Preparation method and application of wall-coated enzyme-loaded microchannel reactor

Abstract

The invention relates to a preparation method and application of a wall coating type enzyme-loaded microchannel reactor. The method comprises the following steps: mixing tetramethyl orthosilicate, methyltrimethoxysilane and methanol to obtain a solution A; deionized water, polyethylene glycol, a sodium fluoride aqueous solution and a dialyzed lipase solution are mixed to obtain a solution B, the solution B and the solution B are mixed for 3-10 minutes in an ice bath environment to obtain a solution C, the solution C is introduced into the pretreated microchannel through an injection pump, after incubation is conducted for 10-60 minutes, nitrogen is adopted for purging, redundant sol-gel is removed, and then the solution C is put into a drying oven to be dried; the catalyst is used for hydrolysis reaction of grease and various ester exchange and esterification reactions. The method can be carried out in a mild environment, and has higher conversion rate and shorter reaction time.

Description

Technical Field

[0001] The invention belongs to the field of chemical synthesis, and in particular relates to a method for continuous catalytic reaction in a wall-coated enzyme-loaded microchannel reactor, and belongs to the technical field of microfluidic chips. Background Art

[0002] (1) Microreactor Microreactors, also known as microstructures or microchannel reactors, can be described as devices with narrow channels (usually with a characteristic size of 10 to 1000 microns). The narrow channels are constructed of stable and inert materials (e.g., glass, silicon, stainless steel, ceramics, or polymers). The "micro" in microreactors means that the channels of the process fluid are at the micron level, and can contain millions of micro channels. The reaction volume in a typical microreactor is about 1000µL or less, and the reaction time is determined and regulated by the pump speed. In this case, the flow is a laminar flow with a low Reynolds number, and the mixing of reagents usually occurs by diffusion.

[0003] The field of microreactors itself is an emerging field, but the concept of microchannels has previously been integrated in capillary components of gas chromatography and capillary electrophoresis devices (made of glass or quartz), or in flow reactors (made of metal). The most important application of microreactor technology in chemistry is the organic or polymer synthesis of various products, such as the synthesis of linear and branched polymers with different structures, including beads, microcapsules and fibers; as well as the synthesis of branched polymers in tubular microreactors and the production of polymer nanoparticles by photopolymerization of methacrylates in borosilicate microreactors. Another field of application of microreactors is energy production. For the synthesis or processing of fuels, different strategies have been developed, among which it is worth noting the methods for the production of methanol, ethanol and biodiesel.

[0004] (2) Bio-enzyme catalyst Bioenzyme is a catalyst, which is a type of protein with catalytic effect in organisms. Bioenzymes can accelerate the rate of chemical reactions, reduce activation energy, and promote metabolic processes and cell functions in organisms. In the process of enzyme-catalyzed reactions, enzymes will form intermediate complexes with substrates through forces such as van der Waals forces, ionic bonds, and hydrogen bonds. Compared with ordinary catalysts, bioenzymes have the characteristic of significantly increasing the rate of chemical reactions with very small amounts. Bioenzyme-catalyzed reactions do not change the equilibrium constant of chemical reactions. At the same time, the specificity, high efficiency, and mild action conditions of enzymes are characteristics that ordinary inorganic catalysts cannot fully possess. It is precisely because of this characteristic of enzyme-catalyzed reactions that the application of enzymes in the fields of chemical production and pollutant treatment has developed rapidly.

[0005] Although biological enzymes have replaced traditional chemical catalysts in many fields due to their advantages such as high efficiency and environmental friendliness, the application of biological enzyme catalysts is limited due to some constraints: (1) Free enzymes are easily affected by environmental conditions, such as temperature and pH, which leads to fluctuations in activity and inactivation. (2) Free enzymes are difficult to recover after the reaction and other methods are needed to separate the enzyme and the product. (3) Since free enzymes are not fixed on a carrier, they are prone to conformational changes or inactivation, resulting in decreased activity. Therefore, suitable conditions are required to maintain the activity and stability of the enzyme in industrial applications. The increasingly developed immobilized enzyme technology is one of the ways to solve the above problems. Immobilized enzymes are the process of fixing enzymes on a support material to increase their stability and reusability. The following are some common methods for immobilizing enzymes: adsorption, covalent binding, encapsulation and cross-linking. Adsorption: Adding the enzyme solution to the support material so that the enzyme is adsorbed on the surface of the support material. Covalent binding: Covalently binding the enzyme to the support material through a chemical reaction. Encapsulation: Encapsulating the enzyme inside the support material to form immobilized enzyme particles. Cross-linking: Enzymes are cross-linked to support materials through cross-linking agents. Immobilized enzyme technology can play an important role in biocatalytic reactions, biosensors and other fields. The possibility of reaction control is enhanced by separating catalysts from reagents and products, preventing products from being contaminated by enzymes, inhibiting undesirable side reactions and reusing catalysts.

