Method for preparing fatty acid esters through enzyme catalysis

Abstract

The invention relates to the technical field of fatty acid ester preparation, and particularly discloses a method for preparing fatty acid esters by enzyme catalysis, which comprises the following steps: immobilizing lipase on a nano material or a porous carrier to form immobilized enzyme; adding the reaction raw materials and the immobilized enzyme into a reaction container to construct a reaction system, and adding a buffer solution and a protective agent into the reaction system; placing the reaction container in a constant-temperature water bath kettle or a shaking table, and installing physical strengthening equipment on the reaction container; reaction can be started after reaction conditions are set; in the reaction process, the viscosity of a reaction system in the reaction container is detected, and when the viscosity of the reaction system is larger than a preset value, an organic solvent or a surfactant is added into the reaction system. According to the method for catalytically preparing the fatty acid esters, the enzyme is immobilized by adopting a nano material or a porous carrier, so that the stability of the enzyme can be enhanced, and the tolerance of the enzyme to temperature and pH value changes can also be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of fatty acid ester preparation, in particular to a method for preparing fatty acid esters by enzyme catalysis. Background Art

[0002] Traditional enzyme-catalyzed methods for preparing fatty acid esters face numerous technical challenges. First, ensuring enzyme activity and stability is difficult. Enzymes are extremely sensitive to the reaction environment; even the slightest fluctuation in temperature, pH, and other conditions can lead to decreased enzyme activity or even inactivation. For example, high temperatures can easily denature enzyme proteins, while extreme pH levels can disrupt the enzyme's spatial structure, thereby affecting catalytic efficiency and resulting in low fatty acid ester yields, making them difficult to meet the demands of large-scale industrial production. Second, the reaction rate is slow. During the enzyme-catalyzed reaction, the binding efficiency between substrate and enzyme is limited, and the high mass transfer resistance in the reaction system prevents substrate molecules from contacting the enzyme's active centers in a timely manner, prolonging the reaction time. This mass transfer difficulty is particularly prominent when processing highly viscous substrates, further slowing the reaction and increasing production costs. Summary of the Invention

[0003] The object of the present invention is to provide a method for preparing fatty acid esters by enzyme catalysis, so as to solve the problems raised in the above background technology.

[0004] To achieve the above object, the present invention provides the following technical solutions: A method for preparing fatty acid esters by enzyme catalysis comprises the following steps: immobilizing the lipase on a nanomaterial or a porous carrier to form an immobilized enzyme; Adding reaction raw materials and immobilized enzyme into a reaction vessel to construct a reaction system, and adding a buffer and a protective agent into the reaction system; The reaction vessel is placed in a constant temperature water bath or a shaker, and a physical strengthening device is installed on the reaction vessel; the reaction can be started after the reaction conditions are set; the viscosity of the reaction system in the reaction vessel is detected during the reaction process, and when the viscosity of the reaction system is greater than a preset value, an organic solvent or a surfactant is added to the reaction system.

[0005] As a further embodiment of the present invention: a method for immobilizing lipase on a nanomaterial to form an immobilized enzyme comprises the following steps: Prepare crude lipase solution: weigh 2.0-5.0 g crude lipase powder and place it in a 100 ml beaker. Pour 6-9 ml of pH 7 phosphate buffer into the beaker containing crude enzyme powder. Stir at low temperature for 1-3 hours to dissolve the lipase in the phosphate buffer, then pour it into a volumetric flask to make up to 100 ml, and then place it in a refrigerator at 4°C for 6-12 hours to obtain the crude lipase solution; Before use, centrifuge the crude lipase solution at 2500-3000 r / min for 5-8 minutes to remove starch carriers and other substances in the crude lipase solution, and store the supernatant at low temperature for later use; Immobilizing lipase on nanomaterials: 3-5 ml of crude lipase solution was placed in a 50 ml stoppered conical flask. 9 ml of acetone precipitant and 0.5 ml of glutaraldehyde cross-linking agent were added sequentially at 4°C. The mixture was shaken and cross-linked at 4°C for 2-4 hours. The mixture was centrifuged at 6000-8000 rpm for 5-10 minutes. The supernatant was discarded, the precipitate was washed with buffer, and then centrifuged three times at 6000-8000 rpm to obtain cross-linked enzyme aggregates. The cross-linked enzyme aggregates were placed in 3 ml of phosphate buffer to obtain a cross-linked enzyme aggregate solution, which was stored in a refrigerator at 4°C for later use. The nanoparticles are added to the cross-linked enzyme aggregate solution, shaken evenly, and then precipitated and cross-linked to obtain the solidified enzyme.

