Immobilized lipase taking modified nano SiO2 as carrier as well as preparation method and application of immobilized lipase

Abstract

The invention relates to the technical field of immobilized lipase, in particular to immobilized lipase taking modified nano SiO2 as a carrier as well as a preparation method and application of the immobilized lipase. The immobilized lipase sold in the market is high in price and poor in mechanical strength, the enzyme loss is too large due to shear force or carrier structure damage in the continuous production process, and the transesterification efficiency is poor. In order to solve the technical problems, the invention provides the immobilized lipase taking the modified nano SiO2 as the carrier, the immobilized lipase is obtained by sequentially grafting a modified nano silicon dioxide carrier with an amino silane coupling agent and an epoxy silane coupling agent and loading lipase on the surface of the modified nano silicon dioxide carrier, according to the method, the immobilized lipase forms an enzyme column in a chromatographic column and then serves as a reactor, damage of mechanical stirring to lipase particles is avoided, the ester exchange degree is more effectively controlled by adjusting the flow speed of a substrate, the production cost of an enterprise can be remarkably reduced, and the product quality is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of immobilized lipase, and particularly relates to an immobilized lipase using modified nano-SiO2 as a carrier, its preparation method and application. Background Art

[0002] With the development of social economy and the improvement of residents' consumption levels, high-fat diets have gradually become one of the mainstream dietary structures of modern people, and the resulting health problems related to lipid metabolism (such as obesity, cardiovascular diseases, diabetes, etc.) have become increasingly prominent. The metabolism of traditional fats and oils in the human body mainly focuses on long-chain fatty acids (LCT), which have a long metabolic cycle and are prone to fat accumulation. Long-term intake may exacerbate lipid metabolism imbalance. Therefore, the development of new healthy fats and oils with unique metabolic pathways and physiological functions has become an important research direction in the fields of food science and nutrition.

[0003] Medium-chain and long-chain fatty acid structural esters (MLCT), as a type of functional structured lipid, because their molecules simultaneously contain the esterification structures of medium-chain fatty acids (MCT) and long-chain fatty acids (LCT), have the dual advantages of rapid energy supply (MCT is directly metabolized through the portal vein) and providing essential fatty acids (LCT participates in cell membrane construction) during in vivo metabolism. Research shows that MLCT can not only reduce body fat accumulation and improve lipid metabolism disorders, but also maintain the normal physiological functions of the body. Therefore, it shows broad application prospects in the fields of healthy edible oils, foods for special medical purposes, etc. In recent years, the global market demand for MLCT has been continuously increasing, driving the research and development of its synthesis technology to become a hot topic in the industry.

[0004] Currently, the industrial production of MLCT mainly relies on chemical synthesis methods and enzymatic synthesis methods.

[0005] Chemical synthesis methods usually use transesterification or esterification reactions under high temperature and high pressure conditions. Although the process is mature, it has significant defects:

[0006] High energy consumption and harsh reaction conditions (requiring strong acid / strong base catalysts);

[0007] Many side reactions, low product purity, and complex subsequent separation and purification steps;

[0008] Large wastewater emissions and prominent environmental protection pressure.

[0009] Enzymatic synthesis methods use immobilized lipase as a catalyst, which has the advantages of mild reaction conditions (room temperature and normal pressure), high selectivity, few by-products, and environmental friendliness, and is considered a more sustainable synthesis route. However, this technology still faces the following bottlenecks in practical applications:

[0010] High cost of immobilized enzymes: The price of commercially available immobilized lipase (such as Novozym 435) is as high as 800 yuan / kg, and its mechanical strength is poor. During continuous production, enzyme loss is likely to occur due to shear force,

[0011] Limited reusability: The existing immobilized enzymes have insufficient stability and limited recycling times (usually < 10 times), significantly increasing the production cost.

