CN115873827A - High-activity immobilized lipase and application thereof in plastic degradation - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and particularly provides a high-activity immobilized lipase and application thereof in plastic degradation. The catalytic activity and the stability of the immobilized enzyme are obviously improved, and the catalytic activity of the immobilized enzyme is 166 percent of that of free enzyme; the activity of free enzyme is rapidly reduced by the same condition treatment at 30 ℃, and the half-life is reached after the immobilized enzyme is incubated for 4 days; in addition, the immobilized enzyme also obtains reusability, and 64 percent of the initial activity is still kept after 6 times of repetition; in the application aspect, the obtained immobilized enzyme can be applied to the fields of food, medicine, energy, agriculture and the like. In the invention, 71% of dibutyl phthalate can be degraded by the immobilized enzyme at 30 ℃ within 24h, and a green and efficient treatment technology is provided for plastic degradation.

Description

High-activity immobilized lipase and application thereof in plastic degradation

Technical Field

The invention belongs to the technical field of immobilized preparation of lipase, and particularly relates to high-activity immobilized lipase and application thereof in plastic degradation.

Background

Phthalate compounds (PAEs) serving as a plastic modifier are widely applied to products such as plastics, pesticides, coatings, cosmetics and the like, are not easy to degrade after entering the environment, can be accumulated in the environment and enriched through biological chains, and have reproductive toxicity at low concentration. With the rapid development of industrial and agricultural industries in recent years, PAEs have become one of the main pollutants in the environment. However, the conventional physical and chemical degradation methods are costly, complicated to operate, and may cause secondary pollution.

At present, the biodegradation method is considered to be the most effective way for degrading PAEs, and has the advantages of low energy consumption, low cost, negligible secondary pollution and the like. Lipase can catalyze phthalic acid ester to carry out hydrolysis reaction to form phthalic acid monoacid and alcohol, and then phthalic acid and alcohol are formed. As an enzyme with multiple catalytic functions, lipase is widely applied to important industrial fields of food chemical industry, medicine manufacturing, biodiesel, pesticide degradation and the like. However, natural lipases are poor in stability, susceptible to various factors such as pH, temperature, etc., and difficult to recover after use, which prevents the lipases from being widely used commercially.

In general, immobilization of lipase is one of effective strategies to solve problems such as high cost, difficulty in recovery, and low stability. The enzyme immobilization refers to that the enzyme is combined with a water-insoluble carrier through a physical or chemical method, so that the enzyme is limited in a certain range of space, the catalytic activity of the enzyme is reserved, and the enzyme can be recycled. Currently, commonly used immobilization support materials can be divided into two main categories: traditional carrier materials and novel carrier materials, wherein the traditional carrier materials comprise inorganic materials and organic high molecular materials. However, conventional support materials do not have good tunability and crystallinity, which may lead to low protein loading efficiency, instability, and enzyme leaching, which in turn affects the activity of the immobilized enzyme.

Metal-organic frameworks (MOFs) are a new type of support material, and have the advantages of various crystal structures, adjustable pore sizes, high porosity and large specific surface area. The surface charge and chemical properties of proteins or enzymes determine their ability to be encapsulated into MOFs, and therefore most immobilized enzymes are not highly enzymatic.

The catalytic activity of the enzyme can be improved by inducing or doping amino acids and derivatives thereof. We have found that the activity of the enzyme can be enhanced by combining it with an appropriate amino acid. The enzyme and proper amino acid are fixed in MOFs, and the captured amino acid can maintain the active conformation of the enzyme, so that the catalytic activity is finally improved. In addition, the existence of the amino acid can also protect the active site under the high-temperature condition, thereby improving the enzyme activity. Glutaraldehyde, the most commonly used bifunctional cross-linking agent in enzyme immobilization, can immobilize proteins by covalent bonding to an amino-activated support, and can also immobilize proteins by cross-linking of protein aggregates, thereby improving the stability of the immobilized enzyme.

Disclosure of Invention

In order to solve the above-mentioned problems, the present invention provides an immobilized lipase (Cys-CRL @ GA @ MAF-7). The immobilized lipase is prepared by taking Candida Rugosa Lipase (CRL) as a target protein and MAF-7 as an immobilized carrier.

Preferably, the immobilized lipase has an optimum reaction pH of 8.5 and an optimum reaction temperature of 45 ℃. Preferably, the immobilized lipase has improved catalytic activity, and the enzyme activity is 166% of that of free enzyme.

