An immobilized enzyme membrane, its preparation method, and its application in the preparation of rare sugars.

Abstract

This invention relates to an immobilized enzyme membrane, its preparation method, and its application in the preparation of rare sugars, belonging to the field of enzyme catalysis technology. This invention prepares an immobilized enzyme membrane by controlling the surface charge of the membrane and then crosslinking it with RDPE and TMC. The positively charged membrane is more conducive to the loading of negatively charged RDPE. This immobilized enzyme membrane greatly improves the stability of the enzyme and enables its reuse and recovery. Free enzyme membranes and immobilized enzyme membranes were tested in positive osmosis mode. The immobilized enzyme membrane can achieve in-situ separation of products and can break the limitations of reaction equilibrium.

Description

Technical Field

[0001] This invention relates to an immobilized enzyme membrane, its preparation method, and its application in the preparation of rare sugars, belonging to the field of enzyme catalysis technology. Background Technology

[0002] Due to the intake of high-fat and high-sugar foods, the incidence of diseases such as hyperglycemia, hyperlipidemia, and diabetes continues to increase worldwide. Rare sugars have attracted widespread attention from researchers because of their low calories and unique biological functions. According to the definition of the International Rare Sugars Association (ISRS), rare sugars are monosaccharides and their derivatives that exist in very small amounts in nature. For example, D-tagatose, D-allulose, and D-allose, although rarely found in nature, have broad application prospects in the food and pharmaceutical fields due to their biological functions such as lowering blood sugar and lipids, anti-inflammation, and improving immunity[1]. Among them, D-allulose was certified by the U.S. Food and Drug Administration in 2011 as a substance that meets the U.S. food additive standards, and can be obtained by reversibly converting inexpensive D-fructose through epimerase.

[0003] Enzyme catalysis technology has become the mainstream method for preparing rare sugars due to its advantages such as mild reaction conditions, high specificity, green environmental protection and high efficiency. However, some inherent disadvantages of enzymes, such as loss of activity due to structural denaturation, difficulty in reuse due to water solubility and self-aggregation, limit their further application [2]. In addition, rare sugars are generally prepared by enzyme catalysis of reversible transformation between monosaccharides. The reversibility of the reaction and the small difference in molecular size between the reactants and products further increase the difficulty.

[0004] [1]Bilal M,Iqbal HMN,Hu H,et al.Metabolic engineering pathways forrare sugars biosynthesis,physiological functionalities,and applications—areview[J].Critical Reviews in Food Science&Nutrition,2017.

[0005] [2]Liu C, Saeki D, Matsuyama H.Anovel strategy to immobilize enzymes onmicroporous membranes via dicarboxylic acid halides[J]. Rsc Advances, 2017, 7(76): 48199-48207. Summary of the Invention

[0006] The purpose of this invention is to address the problems of low reaction efficiency and low enzyme recovery rate in the enzymatic synthesis of rare sugars in existing technologies. This invention proposes an enzyme membrane reactor that immobilizes RDPE on the membrane surface, enabling in-situ product separation and thus promoting the reaction in the forward direction, overcoming the limitations of reaction equilibrium.

[0007] The technical solution is:

[0008] An immobilized enzyme membrane includes a base membrane coated with a cationic polymer layer, the surface of which is loaded with an immobilized enzyme layer obtained by polymerizing a rare sugar synthase with an acyl chloride monomer.

[0009] The base membrane is a porous polymer membrane made of polyimide.

[0010] The cationic polymer layer is a polyethyleneimine layer.

[0011] The rare sugar synthase mentioned is D-allulose 3 epimerase (RDPE).

[0012] The acyl chloride monomer is pyromellitic trimethylolpropionate chloride.

[0013] The above-mentioned method for preparing immobilized enzyme membranes includes the following steps:

[0014] Step 1: Coat the surface of the base film with a solution containing a cationic polymer, so that the cationic polymer crosslinks on the surface of the base film;

[0015] Step 2: Contact the membrane obtained in Step 1 with a solution containing rare sugar synthase, so that the rare sugar synthase is loaded on the surface of the membrane.

