CN111647593A

CN111647593A - Preparation method of biomineralization type immobilized lipase and application of biomineralization type immobilized lipase in catalytic synthesis of OPO

Info

Publication number: CN111647593A
Application number: CN202010691701.XA
Authority: CN
Inventors: 谢恬; 王安明; 李宁宁; 张静
Original assignee: Hangzhou Normal University
Current assignee: Hangzhou Normal University
Priority date: 2020-07-17
Filing date: 2020-07-17
Publication date: 2020-09-11

Abstract

The invention relates to a preparation method of biomineralization type immobilized lipase and application of the biomineralization type immobilized lipase in catalytic synthesis of OPO (oriented ordered).A lipase and a calcium chloride solution in a certain proportion are added into a phosphate buffer solution, then a mixture is stood at 1-8 ℃ to obtain the immobilized lipase, and the immobilized lipase is applied to catalytic synthesis of 1, 3-dioleyl-2-palmitoyl glyceride. The method has simple process, the temperature stability of the prepared immobilized lipase is obviously improved, and the catalytic yield is high when the immobilized lipase is used for catalytically synthesizing 1, 3-dioleyl-2-palmitoyl glyceride; the invention can effectively improve the thermal stability and catalytic activity of lipase.

Description

Preparation method of biomineralization type immobilized lipase and application of biomineralization type immobilized lipase in catalytic synthesis of OPO

Technical Field

The invention relates to an enzyme immobilization method, in particular to preparation of biomineralization type immobilized lipase and application of biomineralization type immobilized lipase in catalytic synthesis of OPO.

Background

An enzyme is a protein that catalyzes biological and chemical reactions, which are widely present in plants, animals, and microorganisms. Has the advantages of high catalytic activity, strong selectivity, good specificity, low cost, mild reaction condition, biodegradability and the like. Therefore, the enzyme has high application in various fields such as fine chemistry, pharmaceutical chemistry, food or energy. The biocatalytic process has received wide attention as a reasonable, efficient conversion process. The enzymatic process plays an important role in supporting sustainable green chemical industry. However, natural enzymes are generally poor in handling and storage stability in practical use, difficult in recovery and reuse of the enzymes, high in cost, and also affected by the reaction solution. Thus, immobilization is a commonly used improved method in enzyme catalysis.

In practical application, the immobilized enzyme has advantages in the aspects of stability, biocatalyst loading, recoverability and the like. Immobilization is a key technology for the successful implementation of enzyme-based industrial processes, in particular for the production of green and sustainable energy or biologically derived catalytic conversion chemicals (industrialization). Immobilization of the enzyme may alter the activity, specificity or selectivity of the enzyme, which provides a good basis for increasing the availability of the enzyme on the substrate. Biomimetic mineralization has now been developed as a convenient method for the preparation of biomimetic materials.

The organic-inorganic hybrid nano material with nano-scale characteristics is a flower-shaped nano material formed by self-assembling metal ions and organic components (protein, polypeptide, amino acid to DNA/RNA and other biomolecules). In 2012, Zare et al discovered and reported for the first time a method of creating protein-inorganic hybrid nanoflowers using copper (II) sulfate as an inorganic ingredient and containing proteins, which ingredient enhances activity and stability. With the continuous development of technology, the inorganic metal ions used for preparing the hybrid inorganic nanoflowers are also from Cu²⁺Extension to Co²⁺，Ca²⁺，Mn²⁺And the like, can be applied to the fields of biomedicine, including biosensing, biocatalysis, cancer treatment and the like. The harm of heavy metals has caused potential harm to human health and environment, which arouses wide attention all over the world, and the green idea is the foundation of chemical development. More than half of the biominerals in nature are calcium-containing minerals, including bone and thus, Ca²⁺It is more biocompatible to immobilize the enzyme. Amjad and collaborators thereof researchProtease-inorganic hybrid nanocomposite (alcalase @ CaHPO)₄) The soybean protein hydrolysate is used for hydrolyzing soybean protein isolate, and the stability and the catalytic capability of the enzyme are improved, and the catalytic capability is 1.5 times of that of free enzyme. Zhang et al studied a novel immobilized lipase ZC12/Ca₃(PO₄)₂A hybrid nanoflower that has a kcat/Km value that is 206% free and 2.31 times enzyme activity and higher specific activity at lower temperatures. Preparation of CaHPO by Tian et al₄PGUS1 hybrid nanocomposite, 1.2mg CaHPO under optimal conditions₄The immobilization efficiency of the mixed nanoprecipitate of PGUS1 was 71.2% with a relative activity of 118%. The nano composite material synthesized by the method has good activity and stability due to Ca²⁺Has great biocompatibility and great application prospect in food industry and biotechnology.

