Preparation method of glass immobilized enzyme with biological activity

A technology of bioactive glass and immobilized enzymes, applied in the direction of fixing on or in inorganic carriers, fixing on/in organic carriers, etc., can solve the problem that the carrier structure is difficult to control, affects catalytic performance and application, and enzyme activity and the reduction of enzyme catalytic efficiency, to avoid product pollution and side effects, improve the immobilization performance of loaded enzymes, and prevent the leakage of enzyme molecules

Inactive Publication Date: 2014-08-20
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The carrier materials used for enzyme immobilization were originally natural polymer materials, such as resins, gels and fibers, etc., but these materials had disadvantages such as low reusability, difficult cleaning, low mechanical strength, poor thermal and chemical stability; Developed to the use of inorganic materials and composite materials, especially porous inorganic carrier materials usually have high enzyme loading capacity, high initial activity of enzyme immobilization, good thermal and chemical stability, high mechanical strength, low cost, and reusable and other advantages, so it has attracted great attention. For example, Luo Wen et al. used porous glass beads as a carrier to immobilize lipase by covalent method, and compared the optimum reaction temperature and pH value of immobilized enzyme and free enzyme. and thermal stability, and using the prepared immobilized enzyme as a catalyst, rapeseed oil was used to synthesize biodiesel in a micro-water system, and the influence of solvent amount, system water content, methanol and other factors on the catalytic performance of the immobilized enzyme was investigated. The operational stability of the immobilized enzyme was studied; Wang Baokang et al. made two kinds of porous glass microbead carriers by phase separation method and filling method, and immobilized α-amylase by physical adsorption method, covalent coupling method and diazo method respectively. , after comparing their immobilization effects, the covalent coupling method was selected to conduct a more detailed study; the optimal immobilization conditions, the best application conditions and the properties of the immobilized enzyme were determined for the covalently coupled α-amylase and studied the impact of porous glass microbeads production conditions on immobilized enzymes and the immobilization effect of porous glass microbeads on catalase and glutamate decarboxylase; patent document CN101451133B (200910044820.X) discloses a method using A method for preparing an immobilized enzyme biocatalyst of silicon-based mesoporous molecular sieve SBA-15. The method uses silicon-based mesoporous molecular sieve SBA-15 as a carrier, horseradish peroxidase as an immobilized object, and the adsorption method is an immobilization method. The property of mesoporous molecular sieve and the characteristic that the pore size of SBA-15 matches the molecular size of horseradish peroxidase realizes simultaneous immobilization of enzyme on the pores and outer surface of silicon-based mesoporous molecular sieve SBA-15; patent document CN101423829A (200810160777.9) discloses a calcium-enriched fly ash microporous glass carrier for immobilized enzymes and a preparation method thereof. The polyurethane foam material is used as a substrate, impregnated with calcium-enriched fly ash slurry, and heat-treated after drying to obtain an immobilized enzyme. Calcium-enriched fly ash porous glass carrier for enzyme
[0003] The above-mentioned documents use inorganic materials such as porous silicate glass, borosilicate glass or silicon, which need to be immobilized by surface modification. Although they have the advantages of recyclable reuse and low production cost, due to the biological activity and relative Defects such as poor compatibility, difficult control of the carrier structure, and poor enzyme binding ability reduce the enzyme activity and catalytic efficiency, and the enzyme is easy to desorb, leak and inactivate; in addition, the surface modification materials are easy to pollute or be toxic to the product Side effects also seriously affect its catalytic performance and application

Method used

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  • Preparation method of glass immobilized enzyme with biological activity
  • Preparation method of glass immobilized enzyme with biological activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Take 3g of Lactobacillus salivarius and dissolve it in 80ml of MRS medium solution, cultivate and emulsify it for 17 hours under the conditions of 42°C and pH value of 6, and the cell concentration in the control solution is 7.9×10 6 unit / ml, it is emulsion A.

[0031] 50mol SiO in molar ratio 2 -4mol P 2 o 5 -31mol CaO-15mol ZnO bioactive glass composition, the precursor raw materials ethyl orthosilicate 11.2ml, triethyl phosphate 1.36ml, calcium nitrate tetrahydrate 7.32g, zinc nitrate hexahydrate 4.46g were added to emulsification In solution A, stir for 30 minutes between each addition of a raw material to form precursor sol solution B.

