CN109439189B - Method for immobilizing antifouling enzyme on surface of inorganic coating - Google Patents
Method for immobilizing antifouling enzyme on surface of inorganic coating Download PDFInfo
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- C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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Abstract
The invention belongs to the technical field of antifouling enzyme immobilization, and particularly relates to an immobilization method of an antifouling enzyme on the surface of an inorganic coating, wherein an inorganic coating with amino groups on the surface is prepared by using alkoxy silane, amino silane and silica sol, and the antifouling enzyme is immobilized on the surface of a transparent silica sol inorganic coating through the connection action of glutaraldehyde, so that the inorganic coating has good corrosion resistance and better antifouling performance, is applied to surface antifouling of marine ships, structures (docks, drilling platforms, piers and piles) and the like and corrosion and antifouling of key parts such as underwater equipment observation windows and the like, and can provide technical support for the preparation of enzyme-based antifouling coating materials; the preparation method is simple and feasible, the used raw materials are cheap and easy to obtain, the preparation cost is greatly reduced, and the silica sol inorganic coating immobilized antifouling enzyme has better transparency and good anticorrosion and antifouling properties, and has wide application prospects in surface antifouling and anticorrosion and antifouling in marine environments.
Description
The technical field is as follows:
the invention belongs to the technical field of antifouling enzyme immobilization, and particularly relates to an immobilization method of antifouling enzyme on the surface of an inorganic coating.
Background art:
marine biofouling organisms, also known as marine biofouling organisms, are a general term for animals, plants and microorganisms that grow on the surface of all facilities at the bottom of ships and in the sea, and the process of biofouling growth is called biofouling. Marine biofouling poses a serious hazard to ships, underwater equipment, and offshore artificial facilities, and effective measures must be taken to prevent fouling and adhesion of marine organisms to the marine equipment. The organic tin antifouling paint is forbidden due to serious harm to marine ecology and environment; copper-containing antifouling paints widely used in the prior art have been restricted in use and are subject to a tendency to be banned in some countries due to the accumulation of copper in gulf, which also causes serious marine ecological problems. Aiming at the background that marine biofouling causes serious harm to ships and offshore artificial facilities and the traditional toxic antifouling paint faces banning and limited use, the development of the environment-friendly marine antifouling paint has important significance, wherein the enzyme-based antifouling coating technology has attracted extensive attention and deep research of all countries in the world due to environmental protection, high efficiency and universality, and becomes one of important directions for the development of novel environment-friendly antifouling materials. The research finds that the attachment basis of the marine fouling organisms is almost the same, and the adhesion substances comprise proteins, polysaccharides, lipids and the like. The adhesion inhibiting effect can be obtained by directly decomposing adhesion substance based on enzyme decomposition, such as protease decomposition protein adhesion substance, amylase decomposition polysaccharide adhesion substance, and lipase decomposition lipid adhesion substance. The development of enzyme-based antifouling coating materials by utilizing the efficient hydrolytic activity of enzyme on fouling organism adhesion substances and good environmental friendliness is an important development trend of a marine antifouling technology, and the immobilization treatment of antifouling enzyme is an important link in the application process of the antifouling enzyme.
The commonly used immobilization methods of the anti-fouling enzyme are classified into a physical adsorption method, an entrapment method, a cross-linking method, and a covalent bonding method according to the acting force. The enzyme immobilization carrier mainly comprises an inorganic carrier and an organic carrier, wherein the inorganic carrier mainly comprises porous inorganic materials such as diatomite, silica gel, porous glass, a mesoporous molecular sieve and the like, and the organic carrier mainly comprises a natural high molecular carrier (such as sodium alginate, cellulose, chitosan and the like) and a synthetic organic high molecular carrier (such as macroporous resin, synthetic fiber and the like). Gatenholm et al demonstrated that bacteria embedded in the coating can still remain viable and produce bioactive components; holmstrom et al discusses factors and conditions required to affect the viability of bacteria immobilized in the gel; the Candida lipase is covalently immobilized on the surfaces of the silicon nanoparticles and the glass microspheres by Dandavate and Yilmaz respectively to obtain an enzyme preparation which can be recycled and used for esterification synthesis; huijs et al immobilized the enzyme by acetoacetoxy group, aldehyde group, chloromethyl group, etc. in the polymer. The results of immobilized enzyme and antifouling test show that the enzyme can be covalently linked and immobilized through the functional group of the coating binder to generate effective antifouling effect, the enzyme applied in the way shows remarkable antifouling effect in the coating, and in addition, the antifouling performance of the enzyme is improved through immobilization. U.S. Pat. No. 2,20080038241, filed by Biolocus, discloses an antifouling paint using an organosilicon-acrylic acid self-polishing resin as a film forming material and a peroxide produced by an enzyme as an antifouling agent; korean university research and commercial fund applied for an antifouling paint using a mixture of enzyme and fiber as an antifouling material; the international famous Danish enzyme preparation company GENECOR and the famous paint manufacturer HEMPEL cooperate to develop a new antifouling technology for producing hydrogen peroxide based on the combined action of glucoamylase and hexose oxidase, and the technology is successfully applied to antifouling paint and starts to be applied to a real ship at present.
