Polydopamine cross-linked lipase aggregate and preparation method thereof

Abstract

The invention provides a polydopamine cross-linked lipase aggregate and a preparation method thereof, and relates to the technical field of enzyme immobilization. The polydopamine cross-linked lipase aggregate is mainly prepared from lipase and a dopamine hydrochloride cross-linking agent through a Schiff base and Michael addition reaction and a cross-linked enzyme aggregate technology. The preparation method of the polydopamine cross-linked lipase aggregate is simple, convenient and controllable; the prepared polydopamine cross-linked lipase aggregate still has excellent enzyme activity in a heavy metal salt solution.

Description

Technical Field

[0001] This application relates to the technical field of enzyme immobilization. Specifically, it relates to a polydopamine-crosslinked lipase aggregate and a preparation method thereof. Background Art

[0002] Enzymes are natural biocatalysts, which have the characteristics of high selectivity, remarkable catalytic efficiency, and mild reaction conditions. However, due to the poor stability of enzymes under harsh process conditions and the limited reusability of free enzymes, the large-scale utilization of enzymes in industry faces great obstacles. To overcome this difficulty, enzyme immobilization strategies have been developed, which can stabilize enzymes, inhibit enzyme inactivation, improve enzyme activity or reduce the sensitivity of enzymes to microbial contamination while improving the reusability of enzymes. Cross-linked enzyme aggregates refer to a method of immobilization in which salts, water-miscible organic solvents or ionic polymers are added to an aqueous protein solution, resulting in the precipitation of proteins into physical aggregates bound together by non-covalent bonds, and then a bifunctional cross-linking agent is added to cross-link the enzyme aggregates. The preparation method of cross-linked enzyme aggregates is simple, easy to optimize, with good retention of enzyme activity and reusability, and has good research prospects.

[0003] Currently, more reports are about using small molecule compounds such as glutaraldehyde as cross-linking agents to synthesize cross-linked enzyme aggregates. By optimizing the cross-linking reaction conditions (time, temperature, cross-linking agent concentration, etc.), cross-linked enzyme aggregates with relatively high retention of enzyme activity are finally obtained. However, the cross-linked enzyme aggregates synthesized in this way often do not have good biocompatibility (Patent Publication No. CN102978269A), and the retention of enzyme activity in heavy metal salt aqueous solutions is poor. Therefore, finding a new cross-linking agent to replace traditional small molecule cross-linking agents and synthesizing a green, non-toxic cross-linked enzyme aggregate that can retain enzyme activity in heavy metal salt aqueous solutions is a requirement for further realizing the industrial application of cross-linked enzyme aggregates. Summary of the Invention

[0004] Aiming at the above problems, the purpose of the present invention is to provide a polydopamine-crosslinked lipase aggregate and a preparation method thereof, which significantly improve the retention of activity of cross-linked lipase aggregates in different heavy metal salt aqueous solutions.

[0005] To achieve the above invention purpose, the technical solution provided by the present invention is as follows:

[0006] A preparation method of an aqueous dispersion of a polydopamine-crosslinked fat aggregate, comprising the following steps:

[0007] (1) Disperse lipase powder in deionized water, stir to dissolve it fully, filter to remove insoluble substances to obtain a crude lipase solution, and quickly add the crude lipase solution to a precipitant under stirring to precipitate, obtaining lipase precipitate aggregates;

[0008] (2) Dissolve dopamine hydrochloride in an organic solvent to obtain a dopamine hydrochloride crosslinking agent solution;

[0009] (3) Add the dopamine hydrochloride crosslinking agent solution to the lipase precipitate aggregates, stir for a period of time, add an organic base for reaction after the solution is mixed evenly, centrifuge after the reaction, wash the precipitate, and then add deionized water to redisperse the precipitate to obtain an aqueous dispersion of polydopamine crosslinked lipase aggregates.

[0010] In one embodiment of the invention, in the step (1), the concentration of the crude lipase solution is 20 - 60 mg / mL, and the precipitant is selected from at least one of methanol, ethanol, and isopropanol; the volume ratio of the precipitant to the crude lipase solution is 5:1 - 11:1.

[0011] In one embodiment of the invention, in the step (2), the concentration of the dopamine hydrochloride crosslinking agent is 3 - 60 mmol / L, and the organic solvent is selected from at least one of methanol, ethanol, and isopropanol.