[0016] Step 3: Contact the membrane obtained in Step 2 with a solution containing acyl chloride monomers to immobilize the rare sugar synthase by cross-linking it with the acyl chloride monomers.

[0017] In step 1, the cationic polymer solution is an alcoholic solution of polyethyleneimine with a concentration of 0.5-5 wt%, and the crosslinking time is 5-30 h.

[0018] In step 2, the solution containing rare sugar synthase is an aqueous solution containing 0.1-5 mg / mL D-allulose 3 epimerase (RDPE).

[0019] In step 3, the solution containing acyl chloride monomers is an organic solution containing 0.05-0.5 wt% trimesoyl chloride, and the contact time is 5-15 min.

[0020] A method for preparing rare sugars by immobilizing enzyme membranes includes the following steps:

[0021] Using the above-mentioned immobilized enzyme membrane, the immobilized enzyme layer was brought into contact with D-fructose solution to carry out the reaction;

[0022] Rare sugars obtained from the osmotic side are collected using forward osmosis.

[0023] The temperature range of the reaction process is 40-80℃.

[0024] During forward osmosis, the draw solution used on the osmotic side of the immobilized enzyme membrane is PBS solution.

[0025] Beneficial effects

[0026] This invention first increases the loading density of RDPE by regulating the surface charge of the membrane to create an electrostatic interaction between it and D-allulose 3-epimerase (RDPE). Then, TMC is used to further cross-link and immobilize the enzyme, thereby improving the stability of the immobilized enzyme membrane. This paper utilizes an electrostatic adsorption-cross-linking method to immobilize RDPE on the membrane surface. Experimental results show that regulating the surface charge of the membrane using a positively charged polyelectrolyte (such as polyethyleneimine (PEI)) can increase the enzyme loading density.

[0027] This immobilized enzyme membrane enables the reuse of enzymes and exhibits high stability. In the forward osmosis process, compared with a free enzyme membrane reactor, the advantages of the immobilized enzyme membrane, such as enabling in-situ product separation and thus promoting the reaction forward and overcoming the limitations of reaction equilibrium, are highlighted.

[0028] The greatest advantage of using membranes as immobilized enzyme carriers lies in their ability to integrate reaction and separation, potentially enabling in-situ product separation and thus promoting the shift of equilibrium in thermodynamically constrained reactions towards the product side. This allows the reaction to proceed in the forward direction, mitigating product inhibition and the occurrence of reversible reactions. Attached Figure Description

[0029] Figure 1 Schematic diagram of the preparation of immobilized enzyme membranes

[0030] Figure 2 (a) Photographs of PI and PI-PDA films, (b) FTIR spectra of PI and PI-PEI films.

[0031] Figure 3 (a) Activity of RDPE immobilized in polyimide (PI) films with different modifications; (b) Zeta potential of PI films with different modifications.

[0032] Figure 4(a) Fluorescence image of PI@RDPE film, (b) Fluorescence image of PI-PDA@RDPE film, (c) Fluorescence image of PI-PEI@RDPE film, (d) RDPE loading density of PI@RDPE, PI-PDA@RDPE and PI-PEI@RDPE films.

[0033] Figure 5 (a) SEM micrograph of PI@RDPE membrane, (b) SEM micrograph of PI-PDA@RDPE membrane, (c) SEM micrograph of PI-PEI@RDPE membrane.

[0034] Figure 6 (a) Schematic diagram of the reaction between RDPE and TMC; (b) FTIR spectra of Pristine, PI-PEI, PI-PEI@RDPR, and PI-PEI@RDPE & TMC films.

[0035] Figure 7 (a) Fluorescence image of PI-PEI@RDPE&TMC membrane before ultrasound, (b) Fluorescence image of PI-PEI@RDPE&TMC membrane after ultrasound, (c) Fluorescence image of PI-PEI@RDPE membrane before ultrasound, (d) Fluorescence intensity of PI-PEI@RDPE membrane after ultrasound, (e) Average fluorescence intensity of PI-PEI@RDPE and PI-PEI@RDPE&TMC membranes before and after ultrasound, (f) Relative activity of PI-PEI@RDPE and PI-PEI@RDPE&TMC membranes before and after ultrasound.

