Immobilized lipase, preparation method and application of immobilized lipase in preparation of structural lipid

Abstract

The invention discloses immobilized lipase, a preparation method and application of the immobilized lipase in preparation of structural lipid. The preparation method comprises the following steps: synthesis of an immobilized carrier, immobilization of enzyme and synthesis of the structural lipid. The lipase is immobilized by the nano mesoporous silica material, so that the reaction time is greatly shortened, the enzyme activity is better reserved, the use frequency of the enzyme is increased, and the problems of high cost and low utilization rate of the lipase in the industrial production process of sterol ester are solved. Two beneficial substances of sterol ester and diglyceride are synthesized at the same time through a one-step reaction, and the product can be directly used as functional grease without purification.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional oils and fats, and particularly relates to an immobilized lipase, a preparation method thereof, and an application thereof in the preparation of structured lipids. Background Art

[0002] Enzymes are naturally occurring biocatalysts with characteristics such as strong catalytic selectivity, fast reaction speed, mild reaction conditions, and being green and pollution-free. They are highly potential catalysts. There are many types of lipases, which can catalyze various reactions such as ester hydrolysis, ester synthesis, and transesterification, and are widely used in many fields such as the food production industry, the oil chemical industry, the agrochemical industry, the paper industry, the wastewater treatment industry, and the synthesis of health products and drugs. However, due to the high cost of using lipases, easy inactivation, difficulty in recovery, and low enzyme activity in organic phase catalytic reactions, the application of lipases is greatly restricted. Therefore, it is necessary to apply enzyme immobilization technology to improve its stability and reusability and reduce the use cost.

[0003] The nano-mesoporous silica immobilization technology is an enzyme immobilization method proposed and widely studied in recent years. Mesoporous silica is a material that is particularly attractive for enzyme immobilization because it has an ordered pore structure, a narrow pore size distribution, a large specific surface area, stable chemical and mechanical properties, and can also be chemically modified with various functional groups to increase its scope of application. Due to the large specific surface area and internal space of mesoporous silica, sufficient immobilization sites are provided for lipases. The "interface activation" effect of lipases can significantly improve their catalytic activity. Compared with lipases immobilized on hydrophilic carriers, lipases immobilized on hydrophobic carriers often can obtain significantly improved catalytic activity and stability. This is because the hydrophobic region near the active center of lipases forms a strong hydrophobic interaction with the surface of the hydrophobic carrier, causing a certain angle of flipping of the "lid" structure of lipases, changing from the "closed" state to the "open" conformation after immobilization, exposing the active center of the enzyme, and making it easier for the substrate to enter the enzyme active site for catalysis. Mesoporous silica can be modified by silanization to improve its surface hydrophobicity, thereby greatly improving the activity of the immobilized enzyme.

[0004] Phytosterols are natural active ingredients present in various vegetable oils, nuts, and plant seeds, and have functions such as reducing cholesterol content, preventing cardiovascular diseases, anti-inflammatory, and anti-cancer. However, the poor solubility and bioavailability of phytosterols limit their practical applications. Therefore, the preparation of phytosterol esters by lipase-catalyzed phytosterol reactions is a current research hotspot, which can significantly improve the oil solubility of sterols. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of the present application, to avoid obscuring the purpose of this section, the abstract, and the title, and such simplifications or omissions shall not be used to limit the scope of the present invention.

[0006] In view of the above and / or problems existing in the prior art, the present invention is proposed.

[0007] Therefore, an object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing immobilized lipase.

[0008] To solve the above technical problems, the present invention provides the following technical solutions: including,

[0009] Prepare a modified mesoporous silica material: concentrated ammonia water is added to an aqueous solution of cetyltrimethylammonium bromide, and a non-polar solvent and tetraethyl orthosilicate are added with stirring to form a homogeneous solution; the product is collected, washed, dried, and then calcined to remove the template to obtain mesoporous silica, which is modified with a silane coupling agent;

[0010] Prepare immobilized enzyme: a lipase solution is placed in a carrier for adsorption, and after centrifuging to remove the solvent, it is dried to obtain immobilized lipase.

[0011] As a preferred embodiment of the method for preparing immobilized lipase according to the present invention, wherein: the stirring speed is 100 - 250 r / min, the stirring temperature is 25 - 40 °C, and the stirring time is 8 - 24 h.

[0012] As a preferred embodiment of the method for preparing immobilized lipase according to the present invention, wherein: the calcination temperature is 400 - 600 °C, and the calcination time is 3 - 6 h.

