system Weigh 22.5 g of dipotassium hydrogen phosphate trihydrate and 11.2 g of citric acid monohydrate, dissolve and dilute to 1000 mL to obtain a pH 4.5 phosphate buffer. Weigh 840 mg of sucrose and dissolve it in 1 mL of phosphate buffer to obtain a saturated sucrose macromolecular solution with a concentration of 12 mM. Dilute the saturated sucrose macromolecular solution to obtain sucrose solutions with concentrations of 2, 4, 6, 8, 10, and 12 mM. Measure the water activity of the above samples using a water activity meter, with the phosphate buffer as the control group, to obtain the water activity of the reaction system of different concentrations of sucrose macromolecular solution.

[0048] 3. Determination of β-galactosidase activity in polysucrose macromolecular systems (1) Hydrolysis activity determination Take the polysaccharide systems prepared in step 1 at various concentrations, dissolve 10 mg of substrate oNPG in 1 mL of phosphate buffer, add 10 μL of substrate oNPG solution and 1 μL of enzyme solution with a concentration of 2 U / mL to a centrifuge tube, and control the concentration of polysaccharide macromolecules in the system during the reaction by adding different volumes of saturated polysaccharide macromolecule solution and buffer, and make up the volume to 100 μL. Measure the absorbance value after 10 min of reaction at 55 ℃ and pH 4.5 using a microplate reader. Plot a standard curve by measuring the absorbance values ​​of substrate ONPG standards at concentrations of 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, and 0.14 mM, thereby converting the absorbance value into hydrolytic activity. The results show that the hydrolytic activity gradually decreases at the 12 mM system, and the hydrolytic activity of the aqueous solution system is 1.86 U / mL. The formula for calculating the hydrolytic activity is as follows:

[0049] In the formula: C represents the concentration of o-nitrophenol in the sample solution, obtained from the standard curve, in mM; Vtotal represents the total volume of the reaction reagents, in μL; D represents the dilution factor; Venzyme represents the volume of enzyme solution involved in the reaction, in μL; t represents the reaction time, in min; and m represents the mass of enzyme powder weighed, in g.

[0050] The experimental temperatures for hydrolysis activity determination were modified to 35, 45, 55, 65, 75, and 85℃, while the rest were performed under the same conditions. The hydrolysis activity of β-galactosidase in the polysucrose macromolecular reaction system at different reaction temperatures was measured.

[0051] (2) Determination of GOS synthesis rate Take the polysaccharide systems prepared in step 1 at various concentrations, prepare 190 mg / mL saturated lactose solutions, and then dissolve 140, 280, 420, 560, 700, and 840 mg of polysaccharide macromolecules respectively to obtain saturated lactose solutions containing 2, 4, 6, 8, 10, and 12 mM polysaccharide macromolecule lactose solutions in centrifuge tubes. Add 100 μL of polysaccharide macromolecule lactose solution to centrifuge tubes, and add β-galactosidase solution at a concentration of 2 U / μL. React at 55 ℃ and pH 5.5 for 30 min, and then inactivate the enzyme by boiling in a water bath for 10 min. Perform thin-layer chromatography analysis on the transglycosylation reaction products, scan the TLC plate, and use ImageJ software to determine the gray values ​​of monosaccharides (glucose, galactose), disaccharides (lactose), and GOS. Calculate the percentage of each sugar in the total sugar content based on the gray value. The results show that the GOS synthesis rate gradually decreases in the 12 mM system. The formula for calculating the GOS synthesis rate is as follows:

[0052] In the formula: the amount of GOS synthesized is the corresponding gray value of the GOS blot in thin-layer chromatography; the total sugar is the sum of the corresponding gray values ​​of monosaccharides, disaccharides, and GOS blots in thin-layer chromatography.

[0053] The reaction time for the GOS synthesis rate determination experiment was modified to 10, 30, 60, 90, 120, and 150 min, while the rest were carried out under the same conditions. The β-galactosidase GOS synthesis rate was measured in the polysucrose macromolecular reaction system at different times.

[0054] 4. Structural verification of β-galactosidase in a polysucrose macromolecular system (molecular dynamics simulation) (1) Construction of simulation system: The crystal structure of Aspergillus oryzae β-galactosidase (PDB:4IUG) was obtained and impurities and water were removed; a polysucrose model with a degree of polymerization of 50 was constructed using Glycam, and 10 polysucrose molecules and 1 enzyme protein were inserted into a 30×30×36 nm virtual box. A 4 mM polysucrose reaction system was constructed by limiting the number of water molecules, with a polysucrose-free aqueous solution system as a control. (2) Simulation conditions: Gromacs software, AMBER99SB-ILDN force field, SPC216 water model, temperature 300 K, pressure 1 bar, Na added + / Cl -Balance the charge; perform 50,000 temperature equilibration steps and 100,000 pressure equilibration steps, and conduct 20 ns molecular dynamics simulations on the equilibrium system with a step size of 0.002 ps. (3) Structural analysis: The three-dimensional structure of the enzyme was analyzed using PyMOL 2.5.4 software, and parameters such as RMSD, Rg, number of hydrogen bonds, proportion of secondary structures, and hydrophilic accessible surface area were calculated. The results showed that in the 4 mM sucrose system, the enzyme RMSD = 0.174 nm and Rg = 3.093 nm, which were lower than those in the aqueous solution system; the number of hydrogen bonds increased to 765; α-helices were 101.11%, β-sheets were 103.27%, and random coils were 97.84%; the hydrophilic accessible surface area decreased to 170.79 nm. 2 The hydrophilic / hydrophobic accessible surface area is 1.03, demonstrating that the polysucrose macromolecular system can directionally regulate the conformation of enzymes.