[0012] In one embodiment of the invention, in the step (3), the organic base is selected from at least one of ethylamine, triethylamine, and N,N - diisopropylethylamine, and the addition amount of the organic base is 1 - 5% of the total volume of the lipase precipitate aggregates, the dopamine hydrochloride crosslinking agent solution, and the organic base.

[0013] In one embodiment of the invention, in the step (3), the reaction conditions are a constant temperature water bath at 20 - 30 °C for 4 - 24 h, and the centrifugation conditions are centrifugation at 8000 - 10000 rpm for 5 - 15 min.

[0014] The present invention also provides an aqueous dispersion of polydopamine crosslinked lipase aggregates prepared by the above preparation method.

[0015] Advantages of the present invention:

[0016] (1) Compared with the traditional crosslinked enzyme aggregates, the polydopamine crosslinked lipase aggregates provided by the present invention mainly use a novel dopamine hydrochloride crosslinking agent in the crosslinking method, and use polydopamine instead of small molecule crosslinking monomers to realize the preparation of novel crosslinked enzyme aggregates.

[0017] (2) The polydopamine crosslinked lipase aggregates still have high enzyme activity retention in different heavy metal salt solutions because polydopamine can effectively complex heavy metal ions, thereby preventing heavy metal ions from directly contacting the enzyme.

[0018] (3) The preparation method of the polydopamine crosslinked lipase aggregates is green and efficient, with high enzyme activity, providing a broad prospect for the industrial application of crosslinked enzyme aggregates. Description of the Drawings

[0019] To more clearly illustrate the technical solutions of the embodiments of the present application, the accompanying drawings required for use in the embodiments of the present application will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0020] Figure 1 Flow chart of a method for preparing polydopamine-crosslinked lipase aggregates according to an embodiment of the present invention;

[0021] Figure 2 Fourier transform infrared spectrum of polydopamine-crosslinked lipase aggregates;

[0022] Figure 3 Comparison chart of activity retention of polydopamine-crosslinked lipase aggregates and glutaraldehyde-crosslinked lipase aggregates in an aqueous solution of K2PdCl4;

[0023] Figure 4 Comparison chart of activity retention of polydopamine-crosslinked lipase aggregates and glutaraldehyde-crosslinked lipase aggregates in an aqueous solution of NaAuCl4. Detailed implementation manners

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchases.

[0025] The polydopamine-crosslinked lipase aggregates and their preparation methods in the embodiments of the present application will be specifically described below.

[0026] The lipase is derived from Aspergillus oryzae, and the enzyme activity is ≥300000 U / g.

[0027] Example 1: Preparation of an aqueous dispersion of polydopamine-crosslinked lipase aggregates

[0028] (1) Disperse 20 mg of lipase powder in 1 mL of deionized water, stir for 30 minutes to fully dissolve it, filter to remove insoluble substances to obtain a crude lipase solution, and quickly add 0.5 mL of the crude lipase solution to 4 mL of isopropanol under stirring conditions for precipitation. The precipitation ratio is 1:8 to obtain lipase precipitate aggregates;

[0029] (2) Dissolve 0.57 mg of dopamine hydrochloride in 1 mL of isopropanol to obtain a dopamine hydrochloride crosslinking agent solution;

[0030] (3) Add 450 μL of hydrochloric acid dopamine cross-linking agent solution to the lipase precipitate aggregate, stir for 2 min, then add 50 μL of ethylamine to initiate the reaction, and react at a constant temperature in a water bath at 30 °C with a stirring speed of 200 rpm for 24 hours; after the reaction, centrifuge at 8000 rpm for 5 min, wash the obtained solid with isopropanol twice to obtain polydopamine cross-linked lipase aggregate, and then disperse it in 5 mL of deionized water to obtain an aqueous dispersion of polydopamine cross-linked lipase aggregate.

