Method for preparing immobilized lipase

Abstract

The invention discloses a method for preparing immobilized lipase, and relates to the technical field of biochemical engineering and biology. The immobilization method comprises the following steps: firstly, fully mixing a chitosan solution and a modified polyvinyl alcohol solution to form gel; then, solidifying the gel by using a sodium tripolyphosphate solution to form an immobilized carrier of the lipase, and activating the immobilized carrier of the lipase by using a glutaraldehyde solution. And finally, soaking the activated lipase immobilization carrier in an emulsion containing a certain amount of lipase, and immobilizing the lipase. The immobilized lipase is simple and convenient to prepare, the loading rate of the lipase is improved while the activity of the lipase is ensured, and the immobilized lipase method with improved storage stability and reusability is formed.

Description

Technical Field

[0001] The invention belongs to the field of biochemical engineering and biotechnology, and specifically relates to a method for preparing immobilized lipase. Background Art

[0002] Lipase (Lip) can catalyze reactions such as hydrolysis, esterification, transesterification and peptide synthesis. It has high catalytic activity and a wide substrate spectrum. It has received extensive attention in both theoretical research on enzymology and actual industrial production. In the food industry, Lip is used for meat defatting, flour improvement, oil modification, cheese processing, winemaking and sauce processing. In the feed industry, Lip is compounded with other carbohydrate degrading enzymes to form a composite enzyme as a feed additive, which can effectively improve feed utilization. Lip is also widely used in the bioresolubilization of chiral compounds in the pharmaceutical and fine chemical industries. In the papermaking industry, Lip can achieve enzymatic removal of rosin from wood pulp. In the biodiesel industry, Lip can be used to catalyze the transesterification reaction of oils and chain-breaking alcohols for enzymatic synthesis of biodiesel. Lip is also widely used in the washing industry, leather processing and textile industries. However, in the actual application process of industry, free Lip has problems such as high cost, poor stability and low reuse rate. Therefore, the key to solving such problems is to find a method that can ensure the catalytic activity of the enzyme and reduce the cost while improving its stability and reuse rate. Immobilized enzyme technology is one of the methods that can effectively solve the problems of poor enzyme stability, low reuse rate and high cost.

[0003] At present, the methods for enzyme immobilization can be divided into embedding method, covalent binding method and adsorption method. Chitosan (CS) is a derivative of chitin, which is the largest biomacromolecule in nature except cellulose. Because it contains a large number of active amino groups on its surface, has biocompatibility and is easy to be chemically modified, it is now widely used in the preparation of enzyme immobilization carriers. Other biopolymers (such as gelatin, collagen, alginate, etc.) are usually selected to form a composite gel with CS to improve the density and mechanical strength of CS. Polyvinyl alcohol (PVA) is a low-cost, well-soluble polymer macromolecule with biocompatibility, high mechanical strength and flexibility. PVA has also been widely used in enzyme immobilization. In addition, the combination of PVA and CS can improve the gel density and mechanical strength of CS, greatly expanding the application of CS. However, the mechanical stability of the mixed gel formed by the combination of CS and PVA is poor, which limits its application. Therefore, it is necessary to find a method to improve the mechanical stability of the mixed gel of CS and PVA. PVA is modified so that the interaction between it and CS changes from ionic interaction to covalent interaction, thereby improving its stability and expanding its application range. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing immobilized lipase, which can improve the loading rate of lipase while ensuring the activity of the enzyme, and form an immobilized lipase method with improved temperature tolerance, pH tolerance, catalytic stability and reusability.