[0036] Figure 8 Effect of TMC concentration on the relative activity of PI-PEI@RDPE & TMC membranes

[0037] Figure 9 (a) The activity of free and immobilized RDPE over time; (b) The reusability of PI-PEI@RDPE&TMC for D-fructose conversion at pH 8.0 and 60°C; (c) The storage stability of PI-PEI@RDPE&TMC.

[0038] Figure 10 (a) Schematic diagram of the reaction-separation coupling process; (b) Amount of D-allulose in the osmotic-side immobilized enzyme membrane reactor and the free enzyme membrane reactor; (c) Distribution of D-allulose on the osmotic and feed sides; (d) Comparison of conversion rates of immobilized and free enzymes. Detailed Implementation

[0039] Material

[0040] Polyimide (PI), polyethylene glycol 400 (PEG 400), N-methylpyrrolidone (NMP), polyethyleneimine 70000 (PEI 70000Da), D-fructose, D-alulose, K2HPO4, KH2PO4, dopamine hydrochloride, trimesoyl chloride (TMC), and n-hexane can all be used directly after purchase without further purification. D-alulose 3-epimerase (RDPE) was provided by Tianjin Institute of Biotechnology.

[0041] Preparation and modification of base film

[0042] The polyimide-based membrane used in this patent is obtained through a phase inversion method in the prior art. The main steps are: preparing a casting solution for the PI-based membrane, allowing it to stand to remove bubbles, scraping the membrane onto a smooth glass plate, and then immersing it in a pure water coagulation bath to form a membrane through phase inversion.

[0043] The PI-based membrane was soaked in pure water overnight to remove excess solvent, and then immersed in a 2 wt% PEI solution (with isopropanol as the solvent) for crosslinking for 16 h. Afterward, it was washed with pure water and stored in pure water for subsequent enzyme immobilization (the PEI crosslinked membrane was named PI-PEI).

[0044] Dopamine (2 g / L, pH 8.5 phosphate buffer) was coated onto the surface of a PI-based membrane under 1 bar pressure, and the membrane was named PI-PDA.

[0045] Enzyme fixation

[0046] The PI-PEI cross-linked membrane was immersed in RDPE enzyme solution (approximately 0.5 mg / mL) for 24 h, followed by washing three times with phosphate buffer (pH 7.5) to obtain a membrane named PI-PEI@RDPE. The immersed enzyme solution and washing solution were collected for subsequent enzyme concentration testing. A 0.1 wt% TMC solution was prepared with n-hexane, and the PI-PEI@RDPE membrane was left to stand for a period to remove excess moisture from the membrane surface. It was then immersed in the TMC solution for 10 min (denoted as PI-PEI@RDPE&TMC). The PI-PDA@RDPE&TMC membrane was prepared following the same steps. The enzyme membrane was then stored in a buffer solution (pH = 7.5) at 4°C for subsequent experiments and characterization.

[0047] Membrane characterization methods

[0048] The surface and cross-sectional morphology of the modified membrane and the immobilized enzyme membrane were observed using field emission scanning electron microscopy (FESEM, S4800, Hitachi, Japan). Zeta potential analysis (SurPASS™ 3, Anton Paar, Austria) was used to analyze the changes in surface charge of the base membrane and the modified membrane with pH. Fourier transform attenuated total reflectance infrared spectroscopy (ATR-FTIR, ThermoScientific, Nicolet iS50) was used to analyze the cross-linking of the base membrane and the condensation reaction between RDPE and TMC. After staining the enzyme with fluorophore isothiocyanate (FTIC), the distribution of the enzyme on the membrane surface was observed using fluorescence microscopy. The staining procedure was as follows: a 1 mg / mL FTIC solution was prepared using dimethyl sulfoxide (DMSO) as the solvent. 0.15 mL of the FTIC solution was added dropwise to 30 mL of RDPE solution (5 mg / mL), and the mixture was stirred at room temperature for 1 h for staining. The reaction was then stopped by adding 2 mL of NH4Cl (50 mM). Finally, unreacted FTIC was removed by dialyzing in 20 mM PBS buffer for 48 h.