[0006] As a further solution of the present invention: the mass volume ratio of nanoparticles to cross-linked enzyme aggregate solution is (1-5 mg):3 ml, and the selected nanoparticles are one of MgO, Cu, Ni, and Fe3O4.

[0007] As a further solution of the present invention: the porous carrier is mesoporous silica.

[0008] As a further embodiment of the present invention, a method for immobilizing lipase on mesoporous silica to form an immobilized enzyme comprises the following steps: The mesoporous SiO2 support was synthesized using a pore-forming agent and ethyl orthosilicate, and then calcined at a high temperature of 550-650°C to remove the pore-forming agent. The calcined mesoporous SiO2 support was immersed in a mixed solution of APTES and ethanol, with the volume proportion of APTES being 5%. Oxygen was removed by bubbling with nitrogen, and then refluxed at 70°C for 8-12 hours. After washing with ethanol three times, the solution was vacuum-dried at 60-80°C to obtain an amino-modified mesoporous support. The amino-modified mesoporous carrier and 20 mg / mL lipase were mixed at a mass ratio of 8:1 and shaken in a pH 7.0 phosphate buffer for 10 hours. After washing and drying, the lipase was immobilized in the amino-modified mesoporous carrier to form an immobilized enzyme.

[0009] As a further solution of the present invention: The method for synthesizing a mesoporous SiO2 carrier using a pore-forming agent and tetraethyl orthosilicate is as follows: 2.5g of the pore-forming agent is added to 120ml of deionized water and dissolved therein, the deionization temperature is 60°C, followed by dropwise addition of 10-12mL of 28wt% ammonia water, and then dropwise addition of 6-8ml of tetraethyl orthosilicate under vigorous stirring, followed by reaction at a temperature of 70-80°C for 12-18h to obtain a mesoporous SiO2 carrier.

[0010] As a further embodiment of the present invention, the reaction raw materials include oleic acid and ethanol, the molar ratio of oleic acid to ethanol is 1:1.2-1.5, the amount of oleic acid added is 30%-40% of the total mass of the reaction system, and the amount of ethanol added is 20%-30% of the total mass of the reaction system.

[0011] As a further embodiment of the present invention, the buffer solution is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution, the pH value of the potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution is 6.5-7.5, the concentration is 0.1-0.2 mol / L, and the amount added is 10%-20% of the total volume of the reaction system; the protective agent includes glycerol and sorbitol, the concentration of the protective agent is 5%-10% (v / v), and the amount of the protective agent added is 5%-10% of the total volume of the reaction system.

[0012] As a further embodiment of the present invention: when the viscosity of the reaction system is greater than >500 mPa·s, it is considered to be high viscosity, the organic solvent includes n-hexane and cyclohexane, the surfactant includes Tween 80 and Span 80, and the amount of the organic solvent and surfactant added is 5%-10% of the total volume of the reaction system.

[0013] As a further solution of the present invention: the physical strengthening equipment includes an ultrasonic device and a microwave device, the power of the ultrasonic device is 100W-500W, the processing time is 10min-60min, and the ultrasonic frequency is 20kHz-1MHz; the power of the microwave device is 100W-1000W, the processing time is 5-25min, and the processing temperature is 30℃-50℃.

[0014] Compared with the prior art, the present invention has the following beneficial effects: the method for catalytically preparing fatty acid esters disclosed in the present invention uses nanomaterials or porous carriers to immobilize the enzyme, which not only enhances the stability of the enzyme but also improves the enzyme's tolerance to changes in temperature and pH value; at the same time, a microenvironment control system is designed to precisely adjust the local environment of the reaction system through buffers, protective agents, etc., to provide stable catalytic conditions for the enzyme; physical field enhancement technologies such as ultrasound and microwaves are used to promote sufficient contact between the substrate and the enzyme, reduce mass transfer resistance, and accelerate the reaction rate; and for high-viscosity substrates, suitable organic solvents or surfactants are added to improve the fluidity of the system and accelerate the reaction process. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The present invention is a flow chart of a method for preparing fatty acid esters by enzyme catalysis. DETAILED DESCRIPTION