[0006] (3) Organic combination of immobilized enzyme technology and microreactor Microchannel immobilized enzyme technology is an immobilized catalytic device prepared by combining biomolecule immobilization technology with microreactors. It is expected to organically combine the technical characteristics of microfluidics and the excellent characteristics of enzyme catalysis, while achieving high efficiency and repeated use of enzymes, and enhancing the efficiency of enzymatic reactions in the entire catalytic process. Microchannel immobilized enzyme technology can separate free enzymes from products and increase the reuse of enzymes. Coating enzymes on microreactors can also effectively protect enzymes and reduce the risk of inactivation due to external environmental influences, thereby improving the stability and activity of enzymes. It has great application potential in the synthesis of high value-added products.

[0007] The combination of microchannel immobilized enzyme technology and the two is different due to the type of microchannel and the nature of the enzyme; there are usually three forms, namely packed bed type, monolithic type and wall coating type. Kundu et al. (Journal of the American Chemical Society vol. 133, 15 (2011): 6006-11) designed a microreactor filled with PMMA beads, in which CaLB was physically immobilized, and studied the enzyme-catalyzed ring-opening polymerization reaction of polycaprolactone, but due to the dense accumulation of particles, the pressure drop in the microchannel is large, and the heat transfer inside the channel is also difficult to control. Compared with the packed bed type, the monolithic type has a high porosity and the fluid is easy to flow. Cheng et al. (Journal of Pharmaceutical and Biomedical Analysis vol. 165, 20 (2019): 0731-7085) developed a new immobilized trypsin microreactor based on a monolithic capillary column for rapid proteolytic digestion, which greatly reduced the hydrolysis time. However, due to the presence of more pores, it is easy to be blocked, the permeability is uneven, and the monolithic structure is time-consuming to make and has poor reproducibility. Compared with the packed structure, the wall-mounted structure has more microreactor channel space, which is more conducive to the integration and intensification of the process; however, due to the smooth inner wall and large curvature of the microchannel, it is difficult to wall-load the enzyme catalyst. Summary of the invention

[0008] The purpose of the present invention is to provide a preparation method and application of a wall-coated enzyme-carrying microchannel reactor in view of the shortcomings of the current lipase catalysis technology. The method uses a sol-gel method to fix the enzyme after pre-treating the microchannel, and passes the sol into the microchannel reactor at a certain flow rate. After aging, a gel is formed and attached to the inner wall of the microchannel to obtain a wall-coated enzyme-carrying microchannel reactor. The present invention can be carried out under a mild environment and has a higher conversion rate and a shorter reaction time.

[0009] The technical solution of the present invention is as follows: A method for preparing a wall-coated enzyme-loaded microchannel reactor comprises the following steps: Methyl orthosilicate, methyltrimethoxysilane and methanol are mixed to obtain solution A; deionized water, polyethylene glycol, sodium fluoride aqueous solution and dialyzed lipase solution are mixed to obtain solution B, the two are mixed in an ice bath environment for 3 to 10 minutes to obtain solution C, and the solution C is introduced into the pretreated microchannel by a syringe pump, incubated for 10 to 60 minutes, purged with nitrogen to remove excess sol gel, and then placed in an oven for drying; The mass ratio of methyl orthosilicate to methyltrimethoxysilane is 1:1~1:5; the mass fraction of methanol in liquid A is 30%~60%; The mass fraction of the dialyzed lipase solution in solution B is 50%-60%, the mass ratio of polyethylene glycol to sodium fluoride aqueous solution is 1:5-1:9; the mass fraction of deionized water in solution B is 20-45%; the concentration of sodium fluoride aqueous solution is 0.8-1.0M; The mass ratio of solution A to solution B is 1:1~1:2; the injection flow rate of solution C is 30~100 μl / min. The microchannel is a chip-type microchannel or a capillary microchannel; Chip-type microchannel: a serpentine microchannel pattern with a cross section of 1 mm 1 mm rectangular channel, total length 1200~1400 mm, total number of right-angle bends 45~60; Capillary microchannel: a hard straight tube, with a quantity of 1 to 50, a length of 20 cm, an outer diameter of 3 mm, a wall thickness of 1 mm, and an inner diameter of 1 mm.