[0013] As a preferred embodiment of the method for preparing immobilized lipase according to the present invention, wherein: the silane coupling agent is one or more of n-octyltrimethoxysilane, n-octyltriethoxysilane, dodecyltrimethoxysilane, dodecyltriethoxysilane, hexadecyltrimethoxysilane, or hexadecyltriethoxysilane.

[0014] As a preferred embodiment of the method for preparing immobilized lipase according to the present invention, wherein: the mass concentration of the lipase solution is 30 - 40 g / L.

[0015] As a preferred embodiment of the method for preparing immobilized lipase according to the present invention, wherein: the addition amount of the immobilized enzyme is 2% - 6%.

[0016] Therefore, another object of the present invention is to overcome the deficiencies in the prior art and provide an immobilized lipase.

[0017] Therefore, another object of the present invention is to overcome the deficiencies in the prior art and provide an application of immobilized lipase in the preparation of structured lipids.

[0018] To solve the above technical problems, the present invention provides the following technical solution: The immobilized lipase is used to catalyze the synthesis of sterol esters and diglycerides from fats, including

[0019] Using a non-polar solvent as the reaction solvent and the immobilized enzyme as the catalyst, sterols and oils are added for reaction.

[0020] As a preferred embodiment of the application of the immobilized lipase in the preparation of structured lipids of the present invention, wherein: the non-polar solvent includes one or more of isooctane, n-hexane, n-heptane or cyclohexane.

[0021] As a preferred embodiment of the application of the immobilized lipase in the preparation of structured lipids of the present invention, wherein: the reaction temperature of the sterols and oils is 40-70 °C.

[0022] Advantages of the present invention:

[0023] The present invention prepares a nano-porous silica material to immobilize lipase, better retaining the enzyme activity and increasing the number of times the enzyme can be used, solving the problems of high cost and low utilization rate of lipase in the industrial production of sterol esters. By a one-step reaction, two beneficial substances, sterol esters and diglycerides, are synthesized simultaneously, and the product can be directly used as a functional oil without purification. By using the immobilized enzyme, the sterol conversion rate is effectively increased, and a better immobilized carrier is made. Description of the Drawings

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings. Among them:

[0025] Figure 1 It is a scanning electron microscope (SEM) image of the nano-porous silica material prepared in Example 1 of the present invention;

[0026] Figure 2 It is a transmission electron microscope (TEM) image of the nano-porous silica material prepared in Example 1 of the present invention;

[0027] Figure 3 It is the N 2 adsorption-desorption isotherm of the nano-porous silica material prepared in Example 1 of the present invention;

[0028] Figure 4It is the thermogravimetric analysis (TGA) diagram of the nano - mesoporous silica material prepared in Example 1 of the present invention;

[0029] Figure 5 It is the Fourier transform infrared spectroscopy (FTIR) diagram of the nano - mesoporous silica material prepared in Example 1 of the present invention. Detailed implementation manners

[0030] To make the above - mentioned objects, features and advantages of the present invention more obvious and understandable, the following describes the detailed implementation manners of the present invention in conjunction with the embodiments of the specification.

[0031] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0032] Secondly, the so - called "one embodiment" or "embodiment" herein refers to a specific feature, structure or characteristic that may be included in at least one implementation manner of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it an individual or alternative embodiment that is mutually exclusive with other embodiments.

[0033] Unless otherwise specified, the raw materials used in the present invention are all commercially available.

[0034] The materials prepared in the embodiments of the present invention are tested for performance according to the following method:

[0035] 1. Analysis method for the synthesized product:

[0036] ① Operating methods and parameters for HPLC - ELSD detection: A normal - phase high - performance liquid chromatograph (RP - HPLC) equipped with an evaporative light - scattering detector is used to detect and analyze the components in the sample oil. Take 2 mg of the sample, dissolve it in 1.0 mL of chromatographic - grade n - hexane, filter it through a membrane, and then place it in the high - performance liquid chromatograph for analysis. High - performance liquid chromatography conditions: silica gel chromatographic column (250 mm×4.6 mm×5 μm); evaporative light - scattering detector temperature 55°C; air flow rate set at 1.8 mL / min, gain value 1; elution flow rate 0.5 mL / min; injection concentration 2 mg / mL, injection volume 10 μL.

[0037] High - performance liquid chromatography program: The mobile - phase elution program is shown in Table 1, and quantitative analysis is carried out in combination with the peak - area normalization method.