[0049] Test methods for determining enzyme loading

[0050] The amount of immobilized enzyme on the membrane was calculated using the mass balance method, and the enzyme concentration was determined at 595 nm using a spectrophotometer (UV-Vis) according to the Bradford method. The mass balance equation is as follows:

[0051]

[0052] Among them, Q e It is the amount of RDPE loaded on a unit area of ​​membrane (mg / cm²). 2 ),C o and C e The enzyme concentrations (mg / mL) in the solution before and after enzyme loading are shown below, respectively. w The enzyme concentration (mg / mL) in the PBS washing solution, V o and V w These represent the volumes (mL) used for loading the enzyme and the volume (mL) of the washing buffer, respectively, where A is the membrane area.

[0053] RDPE activity testing methods under free and immobilized conditions

[0054] 80 μg of free RDPE was added to 80 mL of D-fructose solution (pH = 8) and reacted at 60 °C. Samples were taken at different time points (10 min, 20 min, 30 min, 1 h, 2 h, 3 h, 4 h), and the reaction was terminated by boiling for 5 min. Subsequently, the reaction solution was diluted 10-fold, and the D-allulose content was determined by HPLC to calculate the free enzyme activity. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of D-allulose per minute under the above conditions. The activity of the immobilized RDPE membrane was also tested using the same steps. The kinetic parameters (Km and Vmax) of the free and immobilized enzymes were calculated using the Michaelis-Menten equation and Lineweaver-Burk plot.

[0055]

[0056] Among them, V and V max K represents the initial rate and maximum reaction rate of the enzyme-catalyzed reaction, respectively. m Here, S represents the concentration of D-fructose in the reactant. The kinetic parameters were measured at 60°C and pH 8.0.

[0057] The chromatographic conditions for this experiment were as follows: Agilent 1260 high-performance liquid chromatograph, ELSD detector, Waters Sugar Park 1 column (6.5 mm × 300 mm), and the mobile phase was ultrapure water with a flow rate of 0.48 mL / min. -1 The injection volume was 10 μL, the column temperature was 80℃, and the ELSD conditions were: Evaporator Temperature 80℃, Nebulizer Temperature 80℃, PMT Gain 5. The concentrations of D-fructose and D-allulose were determined by external standard method using a standard curve.

[0058] Stability testing methods for immobilized RDPE membranes

[0059] To test the reusability of the immobilized RDPE membrane, the enzyme membrane was reacted for 10 min under the reaction conditions described in section 2.5, then removed and stored in phosphate buffer at pH 7.5 at 4°C for future use. The reaction solution was diluted 10-fold and the immobilized RDPE activity was tested by HPLC, with the activity from the first test considered 100%. This experiment was performed in triplicate, but only the average value is provided. To determine whether ultrasonic cleaning would damage the interfacial polymer layer between RDPE and TMC, the enzyme membrane was ultrasonically cleaned for 30 min after the first test, followed by a second test.

[0060] In-situ separation and testing methods for immobilized enzyme membranes

[0061] The immobilized RDPE membrane was placed in an H-type permeation apparatus. The feed side contained 40 mL of D-fructose solution (20 mM PBS, pH 8), and the permeation side contained 20 mM PBS solution. The permeation apparatus was heated to 60 °C for reaction. At different time points, 0.2 mL of the solutions from both sides were diluted and the concentrations of D-fructose and D-allulose were determined by HPLC. The D-fructose conversion rate was calculated using the following formula:

[0062]

[0063] Where K is the conversion rate of D-fructose (%), and C o and C p The initial and post-reaction D-fructose concentrations (mg / mL) are shown. The morphology of the modified films and the distribution of immobilized RDPE are also presented.

