A method for ligand coupling immobilized lipase

Abstract

The present application relates to the technical field of enzyme immobilization, and specifically discloses a method for ligand-coupled immobilized lipase. A method for ligand-coupled immobilized lipase, using cobalt sulfate heptahydrate as a soft template to synthesize the metal salt precursor of Co-MOF, by dissolving cobalt sulfate heptahydrate and horseradish peroxidase in an appropriate amount of methanol to form colloidal condensed spheres, and then by coordination reaction with trimesic acid, and dissolving the soft template by aqueous phase, pore formation, obtain HRP@Me-Co-MOF, achieve the fixation of horseradish peroxidase, and then achieve the fixation of Antarctic Candida lipase by covalent bonding, and finally prepare ligand-coupled immobilized lipase. The ligand-coupled immobilized lipase prepared in this application has the characteristics of being able to promote the efficient synthesis of quinoxaline compounds and being reusable.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme immobilization, and particularly relates to a method for ligand-coupled immobilized lipase. Background Art

[0002] Enzymes can catalyze many non-natural reactions, a phenomenon known as "enzyme catalytic non-specificity". This discovery has greatly expanded the application of enzymes in organic synthesis. Heme proteins are a class of proteins containing iron porphyrin (hemoglobin, myoglobin, cytochrome, peroxidase, etc.), which have the ability to catalyze a variety of non-natural reactions, such as oxidation reactions, carbene transfer reactions, and carbene XH insertion reactions. These studies have opened up a new world of organic reaction types catalyzed by heme proteins. Chinese patent 201910954938.X discloses a method for synthesizing quinoxaline compounds by dual-protein catalytic cascade reaction. By coupling the carbene reaction catalyzed by heme proteins with the lipase protein catalytic reaction, a green method for synthesizing quinoxaline compounds by dual-protein one-pot method is constructed. Compared with the traditional step-by-step synthesis method, only one step of series connection is required, and the raw materials can be put into one reaction system to complete the two-step catalysis and obtain the final product. However, this method still has problems such as the difficulty of recycling free heme proteins and lipase proteins in solvents and low activity in the reaction. Summary of the invention

[0003] In order to further improve the catalytic effect and recyclability of heme proteins and lipase proteins, the present application provides a method for ligand-coupled immobilized lipase.

[0004] In a first aspect, the present application provides a method for ligand-coupled immobilized lipase, using the following technical solution:

[0005] A method for ligand-coupled immobilized lipase comprises the following steps: S1, first dissolving trimesic acid in methanol to obtain a mixed solution A; S2, dissolving cobalt sulfate heptahydrate in methanol, then adding horseradish peroxidase (HRP), and mixing evenly to obtain a mixed solution B; S3, mixing the mixed solution A and the mixed solution B evenly, stirring at 38-45°C for 15-20h, centrifuging for the first time, transferring the precipitate to the aqueous phase and standing for 20-30min, centrifuging for the second time, and washing with deionized water to obtain a mesoporous cobalt-based metal organic framework-loaded horseradish peroxidase (HRP). RP@Me-Co-MOF); S4, suspend HRP@Me-Co-MOF in 2-morpholineethanesulfonic acid buffer, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, stir for 40-60 min, then add N-hydroxysuccinimide, continue stirring for 50-70 min, add Antarctic Candida lipase (CALB), continue to react for 10-12 h, then centrifuge, collect the precipitate, rinse with deionized water, and freeze-dry to obtain ligand-coupled immobilized lipase (HRP@Me-Co-MOF@CALB).

[0006] The above technical scheme is adopted, cobalt sulfate heptahydrate is used as a soft template and a metal salt precursor for synthesizing a cobalt-based metal organic framework (Co-MOF), and the cobalt sulfate heptahydrate is dissolved in an appropriate amount of methanol to form colloidal condensed spheres, and then a coordination reaction with trimesic acid is carried out, and the soft template is dissolved in an aqueous phase to form pores, thereby obtaining a mesoporous cobalt-based metal organic framework carrier (Me-Co-MOF) with a shell-core structure having a dense shell and a loose core. Horseradish peroxidase is immobilized by encapsulating it in the preparation of Me-Co-MOF, and then Antarctic Candida lipase is immobilized by a covalent bonding method, and finally a ligand-coupled immobilized lipase capable of promoting the efficient synthesis of quinoxaline compounds is prepared.