[0016] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0017] Traditional enzyme-catalyzed methods for preparing fatty acid esters face numerous technical challenges. First, ensuring enzyme activity and stability is difficult. Enzymes are extremely sensitive to the reaction environment; even the slightest fluctuation in temperature, pH, and other conditions can lead to decreased enzyme activity or even inactivation. For example, high temperatures can easily denature enzyme proteins, while extreme pH levels can disrupt the enzyme's spatial structure, thereby affecting catalytic efficiency and resulting in low fatty acid ester yields, making them difficult to meet the demands of large-scale industrial production. Second, the reaction rate is slow. During the enzyme-catalyzed reaction, the binding efficiency between substrate and enzyme is limited, and the high mass transfer resistance in the reaction system prevents substrate molecules from contacting the enzyme's active centers in a timely manner, prolonging the reaction time. This mass transfer difficulty is particularly prominent when processing highly viscous substrates, further slowing the reaction and increasing production costs.

[0018] Based on this, see Figure 1 A method for preparing fatty acid esters by enzyme catalysis comprises the following steps: S101, immobilizing lipase on a nanomaterial or a porous carrier to form an immobilized enzyme; In step S101, the method of immobilizing lipase on nanomaterials to form an immobilized enzyme includes the following steps: S201, preparing crude lipase solution, specifically comprising the following steps: Weigh 2.0-5.0g of crude lipase powder and place it in a 100ml beaker. Slowly pour 6-9ml of pH 7 phosphate buffer into the beaker containing the crude enzyme powder. Stir at low temperature for 1-3 hours to dissolve the lipase in the phosphate buffer, then pour it into a volumetric flask to make up to 100 ml, and then place it in a refrigerator at 4°C for 6-12 hours to obtain the crude lipase solution; Before use, centrifuge the crude lipase solution at 2500-3000 r / min for 5-8 minutes to remove the starch carrier and other substances in the crude lipase solution. The supernatant is stored at low temperature for later use. It should be noted that during the storage in the refrigerator, the enzyme solution should be shaken every 2 hours to allow the enzyme protein molecules to fully dissolve from the starch carrier. S202, solidifying the lipase on the nanomaterial, specifically comprising the following steps: Place 3-5 ml of crude lipase solution in a 50 ml stoppered conical flask. Add 9 ml of acetone precipitant and 0.5 ml of glutaraldehyde cross-linking agent sequentially at 4°C. Cross-link at 4°C with shaking for 2-4 hours. Centrifuge at 6000-8000 rpm for 5-10 minutes, discard the supernatant, wash the precipitate with buffer, and centrifuge again at 6000-8000 rpm for three times to obtain cross-linked enzyme aggregates. The cross-linked enzyme aggregates were placed in 3 ml of phosphate buffer to obtain a cross-linked enzyme aggregate solution, which was stored in a refrigerator at 4°C for later use. The nanoparticles are added to the cross-linked enzyme aggregate solution, shaken evenly, and then precipitated and cross-linked to obtain the solidified enzyme; the mass volume ratio of the nanoparticles to the cross-linked enzyme aggregate solution is (1-5 mg): 3 ml, and the selected nanoparticles are one of MgO, Cu, Ni, and Fe3O4.

[0019] In addition, when the porous carrier is mesoporous silica, the method for immobilizing the lipase on the mesoporous silica to form an immobilized enzyme comprises the following steps: S301, synthesizing a mesoporous SiO2 carrier using a pore-forming agent and tetraethyl orthosilicate, and then calcining at a high temperature of 550-650° C. to remove the pore-forming agent, specifically, the pore-forming agent is one of cetyltrimethylammonium bromide, polyethylene oxide-polypropylene oxide, and a triblock copolymer; S302, immersing the calcined mesoporous SiO2 support in a mixed solution of APTES and ethanol, wherein the volume proportion of APTES is 5%, bubbling with nitrogen to remove oxygen, and then refluxing at 70°C for 8-12 hours, washing with ethanol three times, and then vacuum drying at 60-80°C to obtain an amino-modified mesoporous support; S303, mixing the amino-modified mesoporous carrier with 20 mg / mL lipase at a mass ratio of 8:1 and oscillating in a pH 7.0 phosphate buffer for 10 hours. After washing and drying, the lipase can be immobilized in the amino-modified mesoporous carrier to form an immobilized enzyme.

