CN112723522B

CN112723522B - Preparation method and application of laccase hybrid gel particles

Info

Publication number: CN112723522B
Application number: CN202011381791.9A
Authority: CN
Inventors: 王生杰; 马宁; 唐勇; 戴良鸿; 崔丙顺; 曹美文; 夏永清
Original assignee: China University of Petroleum East China
Current assignee: China University of Petroleum East China
Priority date: 2020-12-01
Filing date: 2020-12-01
Publication date: 2023-03-17
Anticipated expiration: 2040-12-01
Also published as: CN112723522A

Abstract

The invention provides a preparation method and application of laccase hybrid gel particles. Belongs to the technical field of environmental protection, and can effectively remove chlorophenol pollutants which are difficult to degrade by a common method. Solves the problems of complex system, easy hydrolysis and leakage of enzyme, easy inactivation and the like in the prior laccase immobilization method. The technical scheme comprises the following steps: (1) Dispersing a certain amount of sodium alginate and amino modified silica nanoparticles in water, performing ultrasonic dispersion, adding a certain amount of laccase, uniformly dispersing, dropwise adding the uniformly dispersed laccase into a calcium chloride solution, and reacting for a certain time to obtain laccase hybridized gel particles. The laccase hybrid gel particles obtained by the invention can be used for catalyzing and degrading chlorophenol pollutants, have high catalysis efficiency and can be recycled.

Description

Preparation method and application of laccase hybrid gel particles

Technical Field

The invention belongs to the technical field of environmental protection, and particularly relates to a method for removing chlorophenol pollutants in water by using biological enzyme and application of the method.

Background

In recent years, along with the development of industry, the discharge amount of waste water containing chlorophenols and the content of chlorophenols in the waste water have been increasing year by year. Chlorophenols have been widely used for a long time for the preservation of wood and the production of rust inhibitors, bactericides, insecticides, herbicides and the like, have a strong denaturing effect on biological tissues, strongly stimulate the skin and have corrosiveness. Because of its high toxicity, resistance to Degradation, and greater danger to many organisms including humans, effective removal has become a research issue of great concern (Pandiyan T.et. Al. Complex of Methods for the Photochemical Degradation of Chlorophenols [ J ]. Journal of Photochemistry, 2002, 146-155).

At present, the treatment methods of chlorophenol pollutants in water are mainly divided into a microbial treatment method, a physicochemical method, a chemical reduction method, a chemical oxidation method and the like (Wei Chaohai and the like, and the research progress of a toxic refractory organic pollutant treatment method [ J ]. Chongqing environmental science 1998,20 (4): 22-27). Depending on the source and nature of the various contaminants, different treatment techniques have evolved, each with some advantages and disadvantages. The laccase degradation technology has a specific catalytic degradation effect and mild reaction conditions, and belongs to an environment-friendly water pollutant treatment method, so that more attention of people is attracted.

Laccases are readily soluble in water, creating major challenges for long-term use and recycling. The use of immobilized enzymes, such as alginate (Pan T. Et al, antibiotics of immobilized on catalytic activity of immobilized laccase in Cu-alkaline substrates: inhibition of chloride and activation of acetate [ J ]. Chinese Chemical Letters,2014,25,983-988), chitosan (Chen Hui, etc., laccase catalyzed degradation of chlorophenol organic contaminants [ J ]. Beijing university, 2005,41 (4): 605-611), carbon nanotubes (Costa J. B. Et al. Enhanced biological activity of lacase by immobilized functional activity of carbon nanoparticles/nanoparticles [ J ]. J ], and laccase-immobilized enzymes, 989, which are expected to increase the cost of immobilized enzymes, such as immobilized enzymes, laccase, immobilized on alginate on/nanoparticles [ J. ] and laccase, on the other hand, immobilized enzymes, such as laccase, immobilized on laccase, 989, and the like, is expected to increase the cost of immobilized enzymes. The alginate biomacromolecule has the characteristics of no toxicity, low price, easy control of gel degree and gelation time and biodegradability, thereby becoming one of important candidate materials of the enzyme immobilization carrier. However, alginate gel has strong hydrophilicity and is easy to swell in an aqueous environment, the strength of the gel is not enough only by the chelation of metal ions and carboxyl groups, and laccase is easy to leak from the alginate gel when the alginate gel is used in an aqueous solution, so that immobilization failure is caused. Therefore, how to improve gel strength and increase the control of laccase coating without affecting enzyme activity is one of the important problems that needs to be solved urgently at present.