[0031] (4) Freeze-dry the aqueous dispersion of the above-mentioned polydopamine cross-linked lipase aggregate and test the Fourier transform infrared spectrum, and the results are as Figure 2 shown. Among them, the absorption peak at 3415 cm -1 is attributed to the stretching vibrations of N-H and O-H bonds and the presence of water molecules, and 2975 cm -1 is attributed to the asymmetric stretching vibration of C-H bonds, and the absorption peak at 2927 cm -1 belongs to the symmetric stretching vibration of C-H bonds, the absorption peak at 1616 cm -1 belongs to the amide I band, and the absorption peak at 1384 cm -1 is attributed to the amide Ш band, and the absorption peak at 1498 cm -1 is attributed to indole and its derivatives in the self-polymerization of dopamine. The infrared characteristic peaks of both polydopamine and lipase are simultaneously observed in the infrared spectrum of the polydopamine cross-linked lipase aggregate, which proves the successful preparation of the polydopamine cross-linked lipase aggregate.

[0032] Example 2: Preparation of an aqueous dispersion of polydopamine cross-linked lipase aggregate

[0033] (1) Disperse 40 mg of lipase powder in 1 mL of deionized water, stir for 30 minutes to fully dissolve it, filter to remove insoluble substances to obtain a crude lipase solution, and quickly add 0.5 mL of the crude lipase solution to 5 mL of ethanol under stirring conditions for precipitation, with a precipitation ratio of 1:10, to obtain a lipase precipitate aggregate;

[0034] (2) Dissolve 11.38 mg of hydrochloric acid dopamine in 1 mL of ethanol to obtain a hydrochloric acid dopamine cross-linking agent solution;

[0035] (3) Add 350 μL of hydrochloric acid dopamine cross-linking agent solution to the lipase precipitate aggregate, stir for 2 min, then add 150 μL of triethylamine to initiate the reaction, and react at a constant temperature in a water bath at 25 °C with a stirring speed of 200 rpm for 10 hours; after the reaction, centrifuge at 8000 rpm for 15 min, wash the obtained solid with ethanol twice to obtain a polydopamine cross-linked lipase aggregate, and then disperse it in 6 mL of deionized water to obtain an aqueous dispersion of polydopamine cross-linked lipase aggregate.

[0036] Example 3: Preparation of an aqueous dispersion of polydopamine-crosslinked lipase aggregates

[0037] (1) Disperse 50 mg of lipase powder in 1 mL of deionized water, stir for 30 minutes to fully dissolve it, filter to remove insoluble substances to obtain a crude lipase solution. Under stirring conditions, quickly add 0.5 mL of the crude lipase solution to 3 mL of methanol for precipitation, with a precipitation ratio of 1:6, to obtain lipase precipitate aggregates;

[0038] (2) Dissolve 3.80 mg of dopamine hydrochloride in 1 mL of methanol to obtain a dopamine hydrochloride crosslinking agent solution;

[0039] (3) Add 300 μL of the dopamine hydrochloride crosslinking agent solution to the lipase precipitate aggregates, stir for 2 min, then add 200 μL of ethylamine to initiate the reaction, and carry out a constant temperature reaction in a water bath at 30 °C with a stirring speed of 200 rpm for 8 hours; after the reaction is completed, centrifuge at 10000 rpm for 10 min, wash the obtained solid with methanol twice to obtain polydopamine-crosslinked lipase aggregates, and then disperse it in 4 mL of deionized water to obtain an aqueous dispersion of polydopamine-crosslinked lipase aggregates.

[0040] Example 4: Preparation of an aqueous dispersion of polydopamine-crosslinked lipase aggregates

[0041] (1) Disperse 60 mg of lipase powder in 1 mL of deionized water, stir for 30 minutes to fully dissolve it, filter to remove insoluble substances to obtain a crude lipase solution. Under stirring conditions, quickly add 0.5 mL of the crude lipase solution to 5.5 mL of methanol for precipitation, with a precipitation ratio of 1:11, to obtain lipase precipitate aggregates;

[0042] (2) Dissolve 9.48 mg of dopamine hydrochloride in 1 mL of methanol to obtain a dopamine hydrochloride crosslinking agent solution;

[0043] (3) Add 300 μL of the dopamine hydrochloride crosslinking agent solution to the lipase precipitate aggregates, stir for 2 min, then add 200 μL of ethylamine to initiate the reaction, and carry out a constant temperature reaction in a water bath at 20 °C with a stirring speed of 200 rpm for 6 hours; after the reaction is completed, centrifuge at 8000 rpm for 5 min, wash the obtained solid with methanol twice to obtain polydopamine-crosslinked lipase aggregates, and then disperse it in 6.5 mL of deionized water to obtain an aqueous dispersion of polydopamine-crosslinked lipase aggregates.