The anticorrosive coatings applied in the field of marine corrosion prevention mainly comprise epoxy, polyurethane, rubber, fluororesin, organic silicon resin, polyurea elastomer and the like, and are usually applied in a matched system mode in an underwater marine environment, such as an anticorrosive primer, an intermediate connecting paint and an antifouling finish paint system, namely, an anticorrosive coating and an antifouling coating are bonded through a connecting paint and are applied in a matched mode. The method for immobilizing the antifouling enzyme on the surface of the graphene oxide disclosed in the Chinese patent 201810373368.0 comprises four steps of dispersing, amination, aldehyde group immobilization and immobilization, wherein the key process is the immobilization of the antifouling enzyme on the surface of graphene oxide powder, and the method is used for preparing a marine organism adhesion-preventing material and does not provide corrosion resistance; chinese patent 201610250733.X discloses a preparation method of temperature-sensitive self-polishing resin for covalent immobilization of antifouling enzyme, which comprises two steps of preparation of temperature-sensitive self-polishing resin and covalent immobilization of antifouling enzyme, wherein the covalent immobilization of antifouling enzyme involves immobilization of antifouling enzyme on the surface of resin particles, and the immobilized enzyme-containing resin is redispersed in xylene solution to form coating resin for preventing marine organism adhesion. The above patent methods are not suitable for immobilization of antifouling enzymes on the surface of an organic coating, and if one coating has good anticorrosion performance and antifouling performance, the cost can be reduced to a great extent, the application field can be expanded, and the application in the aspect is rarely reported at present. Therefore, according to the urgent need of the green antifouling coating in marine fouling environments such as marine ships, marine structures, underwater equipment observation windows and the like, a method for immobilizing the antifouling enzyme on the surface of the inorganic coating is provided.
The invention content is as follows:
the invention aims to overcome the defects in the prior art, and develops and designs an immobilization method of an antifouling enzyme on the surface of an inorganic coating, so that the inorganic coating after covalent immobilization of the antifouling enzyme has good corrosion resistance and better antifouling performance.
In order to achieve the purpose, the technological process of the method for immobilizing the antifouling enzyme on the surface of the inorganic coating comprises three steps of preparing the organic coating, cross-linking treatment and immobilizing the antifouling enzyme:
preparing an organic coating: adding alkoxy silane into silica sol with the pH value of 4.0-5.0, hydrolyzing at 45-60 ℃ for 0.5-2h to form a mixed solution, adding amino alkoxy silane into the mixed solution, uniformly mixing, coating on a film coating sheet, and forming an organic coating on the film coating sheet;
the mass ratio of the silica sol to the alkoxy silane is 0.8-1.3, and the mass of the amino alkoxy silane accounts for 0.1-5.0% of the mass of the mixed solution;
(II) crosslinking treatment: adding glutaraldehyde into a phosphate buffer solution with the pH value of 7 to prepare a glutaraldehyde solution with the mass percentage concentration of 5%, immersing the coated membrane into the glutaraldehyde solution, performing glutaraldehyde crosslinking treatment for 5 hours, then cleaning the coated membrane for 3 times by using the phosphate buffer solution with the pH value of 7, removing residual glutaraldehyde, and finishing the crosslinking treatment;
(III) immobilized antifouling enzyme: adding the antifouling enzyme into a phosphate buffer solution with the pH value of 7 to prepare an antifouling enzyme solution with the mass percentage concentration of 5-20%, immersing the coating film sheet subjected to the crosslinking treatment in the step (II) into the antifouling enzyme solution, soaking for 5h at 5-30 ℃, then cleaning the coating film sheet for 3 times by using the phosphate buffer solution with the pH value of 7, and freeze-drying the cleaned sample in a vacuum freeze-drying machine to finish the immobilization of the antifouling enzyme on the surface of the organic coating.
The alkoxy silane is one or more of ethyl orthosilicate alkyl, methyl triethoxysilane and methyl trimethoxysilane.
The amino alkoxy silane related by the invention is one or more of diethylene triamino propyl trimethoxy silane, (3-aminopropyl) trimethoxy silane and (3-aminopropyl) triethoxy silane.
