阅读说明：本技术 一种双亲性酶固定化载体 (Amphiphilic enzyme immobilization carrier ) 是由 朱珣之 高婷 黄犀 罗会婷 王仲伟 汤诗杰 于 2019-12-09 设计创作，主要内容包括：本发明公开了一种双亲性酶固定化载体,该双亲性酶固定化载体由下述步骤制得：取天然植物纤维材料和聚偏氟乙烯,分别干燥备用；配制离子液体溶液；将天然植物纤维材料和聚偏氟乙烯加入离子液体溶液中,然后加入仿酶,加热搅拌处理或者气爆处理,再加入修饰剂搅拌均匀,洗涤、干燥、真空脱气,得到双亲性的载体；对所得载体依次进行生物处理及抗菌修饰,产物经洗涤、干燥、真空脱气,得到双亲性酶固定化载体。本发明的双亲性酶固定化载体兼具亲油和亲水的性能,载体性质稳定性好、酶吸附率高,而且,该酶固定化载体重复使用多次,仍能保持较高的酶活性。(The invention discloses an amphiphilic enzyme immobilization carrier, which is prepared by the following steps: respectively drying natural plant fiber materials and polyvinylidene fluoride for later use; preparing an ionic liquid solution; adding natural plant fiber material and polyvinylidene fluoride into ionic liquid solution, then adding mimic enzyme, heating and stirring or air explosion treatment, adding a modifier, uniformly stirring, washing, drying and vacuum degassing to obtain an amphiphilic carrier; and sequentially carrying out biological treatment and antibacterial modification on the obtained carrier, and washing, drying and vacuum degassing the product to obtain the amphiphilic enzyme immobilized carrier. The amphiphilic enzyme immobilized carrier has both oleophilic and hydrophilic performances, good stability of carrier property and high enzyme adsorption rate, and can still maintain higher enzyme activity after being repeatedly used for many times.)

1. An amphiphilic enzyme immobilization carrier, which is characterized by being prepared by the following method:

(1) respectively drying natural plant fiber materials and polyvinylidene fluoride for later use;

(2) preparing an ionic liquid solution;

(3) adding the natural plant fiber material obtained in the step (1) and polyvinylidene fluoride into an ionic liquid solution, then adding a mimic enzyme, heating and stirring or gas explosion treatment, then adding a modifier, uniformly stirring, washing, drying and vacuum degassing to obtain an amphiphilic carrier;

(4) carrying out biological treatment on the carrier obtained in the step (3) by adopting bacteria;

(5) and (4) carrying out antibacterial modification on the carrier obtained in the step (4), and washing, drying and vacuum degassing the product to obtain the amphiphilic enzyme immobilized carrier.

2. The amphiphilic enzyme immobilization carrier according to claim 1, wherein in step (1), the natural plant fiber material is a mixture of lignin and cellulose, and the mass ratio of lignin to cellulose is 1: 1-6.

3. The amphiphilic enzyme-immobilized carrier according to claim 1, wherein in step (2), the ionic liquid solution is a mixed solution of an ionic liquid and a dimethylsulfoxide solution or N, N-dimethylformamide, wherein the ionic liquid is at least one of 1-ethyl-3-methylimidazole hydrogensulfate, 1-ethyl-3-methylimidazole diethyl phosphate, 1-methyl-3-methylimidazole dimethyl phosphate, 1-methyl-3-methylimidazole diethyl phosphate, 1-ethyl-3-methylimidazole dimethyl phosphate, 1-butyl-3-methylimidazole chloride, and 1-butyl-3-methylpyridine chloride; the mass of the dimethyl sulfoxide solution or the N, N-dimethylformamide solution is 2-8% of that of the ionic liquid.

4. The amphiphilic enzyme immobilization carrier according to claim 1, wherein in step (3), the mass ratio of the natural plant fiber material to the polyvinylidene fluoride is 1: 1-5: 7, and the mass ratio of the natural plant fiber material to the ionic liquid is 1: 15-20.

5. The amphiphilic enzyme immobilized carrier of claim 1, wherein in step (3), the mimic enzyme is any one or a mixture of two of Fe-CA, TPPFe and Fe-EDTA, and the ionic liquid solution after the mimic enzyme is added has a final concentration of 8-30 mmol-kg^-1(ii) a Adding a dummyHeating and stirring treatment at 130-150 ℃ or gas explosion treatment at 100-130 ℃ after the enzyme treatment, wherein the treatment time is 10-30 min.

6. The amphiphilic enzyme immobilized carrier of claim 1, wherein in step (3), the modifier is genipin, tris (hydroxymethyl) phosphonium, maleic anhydride or carbodiimide, and the final concentration of the solution after the modifier is added is 0.05-0.2M.

7. The amphiphilic enzyme immobilization vector according to claim 1, wherein in step (4), the bacteria are Bacillus subtilis and/or Bacillus amyloliquefaciens.

8. The amphiphilic enzyme immobilization carrier according to claim 1, wherein in step (5), the antibacterial modification method is: adding the carrier obtained in the step (4) into an antibacterial reagent, and carrying out oil bath heating reaction; wherein the antibacterial agent is a mixture of chitosan and silver or titanium dioxide.