Preferably, the immobilized lipase has enhanced stability and retains 48% of its initial activity after incubation at 30 ℃ for 4 days.

Preferably, the immobilized lipase has better reusability, and 64% of the initial activity is still retained after 6 times of repeated use.

The invention also provides a preparation method of the immobilized lipase, which is characterized in that the immobilized lipase is prepared by taking the lipase from Candida rugosa as a target protein, MAF-7 as an immobilized carrier, cysteine as an auxiliary agent, glutaraldehyde as a cross-linking agent and ammonia water as an accelerator.

Preferably, the preparation method comprises the following steps: (1) Mixing the amino acid solution and the lipase solution in equal volume; (2) Mixing the solution obtained in the step (1) with 3-methyl-1, 2, 4-triazole, glutaraldehyde, zinc acetate and ammonia water solution in sequence, wherein the amino acid in the mixed solution is cysteine, the final concentration of the amino acid is 5-70mM, the final concentration of the 3-methyl-1, 2, 4-triazole is 25-200mM, the final concentration of the zinc acetate is 5-35mM, the final concentration of lipase is 50-400mg/L, the final concentration of the glutaraldehyde is 0.5-5%, and the final concentration of the ammonia water is 0.3% (volume fraction); mixing, and reacting in a shaking table at 20-40 deg.C for 10-60min; (3) And (3) centrifuging the reaction mixture obtained in the step (2) to obtain a light yellow precipitate, and washing the precipitate for three times to obtain the immobilized lipase.

Most preferably, the preparation method comprises the following steps:

(1) Respectively preparing 30mM cysteine solution, 1mg/mL Candida rugosa lipase solution, 1M 3-methyl-1, 2, 4-triazole solution (ethanol is used as a solvent), zinc acetate solution and 30% ammonia water solution by volume concentration;

(2) 2.5mL of the prepared cysteine and 2.5mL of the prepared lipase solution are mixed in equal volume, then 1mL of 3-methyl-1, 2, 4-triazole, 0.4mL50% glutaraldehyde, 0.25mL of zinc acetate and 0.1mL of ammonia water solution are sequentially added, the mixture is shaken for 30s after each addition, finally deionized water is used for complementing the system to 10mL, and the mixture is reacted for 30min in a shaking table at 37 ℃;

(3) The reaction solution turns turbid by clarification, is centrifuged for 5min at 10,000rpm to obtain light yellow precipitate, and is washed with ultrapure water for three times to obtain the immobilized lipase.

The invention also provides application of the immobilized lipase in the field of plastic degradation.

Preferably, the dibutyl phthalate is uniformly dispersed in the preferably immobilized lipase solution, the reaction mixture is shaken and incubated, and after the reaction is finished, the undegraded dibutyl phthalate is obtained by separation.

Preferably, the reaction temperature is 25-37 ℃, the oscillation speed is 150-200rpm, and the reaction time is 0-24h.

The invention has the advantages that:

1. the metal organic framework is composed of metal ions and organic ligands through strong coordination bonds. The MAF-7 carrier adopted by the invention is an MOFs material with excellent performance, has larger specific surface area, excellent chemical and thermal stability, no toxicity and simple synthesis process.

2. The immobilized lipase is successfully prepared by a biomineralization method under the conditions of normal temperature and normal pressure by adopting cysteine as an auxiliary agent, glutaraldehyde as a cross-linking agent and ammonia water as an accelerating agent.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without inventive labor.

FIG. 1 shows the determination of the molar concentration of 3-methyl-1, 2, 4-triazole during enzyme immobilization (\9632;, relative activity; \9679;, immobilization).

FIG. 2 is a graph showing the determination of the molar concentration of zinc acetate during enzyme immobilization (\9632;, relative activity; \9679;, immobilization).

FIG. 3 shows the determination of the mass concentration of an enzyme protein during enzyme immobilization (\9632;, relative activity; \9679;, immobilization).

FIG. 4 shows the determination of the amino acid type during the enzyme immobilization process (\9632;, relative activity; \9679;, immobilization).

FIG. 5 shows the determination of cysteine concentration during enzyme immobilization (\9632;. Relative activity;. 9679;. Immobilization efficiency).

FIG. 6 shows the determination of glutaraldehyde concentration during enzyme immobilization (\9632;, relative activity; \9679;, immobilization effect).

FIG. 7 is a graph showing the effect of reaction pH on CRL and Cys-CRL @ GA @ MAF-7 enzyme activities.