[0010] The molecular weight of the polyethylene glycol is 400-2000.

[0011] The flow rate of nitrogen purge is 0.1~1 ml / min, and the purge time is 0.5~2 hours.

[0012] The pretreatment is as follows: the microchannel reactor is ultrasonicated in a mixed solution of water and ethanol for 1.5 to 2.5 hours, and then the microchannel is pretreated with a sodium hydroxide aqueous solution with a concentration of 1 to 3 mol / L, which is continuously injected into the microchannel by a syringe pump for a pretreatment time of 0.5 to 2 hours. After the pretreatment is completed, water and ethanol are introduced to remove the residual sodium hydroxide for a treatment time of 5 to 20 minutes, and the microchannel is blown dry with nitrogen; The lipase in the lipase solution is Candida antarctica lipase A (CaLA), Candida antarctica lipase B (CaLB) or Candida plicata lipase (CRL); preferably, when the CaLB enzyme is used, the lipase content is 6%, The composition and mass percentage of lipase solution are as follows:

[0013] The dialysis membrane (MD25-3500) used for dialysis of the lipase solution was 3 days, and the buffer solution was replaced every 12 hours); The composition of the buffer solution is phosphate buffer (Na2HPO4 / NaH2PO4), pH=7.5; The wall-coated enzyme-loaded microchannel reactor prepared by the method is used for the hydrolysis reaction of oils and fats and various transesterification and esterification reactions; At 30-70°C, the reaction solution is injected into the microreactor through a syringe pump to obtain fatty acids and glycerol; or various ester substances; Wherein, the residence time of the reaction solution is controlled at 5 to 30 minutes; When fatty acids and glycerol are obtained, the reaction liquid is composed of oil and water, and the mass of the oil is 50-80% of the mass of the reaction liquid; When vitamin E succinate is obtained, the reaction solution is composed of succinic anhydride and tocopherol, and the mass of vitamin E is 30-50% of the mass of the reaction solution; The oil is one or two of soybean oil, fish oil, peanut oil, rapeseed oil and sunflower oil; The microreactor is any of the following two: The first type, a chip-type microchannel, includes a rectangular substrate, a cover plate, a sealing gasket, two polytetrafluoroethylene films, and a quartz glass plate; The substrate is engraved with a serpentine microchannel, a sealing gasket is placed in a groove around the serpentine microchannel on the substrate, two tetrafluoroethylene films and a quartz glass plate are sequentially covered on the surface engraved with the serpentine pattern, and finally a cover plate is fixedly connected to the substrate; Alternatively, the second type, capillary microchannels, are hard straight tubes, the number of which is 1 to 50, each with a length of 20 cm, an outer diameter of 3 mm, a wall thickness of 1 mm, and an inner diameter of 1 mm.

[0014] The microchannel is a serpentine microchannel pattern with a cross section of 1 mm 1 mm rectangular channel, total length 1200~1400 mm, total number of right-angle bends 45~60.

[0015] The essential features of the present invention are: The precursors used in the present invention are methyl orthosilicate and methyltrimethoxysilane (liquid A), and methanol is a cosolvent. Compared with other methods that use ethyl orthosilicate as a precursor and produce ethanol after hydrolysis to poison biological enzymes, the methanol produced after hydrolysis of methyl orthosilicate has little harm to the enzyme. After adding methyltrimethoxysilane, the gel aging speed is effectively slowed down, so that the coating can be evenly attached to the inner wall; the weak alkaline catalyst sodium fluoride used in liquid B effectively guarantees the enzyme activity compared to other formulas that use hydrochloric acid as a catalyst. The loading method adopted by this formula is wall loading. Compared with the filling type and the integral type, the controllable wall loading of the coating can be achieved by adjusting the formula and other methods.

[0016] In addition, the present invention also removes glycerol, sorbitol and the like in the enzyme solution through dialysis, thereby increasing the amount of enzyme loaded on the coating. The coating dries quickly and will not stick together after aging, thereby ensuring the mechanical properties of the coating, improving the high efficiency and long-term effectiveness of the coating, and improving the reaction efficiency.

[0017] The beneficial effects of the present invention are: The present invention adopts a wall-coated enzyme catalytic microreactor to realize the continuous enzyme catalytic process; the wall coating process is simple and easy, with mild reaction conditions, and the lipase is fixed on the inner wall of the microchannel and can be reused, reducing the production cost and the difficulty of subsequent separation.