Technical Field

The invention relates to an amphiphilic enzyme immobilization carrier, and belongs to the technical field of oil-water amphiphilic materials.

Background

The lipase belongs to carboxyl ester hydrolase, is a special ester bond catalytic enzyme and has biological activity. In the water phase or the organic phase, lipase can catalyze various reactions such as hydrolysis, alcoholysis, esterification, ester exchange and the like. An important property of lipases is that they act only in heterogeneous systems, i.e.at the water-insoluble lipid-water interface, and have no or slow action on homogeneously dispersed and water-soluble substrates. Therefore, the activity and the stability in an oil-water interface or an oil-water amphiphilic material are good.

Lipases are widely used in the fields of tanning, medicine, food processing, and the like. However, the higher structure of the lipase protein is susceptible to environmental influences, and there are many problems such as separation, purification, and recovery. By immobilizing lipase on a carrier material, the catalytic performance of lipase can be greatly improved within a limited micro-area range. However, since the price of the lipase is expensive, the free lipase is generally easily soluble in water, can aggregate in organic solvents, is extremely unstable in nature, easily loses its own activity in the using process and is not easily separated after use, which greatly limits the application of the lipase in large-scale industrial production to a certain extent.

Compared to free lipase, immobilized lipase has: (1) good stability, repeated use, easy control of reaction conditions and low production cost; (2) the immobilized lipase is easy to separate from a substrate and a product, and the product is easy to purify; (3) the immobilized lipase is more suitable for a multi-enzyme reaction system. At present, the research on the immobilized lipase not only has important value on the continuity and automation of industrial production, but also has important significance in the basic theoretical research of biology, clinical medicine and the promotion of the development of other biochemical technologies.

The carrier property can obviously affect the enzyme loading capacity, the enzyme activity and the enzyme loss rate. At present, a lot of carriers are used for immobilized enzymes, wherein silicon dioxide has the characteristics of low cost, acid and alkali resistance, long service life, no toxicity to microorganisms, difficulty in decomposition by microorganisms and the like, and has a large specific surface area and a large protein adsorption capacity, so that the silicon dioxide is an important carrier material. However, silica supports are inert supports and generally have both hydrophilic and lipophilic properties. The polyvinylidene fluoride carrier has good lipophilicity but poor hydrophilicity, and is not beneficial to keeping the activity of the enzyme because water is beneficial to keeping the activity of the enzyme. There is also a carrier prepared by mixing cellulose with polyvinylidene fluoride, the cellulose surface has hydroxyl groups, which is helpful for keeping enzyme activity, but too many hydroxyl groups are not beneficial for enzyme immobilization. In summary, no ideal amphiphilic enzyme immobilization carrier exists at present.

Disclosure of Invention

The purpose of the invention is as follows: in order to overcome the problems of the existing enzyme immobilization carrier, the invention provides an amphiphilic enzyme immobilization carrier which has excellent oleophilic and hydrophilic properties.

The technical scheme is as follows: the invention relates to an amphiphilic enzyme immobilization carrier, which is prepared by the following method:

(1) respectively drying natural plant fiber materials and polyvinylidene fluoride for later use;

(2) preparing an ionic liquid solution;

(3) adding the natural plant fiber material obtained in the step (1) and polyvinylidene fluoride into an ionic liquid solution, then adding a mimic enzyme, heating and stirring or gas explosion treatment, then adding a modifier, uniformly stirring, washing, drying and vacuum degassing to obtain an amphiphilic carrier;

(4) carrying out biological treatment on the carrier obtained in the step (3) by adopting bacteria;

(5) and (4) carrying out antibacterial modification on the carrier obtained in the step (4), and washing, drying and vacuum degassing the product to obtain the amphiphilic enzyme immobilized carrier.

In the step (1), the natural plant fiber material is preferably a mixture of lignin and cellulose, wherein the mass ratio of the lignin to the cellulose is 1: 1-6.

In the step (2), the ionic liquid solution is preferably a mixed solution of an ionic liquid and a dimethyl sulfoxide solution or N, N-dimethylformamide, wherein the ionic liquid can be at least one of 1-ethyl-3-methylimidazole hydrogen sulfate, 1-ethyl-3-methylimidazole diethyl phosphate, 1-methyl-3-methylimidazole dimethyl phosphate, 1-methyl-3-methylimidazole diethyl phosphate, 1-ethyl-3-methylimidazole dimethyl phosphate, 1-butyl-3-methylimidazole chloride and 1-butyl-3-methylpyridine chloride; the mass of the dimethyl sulfoxide solution or the N, N-dimethylformamide solution is 2-8% of that of the ionic liquid.