[0038] Table 1 Mobile - phase elution program for high - performance liquid chromatography

[0039]

[0040] 2. Determination of the protein concentration in the enzyme solution: According to the usage method of the BCA protein concentration assay kit, add the sample and the BCA working solution to the sample wells of a 96-well plate and incubate at 37 °C for 3 min. Measure the absorbance at a wavelength of A562 using an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the protein concentration of the sample based on the measured standard curve.

[0041] 3. Determination of the protein immobilization rate of the immobilized enzyme:

[0042] Protein immobilization rate (%) = (Ca - Cb) / Ca × 100

[0043] Where:

[0044] Ca—Protein concentration in the enzyme solution before adsorption, mg / mL;

[0045] Cb—Protein concentration in the enzyme solution after adsorption, mg / mL.

[0046] Example 1

[0047] Preparation of mesoporous silica material: Slowly add 10 g of cetyltrimethylammonium bromide to 1600 mL of deionized water, then add 30 mL of concentrated ammonia water and stir to form a clear solution; Stir 200 mL of n-hexane and 50 mL of tetraethyl orthosilicate evenly, and slowly add them to the clear solution through a constant pressure funnel at a rate of 4 mL / min. Stir at 35 °C for 12 h at a rotation speed of 200 rpm to form a uniform colloidal solution; Then centrifuge at a rotation speed of 7000 rpm for 15 min to collect the product, wash it three times with deionized water and ethanol respectively, and finally dry it in an oven; Calcinate the dried material at 550 °C for 5 h to remove the template and obtain nano-mesoporous silica.

[0048] Hydrophobic modification: Disperse 1 g of mesoporous silica into 10 mL of toluene, add 15 μL of triethylamine and 10 mM of octyltrimethoxysilane dropwise respectively, react in a high-pressure reaction kettle at 120 °C for 12 h to synthesize hydrophobic mesoporous silica, then centrifuge at a rotation speed of 7000 rpm for 15 min to collect the product, wash it twice with ethanol, and dry it in a vacuum oven at 60 °C for standby. The average particle diameter of the obtained mesoporous silica is 300 nm, and the specific surface area after modification is 500 m 2 / g;

[0049] Preparation of immobilized enzyme: 30 g of Candida rugosa lipase was added to 1 L of phosphate buffer (50 mM, pH 7.0), stirred at 200 rpm for 15 min at 4 °C, and centrifuged (7000 rpm, 10 min) to obtain the supernatant, which was made into a lipase solution. Subsequently, hollow mesoporous silica (pre-infiltrated with ethanol) was added to the lipase solution and mixed evenly by shaking. It was taken out after immobilization at a shaker (30 °C, 180 rpm) for 4 h, centrifuged (7000 rpm, 10 min), the supernatant was reserved for protein measurement, and the precipitate (immobilized enzyme) was washed 3 times with phosphate buffer and then freeze-dried to obtain the immobilized enzyme, and the protein immobilization rate was 75%;

[0050] Catalytic synthesis of sterol esters and diglycerides: 414 g (0.8 M) of phytosterol (sitosterol) and 1744 g (2 M) of soybean oil were weighed and added to the reaction kettle, and then 10 L of isooctane was added to ultrasonically mix the three mixtures for 15 min (ultrasonic power 600 W) to obtain a premix. 5% of the immobilized enzyme was added to the reaction kettle and stirred with a paddle at 50 °C and a rotation speed of 250 rpm for 2 h.

[0051] Product separation: After the reaction, the lipase was separated and recovered by centrifugation at 7000 rpm for 10 min, and the supernatant was rotary evaporated under reduced pressure at a vacuum of -0.08 MPa and 50 °C to remove the reaction solvent to obtain functional oil. The reaction conversion rate was greater than 98%. It shows that the product obtained by the present invention is rich in two active functional components, sterol esters and diglycerides. The lipase still retained 85% of its activity after being reused 10 times.

[0052] Figure 1 This is the scanning electron microscope (SEM) image of the nano-mesoporous silica material prepared in Example 1 of the present invention. As can be seen from the figure, the nano-mesoporous silica is spherical, with uniform particle size and regular shape, and the diameter is about 300 nm, indicating that the synthesized material has good stability and is a nano-scale mesoporous material.