The antifouling enzyme is one or more of protease, amylase, chitinase and lysozyme.
Compared with the prior art, the invention uses the alkoxy silane, the amino silane and the silica sol to prepare the inorganic coating with amino groups on the surface, and fixes the antifouling enzyme on the surface of the transparent silica sol inorganic coating through the connection action of the glutaraldehyde, so that the inorganic coating has good corrosion resistance and better antifouling performance, is applied to surface antifouling of marine ships, structures (docks, drilling platforms, piers and piles) and the like and corrosion and antifouling of key parts such as an observation window of underwater equipment, and can also provide technical support for the preparation of enzyme-based antifouling coating materials; the preparation method is simple and feasible, the used raw materials are cheap and easy to obtain, the preparation cost is greatly reduced, and the silica sol inorganic coating immobilized antifouling enzyme has better transparency and good anticorrosion and antifouling properties, and has wide application prospects in surface antifouling and anticorrosion and antifouling in marine environments.
Description of the drawings:
FIG. 1 is a block diagram of the process flow of the present invention.
The specific implementation mode is as follows:
the invention is further described by way of example with reference to the accompanying drawings.
Example 1:
the technological process of the method for immobilizing the antifouling enzyme on the surface of the inorganic coating comprises three steps of preparing the organic coating, cross-linking treatment and immobilizing the antifouling enzyme:
preparing an organic coating: adding tetraethoxysilane into silica sol with the pH value of 4.0, hydrolyzing for 1h at 45 ℃ to form a mixed solution, adding diethylenetriaminopropyltrimethoxysilane into the mixed solution, uniformly mixing, coating on a film coating sheet, and forming an organic coating on the film coating sheet;
the mass ratio of the silica sol to the tetraethoxysilane is 0.8, and the mass of the diethylenetriaminopropyltrimethoxysilane accounts for 0.1 percent of the mass of the mixed solution;
(II) crosslinking treatment: adding glutaraldehyde into a phosphate buffer solution with the pH value of 7 to prepare a glutaraldehyde solution with the mass percentage concentration of 5%, immersing the coated membrane into the glutaraldehyde solution, performing glutaraldehyde crosslinking treatment for 5 hours, then cleaning the coated membrane for 3 times by using the phosphate buffer solution with the pH value of 7, removing residual glutaraldehyde, and finishing the crosslinking treatment;
(III) immobilized antifouling enzyme: adding protease into phosphate buffer solution with the pH value of 7 to prepare protease solution with the mass percentage concentration of 20%, immersing the cross-linked coating film sheet obtained in the step (II) into the protease solution, soaking for 5h at 5 ℃, then cleaning the coating film sheet for 3 times by using the phosphate buffer solution with the pH value of 7, and freeze-drying the cleaned sample in a vacuum freeze-drying machine to finish the immobilization of the antifouling enzyme on the surface of the organic coating.
Example 2:
the technological process of the method for immobilizing the antifouling enzyme on the surface of the inorganic coating comprises three steps of preparing the organic coating, cross-linking treatment and immobilizing the antifouling enzyme:
preparing an organic coating: adding methyltriethoxysilane into silica sol with pH value of 5.0, hydrolyzing at 50 deg.C for 2h to form mixed solution, adding (3-aminopropyl) trimethoxysilane into the mixed solution, mixing uniformly, coating on a film coating sheet, and forming an organic coating on the film coating sheet;
the mass ratio of the silica sol to the methyltriethoxysilane is 1.3, and the mass of the (3-aminopropyl) trimethoxy silane accounts for 3.0 percent of the mass of the mixed solution;
(II) crosslinking treatment: adding glutaraldehyde into a phosphate buffer solution with the pH value of 7 to prepare a glutaraldehyde solution with the mass percentage concentration of 5%, immersing the coated membrane into the glutaraldehyde solution, performing glutaraldehyde crosslinking treatment for 5 hours, then cleaning the coated membrane for 3 times by using the phosphate buffer solution with the pH value of 7, removing residual glutaraldehyde, and finishing the crosslinking treatment;
(III) immobilized antifouling enzyme: adding protease into phosphate buffer solution with the pH value of 7 to prepare protease solution with the mass percentage concentration of 10%, immersing the cross-linked coating film sheet obtained in the step (II) into the protease solution, soaking for 5h at 10 ℃, then cleaning the coating film sheet for 3 times by using the phosphate buffer solution with the pH value of 7, and freeze-drying the cleaned sample in a vacuum freeze-drying machine to finish the immobilization of the antifouling enzyme on the surface of the organic coating.
