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Preparation method of blocked macroporous material immobilized laccase

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

Inactive Publication Date: 2015-06-10
NINGBO UNIV
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  • Summary
  • Abstract
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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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Abstract

The invention relates to a preparation method of blocked macroporous material immobilized laccase, and belongs to the technical field of the preparation of immobilized enzyme. The immobilized laccase adopts a blocked SiO2 macroporous material with surface functionalized by virtue of an epoxy group as a carrier, and the laccase is immobilized on the carrier by virtue of a covalent binding method. The preparation method comprises the following two steps: preparation of the carrier and immobilization of the laccase. During the preparation of the carrier, gamma-(2,3-epoxy propoxy)propyl-trimethoxysilane (GPTMS) is adopted as a silane coupling agent, and the GPTMS has grafting reaction on the inner surface of a pore of the SiO2 macroporous material under the solvent thermal reaction condition to prepare the epoxy-group functionalized carrier which can be covalently bound with the laccase. During the immobilization of the laccase, the carrier is immersed in a laccase solution, the solution is vibrated at a room temperature to have the reaction, and a reaction product is washed and dried to obtain the immobilized laccase finished product. The novel immobilized laccase has the characteristics that the preparation process is simple, the cost is low, the activity of the immobilized laccase is high, 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 Applications(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
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