[0018] Specifically reflected in: 1. In combination with Example 2, compared with the existing data that an intermittent reactor can be used to prepare vitamin E succinate and complete 80% to 95% conversion in 20 to 72 hours, the reaction time of this invention is less than 30 minutes and can achieve a conversion rate of 40%, greatly reducing the reaction time.

[0019] 2. After the reaction of the established wall-coated enzyme-catalyzed microreactor is completed and cleaned, the immobilized enzyme still remains in the microreactor, reducing the risk of inactivation due to the external environment. Combined with Examples 3 and 4, its activity can still reach 85% of the initial enzyme activity after 180 days, achieving its long-term effectiveness.

[0020] 3. The present invention is simple to operate, has high catalytic efficiency, can realize continuous production, has strong reusability, can be re-coated after enzyme inactivation, etc., and because of its "micro" characteristics, it reduces the amount of enzyme used, greatly saving production costs. By coupling the enzymatic process with microchannel technology, the green preparation and purification of fine chemicals can be achieved, and the synthesis of high value-added products can be realized. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The coating process flow chart of the present invention.

[0022] Figure 2 This is the assembly diagram of the chip-type immobilized enzyme microreactor used.

[0023] Figure 3 This is the channel pattern, feed position and discharge position of the chip-type immobilized enzyme microreactor.

[0024] Among them, 1 is a 316L plate engraved with microchannels; 2 is a sealing gasket; 3 is a polytetrafluoroethylene film, 4 is a quartz glass plate, and 5 is a 316L microchannel cover plate. Figure 3 The middle reaction liquid 6 and the reaction liquid 7 are inlets, and the output solution is the outlet.

[0025] Figure 4 This is the channel pattern, feed position and discharge position of the capillary immobilized enzyme microreactor.

[0026] Among them, 8 is a glass capillary pipe (made of silicate), 9 is a microreactor composed of a plurality of pipes (ranging from 1 to 50), and 10 is a side surface of a microreactor composed of a plurality of pipes; Figure 4 The reaction liquid is the inlet, and the output solution is the outlet. DETAILED DESCRIPTION

[0027] In order to more specifically illustrate the continuous and efficient production of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0028] The chip-type microchannel involved in the present invention is made of 316L material. The chip-type microchannel is composed of a square substrate engraved with a serpentine microchannel pattern and a cover plate, as well as a sealing gasket, two polytetrafluoroethylene films and a quartz glass plate. The cross-section of the serpentine microchannel is 1 mm 1 mm rectangular channel, total length 1306.1 mm (total number of right-angle bends is 53, of which the horizontal adjacent right-angle spacing is 2 mm and the vertical adjacent right-angle spacing is 50 mm); the substrate thickness is 15 mm, and the groove width and depth of the sealing gasket are 2 mm 2 mm. First, place the sealing gasket into the groove engraved around the serpentine microchannel on the substrate, then sequentially cover the surface with the serpentine pattern with two polytetrafluoroethylene films (used to prevent corrosion from corrosive fluids and enhance sealing) and a quartz glass plate, and finally seal the customized cover plate and the microchannel substrate with screws.

[0029] The capillary microchannel involved in the present invention is made of glass (silicate) material. The capillary microchannel is a hard straight tube with a length of 20 cm, an outer diameter of 3 mm, a wall thickness of 1 mm, and an inner diameter of 1 mm. The present invention currently only takes a single capillary as an example.

[0030] Example 1 (1) Chip-type immobilized enzyme microchannel reactor (2) After the microchannel reactor was ultrasonicated in a mixed solution of water and ethanol for 2 h, 8 g of sodium hydroxide was weighed and dissolved in 200 ml of deionized water to obtain a sodium hydroxide aqueous solution with a concentration of 1 mol / L. The sodium hydroxide aqueous solution was continuously passed into the microchannel at a flow rate of 0.5 ml / min for 2 h, and water and ethanol were passed into the microchannel at a flow rate of 0.5 mL / min for 10 min in sequence to remove residual sodium hydroxide. Nitrogen was passed through the microchannel at a flow rate of 100 µL / min for 2 h to purge and dry it.