[0053] Figure 2 This is the transmission electron microscope (TEM) image of the nano-mesoporous silica material prepared in this example. As can be seen from the figure, the mesoporous silica material has a core-shell structure, where the core has mesopores arranged in a cubic structure and the shell has mesopores arranged radially, indicating that the synthesized material has good immobilized enzyme ability and the enzyme can be immobilized in the mesopores.

[0054] Figure 3 This is the N 2 adsorption-desorption isotherm of the nano-mesoporous silica material prepared in this example. It can be calculated that the specific surface area of the modified mesoporous silica is 500 m 2 / g, and the specific surface area after immobilizing the enzyme is 176 m 2 / g.

[0055] Figure 4 The thermogravimetric analysis (TGA) diagrams of the nano-mesoporous silica material and the immobilized enzyme prepared in this example are shown. It can be seen that when the temperature is between 50 - 100 °C, the weights of the free enzyme and the immobilized enzyme decrease by 4.9% and 1.6% respectively. The weight change at this stage is caused by the evaporation of the adsorbed water on the surface of the sample, indicating that the ability of the enzyme to adsorb water weakens after being adsorbed on the hydrophobic carrier. When the temperature is between 400 - 600 °C, compared with the mesoporous silica, the weight of the modified mesoporous silica decreases. This part of the weight change is related to the modification of octyl, indicating that octyl has been successfully modified on the mesoporous silica, and the weight percentage is about 13.1%.

[0056] Figure 5 The infrared spectroscopy analysis (FTIR) of the nano-mesoporous silica material prepared in this example shows that the enzyme has been successfully immobilized on the carrier.

[0057] Example 2

[0058] The difference between this example and Example 1 is that the stirring speed during material synthesis is adjusted. The original condition of stirring at 35 °C for 12 h with a rotation speed of 200 rpm is adjusted to stirring at 35 °C for 12 h with a rotation speed of 150 rpm, and the rest of the processes are the same as those in Example 1.

[0059] Example 3

[0060] The difference between this example and Example 1 is that the stirring speed during material synthesis is adjusted. The original condition of stirring at 35 °C for 12 h with a rotation speed of 200 rpm is adjusted to stirring at 35 °C for 12 h with a rotation speed of 100 rpm, and the rest of the processes are the same as those in Example 1.

[0061] The above examples are tested, and the comparison results with Example 1 are shown in the following table.

[0062] Table 2

[0063]

[0064] It can be seen that adjusting the stirring speed during material synthesis has an impact on the sterol conversion rate of this method. This is because the stirring speed affects the formation of nanoparticles. If the stirring speed is too fast, the particles will be too small and the shape and size will be uneven. If it is too slow, the nano-particle size will be too large and the specific surface area will be large, which does not meet the requirements. According to the results in the above table, when the stirring speed during material synthesis in the present invention is 200 rpm, the best technical effect can be obtained.

[0065] Example 4

[0066] The difference between this example and Example 1 is that the calcination temperature is adjusted from 550 °C to 400 °C, and the rest of the processes are the same as those in Example 1.

[0067] Example 5

[0068] The difference between this example and Example 1 is that the calcination temperature is adjusted from 550 °C to 600 °C, and the remaining processes are the same as those in Example 1.

[0069] The above examples were tested, and the comparison results with Example 1 are shown in the following table.

[0070] Table 3

[0071]

[0072] It can be seen that adjusting the calcination temperature has an impact on the sterol conversion rate of this method. This is because the calcination temperature affects whether the surface template agent in the synthesis of mesoporous silica can be completely removed. Too high a temperature will cause sintering of the material, and too low a temperature will result in incomplete removal of the surface template agent. According to the results in the above table, the best technical effect can be obtained when the calcination temperature in the present invention is 550 °C.

[0073] Example 6

[0074] The difference between this example and Example 1 is that the modified silane coupling agent n-octyltrimethoxysilane is adjusted to dodecyltrimethoxysilane, and the remaining processes are the same as those in Example 1.

[0075] Example 7

[0076] The difference between this example and Example 1 is that the modified silane coupling agent n-octyltrimethoxysilane is adjusted to hexadecyltrimethoxysilane, and the remaining processes are the same as those in Example 1.

[0077] The above examples were tested, and the comparison results with Example 1 are shown in the following table.

[0078] Table 4

[0079]

[0080] It can be seen that adjusting the silane coupling agent used for modification has an impact on the sterol conversion rate of this method. This is because different silane coupling agents have different degrees of hydrophobic modification of mesoporous silica, which will affect the activity of lipase in organic solvents. According to the results in the above table, the best technical effect can be obtained when the silane coupling agent in the present invention is n-octyltrimethoxysilane.