[0005] Compared with the traditional immobilization method, the present invention ensures the activity of the enzyme while increasing the loading rate of the lipase, thereby forming an immobilized lipase with improved temperature tolerance, pH tolerance, catalytic stability and reusability. The present invention uses 3-aminopropyltriethoxysilane (3-Aminopropyltriethoxysilane, APTES) to modify PVA to form PVA*, and mixes the PVA* with CS to form a gel, sodium tripolyphosphate solution (Sodiumtripolyphosphate, STPP) and glutaraldehyde solution (Glutaraldehyde, GA) to form an immobilized carrier CS-PVA*-STPP, thereby greatly improving the defect of low loading rate of CS as an immobilized material.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A method for preparing immobilized lipase, first, using APTES solution to react with PVA solution to modify the hydroxyl group of PVA into amino group to generate modified PVA, recorded as PVA*. Then, CS solution and PVA* solution are fully mixed to form CS-PVA* gel. Then, CS-PVA* gel is solidified with STPP solution to form Lip immobilization carrier CS-PVA*-STTP, and then the carrier is activated with GA solution to form activated Lip immobilization carrier CS-PVA*-STTP. Finally, the activated Lip immobilization carrier CS-PVA*-STTP is immersed in an emulsion containing Lip to immobilize Lip. The specific Lip immobilization conditions are: immobilization temperature is 0-4°C, immobilization time is 2-12h, and shaking is 200-600rpm. The immobilized Lip is washed with a buffer solution to remove unbound free enzymes to obtain immobilized lipase Lip@CS-PVA*-STPP.

[0008] Furthermore, the PVA* is prepared by modifying the hydroxyl groups of a PVA solution with a mass fraction of 1.5%-9% into amino groups by using a 0.02mol / L-2mol / L APTES solution, and after the modification, the pH value of the PVA* is adjusted to a range of 1-6 using hydrochloric acid.

[0009] Furthermore, the mass ratio of CS in the CS-PVA* gel ranges from 40% to 80%.

[0010] Furthermore, the STPP solution is a STPP aqueous solution with a mass fraction of 2%-6%.

[0011] Furthermore, the GA solution is a GA aqueous solution with a volume fraction of 0.1%-3%.

[0012] Furthermore, the emulsion containing Lip is a 7.5U / mL-24U / mL enzyme solution prepared using an emulsified solution.

[0013] Furthermore, the emulsified solution is a Tris-HCl buffer containing 1% gum arabic and 0-4 mM sodium deoxycholate in a mass volume fraction.

[0014] Furthermore, the buffer solution is a Tris-HCl buffer solution with a concentration of 50-400 mM and a pH of 7-9.

[0015] The Lip of the present invention can use commercially available crude Lip, and the immobilized Lip with good thermal stability, pH stability, catalytic stability and reusability is formed through the above-mentioned immobilization technology, which meets the needs of industrial applications. After the Lip is immobilized with the immobilization material, the enzyme is reused multiple times, the stability of the enzyme is improved, and the problems of poor stability of the enzyme preparation, low reuse rate and high cost in the industrial application of Lip are effectively solved.

[0016] The beneficial effects of the present invention are:

[0017] 1. The immobilized Lip prepared by the present invention has the characteristics of simple preparation and good reproducibility. By adding an appropriate amount of PVA*, the shortcomings of poor stability and low loading rate of the immobilized carrier formed by traditional CS and PVA are improved, and the repeated use of Lip is realized, thereby improving the stability of Lip.

[0018] 2. The immobilized Lip prepared by the present invention has good effects on catalytic stability and reusability, and also has good storage stability, which greatly reduces the use cost of the enzyme preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the preparation route of immobilized Lip in the examples of the present invention.

[0020] Figure 2 The preparation conditions of immobilized Lip in the embodiment of the present invention are optimized. Among them, Figure a shows the effect of APTES concentration on the activity of immobilized Lip; Figure b shows the effect of CS concentration on the activity of immobilized Lip; Figure c shows the effect of STPP concentration on the activity of immobilized Lip; Figure d shows the effect of GA concentration on the activity of immobilized Lip.

[0021] Figure 3 The scanning electron microscope of the immobilized carrier of Lip in the embodiment of the present invention. Among them, Figure a is the internal structure of the CS-STPP carrier; Figure b is the internal structure of the CS-STPP-GA carrier; Figure c is the internal structure of the CS-PVA*-STPP carrier; Figure d is the internal structure of the CS-PVA*-STPP-GA carrier; Figure e is the surface structure of the CS-PVA*-STPP carrier; Figure f is the surface structure of the CS-PVA*-STPP-GA carrier.