[0031] (3) Place the dialysis membrane (MD25-3500) in a beaker containing equal volumes of water and ethanol, place it in a 75°C water bath for 15 min, then take it out for use. Place the Antarctic Candida lipase solution (wt% = 6) in the dialysis membrane and place it in a phosphate buffer (pH = 7.5, Na2HPO4 / NaH2PO4) for three days before taking it out. Replace the phosphate buffer every 12 h. The composition and mass percentage of the lipase solution before dialysis are as follows:

[0032] Weigh 0.1g of methyl orthosilicate, 0.38g of methyltrimethoxysilane, and 0.62g of methanol to obtain solution A, weigh 0.48g of deionized water, 0.03g of polyethylene glycol, 0.18g of sodium fluoride aqueous solution (1M), and 0.81g of dialyzed Antarctic Candida lipase solution to obtain solution B. Solution A and solution B were mixed under ice bath for 5min to obtain a homogeneous solution. The solution was injected into the microchannel at a flow rate of 50µL / min through a syringe pump. After continuous flow coating for 30min, nitrogen was introduced at a flow rate of 100 microliters per minute for 2h until the gel aged to obtain a chip-type wall-coated enzyme catalytic microreactor.

[0033] (4) 100 g of refined soybean oil and 40 g of water were degassed with helium for 20 min, respectively. The wall-coated enzyme-catalyzed microreactor was pre-purged with nitrogen for 1 h and then placed in an oil bath at 60 °C for constant temperature. Refined soybean oil at a flow rate of 4 mL / min and water at a flow rate of 1.5 mL / min were injected into the wall-coated enzyme-catalyzed microreactor through a syringe pump, respectively. The residence time was set to 30 min to obtain fatty acids and glycerol. The hydrolysis rate of soybean oil can reach 70%~90%.

[0034] Example 2 (1) Capillary Immobilized Enzyme Microchannel Reactor (2) The other steps were the same as those in Example 1, except that the solution was injected into the microchannel at a flow rate of 100 µL / min through a syringe pump. After continuous flow coating for 20 min, nitrogen gas was introduced at a flow rate of 500 µL / min for 1 h until the gel was aged, thereby obtaining a capillary wall-coated enzyme catalytic microreactor.

[0035] (3) 10 mL of helium-degassed dimethyl sulfoxide and 4.3 g of tocopherol were mixed, and 5 g of succinic anhydride and 10 mL of dimethyl sulfoxide were mixed. Nitrogen was introduced as a protective gas, and the mixture was stirred at 300 r / min until the tocopherol and succinic anhydride were completely dissolved.

[0036] (4) A capillary wall-coated microreactor (single tube) was placed in a 40°C water bath to maintain a constant temperature. Two reaction solutions were injected into the microreactor through syringe pumps, respectively. The flow rate of the two reaction solutions was 15 µL / min. Vitamin E succinate was generated by reaction. The residence time was set to 20 min. The maximum conversion rate was about 40%, and the reaction time was less than 30 min. Compared with the existing data that it takes 20 to 72 hours to complete the conversion using an intermittent reactor, the reaction time was greatly reduced. After the reaction was completed, the microreactor could continue to be used after being rinsed with phosphate buffer and then dried.

[0037] Example 3 The enzyme coating prepared in Example 1 was tested for enzyme activity and long-term stability. The test method is as follows: 20 mg of enzyme coating sol-gel powder was placed in 3 mL of phosphate buffer solution (50 mM, pH = 7.5), incubated in a 37 ° C water bath for 3 minutes, and then 0.2 mL of p-nitrophenyl palmitate (5 mg / mL) was added. After reacting for 3 minutes, remove and quickly add 1.5 mL of acetone to terminate the reaction. Finally, the absorbance was measured at 410 nm using a spectrophotometer. Specific enzyme activity (U / g): Specific enzyme activity refers to the amount of enzyme required to catalyze the substrate to produce 1 μmol of fatty acid per minute. The initial enzyme activity is defined as 100%.

[0038] Example 4 The method is consistent with that in Example 3, and other conditions remain unchanged. The enzyme coating sol-gel powder is stored at room temperature, and the enzyme activity is measured every 4 weeks. After 6 cycles, compared with the initial enzyme activity in Example 3, the relative activity can still reach 85%, which is suitable for practical operation.

[0039] Through the above example analysis, it can be concluded that compared with ordinary intermittent reactors, wall-coated microreactors can greatly reduce reaction time, increase reaction rate, and ensure the retention of enzyme activity, and have certain long-term and high efficiency.

[0040] Matters not covered by the present invention are known technologies.