[0081] Example 8

[0082] The difference between this example and Example 1 is that the immobilized enzyme reaction temperature of 50 °C is adjusted to 45 °C, and the remaining processes are the same as those in Example 1.

[0083] Example 9

[0084] The difference between this embodiment and Embodiment 1 is that the temperature of the immobilized enzyme reaction is adjusted from 50 °C to 60 °C, and the rest of the processes are the same as those in Embodiment 1.

[0085] The above embodiments were tested, and the comparison results with Embodiment 1 are shown in the following table.

[0086] Table 5

[0087]

[0088] It can be seen that adjusting the temperature of the immobilized enzyme reaction has an impact on the sterol conversion rate of this method. This is because the temperature of the immobilized enzyme reaction affects the activity of the enzyme and the solubility of the substrate. Too high a temperature will cause the activity of the enzyme to decrease, and too low a temperature will result in a lower solubility of sterol in the solvent, affecting the collision between the enzyme and the substrate, thereby leading to a lower conversion rate. According to the results in the above table, the best technical effect can be obtained when the temperature of the immobilized enzyme reaction in the present invention is 50 °C.

[0089] Embodiment 10

[0090] The difference between this embodiment and Embodiment 1 is that the addition amount of the immobilized enzyme is adjusted from 5% to 2%, and the rest of the processes are the same as those in Embodiment 1.

[0091] Embodiment 11

[0092] The difference between this embodiment and Embodiment 1 is that the addition amount of the immobilized enzyme is adjusted from 5% to 6%, and the rest of the processes are the same as those in Embodiment 1.

[0093] The above embodiments were tested, and the comparison results with Embodiment 1 are shown in the following table.

[0094] Table 6

[0095]

[0096] It can be seen that adjusting the addition amount of the immobilized enzyme has an impact on the sterol conversion rate of this method. This is because the addition amount of the immobilized enzyme affects the sterol conversion rate. Adding too much does not further increase the sterol conversion rate, and adding too little will result in the enzyme not reaching relative saturation. According to the results in the above table, the best technical effect and economic benefits can be obtained when the addition amount of the immobilized enzyme in the present invention is 5%.

[0097] Embodiment 12

[0098] The difference between this embodiment and Embodiment 1 is that the reaction solvent is adjusted from isooctane to n-hexane, and the rest of the processes are the same as those in Embodiment 1.

[0099] Embodiment 13

[0100] The difference between this example and Example 1 lies in that the reaction solvent isooctane is adjusted to cyclohexane, and the rest of the processes are the same as those in Example 1.

[0101] The above examples were tested, and the comparison results with Example 1 are shown in the following table.

[0102] Table 7

[0103]

[0104]

[0105] It can be seen that adjusting the reaction solvent has an impact on the sterol conversion rate of this method. This is because the reaction solvent affects the activity of the enzyme. The higher the LogP value of the solvent, the stronger the hydrophobicity, and solvents with a LogP value lower than 2 will compete for the water molecules near the enzyme, destroying the hydration layer and causing the enzyme to inactivate. According to the results in the above table, the best technical effect can be obtained when the reaction solvent in the present invention is isooctane.

[0106] Comparative Example 1

[0107] The difference between this comparative example and Example 1 lies in that the enzyme is adjusted to free Candida rugosa lipase, and the rest of the processes are the same as those in Example 1.

[0108] Comparative Example 2

[0109] The difference between this comparative example and Example 1 lies in that the immobilization carrier is adjusted to NKA-9, and the rest of the processes are the same as those in Example 1.

[0110] Comparative Example 3

[0111] The difference between this comparative example and Example 1 lies in that the immobilization carrier is adjusted to Celite 545, and the rest of the processes are the same as those in Example 1.

[0112] Comparative Example 4

[0113] The difference between this comparative example and Example 1 lies in that the synthesized mesoporous silica is not hydrophobically modified and is directly used for immobilizing the enzyme, and the rest of the processes are the same as those in Example 1.

[0114] Comparative Example 5

[0115] The difference between this comparative example and Example 1 lies in that the stirring speed during material synthesis is adjusted to 50 rpm, and the rest of the processes are the same as those in Example 1.

[0116] Comparative Example 6

[0117] The difference between this comparative example and Example 1 lies in that the non-polar solvent is adjusted to chloroform, and the rest of the processes are the same as those in Example 1.