The main process of biomineralization is to selectively deposit inorganic elements onto specific organic macromolecules and to convert ions in solution into solid phase minerals under the precise control of the organism. It is widely found in biological tissues such as bones, teeth and shells. In this process, enzymes can participate in the formation of calcium phosphate in vivo as biomacromolecules and have a tremendous impact on the mineralization process by reducing the solubility of minerals. The protein-inorganic hybrid nano composite material is prepared by a coprecipitation method, and generally comprises four processes of coordination, coprecipitation, self-assembly and size growth. Amine groups of proteins (polypeptides or amino acids) with metal ions (II) (Cu)²⁺、Ca²⁺、Mn²⁺、Zn²⁺、Co²⁺) Interact through coordination reactions to become nucleation sites for the formation of metal (II) phosphate primary nanoparticles. At the same time, the protein acts as a capping agent, helping the particles to retain the plate-like morphology of the nanoflower. The synthesis conditions, including synthesis time, temperature, pH and concentrations of the components, etc., are optimized during synthesis to achieve the desired nanometer size.

The application of biomineralization in immobilization has no influence on the enzyme activity of the immobilized enzyme, and the enzyme activity is improved, thereby showing that the method has better biological activityThe method is capacitive, green and environment-friendly, and the fixing method is simple, convenient and high in feasibility. The nano composite material has good activity and stability, and simultaneously Ca²⁺Has great biocompatibility and great application prospect in food industry and biological medicine industry.

Disclosure of Invention

In order to improve the utilization efficiency of free enzyme and save cost, the invention provides a preparation method of biomineralization type immobilized lipase and application of biomineralization type immobilized lipase in catalytic synthesis of OPO (1, 3-dioleic acid-2-palmitic acid triglyceride).

In order to achieve the purpose, the invention adopts the technical scheme that:

a preparation method of biomineralization type immobilized lipase comprises the steps of adding lipase and a calcium chloride solution in a certain proportion into a phosphate buffer solution, and standing the mixture at 1-8 ℃ to obtain the immobilized lipase.

Preferably, the lipase is Thermomyces Lanuginosus Lipase (TLL).

Preferably, the lipase is obtained by the following method: firstly, a Thermomyces lanuginosus lipase gene is constructed on a pPIC9K vector, then a recombinant plasmid is transferred into a Pichia pastoris strain GS115, and the lipase is obtained after induction expression.

Preferably, the concentration of the phosphate buffer is 0.02 mol.L^-1The pH was 7.0.

Preferably, the lipase is added to a final concentration of 0.05 to 0.25 mg/mL^-1。

Preferably, the concentration of the added calcium chloride solution is 0.2 mol.L^-1The volume ratio of the phosphate buffer solution to the calcium chloride solution is 40-60: 1.

preferably, the preparation method of the biomineralization type immobilized lipase specifically comprises the following steps: to 100mL of phosphate buffer, 0.1 mg/mL of phosphate buffer was added to the final concentration^-1And then placing the mixed solution at 4 ℃ for 24 hours, and centrifuging at 10000rpm for 10min to obtain the immobilized lipase, namely the TLL @ hydroxyapatite nanoflower (TLL @ HAp-NFs). In combination with phosphate bufferingAfter washing, the solution is used for subsequent detection and catalysis.

The invention also provides application of the biomineralization type immobilized lipase in catalytic synthesis of OPO.

Immobilizing lipase TLL by biomineralization, dispersing lipase in phosphate buffer solution, and adding CaCl at certain ratio₂Solution, with obvious white precipitate, then standing the mixture to obtain immobilized enzyme, and applying the immobilized enzyme to catalytic synthesis of 1, 3-dioleyl-2-palmitoyl glyceride (OPO). The reaction chemical formula is as follows:

preferably, the application of the biomineralization type immobilized lipase in the catalytic synthesis of OPO comprises the following steps: adding substrate tripalmitin, oleic acid, immobilized lipase and n-hexane, and reacting in a shaking table to obtain OPO (1, 3-dioleoyl-2-palmitic acid triglyceride).