[0012] The above problems have severely restricted the large-scale promotion of enzymatic synthesis of MLCT. Therefore, developing an immobilized lipase technology with high stability, high activity, and low cost, and optimizing its carrier material and immobilization process have become the key to breaking through the production bottleneck of MLCT. By improving the mechanical strength and reusability of the immobilized enzyme, the cost of enzyme preparations can be significantly reduced, promoting the green and efficient synthesis of MLCT and meeting the urgent needs of the healthy oil market. Summary of the Invention

[0013] The problems existing in the prior art are as follows: Commercially available immobilized lipase has poor mechanical strength, and during continuous production, excessive enzyme loss is likely to occur due to shear force or damage to the carrier structure, resulting in poor efficiency of the transesterification reaction. To address the above technical problems, the present invention provides an immobilized lipase using modified nano-SiO2 as the carrier, and its preparation method includes the following steps:

[0014] (1) Sequentially graft an amino-silane coupling agent and an epoxy-silane coupling agent onto the surface of pretreated nano-silica to form a nano-silica carrier;

[0015] (2) Immobilize free lipase on the surface of the nano-silica carrier through intermolecular forces and covalent bonds.

[0016] Preferably, the mass ratio of the amino-silane coupling agent, the epoxy-silane coupling agent, and the pretreated nano-silica is 1 - 3:1 - 3:1000.

[0017] Preferably, the mass ratio of the amino-silane coupling agent, the epoxy-silane coupling agent, and the pretreated nano-silica is 2:2:1000.

[0018] Preferably, the amino-silane coupling agent includes N-(N-acetyl-leucyl)-3-aminopropyltriethoxysilane and 3-(4-ureidoamino)propyltriethoxysilane, and the mass ratio of N-(N-acetyl-leucyl)-3-aminopropyltriethoxysilane to 3-(4-ureidoamino)propyltriethoxysilane is 0.4 - 0.9:0.6 - 1.1.

[0019] Preferably, the epoxy-silane coupling agent is 3-(2,3-epoxypropoxy)propyltrimethoxysilane.

[0020] Preferably, the preparation method of the pretreated nano-silica in step (1) comprises the following steps:

[0021] Disperse the nano-silica in dilute hydrochloric acid, stir and react for at least 2 h, after normal pressure filtration, repeatedly wash and filter with deionized water to obtain a filter cake, then redisperse the filter cake in an aqueous sodium hydroxide solution, stir and react for at least 2 h, after normal pressure filtration, repeatedly wash and filter with deionized water to obtain a filter cake; finally redisperse the filter cake in deionized water, perform ultrasonic treatment at 40 °C for at least 2 h, after normal pressure filtration, obtain a filter cake, and after drying the obtained filter cake, obtain the pretreated nano-silica.

[0022] Preferably, the average particle size of the nano-silica is 20 - 50 nm.

[0023] Preferably, the lipase includes one or more combinations of Candida antarctica lipase A, Aspergillus oryzae lipase, and Candida rugosa lipase.

[0024] Preferably, the method of step (2) comprises the following steps:

[0025] Immerse the nano-silica carrier into the free lipase solution, after sufficient stirring and adsorption, collect the precipitate by solid-liquid separation, repeatedly wash the obtained precipitate with neutral sodium phosphate buffer solution until no enzyme protein is detected, and then perform freeze-drying to obtain the immobilized lipase.

[0026] A preparation method of medium and long-chain fatty acid structural esters, which uses the above-mentioned immobilized lipase as a biocatalyst. During the synthesis of medium and long-chain fatty acid structural esters, first fill the immobilized lipase into a chromatography column to form an enzyme column, then add medium-chain triglyceride and long-chain triglyceride to the enzyme column, and regulate the residence time of the substrate (a physical mixture of MCT and LCT) in the enzyme column by controlling the flow rate of the substrate, so as to control the degree of transesterification.

[0027] The medium-chain triglyceride includes one or more combinations of palm oil, coconut oil, and industrial food-grade caprylic / capric triglyceride;

[0028] The long-chain triglyceride includes one or more combinations of soybean oil, sunflower oil, corn oil, peanut oil, rapeseed oil, olive oil, and linseed oil.

[0029] The present invention has the following beneficial effects:

[0030] (1) The present invention provides a nano-silica carrier graft-modified successively by an amino-silane coupling agent and an epoxy-silane coupling agent, and loads lipase on its surface to obtain an immobilized lipase for catalyzing transesterification reaction. The structure of the immobilized lipase is stable, the reusability is good, and the catalytic effect is excellent;

[0031] (2) The present invention uses the reaction mode of forming an enzyme column with immobilized lipase in a chromatography column as a reactor, which avoids the damage of mechanical stirring to lipase particles. By adjusting the flow rate, the transesterification degree can be more effectively controlled, the production cost of enterprises can be reduced, and high-quality products can be obtained.