[0055] 5. Determination of the thermostability and kinetics of β-galactosidase in polysucrose macromolecular systems (1) Determination of thermal stability A mixture of 2 U / μL enzyme solution and 4 mM sucrose macromolecular system was prepared, with an enzyme solution-buffer mixture as a control. The mixture was placed in a 55 ℃ water bath for 0, 10, 20, and 30 min, and the hydrolytic activity was measured. The results showed that after 30 min of treatment, the enzyme hydrolytic activity in the 4 mM sucrose system was 0.53 U / mL, which was 2.95 times that of the aqueous solution system (0.18 U / mL), demonstrating that the sucrose macromolecular system significantly improved the enzyme's thermostability.

[0056] (2) Determination of enzyme-catalyzed reaction kinetics In a 12 mM sucrose system and an aqueous control, different lactose substrate concentrations were set, and the reaction was carried out at 55 ℃ and pH 4.5 for 5 min. The reaction rate was measured, and Km, Vmax, and Kcat were calculated by plotting 1 / V against 1 / [S]. The results are shown in Table 1. In the 12 mM sucrose system, Km = 3.84 mM, Vmax = 1.06 mM / min, and Kcat = 31.80 min. -1 This demonstrates that at this concentration, the enzyme has the strongest affinity for the substrate and the highest catalytic efficiency.

[0057] An application of a polysucrose macromolecular system for the controlled synthesis of galactooligosaccharides (GOS) utilizes the size exclusion effect and enzyme conformation regulation of the polysucrose macromolecular enzyme activity modification system to inhibit the hydrolytic side reaction of β-galactosidase and promote the main transglycosylation reaction, thereby achieving the efficient synthesis of GOS. Moreover, the polysucrose is non-toxic and non-immunogenic, meets food-grade safety requirements, and can be directly applied to the industrial production of functional food additive GOS.

[0058] In this invention, the polysucrose macromolecular system can be directly used for: industrial production of food-grade galactooligosaccharides (GOS); resource conversion of whey and lactose waste liquid; preparation of GOS for infant formula and functional food additives; and reference modification of other β-glycosidase transglycoside synthesis systems.

[0059] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for regulating the synthesis of galactooligosaccharides using a polysucrose macromolecular system, characterized in that: The efficient synthesis of galacto-oligosaccharides by β-galactosidase regulated by polysucrose was achieved by changing the aqueous reaction system to a polysucrose macromolecular reaction system. The results were obtained by detecting the enzyme activity, RMSD and Rg values ​​of β-galactosidase, and the hydrogen bonds, α-helices and β-sheets in the secondary structure of β-galactosidase under different concentrations of polysucrose macromolecular reaction system. The materials used for testing are: sucrose with a molecular weight of 70 kDa, silica gel plate, lactose, oNPG, o-nitrophenol, dipotassium hydrogen phosphate trihydrate, citric acid monohydrate, EDTA dihydrate, sodium carbonate, n-butanol, n-propanol, ethanol, diphenylamine, aniline, acetone, and phosphoric acid.

2. The method for regulating the synthesis of galactooligosaccharides using a polysucrose macromolecular system according to claim 1, characterized in that: Prepare polysucrose macromolecular solutions of different concentrations. The preparation method includes the following steps. A. Prepare pH 4.5 phosphate buffer: Weigh 22.5 g of dipotassium hydrogen phosphate trihydrate and 11.2 g of citric acid monohydrate, dissolve and dilute to 1000 mL to obtain pH 4.5 phosphate buffer; B. Preparation of saturated sucrose solution: Weigh 840 mg of sucrose with a molecular weight of 70 kDa and dissolve it in 1 mL of the phosphate buffer obtained in step A to obtain a saturated sucrose macromolecular solution with a concentration of 12 mM. C. Construction of the polysucrose macromolecular enzyme activity modification system: The saturated polysucrose solution obtained in step B was serially diluted with pH 4.5 phosphate buffer to prepare polysucrose macromolecular solutions with concentrations of 2, 4, 6, 8, 10, and 12 mM, thus constructing the polysucrose macromolecular enzyme activity modification system.

3. The method for regulating the synthesis of galactooligosaccharides using a polysucrose macromolecular system according to claim 1, characterized in that: The catalytic reaction conditions for the β-galactosidase in the polysucrose macromolecular enzyme activity modification system are as follows: reaction temperature 55℃, transglycosylation reaction time 60 min, and hydrolysis activity assay reaction time 10 min.

