Laccase immobilization method

Abstract

According to the laccase immobilization method, laccase is immobilized in a positive charge medium composed of aluminum hydroxide and silica gel, the stability and catalytic activity of the laccase can be improved, and after immobilization, the laccase loaded with the positive charge silica gel material (EGM) can be used for decontamination of nerve toxicants such as sarin (GB), soman (GD) and Vyx (VX). The preparation method disclosed by the invention is simple and convenient, low in cost, controllable in condition, good in repeatability, low in requirement on equipment and easy for large-scale production; after the laccase is immobilized on the EGM, the laccase shows good temperature, pH and solvent adaptability, and the storage stability and the cycle use frequency of the laccase are also improved. Within 30 minutes, the catalytic degradation activity of the immobilized laccase on neurotoxic agents such as G, V and the like can reach 95% or above. Compared with the prior art, the free laccase has only 30% of catalytic degradation activity on the G-type neurotoxic agent within 120 minutes.

Description

Technical Field

[0001] The invention belongs to the technical field of stabilization of laccase, and in particular relates to a method for immobilizing laccase for decontamination of nerve agents. Background Art

[0002] Nerve agents are highly toxic organophosphorus compounds in chemical warfare agents (CWA), the most famous of which are sarin (GB) and soman (GD) in the G-type agents, and VX in the V-type. Nerve agents are highly toxic organophosphorus compounds. After entering the body, they quickly and irreversibly bind to acetylcholinesterase in the body to generate phosphorylated cholinesterase, which makes cholinesterase lose the function of hydrolyzing acetylcholine, resulting in a large accumulation of cholinergic neurotransmitters, which act on choline receptors, causing serious neurological disorders, respiratory dysfunction and even fatal consequences. Enzymatic hydrolysis is a common method for nerve agent disinfection. Organophosphorus hydrolases (OPHs) have the ability to cleave the phosphate bonds of nerve agents, but they have the disadvantages of being greatly affected by pH, difficult to prepare, unstable activity and strong catalytic preference. Laccase belongs to the blue copper-containing polyphenol oxidase family. It is ubiquitous in nature and can be widely obtained from fungi, higher plants, bacteria and some insects. It is considered to be a highly specific and ecologically sustainable biocatalyst. However, free laccase has disadvantages such as insufficient stability and low recyclability during application. Summary of the invention

[0003] 1. Technical issues to be resolved

[0004] The invention provides a laccase immobilization method to solve the technical problems of insufficient stability and low recyclability of free laccase during application.

[0005] (II) Technical solution

[0006] In order to solve the above technical problems, the present invention provides a laccase immobilization method, which comprises the following steps:

[0007] S1. The purified laccase is prepared into a laccase solution using a Tris-HCl buffer solution, the positively charged silica gel material EGM is dissolved in deionized water to form an EGM solution, and the laccase solution and the EGM solution are mixed in equal volumes to obtain a mixed solution;

[0008] S2. The mixed solution is incubated, shaken, and centrifuged, and then the supernatant is removed and the precipitate is washed to obtain the immobilized material Lac-EGM of laccase and EGM.

[0009] Furthermore, in step S1, the concentration of the Tris-HCl buffer solution is 50 mmol / L, and the pH value is 9.8.

[0010] Furthermore, in step S1, the concentration of the laccase solution is 1 to 16 mg / mL.

[0011] Furthermore, in step S1, the concentration of the EGM solution is 20 mg / mL.

[0012] Furthermore, in step S2, the mixed solution is incubated at 25° C. for 30 to 90 minutes.

[0013] Furthermore, in step S2, the mixture is vibrated in a vibrator at 200 to 1000 r / min for 2 to 10 hours.

[0014] Furthermore, in step S2, the mixed solution is centrifuged at 13000 r / min for 5 min.

[0015] Furthermore, in step S2, the precipitate is washed with deionized water for 3 to 5 times.