[0022] Figure 4 The infrared spectra of the immobilized carriers of Lip immobilized in the embodiments of the present invention are shown in Figure a. The infrared spectra of PVA and PVA* are shown in Figure b. The infrared spectra of CS, CS-PVA*, CS-PVA*-STPP and CS-PVA*-STPP-GA are shown in Figure b.

[0023] Figure 5 Figure 1 is the performance characterization of the immobilized Lip in the embodiment of the present invention. Figure a is the storage stability of Lip@CS-STPP; Figure b is the storage stability of Lip@CS-PVA*-STPP; Figure c is the reusability of Lip@CS-STPP; Figure d is the reusability of Lip@CS-PVA*-STPP. DETAILED DESCRIPTION

[0024] The present invention will be described in detail below by way of examples, but the present invention is not limited to these examples.

[0025] The relative residual activity of the enzyme in the embodiment is calculated by the following formula:

[0026] Relative residual activity = (immobilized enzyme activity / maximum immobilized enzyme activity under relative conditions) × 100%

[0027] Example 1: Preparation of Lip@CS-PVA*-STPP

[0028] Preparation process Figure 1As shown, first, a certain concentration of APTES is added to the dissolved PVA solution, and a magnetic stirrer is used to mix to form PVA*. Hydrochloric acid is used to adjust the pH value of PVA* to less than 6. The PVA* with the adjusted pH value is mixed with a CS solution fully dissolved in 1% (v / v) acetic acid to form a CS-PVA* gel. Then, an appropriate amount of CS-PVA* gel is added dropwise to a certain concentration of STPP solution at a certain speed for solidification to form an immobilized carrier. After the solidification is completed, 1% (v / v) GA solution is added to activate the Lip immobilized carrier to obtain an activated immobilized carrier CS-PVA*-STPP. The above-mentioned activated immobilized carrier is washed with buffer, and the operation is repeated several times to fully remove the excess GA solution to obtain the immobilized carrier of Lip.

[0029] Example 2: Selection of concentration of each substance

[0030] 2.1 Selection of APTES concentration

[0031] By modifying PVA with APTES, most of the hydroxyl groups on its surface are modified into amino groups, which increases the number of covalently cross-linked active groups between the carrier and the enzyme molecules, ultimately achieving the effect of increasing the enzyme loading capacity and activity.

[0032] Take 2.64mL of distilled water, add 63μL of 0.5mol / L hydrochloric acid standard solution and mix well, then add 2.3537mL of APTES to obtain APTES with a concentration of 2mol / L. Dilute 2mol / L APTES with distilled water containing hydrochloric acid to APTES with a concentration of 0.02mol / L-2mol / L. Take 2mL of PVA solution, add 40μL of APTES solution of different concentrations, stir for 15min, and adjust the pH value to less than 6 with hydrochloric acid. Take 2mL of CS solution, add 1mL of PVA solution modified with APTES of different concentrations, stir vigorously for 2h, and remove bubbles by ultrasound. Use a syringe to inhale an appropriate amount of CS-PVA* gel and add it dropwise to a certain concentration of STPP solution at a certain speed for solidification. After solidification, add a certain concentration of GA solution for activation. Get the activated carrier beads. The solution was washed with buffer and the above operation was repeated 3 times to fully remove the excess GA solution. The solution was immersed in 1.2 mL of an emulsified solution containing a certain amount of Lip for incubation. The immobilized enzyme was placed in a refrigerator at 4°C for later use.

[0033] The results are as follows Figure 2 As shown in (a), the APTES concentration affects the relative residual activity of the immobilized enzyme. When the APTES concentration is 0.2 mol / L, the relative residual activity of the immobilized enzyme reaches the highest.