[0020] Furthermore, in step S301, the method for synthesizing a mesoporous SiO2 carrier using a pore-forming agent and tetraethyl orthosilicate is as follows: 2.5 g of a pore-forming agent is added to 120 ml of deionized water for dissolution, the deionization temperature is 60°C, and then 10-12 mL of 28 wt% ammonia water is added dropwise, and 6-8 ml of tetraethyl orthosilicate is added dropwise under vigorous stirring, and then the reaction is carried out at a temperature of 70-80°C for 12-18 hours to obtain a mesoporous SiO2 carrier.

[0021] S102, adding reaction raw materials and immobilized enzyme into a reaction vessel to construct a reaction system, and adding a buffer and a protective agent into the reaction system; In step S102, the reaction raw materials include fatty acids and alcohols. Depending on the type of target fatty acid ester, the selected reaction raw materials are different, and the specific type is subject to actual application. In this embodiment, when the target fatty acid ester is ethyl oleate, the reaction raw materials include oleic acid and ethanol, and the molar ratio of oleic acid to ethanol is 1:1.2-1.5. The amount of oleic acid added is 30%-40% of the total mass of the reaction system, and the amount of ethanol added is 20%-30% of the total mass of the reaction system.

[0022] Furthermore, in step S102, in order to effectively maintain the acid-base balance of the reaction system, the buffer added to the reaction system is potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, the pH value of potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer is 6.5-7.5, the concentration is 0.1-0.2 mol / L, and the amount added is 10%-20% of the total volume of the reaction system; in addition, in step S102, the protective agents added to the reaction system include glycerol and sorbitol. The protective agents can prevent the enzyme from aggregation and inactivation during the reaction process. The concentration of the protective agent is 5%-10% (v / v), and the amount of the protective agent added is 5%-10% of the total volume of the reaction system.

[0023] S103. Place the reaction vessel in a constant temperature water bath or a shaker, and install a physical strengthening device on the reaction vessel; start the reaction after setting the reaction conditions; detect the viscosity of the reaction system in the reaction vessel during the reaction, and when the viscosity of the reaction system is greater than a preset value, add an organic solvent or a surfactant to the reaction system.

[0024] In step S103, the reaction conditions include reaction temperature and pH value of the reaction system. The reaction temperature is 30°C-50°C, and the pH value of the reaction system is between 6.0-8.0. If necessary, a small amount of acid or base is used for fine-tuning.

[0025] In step S103, when the viscosity of the reaction system is greater than >500 mPa·s, it is considered to be high viscosity. For high-viscosity fatty acid or alcohol substrates, the added organic solvents include n-hexane and cyclohexane, and the surfactants include Tween 80 and Span 80. The amount of organic solvent and surfactant added is 5%-10% of the total volume of the reaction system to improve the fluidity of the system and promote sufficient contact between the substrate and the enzyme.

[0026] The physical strengthening equipment includes an ultrasonic device. The power of the ultrasonic device is 100W-500W, the treatment time is 10min-60min, and the ultrasonic frequency is 20kHz-1MHz. The specific parameters can be optimized according to the actual situation of the reaction system. During the ultrasonic treatment process, the state of the reaction system is observed to ensure that the ultrasonic wave acts evenly on the reaction mixture. The cavitation effect of the ultrasonic wave will generate a local high temperature and high pressure environment inside the reaction system, promote the movement and collision of substrate molecules, enhance the mass transfer efficiency between the substrate and the enzyme, and also enable the substrate molecules to more fully contact with the enzyme active center, thereby accelerating the reaction rate. Furthermore, the physical strengthening equipment also includes a microwave device, the power of the microwave device is 100W-1000W, the processing time is 5-25min, and the processing temperature is 30℃-50℃; during the microwave treatment process, the temperature, pressure and other parameters of the reaction system are monitored in real time to ensure that the reaction is carried out under safe and controllable conditions. The thermal effect and non-thermal effect of the microwave can make the molecules in the reaction system move and vibrate rapidly, destroy the hydrogen bonds and other forces between the substrate molecules, reduce the mass transfer resistance, and promote the full contact and reaction between the substrate and the enzyme.