FIG. 8 is a graph showing the effect of reaction temperature on the enzyme activities of CRL and Cys-CRL @ GA @ MAF-7.

FIG. 9 is a graph of the effect of CRL and Cys-CRL @ GA @ MAF-7 stability in 30 ℃ buffer.

FIG. 10 is a graph showing the effect of CRL and Cys-CRL @ GA @ MAF-7 stability in 45 ℃ buffer.

FIG. 11 is a graph showing the number of repeated use of Cys-CRL @ GA @ MAF-7.

FIG. 12 is a graph of the time gradient of Cys-CRL @ GA @ MAF-7 degradation of dibutyl phthalate.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: preparation of immobilized lipase

2.5mL of 30mM cysteine solution and 2.5mL of 1mg/mL lipase solution are mixed in equal volume, then 1mL of 1M 3-methyl-1, 2, 4-triazole (dissolved in ethanol), 0.4mL of 50% glutaraldehyde, 0.25mL of 1M zinc acetate and 0.1mL of 30% ammonia water solution are mixed in sequence, finally deionized water is used for complementing the system to 10mL, and after uniform mixing, the mixture is subjected to shaking table reaction at 37 ℃ for 30min; the reaction solution becomes turbid by clarification, and is centrifuged at 10,000rpm for 5min to obtain a light yellow precipitate, and the light yellow precipitate is washed with ultrapure water for three times to obtain the immobilized lipase Cys-CRL @ GA @ MAF-7.

Example 2: determination of catalytic Activity

The catalytic activity of free enzyme or immobilized lipase is detected by a p-nitrophenol method, and one enzyme activity unit (U) is defined as the amount of enzyme required for producing 1 mu moL of p-nitrophenol per unit time under the conditions of optimal pH (8.0 for free enzyme and 8.5 for immobilized enzyme) and 25 ℃. The standard reaction system is 1mL, and comprises 10 μ L of CRL with concentration of 0.5mg/mL or immobilized enzyme with the same enzyme amount, 10 μ L of 50mM p-nitrophenol butyrate and 980 μ L of 50mM Tris-HClBuffer (free enzyme is pH8.0, immobilized enzyme is pH8.5), mixing uniformly, incubating in a water bath kettle at 25 deg.C for 5min, immediately determining OD410nm for the free enzyme, centrifuging at 12,000rpm for 30s for the immobilized enzyme, immediately determining OD410nm for the supernatant, and using the mixed solution without enzyme as a control. All experiments were independently repeated three times, and the measured values were the average of three independent experiments.

Example 3: optimization of immobilized lipase 3-methyl-1, 2, 4-triazole molar concentration

Mixing 5mL of 0.5mg/mL lipase solution with 1M 3-methyl-1, 2, 4-triazole (dissolved in ethanol), 0.25mL of 1M zinc acetate and 0.1mL of 30% ammonia water solution in sequence, wherein the final concentration of the 3-methyl-1, 2, 4-triazole is 25, 50, 75, 100, 125, 150, 175 and 200mM, finally supplementing the system to 10mL with deionized water, mixing uniformly, and reacting for 30min at 37 ℃ in a shaking table; the reaction solution becomes turbid by clarification, a white precipitate is obtained by centrifugation at 10,000rpm for 5min, and the immobilized lipase CRL @ MAF-7 is obtained by washing with ultrapure water for three times.

The results of determining the molar concentration of 3-methyl-1, 2, 4-triazole in the synthesis process of the immobilized lipase prepared in this example are shown in FIG. 1, and when the final molar concentration of 3-methyl-1, 2, 4-triazole is 100mM, the relative activity and protein immobilization efficiency of the immobilized lipase are the highest, and the immobilization effect is the best.

Example 4: immobilized lipase zinc acetate molar concentration optimization

Mixing 5mL of 0.5mg/mL lipase solution with 1mL of 1M 3-methyl-1, 2, 4-triazole (dissolved in ethanol), 1M zinc acetate and 0.1mL of 30% ammonia water solution in sequence, wherein the final concentration of the zinc acetate is 5,7.5, 10, 15, 20, 25, 30 and 35mM, finally supplementing the system to 10mL with deionized water, mixing uniformly, and reacting for 30min at 37 ℃ in a shaking table; the reaction solution becomes turbid by clarification, a white precipitate is obtained by centrifugation at 10,000rpm for 5min, and the immobilized lipase CRL @ MAF-7 is obtained by washing with ultrapure water for three times.