[0044] Example 5: Preparation of an aqueous dispersion of polydopamine-crosslinked lipase aggregates

[0045] (1) Disperse 40 mg of lipase powder in 1 mL of deionized water, stir for 30 minutes to fully dissolve it, filter to remove insoluble substances, obtain a crude lipase solution, and quickly add 0.5 mL of the crude lipase solution to 4 mL of ethanol under stirring conditions for precipitation. The precipitation ratio is 1:8 to obtain a lipase precipitate aggregate;

[0046] (2) Dissolve 7.59 mg of dopamine hydrochloride in 1 mL of ethanol to obtain a dopamine hydrochloride cross-linking agent solution;

[0047] (3) Add 450 μL of the dopamine hydrochloride cross-linking agent solution to the lipase precipitate aggregate, stir for 2 min, then add 50 μL of N,N-diisopropylethylamine to initiate the reaction, and carry out a constant-temperature reaction in a water bath at 30 °C with a stirring speed of 200 rpm for 4 hours; after the reaction is completed, centrifuge at 8500 rpm for 10 min, wash the obtained solid with ethanol twice to obtain a polydopamine-cross-linked lipase aggregate, and then disperse it in 5 mL of deionized water to obtain an aqueous dispersion of the polydopamine-cross-linked lipase aggregate.

[0048] Example 6: Preparation of polydopamine-cross-linked lipase aggregate

[0049] (1) Disperse 20 mg of lipase powder in 1 mL of deionized water, stir for 30 minutes to fully dissolve it, filter to remove insoluble substances, obtain a crude lipase solution, and quickly add 0.5 mL of the crude lipase solution to 2.5 mL of methanol under stirring conditions for precipitation. The precipitation ratio is 1:5 to obtain a lipase precipitate aggregate;

[0050] (2) Dissolve 3.17 mg of dopamine hydrochloride in 1 mL of methanol to obtain a dopamine hydrochloride cross-linking agent solution;

[0051] (3) Add 350 μL of the dopamine hydrochloride cross-linking agent solution to the lipase precipitate aggregate, stir for 2 min, then add 150 μL of ethylamine to initiate the reaction, and carry out a constant-temperature reaction in a water bath at 30 °C with a stirring speed of 200 rpm for 8 hours; after the reaction is completed, centrifuge at 8000 rpm for 5 min, wash the obtained solid with methanol twice to obtain a polydopamine-cross-linked lipase aggregate, and then disperse it in 3.5 mL of deionized water to obtain an aqueous dispersion of the polydopamine-cross-linked lipase aggregate.

[0052] Example 7: Preparation of an aqueous dispersion of polydopamine-cross-linked lipase aggregate

[0053] (1) Disperse 40 mg of lipase powder in 1 mL of deionized water, stir for 30 minutes to fully dissolve it, filter to remove insoluble substances to obtain a crude lipase solution, and quickly add 0.5 mL of the crude lipase solution to 4 mL of isopropanol for precipitation under stirring conditions. The precipitation ratio is 1:8 to obtain a lipase precipitate aggregate;

[0054] (2) Dissolve 1.90 mg of dopamine hydrochloride in 1 mL of isopropanol to obtain a dopamine hydrochloride cross-linking agent solution;

[0055] (3) Add 400 μL of the dopamine hydrochloride cross-linking agent solution to the lipase precipitate aggregate, stir for 2 min, then add 100 μL of triethylamine to initiate the reaction, and carry out a constant-temperature reaction in a water bath at 30 °C with a stirring speed of 200 rpm for 8 hours; after the reaction is completed, centrifuge at a speed of 8500 rpm for 5 min, wash the solid obtained after centrifugation with isopropanol twice to obtain a polydopamine-cross-linked lipase aggregate, and then disperse it in 5 mL of deionized water to obtain an aqueous dispersion of the polydopamine-cross-linked lipase aggregate.