[0034] 2.2 Selection of CS concentration

[0035] APTES can modify some hydroxyl groups on PVA and CS molecules into amino groups and form covalently cross-linked Si-O-Si bonds between molecules, thereby forming a hybrid gel for fixation. The proportion of CS in the hybrid gel will affect the state of the hybrid gel.

[0036] Prepare PVA solution with a mass fraction range of 1.5%-9% with distilled water. Take 2mL of PVA solution, add a certain amount of 0.2M APTES solution, stir for 15min, adjust the pH value to 5 with hydrochloric acid, and prepare PVA* solution with different concentrations. Take 2mL of CS solution, add 1mL of modified PVA solution with different concentrations, stir vigorously for 2h, and remove bubbles by ultrasound. Use a syringe to inhale an appropriate amount of mixed solution and add it dropwise to a certain concentration of STPP solution at a certain speed for solidification. After solidification, add a certain concentration of GA solution for activation. Obtain the activated carrier beads. Wash with buffer, repeat the above operation 3 times, fully remove the excess GA solution, soak in 1.2mL of emulsified solution containing a certain amount of Lip for incubation, and the obtained immobilized enzyme is placed in a 4℃ refrigerator for standby use.

[0037] The experimental results are as follows Figure 2 As shown in b, the CS concentration affects the relative residual activity of the immobilized enzyme. When the CS concentration is 70% (w / v), the relative residual activity of the immobilized enzyme reaches the highest.

[0038] 2.3 Selection of STPP concentration

[0039] Since the groups in the STPP solution will undergo interfacial reactions with the structural elements in the CS molecular chains dissolved in acetic acid to form immobilized enzyme carrier beads, the concentration of STPP is crucial to the immobilization effect.

[0040] Prepare CS and PVA* gel according to the above experimental steps, use a syringe to suck an appropriate amount of CS-PVA* gel, and add it dropwise at a certain speed to a STPP solution with a mass fraction range of 2%-6% prepared with distilled water for solidification. After solidification, add a certain amount of GA solution for activation. Get the activated carrier beads, wash them with buffer, repeat the above operation 3 times, fully remove the excess GA solution, immerse them in an emulsified solution containing a certain amount of Lip for incubation, wash them with buffer after incubation, repeat the above operation 3 times, and fully remove the excess Lip solution. The obtained immobilized enzyme is placed in a 4℃ refrigerator for standby use.

[0041] The experimental results are as follows Figure 2 As shown in c, the STPP concentration affects the relative residual activity of the immobilized enzyme. When the STPP concentration is 4% (w / v), the relative residual activity of the immobilized enzyme reaches the highest.

[0042] 2.4 Selection of GA concentration

[0043] The addition of GA, on the one hand, forms cross-links between carriers, reduces the carrier pore size and increases the mechanical strength of the carrier; on the other hand, it forms covalent cross-links between the carrier and the enzyme, forming a covalently cross-linked immobilized enzyme.

[0044] First, use pure water to prepare a GA solution with a volume fraction of 0.1%-3%. Secondly, add the immobilized carrier formed by the above experiment to the same volume of GA solution of different concentrations and activate it for 80 minutes. Finally, wash the activated carrier beads with buffer solution, repeat the above operation 3 times, fully remove the excess GA solution, immerse in 1.2mL of emulsified solution containing a certain amount of Lip for incubation, wash with buffer solution after the incubation, repeat the above operation 3 times, and fully remove the excess Lip solution. The obtained immobilized enzyme is placed in a 4℃ refrigerator for standby use.

[0045] The results are as follows Figure 2 As shown in (d), GA concentration affects the relative residual activity of the immobilized enzyme. When the GA concentration is 0.5% (v / v), the relative residual activity of the immobilized enzyme reaches the highest.

[0046] 2.5 Orthogonal experiment to optimize the preparation of Lip@CS-PVA*-STPP

[0047] According to the results of the single-factor experiment, four factors, namely, CS concentration, APTES concentration, STPP solution concentration and GA solution concentration in the immobilized carrier CS-PVA*-STPP, were selected. The relative residual activity of immobilized lipase was used as the evaluation index to design an orthogonal experiment. The factor levels of the orthogonal experiment are shown in Table 1, and the experimental results are shown in Table 2.