4. The method for regulating the synthesis of galactooligosaccharides using a polysucrose macromolecular system according to claim 1, characterized in that: The steps for detecting β-galactosidase activity are as follows: A. Substrate preparation: Dissolve 10 mg oNPG in 1 mL of pH 4.5 phosphate buffer to prepare the oNPG substrate solution; B. Construction of the reaction system: Add 10 μL of substrate oNPG solution and 1 μL of 2 U / mL β-galactosidase solution to a centrifuge tube, add different volumes of saturated polysucrose macromolecule solution and pH 4.5 phosphate buffer, control the polysucrose concentration and make up the system volume to 100 μL; C. Enzymatic reaction: The reaction system was placed at 55 ℃ for 10 min, and the absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) reader. D. Standard curve plotting: Measure the absorbance of NPG standards with concentrations of 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, and 0.14 mM and plot the standard curve. E. Enzyme activity calculation: Convert the absorbance value into hydrolysis activity according to the standard curve. The calculation formula is: Enzyme activity (U / g) = (C×Vtotal×D) / (Venzyme×t×m); In the formula: C is the concentration of o-nitrophenol in the sample solution, in mM; Vtotal is the total volume of the reaction reagents, in μL; D is the dilution factor; Venzyme is the volume of the enzyme solution involved in the reaction, in μL; t is the reaction time, in min; and m is the mass of the enzyme powder weighed, in g.

5. A method for regulating the synthesis of galactooligosaccharides using a polysucrose macromolecular system according to any one of claims 2-4, characterized in that: The concentration settings of the polysucrose macromolecular system enzyme activity modification system follow these principles: the polysucrose concentration gradient is 2, 4, 6, 8, 10, 12 mM, with a pure aqueous solution reaction system as a control; the substrate oNPG concentration gradient in the hydrolysis activity assay is 0.02-0.14 mM, the lactose substrate concentration in the transglycosylation reaction is 190 mg / mL; the enzyme solution concentration used is 2 U / mL, and the enzyme addition amount in the transglycosylation reaction is 2 U / μL.

6. The method for regulating the synthesis of galactooligosaccharides using a polysucrose macromolecular system according to claim 3, characterized in that: The β-galactosidase was placed in a 4 mM polysucrose macromolecular enzyme activity modification system, and oligogalactose was synthesized by transglycosylation catalysis of the enzyme under the conditions of lactose substrate concentration of 190 mg / mL, reaction temperature of 55 ℃ and reaction time of 60 min.

7. The method for regulating the synthesis of galactooligosaccharides using a polysucrose macromolecular system according to claim 1, characterized in that: To determine the activity of β-galactosidase in the efficient synthesis of oligogalactosidase regulated by sucrose at different reaction temperatures, a pH 4.5 phosphate buffer was prepared, and the hydrolysis activity assay temperatures were modified to 35, 45, 55, 65, 75, and 85 °C. The hydrolysis activity of β-galactosidase in the sucrose macromolecular reaction system at different reaction temperatures was measured.

8. The method for regulating the synthesis of galactooligosaccharides using a polysucrose macromolecular system according to claim 1, characterized in that: To determine the activity of β-galactosidase in the efficient synthesis of oligogalactosidase regulated by sucrose at different reaction times, the water bath temperature was set to 55℃. The enzyme stock solution was mixed with sucrose macromolecular reagent, while the control group was a mixture of enzyme and buffer. One sample was placed in the water bath every 10 minutes until the first sample was in the water bath for 30 minutes. All samples were then removed. Four samples were obtained for each of the 0, 10, 20, and 30 minutes of water bath. The hydrolytic activity of each of the four samples was measured according to the enzyme activity assay method.

9. The method for regulating the synthesis of galactooligosaccharides using a polysucrose macromolecular system according to claim 1, characterized in that: Molecular dynamics simulations were performed using Gromacs software. The crystal structure of β-galactosidase was obtained from the Protein Data Bank (PDB number 4IUG), after removing other molecules and water. The simulation conditions were as follows: an AMBER99SB-ILDN force field was added; the model was a rectangular box; solvation was performed using the SPC216 water model; the temperature was set to 300 K; the pressure was 1 bar to simulate atmospheric pressure; and the ion concentration was determined by adding Na+. + or Cl - Ions are used to balance the charge; the total number of steps for temperature equilibrium is 50,000, with a step size of 0.002 ns; the total number of steps for pressure equilibrium is 100,000, with a step size of 0.002 ns; for the equilibrium system, molecular dynamics simulations are performed for 20 ns with a step size of 0.002 ps.

10. An application of a polysucrose macromolecular system for regulating the synthesis of galactooligosaccharides, characterized in that: By utilizing the size exclusion effect and enzyme conformation regulation of the polysucrose macromolecular enzyme activity modification system, the hydrolytic side reaction of β-galactosidase is inhibited and the main transglycosylation reaction is promoted, thus achieving the efficient synthesis of galactooligosaccharides. Moreover, the polysucrose is non-toxic and non-immunogenic, meets the food-grade safety requirements, and can be directly applied to the industrial production of functional food additive galactooligosaccharides.

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