[0064] RDPE was loaded onto the membrane surface using adsorption. To investigate the effect of membrane surface charge on RDPE loading, different monomers were used to modify the PI-based membrane to regulate the membrane surface charge. Figure 1 First, utilizing the adhesive properties of dopamine (PDA) after oxidative self-polymerization under slightly alkaline conditions, polydopamine was coated onto the surface of a PI-based film under pressure assistance. After coating, a layer of black polydopamine was clearly adhered to the film surface. Figure 2 In addition to (a), a layer of polydopamine particles was clearly visible on the surface of the base film through SEM cross-sectional images. Polyethylene imine (PEI) was used to crosslink the PI base film. Figure 2 The infrared image in (b) shows that after the original PI-based film was cross-linked with PEI, the cross-linked film at 1716 cm⁻¹... -1 and 1361cm -1 The characteristic peaks of the C=O and CN bonds of the imide were significantly weakened, and at 1651 cm⁻¹ -1 and 1542cm -1 The appearance of vibrational peaks for amide C=O and CN bonds indicates successful crosslinking between PEI and the base film PI.

[0065] Zeta potentials were characterized for the original membrane and the two modified membranes, such as... Figure 3As shown in (b), the unmodified PI-based membrane was negatively charged in the pH range of 3-9, and its negative charge was slightly reduced after PDA coating. However, after crosslinking with cationic polyelectrolyte PEI, the positive charge of the crosslinked membrane was significantly increased, exhibiting a positive charge in the pH range of 7-8. This is because the NH2 on the membrane surface is protonated, resulting in a positive charge, and the degree of protonation increases with decreasing pH, thus increasing the positive charge. This allows for electrostatic adsorption with the negatively charged RDPE, making it easier to load onto the membrane surface. Three membranes with different surface charges were subjected to enzyme immobilization tests after RDPE fixation, with the maximum enzyme activity defined as 100%. Figure 3 As shown in (a), the enzyme activity of the PI-PEI membrane after cross-linking with PEI was significantly higher than that of the other two negatively charged membranes. To investigate the reason for its high activity, we subsequently performed SEM characterization on PI@RDPE, PI-PDA@RDPE, and PI-PEI@RDPE, and then characterized them by fluorescence microscopy after staining with fluorophore isothiocyanate (FTIC).

[0066] like Figure 4 As shown in (a, b, c), the enzyme exhibited green fluorescence after FTIC staining and observation under a fluorescence microscope. However, the fluorescence intensity on PI@RDPE and PI-PDA@RDPE membranes was significantly lower than that on PI-PEI@RDPE membranes. This may be because the PI-PEI@RDPE membrane has a higher loading density due to electrostatic adsorption with RDPE. This conclusion is consistent with... Figure 3 The activity test results (a) were consistent with those of the enzyme. This conclusion was also confirmed by quantitative determination of the enzyme loading density on the membrane surface. Figure 4 (d) represents the enzyme loading density on the surface of the three membranes. The enzyme loading density on the PI-PEI membrane surface is 0.18 mg / cm³. 2 This is significantly higher than the 0.07 mg / cm³ of PI membrane and PI-PDA membrane. 2 and 0.05 mg / cm 2 The enzyme loading density was optimal. Therefore, PI-PEI membrane was selected as the carrier for subsequent immobilization of RDPE membrane.

[0067] The effect of TMC crosslinking with enzymes on the stability of immobilized enzyme membranes

[0068] To enhance the stability of the immobilized enzyme membrane, we selected TMC and RDPE crosslinking to form a polyamide active reaction layer on the membrane surface (e.g., Figure 6 As shown in (a). Figure 6In the infrared spectrum of (b), after cross-linking with TMC, the characteristic peak of NH2 in the PI-PEI@RDPE&TMC membrane is smaller than that in the PI-PEI@RDPE membrane. This is because the enzyme and NH2 in PEI react with TMC to form amides, leading to a decrease in the characteristic peak. Generally, when enzymes are immobilized by adsorption, the main reason for the decrease in enzyme activity is leakage. To verify the enhanced stability of the immobilized enzyme after TMC cross-linking, we sonicated the membrane for 30 min, and then compared the catalytic activity of the enzyme membrane and the immobilization of the enzyme on the membrane surface before and after sonication. Figure 7 As shown in (f), the enzyme activity of the PI-PEI@RDPE&TMC membrane did not decrease significantly before and after sonication. On the contrary, the enzyme activity of the PI-PEI@RDPE membrane decreased to 54% of its initial activity after sonication. The significant decrease in activity was mainly due to the non-covalent fixation, which made the enzyme easily detach from the membrane surface. The fluorescence images clearly show that the fluorescence intensity of the PI-PEI@RDPE membrane decreased from the initial 17.58 to 0.98 after sonication, while the fluorescence intensity of the PEI@RDPE&TMC membrane only decreased slightly. Figure 7 (a, b, c, d, e)), where the average fluorescence intensity of the enzyme was calculated using ImageJ. Furthermore, we could clearly observe that the fluorescence intensity of the PI-PEI@RDPE membrane before ultrasound was almost twice that of the PEI@RDPE&TMC membrane. This is likely because FTIC staining utilizes the NH2 on the lysine residues in the enzyme to interact with FTIC. Therefore, after cross-linking with TMC, the reduced number of NH2 residues on the enzyme membrane led to a decrease in fluorescence intensity, which is consistent with the conclusions drawn from the infrared spectra.