[0016] (III) Beneficial effects

[0017] The present invention proposes a laccase immobilization method, wherein the laccase is immobilized in a positively charged medium composed of aluminum hydroxide and silica gel, which can improve the stability and catalytic activity of the laccase. The laccase immobilized on the positively charged silica gel material (Electropositive Granule Media, EGM) can be used for the decontamination of nerve agents sarin (GB), soman (GD) and VX (VX). The preparation method of the present invention is simple, low-cost, controllable conditions, good repeatability, low equipment requirements, and easy to scale up production; after the laccase is fixed on the EGM, it shows good temperature, pH and solvent adaptability, and also improves its storage stability and number of cycles. Within 30 minutes, the catalytic degradation activity of the immobilized laccase on G-type, V-type and other nerve agents can reach more than 95%. In contrast, the catalytic degradation activity of free laccase on G-type nerve agents within 120 minutes is only 30%. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1a is a scanning electron microscope image of Lac-EGM in Example 1, Figure 1b This is the element energy spectrum of the surface of the immobilized enzyme material;

[0019] Figure 2a is a scanning electron microscope image of Lac-EGM in Example 2, Figure 2b This is the element energy spectrum of the surface of the immobilized enzyme material;

[0020] Figure 3a is a scanning electron microscope image of Lac-EGM in Example 3, Figure 3b This is the element energy spectrum of the surface of the immobilized enzyme material;

[0021] Figure 4 The enzyme activities of laccase and Lac-EGM under different environmental conditions of the embodiment of the present invention; (in the figure: a1 and a2 are the effects of temperature on the enzyme activities of laccase and Lac-EGM, respectively, and the curves are the changes of the enzyme activities of laccase and Lac-EGM over time in the range of 20-70°C; b1 and b2 are the effects of pH on the enzyme activities of laccase and Lac-EGM, respectively, and the curves are the changes of the enzyme activities of laccase and Lac-EGM over time in the range of pH 3-11; c1 and c2 are the effects of organic solvents on the enzyme activities of laccase and Lac-EGM, respectively, and the curves are the changes of the enzyme activities of laccase and Lac-EGM over time in the range of organic solvent concentration of 0%-60%; d1 and d2 are the effects of salt concentration on the enzyme activities of laccase and Lac-EGM, respectively, and the curves are the changes of the enzyme activities of laccase and Lac-EGM over time in the range of sodium chloride concentration of 0-2M; the ordinate is the relative activity; the abscissa is the sampling time, unit: hour);

[0022] Figure 5 The efficiency of laccase, EGM and immobilized Lac-EGM in degrading nerve agents according to the present invention changes over time (in the figure: a, b, c are the rates of free laccase, EGM and Lac-EGM degrading GB, GD and VX respectively. The curve is the change of laccase and Lac-EGM enzyme activities over time when the pH is 9.0. The ordinate is the degradation rate, unit: %; the abscissa is the sampling time, unit: minute);

[0023] Figure 6 The storability and reusability of the laccase and Lac-EGM of the present invention under different conditions are shown in the figure (in the figure: a1 and a2 are the stability and storability of laccase and Lac-EGM under different pH conditions for 24 days, the ordinate is the relative activity, unit: %; the abscissa is the storage time, unit: day; b1 and b2 are the relative activities of laccase and Lac-EGM after 5 cycles of use, the ordinate is the relative activity, unit: %; the abscissa is the number of cycles). DETAILED DESCRIPTION

[0024] In order to make the purpose, content and advantages of the present invention more clear, the specific implementation methods of the present invention are further described in detail below in conjunction with the drawings and examples.

[0025] Example 1

[0026] 2 mg / mL laccase solution and 20 mg / mL EGM solution were mixed in a volume ratio of 1:1, and the mixed solution was kept at room temperature for 60 minutes, and then shaken in a shaker at 200 r / min for 2 hours. The mixture was centrifuged at 13000 r / min for 5 minutes, and the supernatant was removed. Finally, the immobilized product was washed several times with buffer solution and stored in a refrigerator at 4°C for later use. The morphology of EGM and Lac-EGM was observed by scanning electron microscopy to determine the morphology of the immobilized enzyme (such as Figure 1a and 1b shown).

[0027] Example 2

[0028] 4 mg / mL laccase solution and 20 mg / mL EGM solution were mixed in a volume ratio of 1:1, the mixed solution was kept at room temperature for 60 min incubation time, and then shaken in a shaker at 200 r / min for 2 h; the mixture was centrifuged at 13000 r / min for 5 min, and the supernatant was removed; the immobilized product was washed several times with buffer solution and stored in a refrigerator at 4°C for later use. The morphology of EGM and Lac-EGM was observed by scanning electron microscopy to determine the morphology of the immobilized enzyme (such as Figure 2a and 2b shown).

[0029] Example 3

[0030] 8 mg / mL laccase solution and 20 mg / mL EGM solution were mixed in a volume ratio of 1:1, the mixed solution was kept at room temperature for 60 min incubation time, and then shaken in a shaker at 200 r / min for 2 h; the mixture was centrifuged at 13000 r / min for 5 min, and the supernatant was removed; the immobilized product was washed several times with buffer solution and stored in a refrigerator at 4°C for later use. The morphology of EGM and Lac-EGM was observed by scanning electron microscopy to determine the morphology of the immobilized enzyme (such as Figure 3a and 3b shown).