[0056] Example 8: Preparation of an aqueous dispersion of a polydopamine-cross-linked lipase aggregate

[0057] (1) Disperse 40 mg of lipase powder in 1 mL of deionized water, stir for 30 minutes to fully dissolve it, filter to remove insoluble substances to obtain a crude lipase solution, and quickly add 0.5 mL of the crude lipase solution to 4 mL of ethanol for precipitation under stirring conditions. The precipitation ratio is 1:8 to obtain a lipase precipitate aggregate;

[0058] (2) Dissolve 2.52 mg of dopamine hydrochloride in 1 mL of ethanol to obtain a dopamine hydrochloride cross-linking agent solution;

[0059] (3) Add 450 μL of the dopamine hydrochloride cross-linking agent solution to the lipase precipitate aggregate, stir for 2 min, then add 50 μL of N,N-diisopropylethylamine to initiate the reaction, and carry out a constant-temperature reaction in a water bath at 30 °C with a stirring speed of 200 rpm for 8 hours; after the reaction is completed, centrifuge at a speed of 8500 rpm for 10 min, wash the solid obtained after centrifugation with ethanol twice to obtain a polydopamine-cross-linked lipase aggregate, and then disperse it in 5 mL of deionized water to obtain an aqueous dispersion of the polydopamine-cross-linked lipase aggregate.

[0060] Example 9: Preparation of an aqueous dispersion of a polydopamine-cross-linked lipase aggregate

[0061] (1) Disperse 40 mg of lipase powder in 1 mL of deionized water, stir for 30 minutes to fully dissolve it, filter to remove insoluble substances to obtain a crude lipase solution, and quickly add 0.5 mL of the crude lipase solution to 4 mL of ethanol under stirring for precipitation with a precipitation ratio of 1:8 to obtain lipase precipitate aggregates;

[0062] (2) Dissolve 3.18 mg of dopamine hydrochloride in 1 mL of ethanol to obtain a dopamine hydrochloride cross-linking agent solution;

[0063] (3) Add 450 μL of the dopamine hydrochloride cross-linking agent solution to the lipase precipitate aggregates, stir for 2 min, then add 50 μL of N,N-diisopropylethylamine to initiate the reaction, and carry out a constant-temperature reaction in a water bath at 30 °C with a stirring speed of 200 rpm for 8 hours; after the reaction, centrifuge at a speed of 8500 rpm for 10 min, wash the solid obtained after centrifugation twice with ethanol to obtain polydopamine-cross-linked lipase aggregates, and then disperse them in 5 mL of deionized water to obtain an aqueous dispersion of polydopamine-cross-linked lipase aggregates.

[0064] Example 10: Preparation of an aqueous dispersion of polydopamine-cross-linked lipase aggregates

[0065] (1) Disperse 40 mg of lipase powder in 1 mL of deionized water, stir for 30 minutes to fully dissolve it, filter to remove insoluble substances to obtain a crude lipase solution, and quickly add 0.5 mL of the crude lipase solution to 4 mL of ethanol under stirring for precipitation with a precipitation ratio of 1:8 to obtain lipase precipitate aggregates;

[0066] (2) Dissolve 3.80 mg of dopamine hydrochloride in 1 mL of ethanol to obtain a dopamine hydrochloride cross-linking agent solution;

[0067] (3) Add 450 μL of the dopamine hydrochloride cross-linking agent solution to the lipase precipitate aggregates, stir for 2 min, then add 50 μL of N,N-diisopropylethylamine to initiate the reaction, and carry out a constant-temperature reaction in a water bath at 30 °C with a stirring speed of 200 rpm for 8 hours; after the reaction, centrifuge at a speed of 9000 rpm for 10 min, wash the solid obtained after centrifugation twice with ethanol to obtain polydopamine-cross-linked lipase aggregates, and then disperse them in 5 mL of deionized water to obtain an aqueous dispersion of polydopamine-cross-linked lipase aggregates.