[0032] (3) The three silane coupling agents selected in the present invention, N-(N-acetylleucyl)-3-aminopropyltriethoxysilane, 3-(4-ureidoamino)propyltriethoxysilane, and 3-(2,3-epoxypropoxy)propyltrimethoxysilane, cooperate with each other and present good effects: amino and ureidoamino provide rich polar groups, enhancing the adsorption ability of lipase; epoxy groups provide covalent bond binding sites to ensure the firm immobilization of the enzyme. The two cooperate with each other to endow the carrier surface with both adsorption and covalent binding abilities, enhancing the multifunctionality of the carrier surface; acetylleucyl provides certain hydrophobicity and steric hindrance, which helps to expose the active center of lipase, thereby improving the catalytic activity of the enzyme; finally, through hydrogen bonding and electrostatic interactions, the binding of lipase and the carrier is further stabilized, improving the stability and mechanical strength of the carrier. These synergistic effects jointly improve the performance of the immobilized lipase, making it show higher efficiency and stability in the catalytic reaction. Description of the Drawings

[0033] Figure 1 : It is a schematic structural diagram of the device used in the transesterification reaction in Example 1.

[0034] In the figure: 1. Outer tube of the chromatography column; 2. Inner tube of the chromatography column; 3. Feed inlet; 4. Discharge outlet; 5. Adjusting knob; 6. Substrate liquid level; 7. Water inlet; 8. Water outlet. Detailed Embodiments

[0035] The present invention will be described in detail below with reference to the embodiments. However, it should be understood that the following embodiments are only illustrative examples of the implementation modes of the present invention, rather than limiting the scope of the present invention.

[0036] The average particle size of the nano-silica in the following embodiments of the present invention is 20 nm.

[0037] The pretreated nano-silica in the following embodiments of the present invention is prepared as follows:

[0038] Disperse nano-silica in dilute hydrochloric acid with a mass concentration of 2%, stir and react for 2 h. After filtration at atmospheric pressure, repeatedly wash and filter with deionized water to obtain a filter cake. Then redisperse the filter cake in an aqueous sodium hydroxide solution with a mass concentration of 2%, stir and react for 2 h. After filtration at atmospheric pressure, repeatedly wash and filter with deionized water to obtain a filter cake. Finally, redisperse the filter cake in deionized water, perform ultrasonic treatment at 40 °C for 2 h, and obtain a filter cake after filtration at atmospheric pressure. After drying the obtained filter cake, pretreated nano-silica is obtained.

[0039] The preparation of the free lipase solution in the following examples of the present invention is as follows:

[0040] Slowly add 1 g of Candida antarctica lipase A powder to 1 L of pre-cooled neutral phosphate buffer solution, gently stir or vortex until the enzyme is completely dissolved to obtain a homogeneous free lipase solution.

[0041] Example 1

[0042] An immobilized lipase using modified nano-SiO2 as a carrier, and its preparation method is as follows:

[0043] (1) Disperse 1 kg of pretreated nano-silica in 10 L of an ethanol aqueous solution (volume ratio of ethanol to water is 1:1), then add 1.3 g of N-(N-acetylleucyl)-3-aminopropyltriethoxysilane and 1.7 g of 3-(4-ureidoamino)propyltriethoxysilane, stir and react at 50 °C and 200 rpm for 6 h. After that, filter, repeatedly wash three times with deionized water and filter to obtain a filter cake. The obtained filter cake is dried at 65 °C for 12 h. Then redisperse the filter cake in 10 L of an ethanol aqueous solution (volume ratio of ethanol to water is 1:1), add 1 g of 3-(2,3-epoxypropoxy)propyltrimethoxysilane, perform ultrasonic treatment for 6 h with an ultrasonic frequency of 35 kHz. After that, filter, repeatedly wash three times with deionized water and filter to obtain a filter cake. The obtained filter cake is dried at 65 °C for 12 h to prepare a nano-silica carrier;

[0044] (2) Immerse 20 g of the nano-silica carrier into 1 L of the free lipase solution, stir and adsorb at 40 °C for 2 h, then collect the precipitate by centrifugation. The obtained precipitate is repeatedly washed with a neutral sodium phosphate buffer solution until no enzyme protein is detected, and then freeze-dried to obtain the immobilized lipase.