[0027] Example 1 In an embodiment of the present invention, a method for preparing fatty acid esters by enzyme catalysis comprises the following steps: S101, immobilizing lipase on nanomaterials to form immobilized enzyme; In step S101, the method of immobilizing lipase on nanomaterials to form an immobilized enzyme includes the following steps: S201, preparing crude lipase solution, specifically comprising the following steps: Weigh 2.0 g of crude lipase powder and place it in a 100 ml beaker. Slowly pour 6 ml of pH 7 phosphate buffer into the beaker containing the crude enzyme powder. Stir at low temperature for 1 hour to dissolve the lipase in the phosphate buffer, then pour it into a volumetric flask to make up to 100 ml, and then place it in a refrigerator at 4°C for 6 hours to obtain the crude lipase solution; Before use, centrifuge the crude lipase solution at 2500 r / min for 5 minutes to remove starch carriers and other substances in the crude lipase solution, and store the supernatant at low temperature for later use; S202, solidifying the lipase on the nanomaterial, specifically comprising the following steps: 3 ml of crude lipase solution was placed in a 50 ml stoppered conical flask. 9 ml of acetone precipitant and 0.5 ml of glutaraldehyde cross-linking agent were added sequentially at 4°C. The mixture was shaken and cross-linked at 4°C for 2 h. After centrifugation at 6000 r / min for 5 min, the supernatant was discarded, the precipitate was washed with buffer, and then centrifuged three times at 6000 r / min to obtain cross-linked enzyme aggregates. The cross-linked enzyme aggregates were placed in 3 ml of phosphate buffer to obtain a cross-linked enzyme aggregate solution, which was stored in a refrigerator at 4°C for later use. The nanoparticles are added to the cross-linked enzyme aggregate solution, shaken evenly, and then precipitated and cross-linked to obtain the solidified enzyme. In this embodiment, the mass volume ratio of the nanoparticles to the cross-linked enzyme aggregate solution is 1 mg:3 ml, and the selected nanoparticles are MgO.

[0028] S102, adding reaction raw materials and immobilized enzyme into a reaction vessel to construct a reaction system, and adding a buffer and a protective agent into the reaction system; In step S102, the reaction raw materials include oleic acid and ethanol, and the molar ratio of oleic acid to ethanol is 1:1.2. The amount of oleic acid added is 30% of the total mass of the reaction system, and the amount of ethanol added is 20% of the total mass of the reaction system.

[0029] Furthermore, in step S102, the buffer solution is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution, the pH value of the potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution is 6.5, the concentration is 0.1 mol / L, and the amount added is 10% of the total volume of the reaction system; in addition, in step S102, the protective agents added to the reaction system include glycerol and sorbitol, the concentration of the protective agent is 5% (v / v), and the amount of the protective agent added is 5% of the total volume of the reaction system.

[0030] S103. Place the reaction vessel in a constant temperature water bath or a shaker, and install a physical strengthening device on the reaction vessel; start the reaction after setting the reaction conditions; detect the viscosity of the reaction system in the reaction vessel during the reaction, and when the viscosity of the reaction system is greater than a preset value, add an organic solvent or a surfactant to the reaction system.

[0031] In step S103, the reaction conditions include reaction temperature and pH value of the reaction system. The reaction temperature is 30°C and the pH value of the reaction system is 6.0. If necessary, a small amount of acid or base is used for fine-tuning.

[0032] In step S103, when the viscosity of the reaction system is greater than >500 mPa·s, it is considered to be high viscosity. For high-viscosity fatty acid or alcohol substrates, the added organic solvents include n-hexane and cyclohexane, and the surfactants include Tween 80 and Span 80. The amount of organic solvent and surfactant added is 5% of the total volume of the reaction system.

[0033] In this embodiment, the physical strengthening equipment includes an ultrasonic device, the power of the ultrasonic device is 100W, the processing time is 10 minutes, and the ultrasonic frequency is 20kHz.

[0034] Example 2 In an embodiment of the present invention, a method for preparing fatty acid esters by enzyme catalysis comprises the following steps: S101, immobilizing lipase on a porous carrier to form an immobilized enzyme; The porous carrier is mesoporous silica, and the method for immobilizing lipase on the mesoporous silica to form an immobilized enzyme comprises the following steps: S301, synthesizing a mesoporous SiO2 carrier using a pore-forming agent and tetraethyl orthosilicate, and then calcining at a high temperature of 550° C. to remove the pore-forming agent. Specifically, in this embodiment, the pore-forming agent is one of cetyltrimethylammonium bromide, polyethylene oxide-polypropylene oxide, and a triblock copolymer; S302, immersing the calcined mesoporous SiO2 support in a mixed solution of APTES and ethanol, wherein the volume proportion of APTES is 5%, bubbling with nitrogen to remove oxygen, and then refluxing at 70°C for 8 hours, washing with ethanol three times, and then vacuum drying at 60°C to obtain an amino-modified mesoporous support; S303, mixing the amino-modified mesoporous carrier with 20 mg / mL lipase at a mass ratio of 8:1 and oscillating in a pH 7.0 phosphate buffer for 10 hours. After washing and drying, the lipase can be immobilized in the amino-modified mesoporous carrier to form an immobilized enzyme.