[0031] The following is an investigation of the effect of the immobilized material Lac-EGM on enzyme activity under different environmental conditions. The enzyme activity results of the mixed solution of ABTS (1mmol / ml) and Lac-EGM (10mg / ml) prepared according to the above process at different temperatures (20, 25, 37, 45, 55, 70°C), pH (3, 5, 7, 9, 11), organic solvents (10%, 20%, 40%, 60% DMSO) and salt concentrations (0.25, 0.5, 1, 1.5, 2M NaCl) are shown in the figure. Figure 4 shown.

[0032] The activity of the immobilized material of laccase and EGM is measured or evaluated, and the method steps are as follows:

[0033] The unit of laccase activity (U) is defined as the amount of enzyme that oxidizes 1 μmol ABTS per minute at 35°C (in 0.1 mol L -1 1 mmol L in phosphate buffer -1 ABTS, 1 mmol L -1 HMB and 1mmol L -1 HDB as substrate).

[0034] Enzyme activity (U / L) = 10 6 ×V T ×ΔA / V Lac ×ε×Δt (1)

[0035] The reaction mixture containing the three substrates was added to a colorimeter and the absorbance values ​​at wavelengths of 420 nm / 465 nm / 525 nm were measured by a spectrophotometer. The enzyme activity was calculated according to formula (1), where V T and V Lac Represent the total volume of the reaction system and enzyme solution respectively, ε is the absorption coefficient of the substrate, ΔA is the difference between the absorption values ​​before and after the reaction, and Δt is the reaction time.

[0036] Determination of the degradation ability of immobilized enzymes on GB, GD and VX:

[0037] The degradation rate of GB, GD and VX (10 mg / mL) using laccase and Lac-EGM prepared by the above immobilization process was evaluated by testing the concentration of substrates and products using a gas chromatograph Agilent 7890A. When the pH of the assay solution was 9, the solution components were: 0.1 M Tris-HCl, 10% glycerol, 10 mg / ml GB, GD and VX. The assay solution (800 μl) was mixed with 200 μl of purified protein (1 mg / ml) for 30 minutes, and then extracted with ml dichloromethane. The extract was tested by an Agilent 7890A gas chromatograph. The degradation rate was calculated according to formula (2), C t and C r are the total amount of initial chemical reagents and the degradation residue, respectively. The results are as follows Figure 5 As shown in a~c.

[0038] Degradation efficiency(%)=(C t -C r ) / C t ×100 (2)

[0039] To further explore the stability and storability of the immobilized enzyme, we stored the Lac-EGM immobilized according to the above process under different pH conditions for 24 days and the reusability of the immobilized enzyme. Figure 6 As shown in a1, a2, b1 and b2.

[0040] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for immobilizing laccase, characterized in that: The laccase immobilization method comprises the following steps: S1. The purified laccase is prepared into a laccase solution using a Tris-HCl buffer solution, the positively charged silica gel material EGM is dissolved in deionized water to form an EGM solution, and the laccase solution and the EGM solution are mixed in equal volumes to obtain a mixed solution; S2. The mixed solution is incubated, shaken, and centrifuged, and then the supernatant is removed and the precipitate is washed to obtain the immobilized material Lac-EGM of laccase and EGM.

2. The laccase immobilization method according to claim 1, characterized in that: In step S1, the concentration of the Tris-HCl buffer solution is 50 mmol / L, and the pH value is 9.

8.

3. The laccase immobilization method according to claim 1, characterized in that: In step S1, the concentration of the laccase solution is 1-16 mg / mL.

4. The laccase immobilization method according to claim 1, characterized in that: In step S1, the concentration of the EGM solution is 20 mg / mL.

5. The laccase immobilization method according to claim 1, characterized in that: In step S2, the mixed solution is incubated at 25°C for 30 to 90 minutes.

6. The laccase immobilization method according to claim 1, characterized in that: In step S2, the mixture is vibrated in a vibrator at 200 to 1000 r / min for 2 to 10 hours.

7. The method for immobilizing laccase according to claim 1, characterized in that: In step S2, the mixed solution is centrifuged at 13000 r / min for 5 min.

8. The method for immobilizing laccase according to claim 1, characterized in that: In step S2, the precipitate is washed with deionized water for 3 to 5 times.

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