Preferably, the molar ratio of the tripalmitin to the oleic acid is 1: 2-10, wherein the water content in the reaction is 2%; the table shaking reaction conditions are as follows: the temperature is 25-55 ℃, and the rotating speed is 220 rpm.

The immobilized enzyme is used for catalytically synthesizing 1, 3-dioleate-2-palmitic acid triglyceride, the water content, the solvent, the reaction temperature, the reaction time, the substrate proportion and the added enzyme amount in the catalytic reaction are changed, and the content of the product is improved.

The invention uses hydroxyapatite which is more compatible with biology to immobilize recombinant protein under mild condition, and is used for improving and enhancing catalytic reaction condition and yield. The fungus pichia pastoris is used for expressing the recombinant protein TLL, the TLL is immobilized based on a biomineralization method and is used for catalytic synthesis of OPO, the selectivity of a lipase catalytic position is improved, and the content of a target product OPO in the reaction is further improved. After the immobilized enzyme is subjected to water bath at 40 ℃ and 50 ℃ for 5 hours, the residual relative enzyme activity can be kept above 90 percent, and the enzyme activity of free enzyme is left at about 40 percent. At 70 ℃, the activity of the free enzyme is rapidly reduced to about 23 percent in the first 1h, and about 30 percent of the enzyme activity is lost by TLL @ HAp-NFs. Under the optimal catalysis condition, the content of OPO in the reaction liquid can reach 50.6 percent. Therefore, the selection of the immobilization method can improve the thermal stability and the catalytic activity of the lipase.

Drawings

FIG. 1 is a flow chart of the preparation of the present invention;

FIG. 2 is a comparative scanning electron microscope image of calcium phosphate of the present invention and immobilized lipase;

FIG. 3 is a graph showing the results of temperature stability of the immobilized enzyme of the present invention;

FIG. 4 is a liquid phase detection spectrum of the reaction solution of the present invention;

FIG. 5 is a schematic of the cyclic catalysis of the present invention.

Detailed Description

The present invention is further described with reference to the following specific examples, which are not intended to be limiting, but are intended to be exemplary only in light of the teachings of the present invention and are not intended to be limiting.

Example 1

Referring to FIG. 1, the lipase TLL is immobilized by biomineralization, the lipase is dispersed in phosphate buffer, and a certain proportion of CaCl is added₂Solution, with formation of a distinct white precipitate, and then standing the mixture at 4 ℃ to obtain immobilized lipase, which was used for catalytic synthesis of 1, 3-dioleyl-2-palmitoyl glyceride (OPO).

In the invention, a TLL-hydroxyapatite nanoflower (TLL @ HAp-NFs) compound is produced by a simple method and calcium phosphate through a biomineralization process and is applied to OPO synthesis, firstly, a TLL gene is constructed on a pPIC9K vector, then a recombinant plasmid is transferred into a Pichia pastoris strain GS115, and lipase TLL is obtained after induced expression.

Preparation of TLL @ hydroxyapatite nano flower (TLL @ HAp-NFs)

The concentration of the purified and concentrated lipase protein was first determined by the Bradford protein assay kit whenThe concentration was found to be 10 mg. multidot.mL^-1When the enzyme was added to 6mL of the enzyme solution, 600mL of phosphate buffer solution (PBS, 20mM, pH 7.0) was added thereto, and mixed well, and 12mL of CaCl was added₂(0.2mol·L^-1) And (3) placing the mixture at 4 ℃ for 24 hours, centrifuging at 10000rpm for 10min, and collecting the obtained mixture which is TLL @ hydroxyapatite nanoflower. After freeze-drying, the nano-particles were examined by scanning electron microscopy, as shown in fig. 2, hydroxyapatite (fig. 2a), TLL @ hydroxyapatite nano-flower (fig. 2 b). After the addition of the biomacromolecule, the same treatment forms the shape of regular petals which are stacked together over time, resulting in an increase in size.

The enzyme activity detection method comprises the following steps: mu.L of the diluted enzyme solution was added to 1.4mL of phosphate buffer solution (20mM, pH 6.5), incubated at 37 ℃ for 3 minutes at 220rpm, and then 75. mu.L of p-nitrophenol acetate (50mM) was added. After 3min of reaction in the shaker, the reaction was stopped by removing and adding 1.5mL acetone quickly. Finally, the reaction solution was detected with a spectrophotometer at 405 nm.