[0035] Furthermore, in step S301, the method for synthesizing a mesoporous SiO2 carrier using a pore-forming agent and tetraethyl orthosilicate is as follows: 2.5 g of the pore-forming agent is added to 120 ml of deionized water for dissolution, the deionization temperature is 60°C, and then 10 mL of 28 wt% ammonia water is added dropwise, and 6 ml of tetraethyl orthosilicate is added dropwise under vigorous stirring, and then the reaction is carried out at a temperature of 70°C for 12 hours to obtain a mesoporous SiO2 carrier.

[0036] S102, adding reaction raw materials and immobilized enzyme into a reaction vessel to construct a reaction system, and adding a buffer and a protective agent into the reaction system; In step S102, the reaction raw materials include oleic acid and ethanol, the molar ratio of oleic acid to ethanol is 1:1.5, the amount of oleic acid added is 40% of the total mass of the reaction system, and the amount of ethanol added is 30% of the total mass of the reaction system.

[0037] Furthermore, in step S102, the buffer solution is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution, the pH value of the potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution is 7.5, the concentration is 0.2 mol / L, and the amount added is 20% of the total volume of the reaction system; in addition, in step S102, the protective agent includes glycerol and sorbitol, the concentration of the protective agent is 10% (v / v), and the amount of the protective agent added is 10% of the total volume of the reaction system.

[0038] S103. Place the reaction vessel in a constant temperature water bath or a shaker, and install a physical strengthening device on the reaction vessel; start the reaction after setting the reaction conditions; detect the viscosity of the reaction system in the reaction vessel during the reaction, and when the viscosity of the reaction system is greater than a preset value, add an organic solvent or a surfactant to the reaction system.

[0039] In step S103, the reaction conditions include reaction temperature and pH value of the reaction system. The reaction temperature is 50°C and the pH value of the reaction system is between 8.0. If necessary, a small amount of acid or base is used for fine-tuning.

[0040] In step S103, when the viscosity of the reaction system is greater than >500 mPa·s, it is considered to be high viscosity. For high-viscosity fatty acid or alcohol substrates, the added organic solvents include n-hexane and cyclohexane, and the surfactants include Tween 80 and Span 80. The amount of organic solvent and surfactant added is 10% of the total volume of the reaction system.

[0041] The physical strengthening equipment includes a microwave device, the power of the microwave device is 1000W, the processing time is 25 minutes, and the processing temperature is -50°C.

[0042] Example 3 In an embodiment of the present invention, a method for preparing fatty acid esters by enzyme catalysis comprises the following steps: S101, immobilizing lipase on a porous carrier to form an immobilized enzyme; The porous carrier is mesoporous silica, and the method for immobilizing lipase on the mesoporous silica to form an immobilized enzyme comprises the following steps: S301, using a pore-forming agent and ethyl orthosilicate to synthesize a mesoporous SiO2 carrier, and then calcining it at a high temperature of 650°C to remove the pore-forming agent. In this embodiment, the pore-forming agent is hexadecyltrimethylammonium bromide; S302, immersing the calcined mesoporous SiO2 support in a mixed solution of APTES and ethanol, wherein the volume proportion of APTES is 5%, and nitrogen is used to remove oxygen. Subsequently, the mixture is refluxed at 70°C for 12 hours, washed with ethanol three times, and then dried in vacuo at 80°C to obtain an amino-modified mesoporous support. S303, mixing the amino-modified mesoporous carrier with 20 mg / mL lipase at a mass ratio of 8:1 and oscillating in a pH 7.0 phosphate buffer for 10 hours. After washing and drying, the lipase can be immobilized in the amino-modified mesoporous carrier to form an immobilized enzyme.

[0043] Furthermore, in step S301, the method for synthesizing a mesoporous SiO2 carrier using a pore-forming agent and tetraethyl orthosilicate is as follows: 2.5 g of the pore-forming agent is added to 120 ml of deionized water for dissolution, the deionization temperature is 60°C, and then 12 mL of 28 wt% ammonia water is added dropwise, and 8 ml of tetraethyl orthosilicate is added dropwise under vigorous stirring, and then the reaction is carried out at a temperature of 80°C for 18 hours to obtain a mesoporous SiO2 carrier.