[0048] Table 1 Orthogonal test factor levels for optimizing the preparation conditions of Lip@CS-PVA*-STPP

[0049]

[0050] Table 2 Orthogonal test results and analysis of the preparation conditions optimization of Lip@CS-PVA*-STPP

[0051]

[0052] As shown in Table 2, the preparation process of the best immobilized lipase is A3B1C3D2. The concentration of CS in the immobilized carrier prepared by the best combination is 80% (w / v), the concentration of APTES is 0.02 mol / L, the concentration of STPP is 6% (w / v) and the concentration of GA is 0.5% (v / v). The verification experiment obtained is 87.03% of the relative residual activity of the enzyme.

[0053] Example 3: Characterization of the immobilized carrier of CS-PVA*-STPP

[0054] In this embodiment, the immobilized material was characterized by scanning electron microscopy and Fourier transform infrared spectrometer, and the specific characteristics are as follows:

[0055] 3.1 Scanning electron microscopy characterization results of carriers

[0056] First, 1 mL of CS solution was added dropwise to a 4% STPP solution to form the carrier CS-STPP. Part of the carrier CS-STPP was added to a 0.5% GA solution and activated for 80 min to form the carrier CS-STPP-GA. 1 mL of CS solution was mixed with 0.5 mol / L PVA* and then added dropwise to a 4% STPP solution to form the carrier CS-PVA*-STPP. Part of the carrier CS-PVA*-STPP was added to a 0.5% GA solution and activated for 80 min to form the carrier CS-PVA*-STPP-GA. Secondly, all the above carriers were freeze-dried in a freeze dryer, and a part of the sample was cut open, and then coated with a thermal evaporation integrated coating machine, and then the surface and internal morphology of the carrier were observed with a field emission scanning electron microscope.

[0057] The results are as follows Figure 3 As shown in a, b, c and d, the addition of PVA* makes the pore size inside the carrier smaller and the pore network denser. After GA activation, the pore size inside the carrier is reduced, and many filamentous substances are formed between the pores. This shows that under the action of GA, covalent cross-linking is also formed inside the carrier.

[0058] The results are as follows Figure 3 As shown in Figures e and f, after GA is added, a cross-linked layer will be formed on the carrier surface. By comparing the surface morphology of the carrier with GA added and the carrier without GA added, it can be found that the surface of the carrier with the cross-linked layer presents very regular wrinkles, while the surface of the carrier without the cross-linked layer forms irregular wrinkles due to water loss, which fully proves that under the action of GA, the carrier surface also forms covalent cross-links.

[0059] 3.2 Infrared characterization results of carrier

[0060] First, prepare a 2% (w / v) CS solution to form material CS; prepare a 2.6% (w / v) PVA solution to form material PVA; use 0.2 mol / L APTES to modify the PVA solution to form material PVA*; take a 3% CS solution and mix it with PVA* to prepare material CS-PVA*. Secondly, take the material CS-PVA* and add it dropwise to a 4% STPP solution to form a carrier CS-PVA*-STPP; add part of the carrier CS-PVA*-STPP to a 0.5% GA solution and activate it for 80 minutes to form a carrier CS-PVA*-STPP-GA. All the above materials (PVA, PVA*, CS, CS-PVA*) and carriers (CS-PVA*-STPP, CS-PVA*-STPP-GA) were washed with distilled water and placed in a culture dish. All the culture dishes were placed in an oven at 60°C and dried for 48 hours. After drying, the materials and carriers were taken out, ground and mixed with potassium bromide, and then the 6 samples were subjected to infrared detection.