In the step (3), the mass ratio of the natural plant fiber material to the polyvinylidene fluoride is preferably 1: 1-5: 7, and the mass ratio of the natural plant fiber material to the ionic liquid is preferably 1: 15-20. The mimic enzyme can be any one or a mixture of two of Fe-CA, TPPFe and Fe-EDTA, and the final concentration of the ionic liquid solution after the mimic enzyme is added is preferably controlled to be 8-30 mmol/kg^-1(ii) a Further, after adding the mimic enzyme, heating and stirring treatment at 130-150 ℃ or gas explosion treatment at 100-130 ℃ for 10-30 min. The modifier can be genipin, trihydroxymethyl phosphorus, maleic anhydride or carbodiimide, and the final concentration of the solution after the modifier is added is preferably 0.05-0.2M.

In the step (4), the bacteria used for biological treatment can be bacillus subtilis and/or bacillus amyloliquefaciens.

In the step (5), the antibacterial modification method specifically comprises the following steps: adding the carrier obtained in the step (4) into an antibacterial reagent, and carrying out oil bath heating reaction; among them, the antibacterial agent is preferably a mixture of chitosan and silver or titanium dioxide.

The invention principle is as follows: according to the method, natural plant fiber materials (lignin and cellulose) and polyvinylidene fluoride are used as raw materials, ionic liquid is used as a reaction medium, enzyme imitation and gas explosion treatment are carried out on a carrier, in the treatment process, a polymerization structure in lignin molecules is partially destroyed, a large number of free groups such as carboxyl, aldehyde, hydroxyl, amino and the like are exposed on the surface, after the treatment is completed, the number of the carboxyl, aldehyde, hydroxyl and amino on the surface is increased by 2-3 times, the free groups can provide various active groups for immobilized enzymes, the enzyme activity is favorably maintained, meanwhile, the cellulose is broken into short fibers by long fibers, the free hydroxyl is increased, the mixing uniformity with the polyvinylidene fluoride is better, the performance of the carrier is better, and the carrier is more uniform; then, by further biological treatment, the biocompatibility of the carrier can be improved, and biologically incompatible substances, such as toxic substances generated by enzyme imitation (gas explosion) treatment, can be destroyed, and meanwhile, the surface treatment effect on cellulose components can be achieved, and the roughness of the carrier can be increased. The treated carrier surface has more laminated structures, protruding structures and channel structures, the utilization rate of the carrier surface is greatly improved, the enzyme can be fixed in gaps inside the carrier and also can be fixed on the surface of the carrier, and the enzyme protein can be well protected, so that the stability, the reusability and the stability in an organic phase can be obviously improved.

Has the advantages that: compared with the prior art, the invention has the advantages that: (1) the amphiphilic enzyme immobilized carrier has both oleophilic and hydrophilic performances, the carrier has good property stability and high enzyme adsorption rate, and the utilization rate of the surface of the carrier can reach 60-95%; moreover, the enzyme immobilization carrier can be repeatedly used for many times, and still can keep higher enzyme activity; (2) in the preparation method, the ionic liquid is adopted to treat the lignocellulose material and the polyvinylidene fluoride, the operation condition in the treatment process is mild, the method is green and environment-friendly, pollution-free and simple in treatment process, the treated ionic liquid can be completely recovered, and the production cost of the carrier can be reduced; moreover, the antibacterial substance is added in the preparation process, so that the pollution problem of the carrier is solved.

Drawings

FIG. 1 is a flowchart showing the preparation of an amphiphilic enzyme-immobilized carrier according to the present invention;

FIG. 2 is a scanning electron micrograph of the resulting carrier after biological treatment in example 1;

FIG. 3 is a scanning electron micrograph of the vector obtained after antibacterial modification in example 1;

FIG. 4 is the reusability of immobilized enzyme to catalyze transesterification of ethyl acetate and butanol;

fig. 5 shows reusability of immobilized enzyme in catalyzing esterification reaction of rapeseed oil.

Detailed Description

The technical scheme of the invention is further explained by combining the attached drawings.

The amphiphilic enzyme immobilized carrier has oleophylic and hydrophilic performances, the surface of the carrier has more laminated structures, protruding structures and channel structures, the utilization rate of the surface of the carrier can reach 60-95%, enzyme can be fixed in gaps inside the carrier and also can be fixed on the surface of the carrier, and enzyme protein can be well protected, so that the stability, the reusability and the stability in an organic phase are greatly improved.

The amphiphilic enzyme immobilization carrier is prepared by the following method, as shown in figure 1, and comprises the following steps:

(1) respectively drying natural plant fiber materials and polyvinylidene fluoride for later use;

after drying, the raw materials can be further processed by adopting the modes of crushing, ball milling and the like to obtain powdery raw materials for later use;

(2) preparing an ionic liquid solution;

(3) performing enzyme imitation treatment in ionic liquid: adding the natural plant fiber material treated in the step (1) and polyvinylidene fluoride powder into an ionic liquid solution, then adding a mimic enzyme, heating and stirring or air-blasting, adding a modifier, uniformly stirring, washing, drying and vacuum degassing to obtain an amphiphilic carrier;

(4) biological treatment: carrying out biological treatment on the carrier obtained in the step (3) by adopting bacteria;

(5) antibacterial modification: and (4) adding the carrier obtained in the step (4) into an antibacterial agent for antibacterial modification, washing, drying and vacuum degassing the product to obtain the amphiphilic enzyme immobilized carrier.