[0068] Comparative Example 1: Preparation of an aqueous dispersion of glutaraldehyde-cross-linked lipase aggregates

[0069] (1) Disperse 40 mg of lipase powder in 1 mL of deionized water, stir for 30 minutes to fully dissolve it, filter to remove insoluble substances to obtain a crude lipase solution, and quickly add 0.5 mL of the crude lipase solution to 4 mL of ethanol under stirring conditions for precipitation with a precipitation ratio of 1:8 to obtain lipase precipitate aggregates;

[0070] (2) Dilute 30% aqueous glutaraldehyde solution with water to a concentration of 0.12 mol / L to obtain a glutaraldehyde cross-linking agent solution;

[0071] (3) Add 50 μL of the glutaraldehyde cross-linking agent solution and 450 μL of ethanol to the lipase precipitate aggregates to start the reaction, and carry out a constant-temperature reaction at a stirring speed of 200 rpm and a water bath temperature of 30 °C for 8 h; after the reaction is completed, centrifuge at a speed of 8500 rpm for 10 min, wash the obtained solid with ethanol twice to obtain glutaraldehyde-cross-linked lipase aggregates, and then disperse them in 5 mL of deionized water to obtain an aqueous dispersion of glutaraldehyde-cross-linked lipase aggregates.

[0072] Comparative Example 2: Preparation of an aqueous dispersion of glutaraldehyde-cross-linked lipase aggregates

[0073] (1) Disperse 40 mg of lipase powder in 1 mL of deionized water, stir for 30 minutes to fully dissolve it, filter to remove insoluble substances to obtain a crude lipase solution, and quickly add 0.5 mL of the crude lipase solution to 4 mL of ethanol under stirring conditions for precipitation with a precipitation ratio of 1:8 to obtain lipase precipitate aggregates;

[0074] (2) Dilute 30% aqueous glutaraldehyde solution with water to a concentration of 0.15 mol / L to obtain a glutaraldehyde cross-linking agent solution;

[0075] (3) Add 50 μL of the glutaraldehyde cross-linking agent solution and 450 μL of ethanol to the lipase precipitate aggregates to start the reaction, and carry out a constant-temperature reaction at a stirring speed of 200 rpm and a water bath temperature of 30 °C for 8 h; after the reaction is completed, centrifuge at a speed of 8500 rpm for 10 min, wash the obtained solid with ethanol twice to obtain glutaraldehyde-cross-linked lipase aggregates, and then disperse them in 5 mL of deionized water to obtain an aqueous dispersion of glutaraldehyde-cross-linked lipase aggregates.

[0076] Comparative Example 3: Preparation of an aqueous dispersion of glutaraldehyde-cross-linked lipase aggregates

[0077] (1) Disperse 40 mg of lipase powder in 1 mL of deionized water, stir for 30 minutes to fully dissolve it, filter to remove insoluble substances to obtain a crude lipase solution, and quickly add 0.5 mL of the crude lipase solution to 4 mL of ethanol under stirring conditions for precipitation with a precipitation ratio of 1:8 to obtain lipase precipitate aggregates;

[0078] (2) Dilute the 30% aqueous glutaraldehyde solution by adding water to a concentration of 0.18 mol / L to obtain a glutaraldehyde cross-linking agent solution;

[0079] (3) Add 50 μL of the glutaraldehyde cross-linking agent solution and 450 μL of ethanol to the lipase precipitate aggregates to start the reaction. React at a constant temperature in a water bath at 30 °C with a stirring speed of 200 rpm for 8 h; after the reaction, centrifuge at 8500 rpm for 10 min, wash the obtained solid with ethanol twice to obtain glutaraldehyde cross-linked lipase aggregates, and then disperse it in 5 mL of deionized water to obtain an aqueous dispersion of glutaraldehyde cross-linked lipase aggregates.

[0080] Test Example 1: Changes in enzyme activity retention of polydopamine cross-linked lipase aggregates and glutaraldehyde cross-linked lipase aggregates over time in an aqueous K2PdCl4 solution

[0081] (1) Take 0.5 mL of the crude lipase solution in Example 8 and add 4.35 mL of deionized water to obtain an aqueous dispersion of free lipase. Disperse the polydopamine cross-linked enzyme aggregates prepared in Example 8 and the glutaraldehyde cross-linked enzyme aggregates prepared in Comparative Example 1 in 4.85 mL of deionized water respectively to obtain aqueous dispersions of polydopamine cross-linked lipase aggregates and glutaraldehyde cross-linked lipase aggregates. Perform enzyme activity tests on the aqueous dispersions of free lipase, polydopamine cross-linked lipase aggregates, and glutaraldehyde cross-linked lipase aggregates respectively to obtain the activity of the aqueous dispersion of free lipase, the activity of the aqueous dispersion of polydopamine cross-linked lipase aggregates, and the activity of the aqueous dispersion of glutaraldehyde cross-linked lipase aggregates.