[0007] Preferably, the usage ratio of trimesic acid to methanol in S1 is (0.5-0.8) mmol: (15-20) mL.

[0008] By adopting the above technical solution, the appropriate amount of methanol added can make cobalt sulfate heptahydrate form colloidal condensed spheres in methanol.

[0009] Preferably, the usage ratio of cobalt sulfate heptahydrate, methanol and horseradish peroxidase in S2 is (0.7-1.0) mmol: (12-18) mL: (12-14) mg.

[0010] By adopting the above technical solution, horseradish peroxidase is added during the formation of Me-Co-MOF, so that the horseradish peroxidase can be coated.

[0011] Preferably, the dosage ratio of the mixed solution A to the mixed solution B in S3 is (18-22) mL: (15-20) mL.

[0012] Preferably, the first centrifugal speed in S3 is 6500-7200 r / min, and the second centrifugal speed is 6000-6800 r / min.

[0013] By adopting the above technical scheme, the Me-Co-MOF prepared at this ratio has the best coating degree and activity of horseradish peroxidase.

[0014] Preferably, the usage ratio of HRP@Me-Co-MOF, 2-morpholineethanesulfonic acid buffer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and Antarctic Candida lipase in S4 is (35-62) mg: (8-10) mL: (28-35) mg: (23-30) mg: (8-10) mg.

[0015] Preferably, the pH value of the 2-morpholineethanesulfonic acid buffer in S4 is 5.5-6.5.

[0016] Preferably, the centrifugal speed in S4 is 6300-7000 r / min.

[0017] By adopting the above technical scheme, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide were used as activating carboxyl agents to covalently attach to the amino groups on the Antarctic Candida lipase, thereby fixing the Antarctic Candida lipase on HRP@Me-Co-MOF.

[0018] In a second aspect, the present application provides a ligand-coupled immobilized lipase, which is prepared using the above-mentioned preparation method.

[0019] In a third aspect, the present application provides an application of a ligand-coupled immobilized lipase, wherein the ligand-coupled immobilized lipase is applied to synthesize quinoxaline compounds.

[0020] In summary, this application has the following beneficial effects:

[0021] 1. The present application adopts the method of dissolving cobalt sulfate heptahydrate in an appropriate amount of methanol solution to form colloidal condensed spheres, and then reacting with trimesic acid and forming pores by aqueous phase dissolution to obtain a Me-Co-MOF carrier with a shell-core structure having a dense shell and a loose core. The Antarctic Candida lipase is immobilized by encapsulating and covalently linking horseradish peroxidase in the Me-Co-MOF preparation, and finally preparing a ligand-coupled immobilized lipase that can promote the efficient synthesis of quinoxaline compounds.

[0022] 2. In the present application, a highly active ligand-coupled immobilized lipase with a mesoporous structure is prepared to solve the mass transfer problem when using enzymes as catalysts in the synthesis of quinoxaline compounds, thereby improving the reaction efficiency.

[0023] 3. The ligand-coupled immobilized lipase prepared in the present application is used to catalyze the synthesis of quinoxaline compounds, which has the characteristics of high reaction activity, fast reaction speed, high immobilization rate and reusability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : SEM image of the ligand-coupled immobilized lipase prepared in Example 3 of the present application, wherein a is Me-Co-MOF, b is HRP@Me-Co-MOF, and c is HRP@Me-Co-MOF@CALB.

[0025] Figure 2 : TEM image of the ligand-coupled immobilized lipase prepared in Example 3 of the present application, wherein a is the mixed solution C, b is Me-Co-MOF, c is HRP@Me-Co-MOF, and d is HRP@Me-Co-MOF@CALB.

[0026] Figure 3 : FTIR graph of the ligand-coupled immobilized lipase prepared in Example 3 of the present application.

[0027] Figure 4 : Nitrogen adsorption and desorption isotherm of Me-Co-MOF prepared in Example 3 of the present application. DETAILED DESCRIPTION

[0028] The present application is further described in detail below with reference to the embodiments.

[0029] Unless otherwise specified, the raw materials used in the examples and comparative examples of the present application are all commercially available.