[0044] S102, adding reaction raw materials and immobilized enzyme into a reaction vessel to construct a reaction system, and adding a buffer and a protective agent into the reaction system; In step S102, the reaction raw materials include oleic acid and ethanol, the molar ratio of oleic acid to ethanol is 1:1.2-1.3, the amount of oleic acid added is 35% of the total mass of the reaction system, and the amount of ethanol added is 25% of the total mass of the reaction system.

[0045] Furthermore, in step S102, the buffer solution is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution, the pH value of the potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution is 7, the concentration is 0.15 mol / L, and the amount added is 15% of the total volume of the reaction system; in addition, in step S102, the protective agents added to the reaction system include glycerol and sorbitol, the concentration of the protective agent is 8% (v / v), and the amount of the protective agent added is 7% of the total volume of the reaction system.

[0046] S103. Place the reaction vessel in a constant temperature water bath or a shaker, and install a physical strengthening device on the reaction vessel; start the reaction after setting the reaction conditions; detect the viscosity of the reaction system in the reaction vessel during the reaction, and when the viscosity of the reaction system is greater than a preset value, add an organic solvent or a surfactant to the reaction system.

[0047] In step S103, the reaction conditions include reaction temperature and pH value of the reaction system. The reaction temperature is 40°C and the pH value of the reaction system is between 7.2 and 7.3. If necessary, a small amount of acid or base is used for fine-tuning.

[0048] In step S103, when the viscosity of the reaction system is greater than >500 mPa·s, it is considered to be high viscosity. For high-viscosity fatty acid or alcohol substrates, the added organic solvents include n-hexane and cyclohexane, and the surfactants include Tween 80 and Span 80. The amount of organic solvent and surfactant added is 8% of the total volume of the reaction system.

[0049] In this embodiment, the physical strengthening equipment includes an ultrasonic device, the power of the ultrasonic device is 300W, the processing time is 30 minutes, and the ultrasonic frequency is 100kHz.

[0050] In summary, the method for catalytically preparing fatty acid esters disclosed in the present invention uses nanomaterials or porous carriers to immobilize enzymes, which not only enhances the stability of the enzymes but also improves the enzymes' tolerance to changes in temperature and pH. Furthermore, a microenvironment control system is designed to precisely regulate the local environment of the reaction system through buffers, protective agents, and the like, thereby providing stable catalytic conditions for the enzymes. Physical field enhancement technologies such as ultrasound and microwaves are used to promote sufficient contact between the substrate and the enzyme, reduce mass transfer resistance, and accelerate the reaction rate. Furthermore, for high-viscosity substrates, suitable organic solvents or surfactants are added to improve the fluidity of the system and accelerate the reaction process.

[0051] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0052] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for preparing fatty acid esters by enzyme catalysis, characterized in that: The following steps are involved: immobilizing the lipase on a nanomaterial or a porous carrier to form an immobilized enzyme; Adding reaction raw materials and immobilized enzyme into a reaction vessel to construct a reaction system, and adding a buffer and a protective agent into the reaction system; The reaction vessel is placed in a constant temperature water bath or a shaker, and a physical strengthening device is installed on the reaction vessel; the reaction can be started after the reaction conditions are set; the viscosity of the reaction system in the reaction vessel is detected during the reaction process, and when the viscosity of the reaction system is greater than a preset value, an organic solvent or a surfactant is added to the reaction system.

2. The method for preparing fatty acid esters by enzyme catalysis according to claim 1, characterized in that: The method for immobilizing lipase on nanomaterials to form an immobilized enzyme comprises the following steps: Prepare crude lipase solution: weigh 2.0-5.0 g crude lipase powder and place it in a 100 ml beaker. Pour 6-9 ml of pH 7 phosphate buffer into the beaker containing crude enzyme powder. Stir at low temperature for 1-3 hours to dissolve the lipase in the phosphate buffer, then pour it into a volumetric flask to make up to 100 ml, and then place it in a refrigerator at 4°C for 6-12 hours to obtain the crude lipase solution; Before use, centrifuge the crude lipase solution at 2500-3000 r / min for 5-8 minutes to remove starch carriers and other substances in the crude lipase solution, and store the supernatant at low temperature for later use; Immobilizing lipase on nanomaterials: 3-5 ml of crude lipase solution was placed in a 50 ml stoppered conical flask. 9 ml of acetone precipitant and 0.5 ml of glutaraldehyde cross-linking agent were added sequentially at 4°C. The mixture was shaken and cross-linked at 4°C for 2-4 hours. The mixture was centrifuged at 6000-8000 rpm for 5-10 minutes. The supernatant was discarded, the precipitate was washed with buffer, and then centrifuged three times at 6000-8000 rpm to obtain cross-linked enzyme aggregates. The cross-linked enzyme aggregates were placed in 3 ml of phosphate buffer to obtain a cross-linked enzyme aggregate solution, which was stored in a refrigerator at 4°C for later use. The nanoparticles are added to the cross-linked enzyme aggregate solution, shaken evenly, and then precipitated and cross-linked to obtain the solidified enzyme.