[0061] The results are as follows Figure 4 As shown in a, PVA* and PVA at 1000 cm -1 -1500cm -1 The peak value of PVA changes. By comparison, it can be seen that PVA has a peak value of 1091cm -1 A sharp peak appeared, which is the characteristic peak of C-OH, and PVA* at 1043cm -1 and 1084cm -1 There is a peak at each of the two locations, which is due to the condensation reaction between the Si-OH formed by the hydrolysis of APTES and the C-OH group and Si-OH on the PVA, which also proves the successful modification of PVA by APTES.

[0062] The results are as follows Figure 4 As shown in b, CS is at 1629cm -1 , 1556cm -1 and 1383cm -1 The absorption peaks at 2800 cm-1 are formed by the stretching vibration of C=O bond, the deformation vibration of NH2 and the bending vibration of -OH bond. Comparing CS and CS-PVA*, it is found that after adding PVA* to CS, the characteristic peak of CS still exists at 2800 cm-1. -1 ~3000cm -1 The characteristic peaks of -CH2 and -CH in the range of 1470 cm-1 and 1471 cm-2 became broader, indicating that PVA* successfully mixed with CS to form a gel. Comparison of CS-PVA* and CS-PVA*-STPP revealed that the immobilized carrier formed by CS and PVA* contained all the characteristic peaks of CS and PVA* materials, among which CS had a peak at 1629 cm-1. -1 and 1556cm-1 The characteristic peak at 1618cm -1 , 1446cm -1 The shift indicates that the amino groups of CS and PVA* interact with the phosphate groups of STPP to form a carrier. Comparison of CS-PVA*-STPP and CS-PVA*-STPP-GA revealed that the carrier activated by GA has a peak at 1596 cm -1 A characteristic peak due to the stretching vibration of the C=N double bond appears at , and the appearance of the characteristic peak indicates that the aldehyde group of GA and the amino group of CS and PVA* undergo cross-linking reaction, and the carrier is successfully activated.

[0063] Example 4: Performance Characterization of Immobilized Lip

[0064] 4.1 Evaluation of the enzymatic properties of Lip@CS-PVA*-STPP

[0065] Lip@CS-STPP was used as the control group and Lip@CS-PVA*-STPP was used as the experimental group. The loading rate and enzyme activity of Lip were detected by Coomassie Brilliant Blue method and p-nitrophenol method, respectively. First, for the loading rate of the enzyme, the protein content of the initial solution, filtrate, carrier residual liquid and washing liquid of Lip was detected, and the protein amount was obtained by comparing with the standard curve. The amount of protein adsorbed on the support was calculated by the difference between the initial protein content in the Lip solution and the residual protein content in the filtrate, carrier residual liquid and washing liquid. Secondly, the relative residual activity of the enzyme was calculated according to the above formula.

[0066]

[0067] Q0 is the initial protein content of the Lip solution; Q1 is the protein content in the Lip filtrate; Q2 is the residual protein content of the carrier; and Q3 is the protein content in the washing solution.

[0068] Table 3 Comparison of enzymatic properties of immobilized enzymes

[0069]

[0070] The results are shown in Table 3. The enzyme loading rate of Lip@CS-PVA*-STPP is 95.58%, and the enzyme loading rate of Lip@CS-STPP is 87.42%. The addition of PVA* effectively increases the loading sites of Lip, and Lip@CS-PVA*-STPP effectively maintains 86.20% of the enzyme activity.

[0071] 4.2 Storage stability of immobilized Lip

[0072] Lip@CS-STPP or Lip@CS-PVA*-STPP was added to a reaction system containing 700 μL of Tris buffer solution and 700 μL of substrate solution to start the reaction in an incubator at 37°C. Lip@CS-STPP or Lip@CS-PVA*-STPP was removed with tweezers and the supernatant was separated. Lip@CS-STPP or Lip@CS-PVA*-STPP was stored, washed with buffer 3 times, stored at 4°C, and re-added to the corresponding substances in the incubation system on the 1st, 3rd, 6th, 9th, 15th, 20th, 25th, and 30th days for re-catalysis. The supernatant solution was taken each time into a 1.5mL EP tube and mixed. Then, 200 μL was added dropwise to a 96-well plate, and the absorbance value of the product was detected in three groups in parallel in an ELISA instrument. Finally, the absorbance value was substituted into the p-nitrophenol standard curve to calculate the product amount to characterize the catalytic stability of the immobilized enzyme.