[0045] The immobilized lipase prepared above was put into a chromatography column to form an enzyme column, and the input amount was 7% of the maximum mass that the chromatography column could accommodate. Soybean oil, coconut oil, and food-grade caprylic / capric triglyceride were used as substrates and put above the enzyme column according to a mass ratio of 12:7:1. The input amount of the substrate was 95% of the maximum mass that the chromatography column could accommodate. The temperature of the outer tube of the chromatography column was set at 50°C. The residence time of the substrate in the enzyme column was adjusted to 25 min by the knob at the bottom of the chromatography column. After the reaction ended, gas chromatography analysis was carried out, and the content of medium- and long-chain fatty acid structural esters (MLCT) in the product reached 72.35%, and the acid value of the product was 2.52 mg KOH / g. The structural schematic diagram of the device used in the above transesterification reaction process is shown in the attached Figure 1 as follows.

[0046] Example 2

[0047] An immobilized lipase using modified nano-SiO2 as a carrier, and its preparation method is as follows:

[0048] (1) Disperse 1 kg of pretreated nano-silica in 10 L of an ethanol aqueous solution (the volume ratio of ethanol to water is 1:1), then add 0.9 g of N-(N-acetylleucyl)-3-aminopropyltriethoxysilane and 1.1 g of 3-(4-ureido)propyltriethoxysilane, and stir and react at 50°C and 200 rpm for 6 h. After that, filter, wash repeatedly with deionized water three times and then filter to obtain a filter cake. The obtained filter cake was dried at 65°C for 12 h. Then the filter cake was redispersed in 10 L of an ethanol aqueous solution (the volume ratio of ethanol to water is 1:1), add 2 g of 3-(2,3-epoxypropoxy)propyltrimethoxysilane, and perform ultrasonic treatment for 6 h with an ultrasonic frequency of 35 kHz. After that, filter, wash repeatedly with deionized water three times and then filter to obtain a filter cake. The obtained filter cake was dried at 65°C for 12 h to prepare a nano-silica carrier;

[0049] (2) Immerse 20 g of the nano-silica carrier into 1 L of a free lipase solution, stir and adsorb at 40°C for 2 h, then collect the precipitate by centrifugation. The obtained precipitate was repeatedly washed with a neutral sodium phosphate buffer solution until no enzyme protein was detected, and then freeze-dried to obtain the immobilized lipase.

[0050] The immobilized lipase prepared above was put into a chromatography column to form an enzyme column, and the input amount was 7% of the maximum mass that the chromatography column could accommodate. Soybean oil, coconut oil, and food-grade caprylic / capric triglyceride were used as substrates and were input above the enzyme column according to a mass ratio of 12:7:1. The input amount of the substrates was 95% of the maximum mass that the chromatography column could accommodate. The temperature of the outer tube of the chromatography column was set at 50 °C, and the residence time of the substrates in the enzyme column was adjusted to 25 min by a knob at the bottom of the chromatography column. After the reaction, gas chromatography analysis was carried out, and the content of medium- and long-chain fatty acid structural esters (MLCT) in the product reached 75.16%, and the acid value of the product was 2.82 mg KOH / g.

[0051] Example 3

[0052] An immobilized lipase using modified nano-SiO₂ as a carrier, and its preparation method is as follows:

[0053] (1) 1 kg of pretreated nano-silica was dispersed in 10 L of an ethanol aqueous solution (the volume ratio of ethanol to water was 1:1), and then 0.4 g of N-(N-acetylleucyl)-3-aminopropyltriethoxysilane and 0.6 g of 3-(4-ureidoamino)propyltriethoxysilane were added. The mixture was stirred and reacted at 50 °C and 200 rpm for 6 h. After that, it was filtered, washed three times with deionized water repeatedly and then suction-filtered to obtain a filter cake. The obtained filter cake was dried at 65 °C for 12 h. Then the filter cake was redispersed in 10 L of an ethanol aqueous solution (the volume ratio of ethanol to water was 1:1), and 3 g of 3-(2,3-epoxypropoxy)propyltrimethoxysilane was added. It was ultrasonically treated for 6 h, and the ultrasonic frequency was 35 kHz. After that, it was filtered, washed three times with deionized water repeatedly and then suction-filtered to obtain a filter cake. The obtained filter cake was dried at 65 °C for 12 h to prepare the nano-silica carrier;

[0054] (2) 20 g of the nano-silica carrier was immersed in 1 L of a free lipase solution. After stirring and adsorbing at 40 °C for 2 h, the precipitate was collected by centrifugation. The obtained precipitate was repeatedly washed with a neutral sodium phosphate buffer solution until no enzyme protein was detected, and then freeze-dried to obtain the immobilized lipase.