[0030] Example 1

[0031] A ligand-coupled immobilized lipase is prepared by the following steps:

[0032] S1, first dissolve 0.5 mmol of trimesic acid in 15 mL of methanol to obtain a mixed solution A;

[0033] S2, dissolving 0.7 mmol of cobalt sulfate heptahydrate in 12 mL of methanol, then adding 12 mg of horseradish peroxidase, and mixing well to obtain a mixed solution B;

[0034] S3, 18 mL of mixed solution A and 15 mL of mixed solution B were mixed evenly, stirred at 38 °C for 15 h, centrifuged for the first time at 6500 r / min for 3 min, the precipitate was transferred to the aqueous phase and allowed to stand for 20 min, centrifuged for the second time at 6000 r / min for 3-5 min, and washed with deionized water to obtain HRP@Me-Co-MOF;

[0035] S4, suspend 35 mg of HRP@Me-Co-MOF in 8 mL of 2-morpholineethanesulfonic acid buffer with a pH value of 5.5, add 28 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, stir for 40 min, then add 23 mg of N-hydroxysuccinimide, continue stirring for 50 min, add 8 mg of Antarctic Candida lipase, continue the reaction for 10 h, then centrifuge at 6300 r / min for 5 min, collect the precipitate, rinse with deionized water, and freeze-dry to obtain ligand-coupled immobilized lipase.

[0036] Example 2

[0037] A ligand-coupled immobilized lipase is prepared by the following steps:

[0038] S1, first dissolve 0.65mmol of trimesic acid in 18mL of methanol to obtain a mixed solution A;

[0039] S2, dissolving 0.85 mmol of cobalt sulfate heptahydrate in 15 mL of methanol, then adding 13 mg of horseradish peroxidase, and mixing well to obtain a mixed solution B;

[0040] S3, 20 mL of mixed solution A and 18 mL of mixed solution B were mixed evenly, stirred at 42 °C for 18 h, centrifuged for the first time at 6800 r / min for 4 min, the precipitate was transferred to the aqueous phase and allowed to stand for 25 min, centrifuged for the second time at 6500 r / min for 4 min, and washed with deionized water to obtain HRP@Me-Co-MOF;

[0041] S4, 50 mg of HRP@Me-Co-MOF was suspended in 9 mL of 2-morpholineethanesulfonic acid buffer with a pH value of 6, 31 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was added, and the mixture was stirred for 50 min. Then 27 mg of N-hydroxysuccinimide was added, and the stirring was continued for 60 min. 9 mg of Antarctic Candida lipase was added, and the reaction was continued for 11 h. Then, the mixture was centrifuged at 6500 r / min for 7 min, the precipitate was collected, rinsed with deionized water, and freeze-dried to obtain ligand-coupled immobilized lipase.

[0042] Example 3

[0043] A ligand-coupled immobilized lipase is prepared by the following steps:

[0044] S1, first dissolve 0.8 mmol of trimesic acid in 20 mL of methanol to obtain a mixed solution A;

[0045] S2, dissolving 1.0 mmol of cobalt sulfate heptahydrate in 18 mL of methanol, then adding 14 mg of horseradish peroxidase (HRP), and mixing well to obtain a mixed solution B;

[0046] S3, 22 mL of mixed solution A and 20 mL of mixed solution B were mixed evenly, stirred at 45 °C for 20 h to obtain mixed solution C, and then centrifuged for the first time at 7200 r / min for 5 min, the precipitate was transferred to the aqueous phase and allowed to stand for 30 min, and centrifuged for the second time at 6800 r / min for 5 min, and washed with deionized water to obtain HRP@Me-Co-MOF;

[0047] S4, suspend 62 mg of HRP@Me-Co-MOF in 10 mL of 2-morpholineethanesulfonic acid buffer with a pH of 6.5, add 35 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, stir for 60 min, then add 30 mg of N-hydroxysuccinimide, continue stirring for 70 min, add 10 mg of Antarctic Candida lipase (CALB), continue the reaction for 12 h, then centrifuge at 7000 r / min for 8 min, collect the precipitate, rinse with deionized water, and freeze-dry to obtain ligand-coupled immobilized lipase (HRP@Me-Co-MOF@CALB).