Disclosure of Invention

Aiming at the problems existing in the prior alginate gel immobilized laccase, the invention provides a preparation method and application of laccase hybrid gel particles. By introducing the functionalized silica nanoparticles to improve the gel network of the sodium alginate, the laccase hybrid gel particles which can stably exist for a long time in various environments are prepared. The preparation method is simple, the reaction condition is mild, and the laccase cannot be damaged. The obtained immobilized material can be used as a catalyst for enzymatic reaction, can keep higher catalytic activity, has better tolerance to the environment, is easy to separate, can be recycled and the like. The specific invention content is as follows:

the invention provides a preparation method of laccase hybrid gel particles, which is characterized by comprising the following steps:

s1: dispersing a certain amount of sodium alginate and amino modified silicon dioxide nano particles in water, performing ultrasonic dispersion, adding a certain mass of laccase, and uniformly mixing to obtain a solution A;

s2: dissolving calcium chloride in water to obtain a solution B;

s3: and dropwise adding the solution A into the solution B at a certain speed, reacting for a certain time, filtering, and washing to obtain laccase hybrid gel particles.

Wherein, the amino modified silica nano-particle in the step (1) is prepared by the following method: taking a proper amount of nano silicon dioxide and aminopropyltriethoxysilane to disperse in anhydrous toluene, taking triethylamine as a reaction promoter, reacting for 3-5 hours at 90-110 ℃, cooling, centrifuging, and washing to obtain the amino modified silicon dioxide nano particles.

Preferably, the concentration of sodium alginate in said solution a is between 0.5 and 5% by weight.

Preferably, the dosage of the amino modified silica nano particles in the solution A is 5-10% of the mass of the sodium alginate.

Preferably, the concentration of the laccase in the solution A is 0.01-0.1mg/L.

Preferably, the concentration of calcium chloride in the solution B is 0.5-3wt%.

Preferably, in the step (3), the reaction time is 3-15min.

The invention provides laccase hybrid gel particles prepared by any one of the preparation methods.

Compared with free enzyme under the same conditions, the laccase hybridized gel particles prepared by the invention have the advantages that the activity is kept above 90%, and the tolerance to temperature, pH and the like is increased.

The invention provides application of laccase hybridized gel particles prepared by the preparation method in degradation of chlorophenol compounds.

The laccase hybrid gel particles prepared by the invention have the degradation rate of 4-chlorophenol reaching more than 60% within 1 hour, and can be recycled.

The preparation method of laccase hybrid gel particles provided by the invention is applied to removal of chlorophenol pollutants in water, and has high removal efficiency and recovery rate. Compared with the prior art, the invention has the advantages and positive effects that:

1. the organic-inorganic hybrid method is used for preparing high-strength gel. The hybrid gel is microscopically in a network structure consisting of silicon dioxide nano particles and sodium alginate nano fibers, has a compact structure, small bulk density and strong compressive property, and has a very large structure and strength regulation space.

2. The organic-inorganic hybrid gel is used as a laccase immobilization carrier, can carry laccase with high efficiency, cannot cause enzyme leakage easily, and can maintain a proper cross-linked network and space, so that the structure maintenance of the enzyme and the diffusion of reactants are facilitated, the catalytic activity of the immobilized enzyme is maintained, and the removal of pollutants is facilitated.

3. The preparation method provided by the invention is simple, green and environment-friendly, the water-based environment is friendly to enzyme, the raw materials are low in price and convenient to obtain, and the industrial production and large-scale popularization are facilitated.

Drawings

FIG. 1 is a photograph of laccase hybridized gel particles prepared in example 3 of the present invention

FIG. 2 is an electron micrograph of amino-modified silica nanoparticles prepared in example 3 of the present invention

FIG. 3 shows the enzyme activity of laccase hybrid gel particles prepared in example 3 of the present invention measured by UV-visible absorption spectroscopy

FIG. 4 is a conversion rate-time curve of catalytic degradation of 4-chlorophenol by laccase hybridized gel particles prepared in example 3 of the present invention

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely below, 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.

The embodiment of the invention provides a preparation method of laccase hybrid gel particles, which comprises the following steps:

s1: dispersing a certain amount of sodium alginate and amino modified silicon dioxide nano particles in water, performing ultrasonic dispersion, adding a certain mass of laccase, and uniformly mixing to obtain a solution A.