[0118] The above embodiments were tested, and the comparison results with Embodiment 1 are shown in the following table.

[0119] Table 8

[0120]

[0121]

[0122] As can be seen from the above table, the enzyme has low activity and a long reaction time in the free state, while the immobilized enzyme can greatly reduce the reaction time, and the enzyme after hydrophobic modification has better reusability. The immobilization of the enzyme is very beneficial to the maintenance of enzyme activity, can increase the number of times of enzyme reuse, and reduce the production cost. Moreover, the hydrophobic mesoporous silica carrier used in the present invention has better protection for enzyme activity compared with conventional carriers and unmodified mesoporous silica carriers. The stirring speed during the synthesis of mesoporous silica materials has a significant impact on the particles of mesoporous silica. If the stirring speed is inappropriate, it will lead to too large particles of mesoporous silica and low yield, unable to meet the experimental requirements.

[0123] In summary, for the nano mesoporous silica material prepared in the present invention, after immobilizing Candida rugosa lipase, the conversion rate of the catalytic transesterification reaction to prepare sterol esters and diglycerides can reach more than 85%. It can better retain the enzyme activity and even improve the enzyme activity. The ester synthesis activity increases with the increase of the hydrophobicity of the carrier surface. In the organic phase, free enzyme molecules tend to aggregate into clusters, resulting in the inability of the inner enzyme molecules to contact the substrate molecules, thus leading to low activity. While the immobilized enzyme can distribute the enzyme molecules on the surface of the carrier, fully contact the substrate molecules, and improve the activity. In addition, based on the "interface activation" characteristic of lipase, with the increase of the hydrophobic interaction on the carrier surface, the "lid" structure in the active center region of the lipase molecule opens wider, which is more conducive to the entry of substrate molecules, thus improving the enzyme activity. The hydrophobic carrier is more conducive to enriching hydrophobic substrates, which is also one of the reasons for the increase in the activity of lipase molecules.

[0124] The cost of the enzyme in this synthesis reaction is high and the enzyme reuse rate is poor. After immobilization, it can be reused, the cost is reduced, the reuseability is improved, the reaction time is short, the efficiency is improved, and the efficiency of multiple repeated uses is high. The conversion rate after reacting for two hours exceeds 90%.

[0125] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing immobilized lipase, characterized in that: comprising, preparing a modified mesoporous silica material: concentrated ammonia water is added to an aqueous solution of cetyltrimethylammonium bromide, stirred, and then a non-polar solvent and tetraethyl orthosilicate are added, and stirred to form a homogeneous solution; collecting the product, washing, drying and then calcining to remove the template to obtain mesoporous silica, and modifying it with a silane coupling agent; preparing immobilized enzyme: the lipase solution is placed in a carrier for adsorption, centrifuged to remove the solvent, and dried to obtain immobilized lipase.

2. The method for preparing immobilized lipase according to claim 1, characterized in that: the stirring speed is 100-250 r / min, the stirring temperature is 25-40 °C, and the stirring time is 8-24 h.

3. The method for preparing immobilized lipase according to claim 1, characterized in that: the calcination temperature is 400-600 °C, and the calcination time is 3-6 h.

4. The method for preparing immobilized lipase according to claim 1, characterized in that: the silane coupling agent is one or more of n-octyltrimethoxysilane, n-octyltriethoxysilane, dodecyltrimethoxysilane, dodecyltriethoxysilane, hexadecyltrimethoxysilane or hexadecyltriethoxysilane.

5. The method for preparing immobilized lipase according to claim 1, characterized in that: the mass concentration of the lipase solution is 30-40 g / L.

6. The method for preparing immobilized lipase according to claim 1, characterized in that: the addition amount of the immobilized enzyme is 2%-6%.

7. An immobilized lipase prepared by the method according to any one of claims 1-6.

8. An application of the immobilized lipase according to claim 1 in the preparation of structured lipids, characterized in that: the immobilized lipase is used to catalyze the synthesis of sterol esters and diglycerides from fats, including, using a non-polar solvent as the reaction solvent, the immobilized enzyme as the catalyst, and adding sterols and oils for reaction.

9. The application of the immobilized lipase according to claim 8 in the preparation of structured lipids, characterized in that: the non-polar solvent includes one or more of isooctane, n-hexane, n-heptane or cyclohexane.

10. The application of the immobilized lipase according to claim 8 in the preparation of structured lipids, characterized in that: the reaction temperature of the sterols and oils is 40-70 °C.

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