[0048] Without adding horseradish peroxidase, prepare in the same manner as S1, S2 and S3:

[0049] S1, first dissolve 0.8 mmol of trimesic acid in 20 mL of methanol to obtain a mixed solution A;

[0050] S2, dissolving 1.0 mmol of cobalt sulfate heptahydrate in 18 mL of methanol to obtain a mixed solution B;

[0051] S3, 22 mL of mixed solution A and 20 mL of mixed solution B were mixed evenly, stirred at 45 °C for 20 h to obtain mixed solution C, and then centrifuged at 7200 r / min for 5 min, the precipitate was transferred to the aqueous phase and allowed to stand for 30 min, and centrifuged at 6800 r / min for a second time for 5 min, and washed with deionized water to obtain Me-Co-MOF;

[0052] The prepared Me-Co-MOF, HRP@Me-Co-MOF, and HRP@Me-Co-MOF@CALB were observed using a scanning electron microscope. Figure 1 As shown. Figure 1It can be seen that Me-Co-MOF is spherical in distribution, and the surface is composed of many small Co-MOF particles with a particle size of 300-500nm. After enzyme immobilization, the morphology of HRP@Me-Co-MOF and HRP@Me-Co-MOF@CALB is basically unchanged compared with Me-Co-MOF, indicating that the immobilization of HRP and CALB enzymes has little effect on the morphology of the Me-Co-MOF carrier.

[0053] The microstructures of the prepared mixed solution C, Me-Co-MOF, HRP@Me-Co-MOF and HRP@Me-Co-MOF@CALB were observed using transmission electron microscopy. Figure 2 As shown, from Figure 2 It can be seen that cobalt sulfate heptahydrate forms condensed colloidal spheres in methanol solution. After the mixed solution A is added, it gradually reacts to form large spheres composed of small particles of Co-MOF. After being dissolved in water, the formed Me-Co-MOF has a shell-core structure with a dense shell and a loose core. At the same time, by observing HRP@Me-Co-MOF and HRP@Me-Co-MOF@CALB, it is confirmed again that the immobilization of HRP and CALB enzymes has little effect on the morphology of the Me-Co-MOF carrier.

[0054] Co-MOF was prepared by the traditional method: S1, 0.8 mmol of trimesic acid was first dissolved in 20 mL of N,N-dimethylformamide to obtain a mixed solution A; S2, 1.0 mmol of cobalt sulfate heptahydrate was dissolved in 18 mL of N,N-dimethylformamide, and then 14 mg of horseradish peroxidase was added and mixed evenly to obtain a mixed solution B; S3, 22 mL of mixed solution A and 20 mL of mixed solution B were mixed evenly, stirred at 100°C for 20 h, centrifuged at 7000 r / min for 5 min, and washed three times with N,N-dimethylformamide and ethanol in turn to obtain Co-MOF.

[0055] Fourier transform infrared spectrometer was used to perform infrared tests on HRP, CALB, Co-MOF, Me-Co-MOF, HRP@Me-Co-MOF and HRP@Me-Co-MOF@CALB. The results are as follows Figure 3 As shown. Figure 3 It can be seen that Co-MOF and Me-Co-MOF have -1 ,1431cm -1 The characteristic peak of the symmetrical stretching vibration of the carboxyl group in the ligand appeared at 1655 cm -1The stretching vibration peak of the enzyme protein amide I bond is only present in HRP, CALB, HRP@Me-Co-MOF and HRP@Me-Co-MOF@CALB, indicating that the enzyme was successfully immobilized on the Me-Co-MOF carrier. In HRP@Me-Co-MOF@CALB, it can be seen that the peak at about 3485 cm -1 The enhanced absorption band at 40° may be due to the formation of a large number of amide bonds when CALB is covalently fixed on Me-Co-MO.

[0056] The nitrogen adsorption and desorption isotherms of Co-MOF and Me-Co-MOF were measured using a specific surface area and pore size analyzer. Figure 4 As shown. Figure 4 It can be seen that the isotherm trend of Co-MOF is a type I isotherm, indicating that its internal pore structure is mostly composed of micropores. The isotherm trend of Me-Co-MOF is a type IV isotherm, and an obvious retention loop appears in the nitrogen desorption stage, indicating that there are mesopores in the internal pore structure. After DFT mode calculation, the micropore diameter of Co-MOF is concentrated at 0.94nm, and the specific surface area is 1730m 2 / g, and the total pore volume is 0.785cm 3 / g; the micropore diameter of Me-Co-MOF is concentrated at 0.88nm, the mesopore diameter is concentrated at 12nm, and the specific surface area is 1538m 2 / g, total pore volume 1.034cm 3 / g.