[0069] The effect of cross-linking degree on enzyme activity was investigated by varying the concentration of TMC, such as... Figure 8 As the TMC concentration increased, the activity of the immobilized RDPE decreased significantly. This is because the cross-linking of the PI-based membrane with PEI in this experiment resulted in the residual NH2 on the membrane surface participating in the cross-linking reaction. The activity of NH2 on PEI is higher than that on the macromolecular enzyme, leading to the formation of a polyamide thin layer that encapsulates the enzyme. As the TMC concentration increased, the polyamide thin layer became increasingly dense, which hindered the contact between the enzyme and the substrate, thereby reducing the activity.

[0070] Stability and reusability of PI-PEI@RDPE&TMC membranes

[0071] To investigate the activity decay of free and immobilized enzymes, both were placed at 60°C to catalyze the conversion of D-fructose. Enzyme activity was determined by measuring the amount of D-allulose in the product at different time points. Figure 9In (a), we can clearly see that the stability of the immobilized enzyme is significantly improved compared to the free enzyme. After 4 hours of continuous reaction, the activity of the free enzyme dropped to less than 20% of its initial value, while the activity of the immobilized enzyme membrane remained above 90% of its initial activity. The significant improvement in the stability of the immobilized enzyme is mainly attributed to the use of TMC and RDPE polymerization to confine it to the membrane surface, which largely restricts changes in the enzyme's three-dimensional conformation, thereby mitigating its inactivation. Immobilization not only greatly improves the stability of the enzyme, but it also makes it easy to reuse. Figure 9 (b) shows that after 50 cycles of PI-PEI@RDPE&TMC membrane, the immobilized enzyme membrane retained 95% of its initial activity. Because the membrane was cleaned with buffer and ultrasonically after each reaction to prevent reactant accumulation and membrane fouling, the enzyme activity did not decrease significantly even after 50 cycles. Another possible reason is the cross-linking of PEI with the base membrane. During the polymerization reaction of TMC and RDPE, TMC also reacts with PEI, thus enhancing the adhesion between the interfacial polymer layer and the base membrane. This prevented enzyme leakage and detachment of the interfacial polymer layer during the cycle. Figure 9 (c) tests the storage stability of the PI-PEI@RDOPE&TMC membrane. The experimental results show that the enzyme activity of the immobilized membrane still remains at 60% of the initial enzyme activity after two months of storage (activity was tested every other day).

[0072] Free enzyme membrane reactors and immobilized enzyme membrane reactors – reaction and separation