[0055] The immobilized lipase prepared above was put into a chromatography column to form an enzyme column, and the input amount was 7% of the maximum mass that the chromatography column could accommodate. Soybean oil, coconut oil, and food-grade caprylic / capric triglyceride were used as substrates and were input above the enzyme column according to a mass ratio of 12:7:1. The input amount of the substrates was 95% of the maximum mass that the chromatography column could accommodate. The temperature of the outer tube of the chromatography column was set at 50 °C, and the residence time of the substrates in the enzyme column was adjusted to 25 min by a knob at the bottom of the chromatography column. After the reaction, gas chromatography analysis was carried out, and the content of medium- and long-chain fatty acid structural esters (MLCT) in the product reached 73.68%, and the acid value of the product was 3.12 mg KOH / g.

[0056] Example 4

[0057] An immobilized lipase using modified nano-SiO2 as a carrier, and its preparation method is as follows:

[0058] (1) Disperse 1 kg of pretreated nano-silica in 10 L of an ethanol aqueous solution (volume ratio of ethanol to water is 1:1), then add 0.9 g of N-(N-acetylleucyl)-3-aminopropyltriethoxysilane and 1.1 g of 3-(4-ureidoamino)propyltriethoxysilane, and stir and react at 50 °C and 200 rpm for 6 h. After that, filter, wash three times with deionized water repeatedly and then perform suction filtration to obtain a filter cake. The obtained filter cake is dried at 65 °C for 12 h. Then, redisperse the filter cake in 10 L of an ethanol aqueous solution (volume ratio of ethanol to water is 1:1), add 2 g of 3-(2,3-epoxypropoxy)propyltrimethoxysilane, perform ultrasonic treatment for 6 h with an ultrasonic frequency of 35 kHz. After that, filter, wash three times with deionized water repeatedly and then perform suction filtration to obtain a filter cake. The obtained filter cake is dried at 65 °C for 12 h to prepare the nano-silica carrier;

[0059] (2) Immerse 20 g of the nano-silica carrier into 1 L of the free lipase solution, stir and adsorb at 40 °C for 2 h, then collect the precipitate by centrifugation. The obtained precipitate is repeatedly washed with a neutral sodium phosphate buffer solution until no enzyme protein is detected, and then freeze-dried to obtain the immobilized lipase.

[0060] Put the above-prepared immobilized lipase into a chromatography column to form an enzyme column, and the input amount is 7% of the maximum mass that the chromatography column can accommodate. Use soybean oil, coconut oil, and food-grade caprylic / capric triglyceride as substrates and input them above the enzyme column according to a mass ratio of 10:9:1. The input amount of the substrate is 95% of the maximum mass that the chromatography column can accommodate. Set the temperature of the outer tube of the chromatography column to 50 °C, and adjust the residence time of the substrate in the enzyme column to 25 min through the knob at the bottom of the chromatography column. After the reaction is completed, perform gas chromatography analysis. The content of medium- and long-chain fatty acid structural esters (MLCT) in the product is measured to be 80.56%, and the acid value of the product is 2.92 mg KOH / g.

[0061] Comparative Example 1 is the same as Example 3, except that in step (2) of Comparative Example 1, 50 g of the nano-silica carrier is immersed into 1 L of the free lipase solution.

[0062] After the transesterification reaction is completed, perform gas chromatography analysis. The content of medium- and long-chain fatty acid structural esters (MLCT) in the product is measured to be 79.55%, and the acid value of the product is 2.35 mg KOH / g.

[0063] Comparative Example 2 is the same as Example 2, except that in Comparative Example 2, the temperature of the outer tube of the chromatography column is set to 30 °C.

[0064] After the transesterification reaction, gas chromatography analysis was carried out. The content of medium and long-chain fatty acid structural esters (MLCT) in the product was measured to be 61.52%, and the acid value of the product was 3.7 mg KOH / g.