[0057] Comparative Example 1

[0058] Same as Example 3, except that:

[0059] The horseradish peroxidase was added at the same time as the Candida antarctica lipase was added to S4.

[0060] Comparative Example 2

[0061] The same as Example 3, except that the order of adding horseradish peroxidase and Candida antarctica lipase was changed, that is: S1, firstly dissolving 0.8 mmol of trimesic acid in 20 mL of methanol to obtain a mixed solution A;

[0062] S2, dissolving 1.0 mmol of cobalt sulfate heptahydrate in 18 mL of methanol, then adding 10 mg of Candida antarctica lipase, and mixing well to obtain a mixed solution B;

[0063] S3, 22 mL of mixed solution A and 20 mL of mixed solution B were mixed evenly, stirred at 45 °C for 20 h to obtain mixed solution C, and then centrifuged for the first time at 7200 r / min for 5 min, the precipitate was transferred to the aqueous phase and allowed to stand for 30 min, and centrifuged for the second time at 6800 r / min for 5 min, and washed with deionized water to obtain CALB@Me-Co-MOF;

[0064] S4, suspend 62 mg of HRP@Me-Co-MOF in 10 mL of 2-morpholineethanesulfonic acid buffer with a pH value of 6.5, add 35 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, stir for 60 min, then add 30 mg of N-hydroxysuccinimide, continue stirring for 70 min, add 14 mg of horseradish peroxidase, continue the reaction for 12 h, then centrifuge at 7000 r / min for 8 min, collect the precipitate, rinse with deionized water, and freeze-dry to obtain ligand-coupled immobilized lipase.

[0065] Comparative Example 3

[0066] The same as Example 3, except that the ligand-coupled immobilized lipase was prepared by adsorption method:

[0067] 62 mg of HRP@Me-Co-MOF was suspended in 10 mL of 2-morpholineethanesulfonic acid buffer with a pH value of 6.5, and then 10 mg of Antarctic Candida lipase was added. The reaction was continued for 12 h, and then centrifuged at 7000 r / min for 8 min. The precipitate was collected and rinsed with 2-morpholineethanesulfonic acid buffer to obtain HRP@Me-Co-MOF.

[0068] Comparative Example 4

[0069] The same as Example 3, except that the horseradish peroxidase was replaced with an equal mass of bovine hemoglobinase.

[0070] Comparative Example 5

[0071] The same as Example 3, except that the Candida antarctica lipase was replaced with an equal mass of porcine chymotrypsin lipase.

[0072] Performance testing

[0073] The ligand-coupled immobilized lipase prepared in Examples 1 to 3 and Comparative Examples 1 to 5 was used to synthesize quinoxaline compounds:

[0074] (1) Add 60 mg of ligand-coupled immobilized lipase to 8 mL of water, then add 177.0 mg of 4,5-dichloro-o-phenylenediamine, 139.2 mg of methyl acetoacetate, and 197.2 mg of p-toluenesulfonyl azide. Stir and react at 45 °C for 4 h. After the reaction, add ethyl acetate for extraction, then wash with hydrochloric acid, dry, concentrate, and purify to obtain 3-methyl-6,7-dichloroquinoxaline-2-carboxylic acid methyl ester. Calculate the yield a1 of the product when it is used for the first time.

[0075] The reaction system containing the ligand-coupled immobilized lipase was centrifuged at 7000 r / min for 8 min, the supernatant was discarded, the ligand-coupled immobilized lipase was recovered, washed with deionized water for 3 times, and added back into a new reaction system. The cycle was repeated 10 times, and the yield a10 of the product at the 10th use was determined.

[0076] (2) In the same manner, 108.0 mg of o-phenylenediamine, 172.8 mg of ethyl propionylacetate and 197.2 mg of p-toluenesulfonyl azide were used to prepare ethyl 3-ethylquinoxaline-2-carboxylate. The yield b1 of the product at the first use and the yield b10 of the product at the tenth use were calculated.