TLL @ HAp-NFs temperature stability detection

10mL of 2 mg/mL^-1The free enzyme and the immobilized enzyme of (2) were placed in water baths at 40 ℃, 50 ℃, 60 ℃ and 70 ℃, respectively, 500. mu.L of the free enzyme and TLL @ HAp-NFs were taken out at intervals of 1 hour and diluted to 1mL (final enzyme concentration of 1 mg. mL)^-1) For measuring enzyme activity. Each set of data was tested in parallel three times. As shown in FIG. 3, immobilization of lipase by biomineralization is an effective immobilization method. As can be seen from FIG. 3a, at 40 ℃ some of the experimental results resulted in some fluctuations in the data, but TLL @ HAp-NFs ensured that there was almost no loss of enzyme activity within 5h, and the relative residual activity of the free enzyme was only 54%. FIG. 3b shows that the relative residual enzyme activity of the immobilized enzyme is stabilized at about 90% within 5h in a 50 ℃ water bath, while the relative residual enzyme activity of the free enzyme is only 41.7%. The comparison of activity between free and immobilized enzymes was more evident at higher temperatures of 60 ℃ (fig. 3c) and 70 ℃ (fig. 3 d). At 70 ℃, the activity of the free enzyme is rapidly reduced to about 23 percent in the first 1h, about 30 percent of the enzyme activity is lost by TLL @ HAp-NFs, and after 6 hours of incubation, the residual relative enzyme activityThe activity was still 63.6%, while the free enzyme remained only 16.1%. After immobilization, the temperature stability is significantly improved, the activity of the enzyme is not affected, but is higher than that of the free enzyme. Probably due to the higher surface area in the nanoflower and the restriction of the enzyme, the nanoflower increases the surface area of the immobilized carrier, can make the active site of the lipase more easily contact with the substrate, and reduces the restriction of the enzyme.

Example 2

Catalytic synthesis of OPO (oriented polystyrene) from TLL @ hydroxyapatite nanoflower

TLL @ HAp-NFs was used as a biocatalyst for the enzymatic acid hydrolysis of tripalmitin (PPP) with Oleic Acid (OA) to produce OPO. The effect of different reaction conditions on the product, including water content, reaction temperature, molar ratio of reactants, etc., was investigated.

The catalytic transesterification reaction was as follows: PPP (0.24mmol), OA (0.48-2.40 mmol), TLL @ hydroxyapatite nano flower (5-25 mg/mL)^-1) Adding different solvents (6 mL of total volume, n-pentane, n-hexane, n-heptane, n-octane, cyclohexane and acetone) for dissolving, wherein the water content in the reaction is 2%, the reaction temperature is 25-55 ℃, and the reaction is carried out in a shaking table at the speed of 220rpm for 12 hours. Molecular sieve is needed for solvents such as n-hexane used in the reaction

Drying for more than 24 hours. 500. mu.L of each sample was taken and mixed with 500. mu.L of chromatographic grade n-hexane. All experiments were repeated three times. The mixed solution was filtered through a 0.22 μm filter and detected by high performance liquid chromatography (HPLC, Agilent 1260). The mobile phase was a mixed solution of n-hexane and acetonitrile (99.15: 0.85v/v, ultrasonic mixing), and the column used was a silver ion column (ChromSpher 5Lipids,250X4.6mm, particle size 5 μm, Agilent Technologies) with a flow rate of 1mL min^-1The sample introduction amount was 10. mu.L, and the column oven temperature was 30 ℃. All products were plotted against peak area versus standard concentration using a standard to obtain a calibration curve. Each sample is measured in parallel for three times, a liquid phase detection spectrogram of the reaction solution is shown in figure 4, the water content in the reaction is 2%, and the substrate PPP/OA molar ratio is 1: 6, adding 6mL of n-hexane for dissolution, and adding fat20mg/mL (20%) of enzyme, reacting at 35 ℃ for 12h, and the peak positions of the products in the reaction solution reflect various substances in the reaction solution.