[0082] Enzyme activity test method:

[0083] Respectively take 50 μL of the above-mentioned aqueous dispersions of free lipase / polydopamine cross-linked lipase aggregates / glutaraldehyde cross-linked lipase aggregates, and add 100 μL of an acetonitrile solution of 4-nitrophenyl acetate (10 mmol / L) to 4.85 mL of a phosphate buffer solution (100 mmol / L, pH = 7.4), and react under stirring at 200 rpm and 40 °C. Use a UV-visible spectrophotometer to measure the absorbance value at 400 nm at regular intervals and calculate the enzyme activity.

[0084] The enzyme activity of this reaction system is defined as:

[0085] Enzyme activity = ΔAbs / (K * Δt);

[0086] In the formula, ΔAbs is the increase in absorbance at 400 nm, Δt is the reaction time (s), and K is the slope of the absorbance value-concentration standard curve of p-nitrophenol at 400 nm (1.284×10 4 M -1 );

[0087] Enzyme activity retention (%) = (enzyme activity of the aqueous dispersion of polydopamine-crosslinked lipase aggregates or enzyme activity of the aqueous dispersion of glutaraldehyde-crosslinked lipase aggregates / enzyme activity of the aqueous dispersion of free lipase) × 100%.

[0088] (2) Add 150 μL of aqueous K2PdCl4 solution (10 mmol / L) to the above aqueous dispersions of polydopamine-crosslinked lipase aggregates / glutaraldehyde-crosslinked lipase aggregates respectively, stir at a stirring speed of 200 rpm, and perform enzyme activity tests on the aqueous dispersions of polydopamine-crosslinked lipase aggregates and glutaraldehyde-crosslinked lipase aggregates after stirring for 1 hour, 2 hours, and 3 hours respectively. The test method refers to step (1).

[0089] Appendix Figure 3 For the above test results, the results show that after 3 hours in the aqueous K2PdCl4 solution, the activity retention of the polydopamine-crosslinked lipase aggregates is 70%.

[0090] Test Example 2: Changes in enzyme activity retention of polydopamine-crosslinked lipase aggregates and glutaraldehyde-crosslinked lipase aggregates with time in aqueous NaAuCl4 solution

[0091] (1) Take 0.5 mL of the crude lipase solution in Example 9, add 4.35 mL of deionized water to obtain an aqueous dispersion of free lipase. Disperse the polydopamine-crosslinked enzyme aggregates prepared in Example 9 and the glutaraldehyde-crosslinked enzyme aggregates prepared in Comparative Example 2 in 4.85 mL of deionized water respectively to obtain aqueous dispersions of polydopamine-crosslinked lipase aggregates and glutaraldehyde-crosslinked lipase aggregates. Perform enzyme activity tests on the aqueous dispersions of free lipase, polydopamine-crosslinked lipase aggregates, and glutaraldehyde-crosslinked lipase aggregates respectively to obtain the enzyme activity of the aqueous dispersion of free lipase, the enzyme activity of the aqueous dispersion of polydopamine-crosslinked lipase aggregates, and the enzyme activity of the aqueous dispersion of glutaraldehyde-crosslinked lipase aggregates. The enzyme activity test method refers to Test Example 1.

[0092] (2) Add 150 μL of aqueous NaAuCl4 solution (10 mmol / L) to the above aqueous dispersions of polydopamine-crosslinked lipase aggregates / glutaraldehyde-crosslinked lipase aggregates respectively, stir at a stirring speed of 200 rpm, and perform enzyme activity tests on the aqueous dispersions of polydopamine-crosslinked lipase aggregates and glutaraldehyde-crosslinked lipase aggregates after stirring for 1 hour, 2 hours, and 3 hours respectively. The test method refers to Test Example 1.

[0093] Appendix Figure 4 For the above test results, the results show that after 3 hours in the aqueous NaAuCl4 solution, the activity retention of the polydopamine-crosslinked lipase aggregates is 70%.