[0073] The results are as follows Figure 5 As shown in a and b, the residual activity of Lip@CS-PVA*-STPP was still retained at about 30% on the 30th day, which was higher than that of the Lip@CS-STPP group, indicating that Lip@CS-PVA*-STPP had better storage stability.

[0074] 4.3 Reusability of immobilized Lip

[0075] The steps in Example 4.2 were repeated, except that after incubation for 20 min, Lip@CS-PVA*-STPP and Lip@CS-STPP were removed with tweezers, the supernatant was separated for detection by ELISA, the immobilized enzyme was retained, washed with buffer three times, and the corresponding substances in the incubation system were directly re-added for re-catalysis. This was repeated seven times, and the absorbance values ​​of the products were detected seven times to characterize the reusability of the immobilized enzyme.

[0076] The results are as follows Figure 5 As shown in Figures c and d, for Lip@CS-STPP, the enzyme activity of Lip remained 50% after the fourth reuse, and Lip@CS-PVA*-STPP could still maintain more than 50% enzyme activity after being reused seven times.

Claims

1. A method for preparing immobilized lipase, characterized in that: First, 3-aminopropyltriethoxysilane APTES solution was reacted with polyvinyl alcohol PVA solution to modify the hydroxyl groups of PVA into amino groups to generate modified PVA, which was recorded as PVA*. Then, chitosan CS solution and PVA* solution were fully mixed to form CS-PVA* gel. Then, the CS-PVA* gel was solidified with sodium tripolyphosphate (STPP) solution to form the Lip immobilization carrier CS-PVA*-STTP, and then the carrier was activated with glutaraldehyde (GA) solution to form the activated Lip immobilization carrier CS-PVA*-STTP. Finally, the activated Lip immobilization carrier CS-PVA*-STTP was immersed in an emulsion containing Lip to immobilize Lip. The immobilized Lip was washed with a buffer solution to remove the unbound free enzyme, and the immobilized lipase Lip@CS-PVA*-STPP was obtained.

2. The method for preparing immobilized lipase according to claim 1, characterized in that: The specific Lip immobilization conditions are: immobilization temperature is 0-4°C, immobilization time is 2-12h, and shaking is 200-600rpm.

3. A method for preparing immobilized lipase according to claim 1 or 2, characterized in that: The PVA* is prepared by modifying the hydroxyl groups of a PVA solution with a mass fraction of 1.5% to 9% into amino groups by using a 0.02 mol / L to 2 mol / L APTES solution. After the modification, the pH value of the PVA* is adjusted to a range of 1 to 6 using hydrochloric acid.

4. A method for preparing immobilized lipase according to claim 1 or 2, characterized in that: The mass ratio of CS in the CS-PVA* gel is in the range of 40%-80%.

5. A method for preparing immobilized lipase according to claim 1 or 2, characterized in that: The STPP solution is a STPP aqueous solution with a mass fraction of 2%-6%.

6. A method for preparing immobilized lipase according to claim 1 or 2, characterized in that: The GA solution is a GA aqueous solution with a volume fraction of 0.1%-3%.

7. A method for preparing immobilized lipase according to claim 1 or 2, characterized in that: The emulsion containing Lip is a 7.5U / mL-24U / mL enzyme solution prepared using an emulsified solution.

8. A method for preparing immobilized lipase according to claim 1 or 2, characterized in that: The emulsified solution is a Tris-HCl buffer solution containing 1% gum arabic and 0-4mM sodium deoxycholate in a mass volume fraction.

9. A method for preparing immobilized lipase according to claim 1 or 2, characterized in that: The buffer solution is a Tris-HCl buffer solution with a pH of 7-9 and a 50-400 mM buffer.

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