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A kind of preparation method of bulk macroporous material immobilized laccase

A technology for immobilizing laccase and macroporous materials, which is applied in the direction of immobilization on or in the inorganic carrier, can solve the problems of poor separation effect, complicated preparation process, and high production cost, and achieve low cost, simple preparation process, and lacquer The effect of high recovery rate of enzyme activity

Inactive Publication Date: 2018-07-10
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing immobilized laccases generally have the disadvantages of high production cost, complicated preparation process, and poor separation effect.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] (1) Using cyclohexane as a solvent, a GPTMS solution with a volume percentage of 10% was prepared in a polytetrafluoroethylene lining of a pressure reactor. Weigh 1.0g bulk SiO 2 Dry the macroporous material at 80°C for 2 hours. After cooling, immerse in GPTMS solution, seal the pressure reactor, and react at 80°C for 24 hours. Rinse 2 to 3 times, then rinse repeatedly with deionized water, and dry to obtain epoxy-functionalized SiO 2 Macroporous material EMS.

[0014] (2) Weigh 0.1g of EMS, immerse in 10ml of laccase solution prepared with pH=4.5 buffer solution, at 20°C, the speed is 50r.min -1 Shake in a shaker for 5 hours, filter out with an 8-10 mesh screen, wash with buffer until no protein can be detected in the supernatant, and vacuum-dry to obtain the immobilized laccase.

Embodiment 2

[0016] (1) Using cyclohexane as a solvent, a GPTMS solution with a volume percentage of 20% was prepared in a polytetrafluoroethylene liner of a pressure reactor. Weigh 1.0g bulk SiO 2 Dry the macroporous material at 80°C for 2 hours. After cooling, immerse in GPTMS solution, seal the pressure reactor, and react at 120°C for 24 hours. Rinse 2 to 3 times, then rinse repeatedly with deionized water, and dry to obtain epoxy-functionalized SiO 2 Macroporous material EMS.

[0017] (2) Weigh 0.1g of EMS, immerse in 10ml and use pH=4.5 buffer solution to prepare a concentration of 25mg.ml -1 In the industrial grade laccase (Novozymes DeniLite IIS) solution, at 30°C, the rotation speed is 50r.min -1 Shake in a shaker for 4 hours, filter out, and wash with buffer until no protein can be detected in the supernatant to obtain immobilized laccase. Its enzyme activity is 112U.g -1 (Define the amount of enzyme needed to oxidize 1 μmol of ABTS in one minute as one enzyme activity unit U...

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PUM

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Abstract

The invention relates to a method for preparing immobilized laccase in block macroporous materials, and belongs to the technical field of immobilized enzyme preparation. The immobilized laccase is based on a bulk SiO2 macroporous material whose surface is functionalized with epoxy groups, and the laccase is fixed on the carrier through a covalent bonding method. The preparation method comprises two steps: preparation of carrier and immobilization of laccase. Preparation of the support: use γ‑(2,3 glycidoxy) propyltrimethoxysilane (GPTMS) as the silane coupling agent, and graft reaction on the inner surface of the SiO2 macroporous material through GPTMS under solvothermal reaction conditions , to prepare an epoxy-functionalized carrier that can be covalently bonded to laccase. Immobilization of laccase: immerse the carrier in laccase solution, shake and react at room temperature, wash and dry to obtain the finished product of immobilized laccase. The invention provides a novel immobilized laccase, which has the characteristics of simple preparation process, low cost, high activity of the immobilized laccase and the like.

Description

technical field [0001] The invention belongs to the technical field of immobilized enzyme preparation, in particular to a bulk SiO 2 Preparation method of immobilized laccase based on macroporous material. Background technique [0002] Laccase is a copper-containing polyphenol oxidase, which belongs to the cerulus oxidase protein, and widely exists in nature. According to its main source, it can be divided into two categories: sumac laccase and fungal laccase. Laccase has broad substrate specificity and good stability. It has important application value in the control of organic pollutants in water, the construction of biosensors, biopulping, food processing, and organic synthesis. Therefore, laccase has always been a very active research hotspot in the fields of biology, chemistry and environmental science, and has attracted widespread attention of researchers at home and abroad. [0003] Laccase can catalyze the oxidation of a variety of refractory organic pollutants, in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/14
Inventor 梁云霄刘晓贞张瑞丰张育淇李云郑璐梦江思
Owner NINGBO UNIV