[0065] Comparative Example 3 was the same as Example 4, except that the immobilized lipase in Comparative Example 3 was prepared as follows:

[0066] (1) 1 kg of pretreated nano-silica was dispersed in 10 L of an ethanol aqueous solution (volume ratio of ethanol to water was 1:1), and then 2 g of N-(N-acetylleucyl)-3-aminopropyltriethoxysilane was added. The mixture was stirred and reacted at 50 °C and 200 rpm for 6 h. After that, it was filtered, washed three times with deionized water repeatedly and then filtered by suction to obtain a filter cake. The obtained filter cake was dried at 65 °C for 12 h. Then the filter cake was redispersed in 10 L of an ethanol aqueous solution (volume ratio of ethanol to water was 1:1), and 2 g of 3-(2,3-epoxypropoxy)propyltrimethoxysilane was added. It was ultrasonically treated for 6 h with an ultrasonic frequency of 35 kHz. After that, it was filtered, washed three times with deionized water repeatedly and then filtered by suction to obtain a filter cake. The obtained filter cake was dried at 65 °C for 12 h to prepare the nano-silica support;

[0067] (2) 20 g of the nano-silica support was immersed in 1 L of a free lipase solution. After stirring and adsorbing at 40 °C for 2 h, the precipitate was collected by centrifugation. The obtained precipitate was repeatedly washed with a neutral sodium phosphate buffer solution until no enzyme protein was detected, and then freeze-dried to obtain the immobilized lipase.

[0068] After the transesterification reaction, gas chromatography analysis was carried out. The content of medium and long-chain fatty acid structural esters (MLCT) in the product was measured to be 59.92%, and the acid value of the product was 2.57 mg KOH / g.

[0069] Comparative Example 4 was the same as Example 4, except that the immobilized lipase in Comparative Example 4 was prepared as follows:

[0070] (1) 1 kg of pretreated nano-silica was dispersed in 10 L of an ethanol aqueous solution (volume ratio of ethanol to water was 1:1), and then 2 g of 3-(4-ureidoamino)propyltriethoxysilane was added. The mixture was stirred and reacted at 50 °C and 200 rpm for 6 h. After that, it was filtered, washed three times with deionized water repeatedly and then filtered by suction to obtain a filter cake. The obtained filter cake was dried at 65 °C for 12 h. Then the filter cake was redispersed in 10 L of an ethanol aqueous solution (volume ratio of ethanol to water was 1:1), and 2 g of 3-(2,3-epoxypropoxy)propyltrimethoxysilane was added. It was ultrasonically treated for 6 h with an ultrasonic frequency of 35 kHz. After that, it was filtered, washed three times with deionized water repeatedly and then filtered by suction to obtain a filter cake. The obtained filter cake was dried at 65 °C for 12 h to prepare the nano-silica support;

[0071] (2) Immerse 20 g of the nano-silica carrier into 1 L of the free lipase solution, stir and adsorb at 40 °C for 2 h, then collect the precipitate by centrifugation. The obtained precipitate is repeatedly washed with a neutral sodium phosphate buffer solution until no enzyme protein is detected, and then freeze-dried to obtain the immobilized lipase.

[0072] After the transesterification reaction, gas chromatography analysis was carried out, and the content of medium and long-chain fatty acid structural esters (MLCT) in the product was measured to be 57.26%, and the acid value of the product was 3.49 mg KOH / g.

[0073] Comparative Example 5 is the same as Example 4, except that the immobilized lipase in Comparative Example 7 was prepared as follows:

[0074] (1) Disperse 1 kg of pretreated nano-silica in 10 L of an ethanol aqueous solution (volume ratio of ethanol to water is 1:1), then add 0.9 g of N-(N-acetylleucyl)-3-aminopropyltriethoxysilane and 1.1 g of cyclohexyltrimethoxysilane, stir and react at 50 °C and 200 rpm for 6 h. After that, filter, wash repeatedly with deionized water three times and then suction filter to obtain a filter cake. The obtained filter cake is dried at 65 °C for 12 h. Then, the filter cake is redispersed in 10 L of an ethanol aqueous solution (volume ratio of ethanol to water is 1:1), add 2 g of 3-(2,3-epoxypropoxy)propyltrimethoxysilane, and perform ultrasonic treatment for 6 h with an ultrasonic frequency of 35 kHz. After that, filter, wash repeatedly with deionized water three times and then suction filter to obtain a filter cake. The obtained filter cake is dried at 65 °C for 12 h to prepare the nano-silica carrier;

[0075] (2) Immerse 20 g of the nano-silica carrier into 1 L of the free lipase solution, stir and adsorb at 40 °C for 2 h, then collect the precipitate by centrifugation. The obtained precipitate is repeatedly washed with a neutral sodium phosphate buffer solution until no enzyme protein is detected, and then freeze-dried to obtain the immobilized lipase.