In the step, the alginate biomacromolecules are used as a skeleton material of the gel, and the amino modified silica particles exist as physical crosslinking points so as to improve the strength of the gel. The purpose of the amino modification is to hopefully enable non-covalent bond interaction, such as electrostatic interaction, between the silicon dioxide nano particles and sodium alginate molecules through introducing the amino to improve the crosslinking strength of the gel. By forming the gel in situ near the laccase, the conformation of the laccase can be better maintained, thereby maintaining higher activity.

S2: calcium chloride was dissolved in water to give solution B.

In this step, calcium ion (Ca) ²⁺ ) As metal chelating ion to interact with carboxyl in sodium alginateAnd (3) fixing the segment of the sodium alginate to form gel. Other polyvalent metal ions, e.g. Cu ²⁺ ,Mg ²⁺ ,Al ³⁺ A similar effect is also achieved. In the present invention, calcium ions are used for the crosslinking reaction, considering that calcium ions are cheap and easily available, and the gelling effect is easily controlled.

S3: and dropwise adding the solution A into the solution B at a certain speed, reacting for a certain time, filtering, and washing to obtain laccase hybridized gel particles.

In this step, solution a is introduced into solution B, and a gel particle structure is gradually formed by calcium ions in solution B penetrating into droplets of solution a, as shown in fig. 1. The internal diameter of the dropping device and the rate of dropping affect the size of the gel particles formed, which in turn affects the path of reactant and product diffusion, and thus the rate of catalytic degradation. The diameter of the laccase gel particles provided by the invention is adjustable between 1 mm and 5 mm.

In this step, silica nanoparticles are provided with surface amino groups by aminopropyltriethoxysilane. Triethylamine is used as a reaction accelerator, and can effectively accelerate the polycondensation reaction between alkoxy on aminopropyltriethoxysilane and silicon hydroxyl on the surface of the silicon dioxide nano-particle. Toluene is a non-polar solvent and has a very low water content, and the reaction is carried out in toluene in order to allow all reactions to occur on the surface of the silica nanoparticles and to prevent the polycondensation reaction between aminopropyltriethoxysilane. In order to ensure that the reaction is more complete, the surface of the silica nano particle can have higher amino grafting rate by the treatment.

In one embodiment of the invention, the concentration of sodium alginate in said solution a is between 0.5 and 5% by weight. Too small concentration of sodium alginate can not form gel, too large concentration and too high viscosity of the solution can result in poor dispersibility of the amino-modified silica nanoparticles and laccase. Studies have shown that suitable alginate gel particles can be obtained when the sodium alginate concentration is between 0.5 and 5wt%, including but not limited to 0.5, 1, 2, 3, 4, 5wt%.

In an embodiment of the invention, the amount of the amino-modified silica nanoparticles in the solution a is 5-10% of the mass of sodium alginate. The amino modified silica nano particles mainly have the functions of providing physical cross-linking points in gel, thereby improving the gel strength, inhibiting the gel hydrolysis, preventing laccase from escaping in the use process and maintaining the long-acting property and the recycling property of laccase hybridized gel particles. The concentration of the amino modified silica nanoparticles is too low, and the contribution to the gel strength is not obvious; too high a concentration may result in too high a gel density, limiting the access of reactants and products, and reducing the catalytic activity of the laccase. Researches prove that laccase hybrid gel particles with proper crosslinking degree and strength can be obtained when the using amount of the amino modified silica nano particles is 5-10 wt%.

In one embodiment of the invention, the concentration of laccase in the solution A is 0.01-0.1mg/L. The concentration of laccase is not as high as possible, and the concentration of laccase can affect the conformational stretching and the loading rate in the gel. Research shows that when the concentration of the laccase is 0.01-0.1mg/L, the laccase serving as a catalyst has better loading rate and catalytic conversion effect when catalyzing the degradation of 4-chlorophenol.

In one embodiment of the present invention, the concentration of calcium chloride in the solution B is 0.5-3wt%. The concentration of calcium chloride mainly affects the rate of gel formation and the degree of gelation. The calcium ion concentration is too high, gel is rapidly formed on the surface of the alginate liquid drop, the calcium ion entering the interior of the liquid drop is influenced, and the surface and the interior of the particle have larger difference in gel degree. Too low calcium ion concentration results in insufficient gelation, and laccase is easy to escape, which is not favorable for repeated use. Studies have shown that calcium ion concentrations of 0.5-3wt% in solution B can lead to laccase hybrid gel particles with the appropriate degree of gelation and gel strength.