[0094] Test Example 3: Variation of Enzyme Activity Retention over Time of Polydopamine-Crosslinked Lipase Aggregates and Glutaraldehyde-Crosslinked Lipase Aggregates in Aqueous K2PtCl4 Solution

[0095] (1) Take 0.5 mL of the crude lipase solution in Example 10 and add 4.26 mL of deionized water to obtain an aqueous dispersion of free lipase. Disperse the polydopamine-crosslinked enzyme aggregates prepared in Example 10 and the glutaraldehyde-crosslinked enzyme aggregates prepared in Comparative Example 2 in 4.76 mL of deionized water respectively to obtain aqueous dispersions of polydopamine-crosslinked lipase aggregates and glutaraldehyde-crosslinked lipase aggregates. Conduct enzyme activity tests on the aqueous dispersions of free lipase, polydopamine-crosslinked lipase aggregates, and glutaraldehyde-crosslinked lipase aggregates respectively to obtain the activity of the aqueous dispersion of free lipase, the activity of the aqueous dispersion of polydopamine-crosslinked lipase aggregates, and the activity of the aqueous dispersion of glutaraldehyde-crosslinked lipase aggregates. The enzyme activity test method refers to Test Example 1.

[0096] (2) Add 240 μL of aqueous K2PtCl4 solution (50 mmol / L) to the above aqueous dispersions of polydopamine-crosslinked lipase aggregates / glutaraldehyde-crosslinked lipase aggregates respectively, stir at a stirring speed of 200 rpm, and conduct enzyme activity tests on the aqueous dispersions of polydopamine-crosslinked lipase aggregates and glutaraldehyde-crosslinked lipase aggregates respectively after stirring for 1 hour, 2 hours, and 3 hours. The test method refers to Test Example 1. The results show that after 3 hours in the aqueous K2PtCl4 solution, the activity retention of the polydopamine-crosslinked lipase aggregates is 85%.

[0097] The above are only the embodiments of the present application and are not used to limit the protection scope of the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application. Each component not clearly defined in this embodiment can be realized by the prior art.

Claims

1. A method for preparing an aqueous dispersion of polydopamine cross-linked lipase aggregates, characterized in that: The following steps are involved: (1) dispersing lipase powder in deionized water, stirring to fully dissolve it, filtering to remove insoluble matter, obtaining a crude lipase enzyme solution, and rapidly adding the crude lipase enzyme solution to a precipitant under stirring to obtain lipase precipitate aggregates; (2) dissolving dopamine hydrochloride in an organic solvent to obtain a dopamine hydrochloride cross-linking agent solution; (3) adding the dopamine hydrochloride crosslinker solution to the lipase precipitate aggregates, stirring for a period of time, adding an organic base to react after the solution is evenly mixed, centrifuging after the reaction is completed, washing the precipitate, and then adding deionized water to redisperse the precipitate to obtain an aqueous dispersion of polydopamine crosslinked lipase aggregates.

2. The method for preparing the aqueous dispersion of polydopamine cross-linked lipase aggregates according to claim 1, characterized in that: In the step (1), the concentration of the crude lipase solution is 20 to 60 mg / mL, the precipitant is selected from at least one of methanol, ethanol, and isopropanol; and the volume ratio of the precipitant to the crude lipase solution is 5:1 to 11:

1.

3. The method for preparing the aqueous dispersion of polydopamine cross-linked lipase aggregates according to claim 1, characterized in that: In the step (2), the concentration of the dopamine hydrochloride cross-linking agent is 3 to 60 mmol / L, and the organic solvent is selected from at least one of methanol, ethanol and isopropanol.

4. The method for preparing the aqueous dispersion of polydopamine cross-linked lipase aggregates according to claim 1, characterized in that: In the step (3), the organic base is selected from one of ethylamine, triethylamine and N,N-diisopropylethylamine, and the amount of the organic base added is 1-5% of the total volume of the lipase precipitate aggregate, the dopamine hydrochloride crosslinker solution and the organic base.

5. The method for preparing the aqueous dispersion of polydopamine cross-linked lipase aggregates according to claim 1, characterized in that: In the step (3), the reaction conditions are 20-30° C. constant temperature water bath for 4-24 hours, and the centrifugation conditions are 8000-10000 rpm for 5-15 minutes.

6. An aqueous dispersion of polydopamine cross-linked lipase aggregates, characterized in that: The polydopamine cross-linked lipase aggregate is prepared by the method for preparing the aqueous dispersion of the polydopamine cross-linked lipase aggregate as described in any one of claims 1 to 5.

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