[0076] After the transesterification reaction, gas chromatography analysis was carried out, and the content of medium and long-chain fatty acid structural esters (MLCT) in the product was measured to be 65.75%, and the acid value of the product was 2.56 mg KOH / g.

[0077] Comparative Example 6 is the same as Example 4, except that the immobilized lipase in Comparative Example 8 was prepared as follows:

[0078] (1) Disperse 1 kg of pretreated nano-silica in 10 L of an ethanol aqueous solution (the volume ratio of ethanol to water is 1:1), then add 0.9 g of dodecyltriethoxysilane and 1.1 g of 3-(4-ureidoamino)propyltriethoxysilane, and stir and react at 50 °C and 200 rpm for 6 h. After that, filter, wash three times repeatedly with deionized water and then perform suction filtration to obtain a filter cake. The obtained filter cake is dried at 65 °C for 12 h. Then, redisperse the filter cake in 10 L of an ethanol aqueous solution (the volume ratio of ethanol to water is 1:1), add 2 g of 3-(2,3-epoxypropoxy)propyltrimethoxysilane, and perform ultrasonic treatment for 6 h with an ultrasonic frequency of 35 kHz. After that, filter, wash three times repeatedly with deionized water and then perform suction filtration to obtain a filter cake. The obtained filter cake is dried at 65 °C for 12 h to prepare a nano-silica support;

[0079] (2) Immerse 20 g of the nano-silica support into 1 L of a free lipase solution, stir and adsorb at 40 °C for 2 h, then collect the precipitate by centrifugation. The obtained precipitate is repeatedly washed with a neutral sodium phosphate buffer solution until no enzyme protein is detected, and then freeze-dried to obtain the immobilized lipase.

[0080] After the transesterification reaction, gas chromatography analysis is carried out. The content of medium and long-chain fatty acid structural esters (MLCT) in the product is measured to be 63.96%, and the acid value of the product is 3.15 mg KOH / g.

[0081] Comparative Example 7 is the same as Example 4, except that for the immobilized lipase in Comparative Example 9, the preparation method is as follows:

[0082] (1) Disperse 1 kg of pretreated nano-silica in 10 L of an ethanol aqueous solution (the volume ratio of ethanol to water is 1:1), then add 0.9 g of N-(N-acetylleucyl)-3-aminopropyltriethoxysilane and 1.1 g of 3-(4-ureidoamino)propyltriethoxysilane, and stir and react at 50 °C and 200 rpm for 6 h. After that, filter, wash three times repeatedly with deionized water and then perform suction filtration to obtain a filter cake. The obtained filter cake is dried at 65 °C for 12 h to prepare a nano-silica support;

[0083] (2) Immerse 20 g of the nano-silica support into 1 L of a free lipase solution, stir and adsorb at 40 °C for 2 h, then collect the precipitate by centrifugation. The obtained precipitate is repeatedly washed with a neutral sodium phosphate buffer solution until no enzyme protein is detected, and then freeze-dried to obtain the immobilized lipase.

[0084] After the transesterification reaction, gas chromatography analysis is carried out. The content of medium and long-chain fatty acid structural esters (MLCT) in the product is measured to be 51.16%, and the acid value of the product is 3.58 mg KOH / g.

[0085] Inspired by the above-described ideal embodiments of the present invention, through the above description, relevant staff can completely make various changes and modifications without departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

[0086] Comparative Example 8 is the same as Example 4, except that the immobilized lipase in Comparative Example 6 is prepared as follows:

[0087] (1) Disperse 1 kg of pretreated nano-silica in 10 L of an ethanol aqueous solution (volume ratio of ethanol to water is 1:1), add 4 g of 3-(2,3-epoxypropoxy)propyltrimethoxysilane, and ultrasonically treat for 6 h at an ultrasonic frequency of 35 kHz. After filtration, repeated washing three times with deionized water and suction filtration, a filter cake is obtained. The obtained filter cake is dried at 65 °C for 12 h to prepare a nano-silica carrier;

[0088] (2) Immerse 20 g of the nano-silica carrier into 1 L of a free lipase solution, stir and adsorb at 40 °C for 2 h, then collect the precipitate by centrifugation. The obtained precipitate is repeatedly washed with a neutral sodium phosphate buffer solution until no enzyme protein is detected, and then freeze-dried to obtain the immobilized lipase.