Example 3

Influence of reaction temperature on the catalytic reaction

Catalytic results at reaction temperatures of 25 ℃, 35 ℃, 45 ℃ and 55 ℃ were mainly explored and compared. In this experiment, we found that not all reactions increased in yield with increasing temperature, but that the free enzyme TTL was more sensitive to increasing temperature. The OPO content in the free enzyme catalyzed reaction can be maintained above 40% at 25 ℃ and 35 ℃, but is significantly reduced at 45 ℃ and 55 ℃. The content of OPO in the catalytic reaction liquid of the TLL @ hydroxyapatite nanoflower is changed more stably, and the content of OPO is at least over 35 percent. However, too high a temperature may also have a negative effect on the immobilized enzyme. The reason for this analysis may be that too high a temperature affects the activity of the enzyme, while too low a temperature may result in incomplete dissolution of the substrate tripalmitin in the reaction solvent, rendering the enzyme and substrate ineffectively accessible. The optimum reaction temperature for the final catalytic reaction was chosen to be 35 ℃.

Example 4

Effect of Water content on catalytic reactions

Lipases are water-soluble, and trace amounts of water are essential to maintain the hydration layer around the lipase (to maintain lipase activity). Therefore, we investigated the effect of water content on the esterification reaction. Different amounts of water (0% to 10%) were added to the parallel experiments, the remaining reaction conditions were kept constant. The water content has a great influence on the product content of the reaction, and for anhydrous reactions, the OPO content in the reaction solution is at least 15%, and when the water content reaches 10%, the OPO content is about 15%. However, when the water content is 2%, the OPO content is at the highest, about 40%. The reason may be that when the water content in the reaction is higher, the contact between the enzyme and the hydrophobic substrate is reduced, resulting in a decrease in the enzyme activity. Therefore, the optimum water content in the catalytic reaction is 2%.

Example 5

Effect of reaction solvent on catalytic reaction

One of the possible interactions between lipase and solvent is that the solvent can act as a competitive inhibitor in the catalytic reaction, using n-pentane, n-hexane, n-heptane, n-octane, cyclohexane and acetone as reaction solvents, respectively. In this experiment, the lipase activity in n-hexane as a solvent is stronger than that in other solvents, such as isooctane, acetone, petroleum ether, etc., and the solvent is easily removed. Although the OPO content of n-octane is higher than that of n-hexane, n-octane has a higher boiling point and is not easily removed from n-hexane. By comprehensive consideration, n-hexane is selected as a reaction solvent.

Example 6

Effect of Lipase dosage on catalytic reaction

To examine the effect of the amount of enzyme used in the reaction on the OPO content, 5 mg/mL was added to each of five parallel experiments^-1，10mg·mL^-1，15mg·mL^-1，20mg·mL^-1And 25 mg. mL^-1Enzyme catalyst was used as a control experiment. The reaction conditions are 2% water content, PPP: molar ratio of OA 1: 3, 6mL of anhydrous n-hexane, at a reaction temperature of 35 ℃ for 12 hours. The OPO content in the reaction solution gradually increased and gradually stabilized with increasing enzyme concentration. When the enzyme was added at a concentration of 5 mg/mL^-1At this time, the OPO content was only 39%. When the concentration of the enzyme added during the reaction was 20mg/mL^-1In this case, the OPO content is 45% at the maximum. At this time, if the amount of the enzyme is further increased, not only the waste of the enzyme is caused but also the OPO content is decreased. This result may be attributed to an increased acyl migration, a hydrolysis side reaction caused by excessive contact between lipase and substrate. Considering OPO content and economic benefit, the final dosage of the enzyme catalyst is 20 mg-mL^-1。

The optimal catalytic conditions of the invention are: the water content in the reaction is 2%, the substrate PPP/OA molar ratio is 1: 6, 6mL of n-hexane was added to dissolve the mixture, and 20mg/mL (20%) of lipase was added to the mixture to react at 35 ℃ for 12 hours.

Example 7

Cyclic catalysis

0.24mmol of tripalmitin and 1.44mmol of oleic acid are dissolved in 6mL of n-hexane solvent, 120. mu.L of water and 120mg of immobilized enzyme are added, reaction is carried out at 35 ℃ for 12 hours, and centrifugation is carried out. A500. mu.L sample of the supernatant was taken and mixed with 500. mu.L of n-hexane, and the resulting content of OPO in the reaction solution was measured by HPLC after filtration. The OPO content in the single catalytic reaction liquid can reach 50.6 percent, which is about 1.3 times higher than the catalytic yield of free TLL. In the cyclic catalysis, the precipitate obtained by centrifugation is washed 3 times with n-hexane and dried for the next cyclic reaction. In the circulation experiment, as shown in fig. 5, after 5 cycles, the relative content of OPO in each circulation product is close to and kept above 90%, and the loss of enzyme activity is small.