In one embodiment of the present invention, the reaction time in the step (3) is 3-15min. The reaction time is mainly used for controlling the gelation degree of the laccase hybridized gel particles through calcium ion infiltration, the influence process is similar to the calcium ion concentration, and the appropriate reaction time can ensure that the laccase hybridized gel particles with the appropriate gelation degree can be obtained.

Another embodiment of the invention provides laccase hybrid gel particles prepared by the method for preparing laccase hybrid gel particles according to any one of the preceding embodiments. On one hand, the in-situ encapsulation of the laccase does not cause obvious change of the laccase conformation, thereby ensuring that the laccase activity before and after gelation has no obvious difference. On the other hand, the introduction of the amino modified silica nanoparticles provides physical crosslinking points, improves the strength of the gel, inhibits the hydrolysis of the gel and the escape of laccase, and enables long-term use and recycling use to be possible.

In another embodiment of the invention, the laccase hybrid gel particles prepared have laccase activity maintained above 90% and increased tolerance to temperature, pH, etc., compared to the free enzyme under the same conditions. The design concept of the invention is verified, and the laccase hybrid gel particles with higher enzyme activity and good stability are obtained.

The invention further provides application of the laccase hybrid gel particles in catalytic degradation of chlorophenol pollutants, and the difficultly-degraded chlorophenol pollutants are effectively removed in a mild aqueous environment. The degradation rate of the chlorophenol reaches more than 60% within 3 hours, and the chlorophenol can be recycled, and has high pollutant removal effect and popularization and application values.

In order to more clearly describe the preparation method of the iron ion doped titanium dioxide nano material provided by the embodiment of the invention, the following description will be made with reference to specific examples.

Example 1

(1) Taking certain amount of nanosilica and aminopropyltriethoxysilane (10wt% ₂ ) Dispersing into 50mL of toluene, adding a few drops of triethylamine as an accelerant, condensing and refluxing for 5h at 110 ℃, cooling, centrifuging, and washing with water to obtain amino modified silicon dioxide nano particlesAnd (3) granules.

(2) To a sodium alginate solution (0.5 wt%), amino-modified silica nanoparticles (5 wt%) were added and dispersed with ultrasound. A certain amount of laccase is dispersed in a silicon dioxide-sodium alginate solution (0.01 mg/L) to obtain a solution A1.

(3) Adding 0.5wt% of CaCl by a peristaltic pump ₂ And dropwise adding the solution A1 into the solution (solution B), soaking for 15min, filtering and washing to obtain the laccase hybrid gel particles 1.

Example 2:

(1) Taking certain amount of nanosilica and aminopropyltriethoxysilane (10wt% ₂ ) Dispersing the amino modified silica nanoparticles into 50mL of toluene, adding a few drops of triethylamine as an accelerant, condensing and refluxing for 5h at 110 ℃, cooling, centrifuging, and washing with water to obtain the amino modified silica nanoparticles.

(2) To a sodium alginate solution (5 wt%), amino-modified silica nanoparticles (10 wt%) were added and dispersed by sonication. A certain amount of laccase is dispersed in a silicon dioxide-sodium alginate solution (0.1 mg/L) to obtain a solution A2.

(3) To 3wt% CaCl by a peristaltic pump ₂ And (3) dropwise adding the solution A2 into the solution (solution B), soaking for 3min, filtering and washing to obtain laccase hybrid gel particles 2.

Example 3:

(1) Taking a certain amount of nanosilica and aminopropyltriethoxysilane (10wt%) ₂ ) Dispersing into 50mL of toluene, adding a few drops of triethylamine as an accelerant, condensing and refluxing for 5h at 110 ℃, cooling, centrifuging, and washing with water to obtain the amino modified silicon dioxide nano-particles.

(2) To a sodium alginate solution (5 wt%), amino-modified silica nanoparticles (7 wt%) were added and dispersed with ultrasound. A certain amount of laccase is dispersed in a silicon dioxide-sodium alginate solution (0.05 mg/L) to obtain a solution A3.

(3) Adding 1wt% of CaCl by a peristaltic pump ₂ And (3) dropwise adding the solution A3 into the solution (solution B), soaking for 7min, filtering and washing to obtain laccase hybrid gel particles 3.

Example 4

Morphology characterization of amino-modified silica nanoparticles

Transmission Electron Microscope (TEM), model: JEM-2100UHR, instrument manufacturer: JEOL Ltd, acceleration voltage 200kV, test temperature 25 ℃.