The result of determining the molar concentration of zinc acetate in the synthesis process of the immobilized lipase prepared in this example is shown in fig. 2, and when the final molar concentration of zinc acetate is 25mM, the relative activity and protein immobilization efficiency of the immobilized lipase are the highest, and the immobilization effect is the best.

Example 5: optimization of immobilized lipase CRL concentration

Mixing a lipase solution of 0.5mg/mL with 1mL of 1M 3-methyl-1, 2, 4-triazole (dissolved in ethanol), 0.25mL of 1M zinc acetate and 0.1mL of 30% ammonia water solution in sequence, wherein the final concentration of CRL is 0.05,0.1,0.15,0.2,0.25,0.3,0.35 and 0.4mg/mL, finally supplementing the system to 10mL with deionized water, mixing uniformly, and reacting for 30min at 37 ℃ by a shaking table; the reaction solution becomes turbid by clarification, a white precipitate is obtained by centrifugation at 10,000rpm for 5min, and the immobilized lipase CRL @ MAF-7 is obtained by washing with ultrapure water for three times.

The determination result of the mass concentration of the enzyme protein in the synthesis process of the immobilized lipase prepared in the example is shown in fig. 3, and when the mass final concentration of the candida rugosa lipase is 0.25mg/mL, the relative activity and the protein immobilization efficiency of the immobilized lipase are the highest, and the immobilization effect is the best.

Example 6: immobilized lipase amino acid species optimization

Mixing 2.5mL of 30mM amino acid solution and 2.5mL of 1mg/mL lipase solution in equal volume, wherein the amino acid is glutamic acid, histidine, cysteine, lysine, proline, glycine and tyrosine, then sequentially mixing with 1mL of 1M 3-methyl-1, 2, 4-triazole (dissolved in ethanol), 0.25mL of 1M zinc acetate and 0.1mL of 30% ammonia water solution, finally complementing the system to 10mL by deionized water, and reacting in a shaking table at 37 ℃ for 30min after uniform mixing; the reaction solution was clarified to become turbid, centrifuged at 10,000rpm for 5min to obtain a white precipitate, and washed with ultrapure water three times to obtain immobilized lipase Aa-CRL @ MAF-7.

The result of determining the amino acid type of the immobilized lipase prepared in this example during the synthesis process is shown in fig. 4, and when the amino acid type is cysteine, the immobilized lipase has the highest relative activity and protein immobilization efficiency, and the best immobilization effect.

Example 7: immobilized lipase cysteine concentration optimization

2.5mL of cysteine solution and 2.5mL of 1mg/mL lipase solution are mixed in equal volumes, wherein the concentration of the cysteine solution is 5, 10, 20, 30, 40, 50, 60 and 70mM, then 1mL of 1M 3-methyl-1, 2, 4-triazole (dissolved in ethanol), 0.25mL of 1M zinc acetate and 0.1mL of 30% ammonia water solution are mixed in sequence, finally deionized water is used for complementing the system to 10mL, and after uniform mixing, the shaking table reaction is carried out at 37 ℃ for 30min; the reaction solution becomes turbid by clarification, white precipitate is obtained by centrifugation at 10,000rpm for 5min, and the immobilized lipase Cys-CRL @ MAF-7 is obtained by washing with ultrapure water for three times.

The result of determining the cysteine concentration of the immobilized lipase prepared in this example during the synthesis process is shown in fig. 5, and when the cysteine concentration is 30mM, the relative activity and protein immobilization efficiency of the immobilized lipase are the highest, and the immobilization effect is the best.

Example 8: immobilized lipase glutaraldehyde concentration optimization

Mixing 2.5mL of 30mM cysteine solution and 2.5mL of 1mg/mL lipase solution in equal volume, sequentially mixing with 1mL of 1M 3-methyl-1, 2, 4-triazole (dissolved in ethanol), 1mL of 50% glutaraldehyde, 0.25mL of 1M zinc acetate and 0.1mL of 30% ammonia water solution, wherein the final concentration of the glutaraldehyde is 0%,0.5%,1%,1.5%,2%,3%,4%,5%, and finally supplementing the system to 10mL with deionized water, and reacting in a shaking table at 37 ℃ for 30min after uniform mixing; the reaction solution becomes turbid by clarification, and is centrifuged at 10,000rpm for 5min to obtain a light yellow precipitate, and the light yellow precipitate is washed with ultrapure water for three times to obtain the immobilized lipase Cys-CRL @ GA @ MAF-7.