[0077] The following control group was prepared, and the enzyme was directly added as a catalyst for catalytic reaction: 3 mg of horseradish peroxidase, 20 mg of Antarctic Candida lipase, and 0.1 mL of TritonX-100 were added to 8 mL of water, and then 177.0 mg of 4,5-dichloro-o-phenylenediamine, 139.2 mg of methyl acetoacetate, and 197.2 mg of p-toluenesulfonyl azide were added, and the reaction was stirred at 45°C for 8 hours. After the reaction was completed, ethyl acetate was added for extraction, and then washed with hydrochloric acid, dried, concentrated, and purified to obtain 3-methyl-6,7-dichloroquinoxaline-2-carboxylic acid methyl ester, and the yield of the product was calculated to be a1. In the same way, 108.0 mg of o-phenylenediamine, 172.8 mg of ethyl propionyl acetate, and 197.2 mg of p-toluenesulfonyl azide were directly added with the enzyme as a catalyst to prepare ethyl 3-ethylquinoxaline-2-carboxylate, and the yield of the product was calculated to be b1.

[0078] The results are shown in Table 1:

[0079] Table 1 Yields of quinoxaline compounds

[0080]

[0081] As can be seen from Table 1, compared with directly adding horseradish peroxidase and Antarctic Candida lipase as catalysts, the product yield obtained by using Me-Co-MOF as an enzyme carrier for catalytic reaction is greatly improved, and the ligand-coupled immobilized lipase can be reused. Combining Comparative Examples 1 and 3, it can be seen that in terms of the loading order and loading method of the enzyme, the catalytic efficiency of the ligand-coupled immobilized lipase obtained by using Me-Co-MOF to coat horseradish peroxidase and covalently crosslink Antarctic Candida lipase with Me-Co-MOF is the highest; Combining Comparative Examples 4 and 5, it can be seen that in terms of the combination selection of enzymes and compatibility with Me-Co-MOF, the catalytic efficiency obtained by combining horseradish peroxidase and Antarctic Candida lipase loaded on Me-Co-MOF is higher.

[0082] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing ligand-coupled immobilized lipase, characterized in that: The following steps are involved: S1, first dissolve trimesic acid in methanol to obtain a mixed solution A; S2, dissolve cobalt sulfate heptahydrate in methanol, then add horseradish peroxidase, mix well, and obtain a mixed solution B; S3, mix the mixed solution A and the mixed solution B well, stir at 38-45°C for 15-20h, centrifuge for the first time, transfer the precipitate to the aqueous phase and let it stand for 20-30min, centrifuge for the second time, wash with deionized water, and obtain HRP@Me-Co-MOF; S4, suspend HRP@Me-Co-MOF in 2-morpholineethanesulfonic acid buffer, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, stir for 40-60min, then add N-hydroxysuccinimide, continue stirring for 50-70min, add Antarctic Candida lipase, continue to react for 10-12h, then centrifuge, collect the precipitate, rinse with deionized water, and freeze-dry to obtain ligand-coupled immobilized lipase.

2. The method for preparing a ligand-coupled immobilized lipase according to claim 1, characterized in that: The usage ratio of trimesic acid and methanol in the S1 is (0.5-0.8) mmol: (15-20) mL.

3. The method for preparing a ligand-coupled immobilized lipase according to claim 1, characterized in that: The dosage ratio of cobalt sulfate heptahydrate, methanol and horseradish peroxidase in S2 is (0.7-1.0) mmol: (12-18) mL: (12-14) mg.

4. The method for preparing a ligand-coupled immobilized lipase according to claim 1, characterized in that: The dosage ratio of the mixed solution A to the mixed solution B in S3 is (18-22) mL: (15-20) mL.

5. The method for preparing a ligand-coupled immobilized lipase according to claim 1, characterized in that: In S3, the first centrifugal speed is 6500-7200 r / min, and the second centrifugal speed is 6000-6800 r / min.

6. The method for preparing a ligand-coupled immobilized lipase according to claim 1, characterized in that: The usage ratio of HRP@Me-Co-MOF, 2-morpholineethanesulfonic acid buffer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and Antarctic Candida lipase in S4 is (35-62) mg: (8-10) mL: (28-35) mg: (23-30) mg: (8-10) mg.

7. The method for preparing a ligand-coupled immobilized lipase according to claim 1, characterized in that: The pH value of the 2-morpholineethanesulfonic acid buffer in the S4 is 5.5-6.

5.

8. The method for preparing a ligand-coupled immobilized lipase according to claim 1, characterized in that: The centrifugal speed in S4 is 6300-7000 r / min.

9. A ligand-coupled immobilized lipase, characterized in that: The lipase is prepared by the method for preparing a ligand-coupled immobilized lipase according to any one of claims 1 to 8.

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