1, 3-dioleyl-2-palmitoyl glyceride (OPO) belongs to triacylglycerol, is one of important components of human milk fat, and is one of important additives commonly used in infant formula milk powder. The invention selects Pichia pastoris strain GS115 to express TLL lipase, develops a simple method, and synthesizes TLL @ hydroxyapatite nanoflower (TLL @ HAp-NFs) by coprecipitation immobilized lipase under the action of biomineralization. The TLL @ HAp-NFs ensured that almost no loss of enzyme activity was observed after being left in the water bath at 40 ℃ for 5h, and the relative residual activity of the free enzyme was only 54%. At 70 ℃, the activity of the free enzyme in the first 1h is rapidly reduced to about 23 percent, TLL @ HAp-NFs only loses about 30 percent of the enzyme activity, and the temperature stability is obviously improved. Under the optimal catalytic conditions, the water content in the reaction is 2%, the PP/OA molar ratio of the substrate is 1: 6, 6mL of n-hexane was added to dissolve the mixture, and 20mg/mL of lipase was added^-1(20%) and reaction at 35 deg.C for 12 hr to obtain product with OPO content up to 50.6% and catalytic yield about 1.3 times higher than that of free TLL. After 5 times of circulating catalysis, the content of OPO in the reaction liquid is still more than 90 percent. Therefore, the selection of the immobilization method improves the thermal stability and the catalytic activity of the lipase. The excellent stability, good catalytic ability and calcium binding ability of the TLL @ hydroxyapatite nanoflower play an important role in the synthesis of additives in infant formula milk powder.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention and are not to be construed as limiting the invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the scope of the invention as defined by the appended claims. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. A preparation method of biomineralization type immobilized lipase is characterized by comprising the following steps: adding lipase and a calcium chloride solution in a certain proportion into a phosphate buffer solution, and standing the mixture at 1-8 ℃ to obtain the immobilized lipase.

2. The method for preparing biomineralization-type immobilized lipase according to claim 1, wherein: the lipase is thermomyces lanuginosus lipase.

3. The method for preparing biomineralization type immobilized lipase according to claim 2, wherein the lipase is obtained by the following method: firstly, a Thermomyces lanuginosus lipase gene is constructed on a pPIC9K vector, then a recombinant plasmid is transferred into a Pichia pastoris strain GS115, and the lipase is obtained after induction expression.

4. The method for preparing biomineralization-type immobilized lipase according to claim 1 or 3, wherein: the concentration of the phosphate buffer solution is 0.02 mol.L^-1The pH was 7.0.

5. The method for preparing biomineralization-type immobilized lipase according to claim 1, wherein: the final concentration of the added lipase is 0.05-0.25 mg/mL^-1。

6. The method for preparing biomineralization-type immobilized lipase according to claim 1, wherein: the concentration of the added calcium chloride solution is 0.2 mol.L^-1The volume ratio of the phosphate buffer solution to the calcium chloride solution is 40-60: 1.

7. the biomineralization of claim 4The preparation method of the immobilized lipase is characterized by comprising the following steps: to 100mL of phosphate buffer, 0.1 mg/mL of phosphate buffer was added to the final concentration^-1And 2mL of a calcium chloride solution, and then the mixed solution was left at 4 ℃ for 24 hours and centrifuged at 10000rpm for 10 minutes to obtain an immobilized lipase.

8. Use of the biomineralized immobilized lipase of claim 1 for the catalytic synthesis of OPO.

9. The use of the biomineralized immobilized lipase according to claim 8 for the catalytic synthesis of OPO, characterized by comprising the following steps: adding substrate tripalmitin, oleic acid, immobilized lipase and n-hexane, and reacting in a shaking table to obtain OPO.

10. The use of the biomineralized immobilized lipase in the catalytic synthesis of OPO according to claim 9, characterized by comprising the following steps: the molar ratio of the tripalmitin to the oleic acid is 1: 2-10, wherein the water content in the reaction is 2%; the table shaking reaction conditions are as follows: the temperature is 25-55 ℃, and the rotating speed is 220 rpm.