The result of determining the glutaraldehyde concentration of the immobilized lipase prepared in this example during the synthesis process is shown in fig. 6, where the relative activity of the immobilized lipase is the highest when the final concentration of glutaraldehyde is 0%, but at this time Cys-crl @ ga @ maf-7 cannot be reused, and when the final concentration of glutaraldehyde is 2%, the relative activity of the immobilized lipase is the second highest, the protein immobilization efficiency is higher, and the reusability is obtained, so the immobilization effect is the best.

Example 9: effect of reaction pH on the enzyme activities of CRL and Cys-CRL @ GA @ MAF-7 in example 1

CRL and Cys-CRL @ GA @ MAF-7 activities were measured at different pH conditions, and the buffer formulations were as follows: 50mM SiO 3 trap buffer (pH 5.5-7.0); 50mM Tris-HCl Buffer (pH 7.0-9.0); 50mM Na ₂ HPO ₄ NaOHBuffer (pH 9.0-10.5). The enzyme activity under the optimum pH condition is measuredThe relative enzyme activity was calculated at 100%. As shown in FIG. 7, the optimum pH of CRL was 8.0 (Tris-HClBuffer), and the optimum pH of Cys-CRL @ GA @ MAF-7 was 8.5 (Tris-HClBuffer);

example 10: effect of reaction temperature on the enzyme activities of CRL and Cys-CRL @ GA @ MAF-7 in example 1

The activities of CRL and Cys-CRL @ GA @ MAF-7 at a temperature range of 25-65 ℃ were measured by using a standard reaction system (the standard reaction system was the same as in example 1) under conditions of pH8.0 and pH8.5, respectively, and the relative enzyme activities were calculated with the enzyme activity at the optimum temperature measured as 100%. As shown in FIG. 8, the optimum reaction temperature was 25 ℃ for Cys-CRL @ GA @ MAF-7 as compared with CRL.

Example 11: effect of CRL and Cys-CRL @ GA @ MAF-7 stability of example 1 in 30 ℃ buffer

CRL and Cys-CRL @ GA @ MAF-7 were prepared into solutions with buffers of pH8.0 and pH8.5, respectively, and the solutions were placed in a 30 ℃ water bath, samples were taken at intervals, and the activities of CRL and Cys-CRL @ GA @ MAF-7 were measured using a standard reaction system (the standard reaction system was the same as in example 1), and the relative enzyme activities were calculated with the enzyme activity without heat treatment as 100%. The results are shown in FIG. 9, where CRL reached half-life after 6h of treatment at 30 ℃ and Cys-CRL @ GA @ MAF-730 ℃ reached half-life after 4 days of treatment.

Example 12: effect of CRL and Cys-CRL @ GA @ MAF-7 stability of example 1 in 45 ℃ buffer

CRL and Cys-CRL @ GA @ MAF-7 were prepared into solutions with buffers of pH8.0 and pH8.5, respectively, and the solutions were placed in a water bath at 45 ℃ and samples were taken at intervals, and the activities of CRL and Cys-CRL @ GA @ MAF-7 were measured using a standard reaction system (the standard reaction system was the same as in example 1), and the relative enzyme activities were calculated with the enzyme activity without heat treatment as 100%. As shown in FIG. 10, the half-life was reached after treatment at CRL45 ℃ for 1 hour, and after treatment at Cys-CRL @ GA @ MAF-745 ℃ for 8 hours.

Example 13: cys-CRL @ GA @ MAF-7 of example 1

Detection in a Standard reaction System (the same Standard reaction System as in example 1) at 25 ℃ and pH8.5

The hydrolytic activity of Cys-CRL @ GA @ MAF-7, after the reaction is finished, centrifuging for 5min at the speed of 12,000rpm, separating the immobilized enzyme from the reaction liquid, washing once by using ultrapure water, repeating the reaction process again, analogizing, recording the value of the OD410 of the supernatant after each reaction, and calculating the relative enzyme activity of the reaction process after each reaction by taking the enzyme activity of the first reaction as 100 percent so as to determine the repeated utilization rate of the Cys-CRL @ GA @ MAF-7. As shown in FIG. 11, cys-CRL @ GA @ MAF-7 retained about 64% of the residual enzyme activity after 6 consecutive uses. The results show that CRL has good reusability after immobilization.