Example 3:
the technological process of the method for immobilizing the antifouling enzyme on the surface of the inorganic coating comprises three steps of preparing the organic coating, cross-linking treatment and immobilizing the antifouling enzyme:
preparing an organic coating: adding methyltrimethoxysilane into silica sol with the pH value of 5.0, hydrolyzing at 60 ℃ for 0.5h to form a mixed solution, adding (3-aminopropyl) triethoxysilane into the mixed solution, uniformly mixing, coating on a film coating sheet, and forming an organic coating on the film coating sheet;
the mass ratio of the silica sol to the methyltrimethoxysilane is 1.0, and the mass of the (3-aminopropyl) triethoxysilane accounts for 5.0 percent of the mass of the mixed solution;
(II) crosslinking treatment: adding glutaraldehyde into a phosphate buffer solution with the pH value of 7 to prepare a glutaraldehyde solution with the mass percentage concentration of 5%, immersing the coated membrane into the glutaraldehyde solution, performing glutaraldehyde crosslinking treatment for 5 hours, then cleaning the coated membrane for 3 times by using the phosphate buffer solution with the pH value of 7, removing residual glutaraldehyde, and finishing the crosslinking treatment;
(III) immobilized antifouling enzyme: adding protease into phosphate buffer solution with the pH value of 7 to prepare protease solution with the mass percentage concentration of 5%, immersing the cross-linked coating film sheet obtained in the step (II) into the protease solution, soaking for 5h at 30 ℃, then cleaning the coating film sheet for 3 times by using the phosphate buffer solution with the pH value of 7, and freeze-drying the cleaned sample in a vacuum freeze-drying machine to finish the immobilization of the antifouling enzyme on the surface of the organic coating. The result of the corrosion resistance test of the immobilized antifouling enzyme prepared by the embodiment by the neutral salt spray method is that no obvious corrosion exists within 1000 h.
In the test for the diatom adhesion inhibition performance of the immobilized antifouling enzyme prepared in this example, a slide glass without protease was used as a control blank, and the concentration of benthic diatoms was diluted to 1.0X 10 with sterilized seawater6Piece/ml, the amount of diatoms attached to the surface of control blank slides after 2 hours was 144 pieces/mm2The attachment amount of diatom on the surface of the immobilized antifouling enzyme glass slide is 31/cm2The test result shows that the immobilized protease has good inhibition effect on the adhesion of the diatom.
Claims (3)
1. The method for immobilizing the antifouling enzyme on the surface of the inorganic coating is characterized in that the process comprises three steps of preparing the organic coating, cross-linking treatment and immobilizing the antifouling enzyme:
preparing an organic coating: adding alkoxy silane into silica sol with the pH value of 4.0-5.0, hydrolyzing at 45-60 ℃ for 0.5-2h to form a mixed solution, adding amino alkoxy silane into the mixed solution, uniformly mixing, coating on a film coating sheet, and forming an organic coating on the film coating sheet;
the mass ratio of the silica sol to the alkoxy silane is 0.8-1.3, and the mass of the amino alkoxy silane accounts for 0.1-5.0% of the mass of the mixed solution;
(II) crosslinking treatment: adding glutaraldehyde into a phosphate buffer solution with the pH value of 7 to prepare a glutaraldehyde solution with the mass percentage concentration of 5%, immersing the coated membrane into the glutaraldehyde solution, performing glutaraldehyde crosslinking treatment for 5 hours, then cleaning the coated membrane for 3 times by using the phosphate buffer solution with the pH value of 7, removing residual glutaraldehyde, and finishing the crosslinking treatment;
(III) immobilized antifouling enzyme: adding the antifouling enzyme into a phosphate buffer solution with the pH value of 7 to prepare an antifouling enzyme solution with the mass percentage concentration of 5-20%, immersing the coated membrane subjected to the crosslinking treatment in the step (II) into the antifouling enzyme solution, soaking for 5 hours at 5-30 ℃, then cleaning the coated membrane for 3 times by using the phosphate buffer solution with the pH value of 7, and placing the cleaned sample in a vacuum freeze dryer for freeze drying to complete the immobilization of the antifouling enzyme on the surface of the organic coating; the alkoxy silane is one or more of ethyl orthosilicate alkyl, methyl triethoxysilane and methyl trimethoxysilane.
2. The method for immobilizing the antifouling enzyme on the surface of the inorganic coating according to claim 1, wherein the aminoalkoxysilane is one or more selected from the group consisting of diethylenetriaminopropyltrimethoxysilane, (3-aminopropyl) trimethoxysilane, and (3-aminopropyl) triethoxysilane.
3. The method for immobilizing the antifouling enzyme on the surface of the inorganic coating according to claim 1, wherein the antifouling enzyme is one or more selected from the group consisting of protease, amylase, chitinase and lysozyme.
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