3. The method for preparing fatty acid esters by enzyme catalysis according to claim 2, characterized in that: The mass volume ratio of the nanoparticles to the cross-linked enzyme aggregate solution is (1-5 mg): 3 ml, and the selected nanoparticles are one of MgO, Cu, Ni, and Fe3O4.

4. The method for preparing fatty acid esters by enzyme catalysis according to claim 1, characterized in that: The porous carrier is mesoporous silica.

5. The method for preparing fatty acid esters by enzyme catalysis according to claim 4, characterized in that: The method for immobilizing lipase on mesoporous silica to form an immobilized enzyme comprises the following steps: The mesoporous SiO2 support was synthesized using a pore-forming agent and ethyl orthosilicate, and then calcined at a high temperature of 550-650°C to remove the pore-forming agent. The calcined mesoporous SiO2 support was immersed in a mixed solution of APTES and ethanol, with the volume proportion of APTES being 5%. Oxygen was removed by bubbling with nitrogen, and then refluxed at 70°C for 8-12 hours. After washing with ethanol three times, the solution was vacuum-dried at 60-80°C to obtain an amino-modified mesoporous support. The amino-modified mesoporous carrier and 20 mg / mL lipase were mixed at a mass ratio of 8:1 and shaken in a pH 7.0 phosphate buffer for 10 hours. After washing and drying, the lipase was immobilized in the amino-modified mesoporous carrier to form an immobilized enzyme.

6. The method for preparing fatty acid esters by enzyme catalysis according to claim 5, characterized in that: The method for synthesizing a mesoporous SiO2 carrier using a pore-forming agent and ethyl orthosilicate is as follows: 2.5 g of a pore-forming agent is added to 120 ml of deionized water and dissolved at a deionization temperature of 60°C, followed by dropwise addition of 10-12 mL of 28 wt% ammonia water, and then dropwise addition of 6-8 ml of ethyl orthosilicate under vigorous stirring, followed by reaction at a temperature of 70-80°C for 12-18 hours to obtain a mesoporous SiO2 carrier.

7. The method for preparing fatty acid esters by enzyme catalysis according to claim 1, characterized in that: The reaction raw materials include oleic acid and ethanol, the molar ratio of oleic acid to ethanol is 1:1.2-1.5, the amount of oleic acid added is 30%-40% of the total mass of the reaction system, and the amount of ethanol added is 20%-30% of the total mass of the reaction system.

8. The method for preparing fatty acid esters by enzyme catalysis according to claim 1, characterized in that: The buffer is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, the pH value of the potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer is 6.5-7.5, the concentration is 0.1-0.2 mol / L, and the added amount is 10%-20% of the total volume of the reaction system; the protective agent includes glycerol and sorbitol, the concentration of the protective agent is 5%-10% (v / v), and the added amount of the protective agent is 5%-10% of the total volume of the reaction system.

9. The method for preparing fatty acid esters by enzyme catalysis according to claim 1, characterized in that: When the viscosity of the reaction system is greater than 500 mPa·s, it is considered to be high viscosity. The organic solvent includes n-hexane and cyclohexane, and the surfactant includes Tween 80 and Span 80. The amount of the organic solvent and the surfactant added is 5%-10% of the total volume of the reaction system.

10. The method for preparing fatty acid esters by enzyme catalysis according to claim 1, characterized in that: The physical strengthening equipment includes an ultrasonic device and a microwave device. The power of the ultrasonic device is 100W-500W, the processing time is 10min-60min, and the ultrasonic frequency is 20kHz-1MHz; the power of the microwave device is 100W-1000W, the processing time is 5-25min, and the processing temperature is 30℃-50℃.

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