Claims

1. A method for preparing a wall-coated enzyme-loaded microchannel reactor, characterized in that the method comprises the following steps: Methyl orthosilicate, methyltrimethoxysilane and methanol are mixed to obtain solution A; deionized water, sodium fluoride aqueous solution, polyethylene glycol and dialyzed lipase solution are mixed to obtain solution B, the two are mixed in an ice bath environment for 3 to 10 minutes to obtain solution C, which is introduced into the pretreated microchannel by a syringe pump, incubated for 0.5 to 2 hours, purged with nitrogen to remove excess sol gel, and then placed in an oven for drying; in, The mass ratio of methyl orthosilicate to methyltrimethoxysilane is 1:1~1:5; The mass fraction of methanol in liquid A is 30%~60%; The mass fraction of the dialyzed lipase solution in solution B is 50%-60%, the mass ratio of polyethylene glycol to sodium fluoride aqueous solution is 1:5-1:9; the mass fraction of deionized water in solution B is 20-45%; the concentration of sodium fluoride aqueous solution is 0.8-1.0M; The mass ratio of solution A to solution B is 1:1~1:2; the injection flow rate of solution C is 30~100 μl / min; The lipase in the lipase solution is Candida antarctica lipase A (CaLA), Candida antarctica lipase B (CaLB) or Candida plicata lipase (CRL); The microchannel is any of the following two: The first type, a chip-type microchannel, includes a rectangular substrate, a cover plate, a sealing gasket, two polytetrafluoroethylene films, and a quartz glass plate; The substrate is engraved with a serpentine microchannel, a sealing gasket is placed in the groove around the serpentine microchannel on the substrate, two polytetrafluoroethylene films and a quartz glass plate are sequentially covered on the surface engraved with the serpentine pattern, and finally the cover plate is fixed to the substrate; the microchannel is a serpentine microchannel pattern with a cross-section of 1 mm 1 mm rectangular channel, total length 1200~1400 mm, total number of right-angle bends 45~60; Alternatively, the second type, capillary microchannels, are hard straight tubes, the number of which is 1 to 50, each with a length of 20 cm, an outer diameter of 3 mm, a wall thickness of 1 mm, and an inner diameter of 1 mm.

2. The method for preparing a wall-coated enzyme-loaded microchannel reactor as claimed in claim 1, characterized in that the molecular weight of the polyethylene glycol is 400-2000.

3. The method for preparing a wall-coated enzyme-loaded microchannel reactor as claimed in claim 1, characterized in that the flow rate of nitrogen purge is 0.1-1 ml / min and the purge time is 0.5-2 hours.

4. The preparation method of the wall-coated enzyme-loaded microchannel reactor as described in claim 1 is characterized in that the pretreatment is: after the microchannel reactor is ultrasonically treated in a mixed solution of water and ethanol for 1.5 to 2.5 hours, the microchannel is pretreated with an aqueous sodium hydroxide solution with a concentration of 1 to 3 mol per liter, which is continuously injected into the microchannel using a syringe pump for a pretreatment time of 0.5 to 2 hours; after the pretreatment is completed, water and ethanol are introduced to remove residual sodium hydroxide for 5 to 20 minutes, and the microchannel is blown dry with nitrogen.

5. The method for preparing a wall-coated enzyme-loaded microchannel reactor according to claim 1, wherein the composition and mass percentage of the lipase solution before dialysis are as follows: The dialysis membrane (MD25-3500) used for dialysis of the lipase solution was 3 days, and the buffer solution was replaced every 12 hours); The composition of the buffer solution is phosphate buffer (Na2HPO4 / NaH2PO4), pH=7.

5.

6. The application of the wall-coated enzyme-loaded microchannel reactor prepared by the method of claim 1, characterized in that it is used for the hydrolysis reaction of oils and fats and various transesterification and esterification reactions.

7. The use according to claim 6, characterized in that it comprises the following steps: At 30-70°C, the reaction solution is injected into the microreactor through a syringe pump to obtain fatty acids and glycerol; or various ester substances; in, The residence time of the reaction solution is 5 to 30 minutes; When fatty acids and glycerol are obtained, the reaction liquid is composed of oil and water, and the mass of the oil is 50-80% of the mass of the reaction liquid; When vitamin E succinate is obtained, the composition of the reaction solution is succinic anhydride and tocopherol, and the mass of vitamin E is 30-50% of the mass of the reaction solution.

8. The use according to claim 7, characterized in that The oil is one or two of soybean oil, fish oil, peanut oil, rapeseed oil and sunflower oil; When soybean oil is selected as the oil, fatty acids and glycerol are obtained, and the soybean oil hydrolysis rate can reach 70%~90%.

9. The use as claimed in claim 7, characterized in that the yield of the vitamin E succinate can reach 40% within 30 minutes.

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