[0032] Put the precursor sol solution B in a sealed container for hydrothermal aging at 70oC for 48h to obtain a wet gel, then dry it at 80°C for 18h to obtain a dry gel; grind it and heat-treat it at 700oC for 3h to obtain a bioactive glass carrier Material, white powder; the material has a porous structure with a macropore diam...

Embodiment 2

[0034] Take 1 g of Lactobacillus salivarius and dissolve it in 50 ml of MRS medium solution, culture and emulsify it for 12 hours under the conditions of 32°C and pH value of 7, and control the cell concentration in the solution to be 7.0×10 6 unit / ml, it is emulsion A.

[0035] 50mol SiO in molar ratio 2 -4mol P 2 o 5 -31mol CaO-15mol ZnO bioactive glass composition, the precursor raw materials ethyl orthosilicate 11.2ml, triethyl phosphate 1.36ml, calcium nitrate tetrahydrate 7.32g, zinc nitrate hexahydrate 4.46g were added to emulsification In solution A, stir for 30 minutes between each addition of a raw material to form precursor sol solution B.

[0036] Put the precursor sol solution B in a sealed container and age at 60oC for 72h to obtain a wet gel, then dry it at 60oC for 24h to obtain a dry gel; grind it and heat-treat it at 600oC for 5h to obtain a white bioactive glass Powder; the powder has a porous structure with a macropore diameter of 80-150 nm and a mesopo...

Embodiment 3

[0038] Take 5 g of Lactobacillus salivarius and dissolve it in 100 ml of MRS medium solution, cultivate and emulsify it for 24 hours under the conditions of 58° C. and pH value of 4, and control the cell concentration in the solution to be 10.0×10 6 unit / ml, it is emulsion A.

[0039] 50mol SiO in molar ratio 2 -4mol P 2 o 5 -31mol CaO-15mol ZnO bioactive glass composition, the precursor raw materials ethyl orthosilicate 11.2ml, triethyl phosphate 1.36ml, calcium nitrate tetrahydrate 7.32g, zinc nitrate hexahydrate 4.46g were added to emulsification In solution A, stir for 30 minutes between each addition of a raw material to form precursor sol solution B.

[0040] Put the precursor sol solution B in a sealed container and age at 80oC for 12h to obtain a wet gel, then dry it at 100oC for 12h to obtain a dry gel; grind it and heat-treat it at 800oC for 2h to obtain a white bioactive glass Powder; the powder has a porous structure, the diameter of the macropore is 120-240nm,...

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Abstract

The invention relates to a preparation method of glass immobilized enzyme with a biological activity. The method comprises the following steps of: preparing a microbial emulsion by lactobacillus salivarius and an MRS culture medium; mixing the microbial emulsion, a silicon source, a phosphorus source, calcium salt and MO salt to sol; hydrothermally aging to form gel; drying to obtain xerogel; thermally treating to obtain a porous glass carrier material with biological activity. Enzyme in the duct is directly bonded and fixed with surface by the characteristics of biological activity and compatibility of the porous glass carrier material with biological activity, excellent surface chemical performance, adjustable multi-pore structure and enzyme molecular size matched so as to obtain a porous glass immobilized enzyme biological catalyst with biological activity. The method has the characteristics of high enzyme load, high enzyme activity, good thermal and chemical stabilities, repeated use, no pollution to products and the like, and can be used to the fields of fine chemical industry, food industry, biological chemicals, wastewater treatment, medicines and the like.

Description

(1) Technical field [0001] The invention relates to a method for preparing biologically active glass-immobilized enzymes, belonging to the technical field of biological materials. (2) Background technology [0002] The new technology of enzyme immobilization has broad application prospects in fine chemical industry, food industry, chemical analysis, wastewater treatment, especially chiral compounds, biosensors and medicine. The carrier materials used for enzyme immobilization were originally natural polymer materials, such as resins, gels and fibers, etc., but these materials had disadvantages such as low reusability, difficult cleaning, low mechanical strength, poor thermal and chemical stability; Developed to the use of inorganic materials and composite materials, especially porous inorganic carrier materials usually have high enzyme loading capacity, high initial activity of enzyme immobilization, good thermal and chemical stability, high mechanical strength, low cost, an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/14C12N11/04
Inventor 何文张旭东闵丹丹
Owner QILU UNIV OF TECH
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