Example 14: cys-CRL @ GA @ MAF-7 of example 1 time gradient for dibutyl phthalate degradation

The degradation capability of Cys-CRL @ GA @ MAF-7 on dibutyl phthalate is explored by adopting a high performance liquid chromatography. First, 100mM dibutyl phthalate solution (methanol as solvent) and 2mg/mL Cys-CRL @ GA @ MAF-7 solution (50mMpH8.5Tris-HClBuffer as solvent) were prepared and stored in a refrigerator at 4 ℃ until use. A reaction system was prepared in the following manner (3 replicates per time gradient) using 15 2mL centrifuge tubes: contains 10. Mu.L of the dibutyl phthalate solution prepared above and 490. Mu.L of the immobilized enzyme solution prepared above. The reaction was carried out at 30 ℃ and incubated under the same conditions using a mixture containing no enzyme as a control. The total reaction time is 24h, starting from 0h, taking out a sample every 6h, adding 50 mu L of 1MHCl to terminate the reaction, then adding 0.3g of NaCl solid particles to saturate the sample, then adding isovolumetric n-hexane, oscillating for 15min, centrifuging for 5min at 10,000rpm, obviously layering the liquid in the Ep tube at the moment, recovering the upper organic phase, filtering the upper organic phase by using an organic phase filter membrane of 0.22 mu m, removing impurities, and then using high performance liquid chromatography to measure. The results are shown in FIG. 12, cys-CRL @ GA @ MAF-7 can degrade 71% of dibutyl phthalate within 24h.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.

Claims (10)

1. The immobilized lipase is characterized in that candida rugosa lipase is used as a target protein, and a metal organic framework material MAF-7 is used as an immobilized carrier.

2. The immobilized lipase according to claim 1, characterized in that the immobilized lipase has high catalytic activity, which is 166% of the free enzyme.

3. The immobilized lipase according to claim 1, wherein the immobilized lipase has a strong stability and retains 48% of the original activity after incubation at 30 ℃ for 4 days.

4. The immobilized lipase according to claim 1, wherein the immobilized lipase has better reusability and retains 64% of the original activity after 6 times of repetition.

5. The process according to any of claims 1 to 4, wherein the immobilized lipase is prepared by using Candida rugosa-derived lipase as a target protein, MAF-7 as an immobilized carrier, cysteine as an auxiliary agent, glutaraldehyde as a crosslinking agent, and ammonia water as an accelerator.

6. Preparation process according to claim 5, characterized in that it comprises the following steps: (1) mixing the amino acid and the lipase solution in equal volume; (2) Mixing the solution obtained in the step (1) with 3-methyl-1, 2, 4-triazole, glutaraldehyde, zinc acetate and an ammonia solution in sequence, wherein the amino acid in the mixed solution is cysteine, the final concentration of the amino acid is 5-70mM, the final concentration of the 3-methyl-1, 2, 4-triazole is 25-200mM, the final concentration of the zinc acetate is 5-35mM, the final concentration of the lipase is 50-400mg/L, the final concentration of the glutaraldehyde is 0.5-5%, and the final concentration of the ammonia is 0.3% (volume fraction); mixing, and reacting in a shaking table at 20-40 deg.C for 10-60min; (3) And (3) centrifuging the reaction mixture obtained in the step (2) to obtain a light yellow precipitate, namely the immobilized lipase.

7. The method of preparation according to claim 6, characterized in that it comprises the following steps:

s1, respectively preparing 30mM cysteine solution, 1mg/mL Candida rugosa lipase solution, 1M 3-methyl-1, 2, 4-triazole solution (ethanol is used as a solvent) and zinc acetate solution, and 30% ammonia water solution by volume;

s2, mixing 2.5mL of the prepared cysteine solution and 2.5mL of the prepared lipase solution in equal volume, sequentially mixing with 1mL of 3-methyl-1, 2, 4-triazole, 0.4mL of 50% glutaraldehyde, 0.25mL of zinc acetate and 0.1mL of ammonia water solution, finally complementing the system to 10mL with deionized water, and reacting for 30min in a shaking table at 37 ℃ after uniform mixing;

s3, clarifying the reaction solution to become turbid, centrifuging at 10,000rpm for 5min to obtain a light yellow precipitate, and washing with ultrapure water for three times to obtain the immobilized lipase.

8. Use of the immobilized lipase according to any of claims 1-7 in the field of plastic degradation.

9. The use according to claim 8, characterized in that dibutyl phthalate is uniformly dispersed in the immobilized lipase solution, the reaction mixture is incubated with shaking, and after the reaction is finished, the undegraded dibutyl phthalate is obtained by separation.

10. Use according to claim 9, wherein the reaction temperature is 25-37 ℃,

the oscillation speed is 150-200rpm, and the reaction time is 0-24h.

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