[0073] like Figure 10 As shown in (a), the enzyme membrane was placed in the middle of the permeate apparatus. Driven by the concentration gradient, D-fructose diffused from the bulk solution to the enzyme membrane surface and was converted into D-allulose under the catalysis of RDPE. Subsequently, D-allulose permeated through the enzyme membrane and finally reached the permeate side. Samples were taken from the feed side and the permeate side at different times to detect the content of D-fructose and D-allulose. The experimental results showed that no D-allulose was detected on the feed side during the test, while the amount of D-allulose on the permeate side accumulated over time. This indicates that after the substrate was converted into a product by the enzyme catalysis, all the product permeated to the permeate side, achieving in-situ separation of D-allulose. Figure 10(c)). To compare with free enzyme membranes, an equivalent amount of RDPE was placed on the feed side of the immobilized enzyme membrane. After reacting for 4 hours under the same conditions, the ratio of D-fructose to D-allulose on the permeate side was found to be 6:1, while the ratio of feed to product on the permeate side of the immobilized enzyme membrane was 1.5:1. This indicates that immobilizing the enzyme on the membrane surface allows for timely separation of products in reversible reactions, thereby increasing the proportion of product on the permeate side. In addition, during the reaction, the amount of D-allulose on the permeate side was also higher than that in enzyme membrane reactors in the free enzyme state (e.g., free enzyme membranes). Figure 10 As shown in (b)). For the free enzyme membrane reactor, the conversion rate remained at 23% after 4 hours of reaction. This is mainly due to two reasons: firstly, the reaction itself is subject to thermodynamic equilibrium, resulting in an equilibrium reaction rate; secondly, the free enzyme is easily deactivated, causing the conversion rate to stop increasing in subsequent times. However, the immobilized enzyme membrane, by enabling in-situ separation of the product, promotes the forward reaction, leading to an increase in the conversion rate and breaking the equilibrium conversion rate limitation. Figure 10 (d)

[0074] In summary, this invention prepared an immobilized enzyme membrane by crosslinking RDPE and TMC after controlling the membrane surface charge. Experimental results show that the positively charged membrane is more conducive to the loading of negatively charged RDPE, increasing its loading density by 6 times. This immobilized enzyme membrane greatly improves enzyme stability and enables enzyme reuse and recycling. After 50 cycles, the immobilized enzyme membrane still retains 95% of its initial activity, and after two months of storage and testing, the enzyme activity remains above 60% of its initial activity. Free enzyme membranes and immobilized enzyme membranes were tested in forward osmosis mode. Experimental data demonstrate that the immobilized enzyme membrane can achieve in-situ separation of products, thereby reducing the feed-to-product ratio on the osmotic side from 6:1 to 1.5:1. Furthermore, the immobilized enzyme membrane can break the reaction equilibrium limitation, increasing the reaction conversion rate from 23% to 33%.

Claims

1. A method for preparing rare sugars via immobilized enzyme membranes, characterized in that, The process includes the following steps: using an immobilized enzyme membrane, contacting the immobilized enzyme layer with a D-fructose solution to carry out the reaction; and collecting the rare sugars obtained on the osmotic side using a forward osmosis method. The immobilized enzyme membrane includes a base membrane, the surface of which is coated with a cationic polymer layer, and the surface of the cationic polymer layer is loaded with an immobilized enzyme layer obtained by polymerizing a rare sugar synthase with an acyl chloride monomer. The cationic polymer layer is a polyethyleneimine layer; the rare sugar synthase is D-allulose 3 epimerase; The method for preparing the immobilized enzyme membrane includes the following steps: Step 1: Coat the surface of the base film with a solution containing a cationic polymer, so that the cationic polymer crosslinks on the surface of the base film; Step 2: Contact the membrane obtained in Step 1 with a solution containing rare sugar synthase, so that the rare sugar synthase is loaded on the surface of the membrane. Step 3: Contact the membrane obtained in step 2 with a solution containing acyl chloride monomers to immobilize the rare sugar synthase by cross-linking with the acyl chloride monomers; In step 1, the cationic polymer solution is an alcoholic solution of polyethyleneimine with a concentration of 0.5-5 wt%, and the crosslinking time is 5-30 h. In step 2, the solution containing rare sugar synthase is an aqueous solution containing 0.1-5 mg / mL D-allulose 3 epimerase; In step 3, the solution containing acyl chloride monomers is an organic solution containing 0.05-0.5 wt% trimesoyl chloride, and the contact time is 5-15 min; The temperature range of the reaction process is 40-80℃. During the forward osmosis process, the draw solution used on the osmotic side of the immobilized enzyme membrane is PBS solution.

2. The method according to claim 1, characterized in that, The base membrane is a porous polymer membrane made of polyimide.

Images