In this embodiment, the morphology of the amino-modified silica nanoparticles is observed by using a transmission electron microscope, specifically, a sample is dispersed in a certain amount of absolute ethanol, 20 μ L of the mixture is dropped onto a 200-mesh copper mesh covered with a carbon support film, the mixture is left at room temperature for a period of time, and after the ethanol is completely volatilized, the mixture is placed in the transmission electron microscope and is characterized under an acceleration voltage of 200 kV. As can be seen from FIG. 2, the obtained amino-modified silica nanoparticles have a uniform size and a diameter of about 120 nm.

Example 5

Enzyme activity determination of laccase hybrid gel particles

Ultraviolet-visible spectrophotometer, model: UV-1700PharmaSpec, 25 ℃ experimental temperature.

Adding laccase hybridized gel particles into a 1.0mM 2,2-linked nitrogen-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium Salt (ABTs) solution, reacting for 4min at 30-40 ℃, measuring the absorbance at the wavelength of 420nm in an ultraviolet-visible spectrometer, and taking the linear part of the absorbance change. Defining: the amount of enzyme required to catalyze the oxidation of 1. Mu.M ABTs per minute is one unit of enzyme activity, expressed in U/g. As can be seen from FIG. 3, the UV absorption at 420nm in the UV-visible spectrum increases gradually over time, showing better enzymatic activity.

Example 6

Characterization of laccase hybridized gel particles for catalytic degradation of 4-chlorophenol

Dissolving a certain amount of 4-chlorophenol in water, mixing a proper amount of laccase hybridized gel particles with the gel particles, putting the gel particles into a shaking incubator at 35 ℃, and carrying out reaction in shaking at 120 r/min. After 0.5h of reaction, the reaction mixture was filtered, and 10mL of the filtrate was taken out and diluted to 50mL. Adjusting the pH value to 7.0, sequentially adding 0.15mL of 2wt% 4-aminoantipyrine aqueous solution and 0.15mL of 80g/L potassium ferricyanide aqueous solution, stirring for about 30s, standing for 15min, and measuring the absorbance of chlorophenol in the system by using an ultraviolet-visible spectrophotometer. As shown in FIG. 4, the concentration of 4-chlorophenol is gradually reduced along with the time, the degradation rate calculated according to the concentration is gradually increased, and the degradation rate of 4-chlorophenol reaches more than 60% when the time reaches 180 minutes (3 hours), which shows that the degradation effect on the difficultly-degradable chlorophenol pollutants is better.

The result shows that by introducing the amino modified silicon dioxide nanoparticles and controlling the cross-linked network, the laccase hybrid gel particles provided by the invention have higher enzyme activity retention rate and environmental tolerance, show higher catalytic efficiency in the aspect of catalytically degrading difficultly-degraded chlorophenols pollutants, can be recycled, have higher comprehensive utilization value, and are suitable for industrial popularization.

Claims

1. The application of laccase hybrid gel particles in degradation of chlorophenol compounds is characterized in that the laccase hybrid gel particles are prepared through the following steps:

(1) Dispersing a certain amount of sodium alginate and amino modified silicon dioxide nano particles in water, performing ultrasonic dispersion, adding a certain mass of laccase, and uniformly dispersing to obtain a solution A; wherein, the concentration of the laccase in the solution A is 0.01-0.1mg/L, and the dosage of the amino modified silicon dioxide nano particles is 5-10% of the mass of the sodium alginate;

(2) Dissolving calcium chloride in water to obtain a solution B;

(3) Dropwise adding the solution A into the solution B at a certain speed, reacting for 3-15min, filtering, and washing to obtain laccase hybridized gel particles, wherein the obtained laccase hybridized gel particles are used as a catalyst, so that the degradation rate of 4-chlorophenol within 3 hours reaches more than 60%, and the gel particles can be recycled;

wherein, the amino modified silica nano-particle in the step (1) is prepared by the following method:

taking a proper amount of nano silicon dioxide and aminopropyltriethoxysilane to disperse in anhydrous toluene, taking triethylamine as a reaction promoter, reacting for 3-5 hours at 90-110 ℃, cooling, centrifuging, and washing to obtain the amino modified silicon dioxide nano particles.

2. Use according to claim 1, characterized in that the concentration of sodium alginate in solution a is between 0.5 and 5% by weight.

3. Use according to claim 1, wherein the calcium chloride concentration in solution B is between 0.5 and 3wt%.

4. The use according to any of claims 1-3, characterized in that the laccase hybrid gel particles have a retention of activity above 90% and an increased tolerance to temperature, pH etc. compared to free laccase under the same conditions.