[0089] After the transesterification reaction, gas chromatography analysis was carried out, and the content of medium and long-chain fatty acid structural esters (MLCT) in the product was measured to be 50.34%, and the acid value of the product was 3.26 mg KOH / g.

[0090] Inspired by the above-described ideal embodiments of the present invention, through the above description, relevant staff can completely make various changes and modifications without departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. An immobilized lipase using modified nano-SiO2 as a carrier, characterized in that: The preparation method comprises the following steps: (1) sequentially grafting an aminosilane coupling agent and an epoxysilane coupling agent onto a nano-silica carrier formed on the surface of pretreated nano-silica; (2) Free lipase is fixed on the surface of nano-silica carrier through intermolecular forces and covalent bonds.

2. The immobilized lipase using modified nano-SiO2 as a carrier according to claim 1, characterized in that: The mass ratio of the aminosilane coupling agent to the epoxysilane coupling agent and the pretreated nano-silica is 1-3:1-3:1000.

3. The immobilized lipase using modified nano-SiO2 as a carrier according to claim 2, characterized in that: The mass ratio of the aminosilane coupling agent to the epoxysilane coupling agent and the pretreated nano-silica is 2:2:1000.

4. The immobilized lipase using modified nano-SiO2 as a carrier according to claim 1, characterized in that: The aminosilane coupling agent includes N-(N-acetylleucyl)-3-aminopropyltriethoxysilane and 3-(4-ureaamino)propyltriethoxysilane, and the mass ratio between the N-(N-acetylleucyl)-3-aminopropyltriethoxysilane and the 3-(4-ureaamino)propyltriethoxysilane is 0.4-0.9:0.6-1.

1.

5. The immobilized lipase using modified nano-SiO2 as a carrier according to claim 1, characterized in that: The epoxysilane coupling agent is 3-(2,3-epoxypropoxy)propyltrimethoxysilane.

6. The immobilized lipase using modified nano-SiO2 as a carrier according to claim 1, characterized in that: The preparation method of pretreated nano-silicon dioxide in step (1) comprises the following steps: The nano-silicon dioxide is dispersed in dilute hydrochloric acid, stirred for reaction for at least 2 hours, filtered at normal pressure, repeatedly washed with deionized water and filtered to obtain a filter cake, and then the filter cake is redispersed in a sodium hydroxide aqueous solution, stirred for reaction for at least 2 hours, filtered at normal pressure, repeatedly washed with deionized water and filtered to obtain a filter cake; finally, the filter cake is redispersed in deionized water, ultrasonically treated at 40° C. for at least 2 hours, filtered at normal pressure to obtain a filter cake, and the obtained filter cake is dried to obtain pretreated nano-silicon dioxide.

7. The immobilized lipase using modified nano-SiO2 as a carrier according to claim 6, characterized in that: The average particle size of the nano silicon dioxide is 20-50 nm.

8. The immobilized lipase using modified nano-SiO2 as a carrier according to claim 1, characterized in that: The lipase comprises one or a combination of two or more of Candida antarctica lipase A, Aspergillus oryzae lipase and Candida plicata lipase.

9. The immobilized lipase using modified nano-SiO2 as a carrier according to claim 1, characterized in that: The method of step (2) comprises the following steps: The nano-silica carrier is immersed in the free lipase solution, fully stirred for adsorption, and the precipitate is collected by solid-liquid separation. The precipitate is repeatedly washed with a neutral sodium phosphate buffer solution until no enzyme protein is detected, and then freeze-dried to obtain the immobilized lipase.

10. A method for preparing a medium- and long-chain fatty acid structure ester, characterized in that: The immobilized lipase described in any one of claims 1 to 9 is used as the immobilized lipase for synthesizing medium- and long-chain fatty acid structure esters by the ester exchange method. During the synthesis of the medium- and long-chain fatty acid structure esters, the immobilized lipase is first filled into a chromatography column to form an enzyme column, and then medium-chain fatty acid triglycerides and long-chain fatty acid triglycerides are added as substrates to the upper layer of the enzyme column. The residence time of the substrate in the enzyme column is adjusted by regulating the flow rate of the substrate, thereby controlling the degree of ester exchange.

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