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Method for preparing immobilized laccase

A technology for immobilizing laccase and laccase, applied in directions such as being immobilized on/in an organic carrier, can solve problems such as difficult preparation and weak binding force, and achieve the effect of solving slow clarification

Inactive Publication Date: 2012-02-29
尉亚辉 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] Obviously, the physical adsorption method is easy to prepare and maintains high enzyme activity, but the binding force is weak; while the covalent cross-linking method has strong binding force, maintains moderate enzyme activity, but is difficult to prepare.

Method used

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  • Method for preparing immobilized laccase

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Embodiment 1

[0022] Step 1, with CuBr 2 As a catalyst, 2,2'-bipyridine as a ligand, ethyl 2-bromopropionate as a free initiator, and a porous sintered glass ring as a carrier to prepare a porous sintered glass with glycidyl methacrylate graft polymerization function ring, the specific process is as follows:

[0023] First take 5Kg of porous sintered glass ring and add 15L of dichloromethane solvent, then stir and vacuum degassing for 15 minutes, then add 0.5Kg of triethylamine and 1Kg of initiator 2-bromoisobutyryl bromide, and react at room temperature for 24 hours. After the reaction, the solid was separated by centrifugation, washed successively with dichloromethane, tetrahydrofuran and acetone solvents, and vacuum-dried at room temperature to obtain a porous sintered glass ring chemically bonded with the initiator 2-bromoisobutyryl bromide. Then take 2Kg of the porous sintered glass ring and add it to 5L of cyclohexanone solvent, stir and vacuum degassing for 15 minutes, then add 1.5K...

Embodiment 2

[0027] Step 1, with CuBr 2 As a catalyst, 2,2'-bipyridyl as a ligand, ethyl 2-bromopropionate as a free initiator, and a porous sintered glass ring as a carrier to prepare a porous sintered glass with glycidyl methacrylate graft polymerization function ring, the specific process is as follows:

[0028]First take 10Kg of porous sintered glass ring and add 20L of dichloromethane solvent, then stir and vacuum degassing for 15 minutes, then add 1.6Kg of 2-bromoisobutyryl bromide and 1Kg of triethylamine, react at room temperature for 24 hours, and the reaction is over Afterwards, the solid was separated by centrifugation, washed successively with dichloromethane, tetrahydrofuran and acetone solvents, and vacuum-dried at room temperature to obtain a porous sintered glass ring chemically bonded with the initiator 2-bromoisobutyryl bromide. Then take 5Kg of this porous sintered glass ring and add it to 10L cyclohexanone solvent, stir and vacuum degassing for 15 minutes, then add 3Kg...

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Abstract

The invention discloses a method for preparing immobilized laccase, comprising the following steps of: taking CuBr2 as a catalyst, taking 2,2'-bipyridine as a ligand, taking ethyl 2-bromopropanoate as a free initiator, and taking a porous sintering glass ring as a vector to prepare a porous sintering glass ring with the function of glycidyl methacrylate graft polymerization; and adding the poroussintering glass ring into a laccase solution, shaking or stirring for 16-24h, then adding a glutaraldehyde solution to crosslink for 1h, filtering, washing by pure water, removing unadsorbed free enzyme, and then centrifugally removing surface moisture to obtain the immobilized laccase adsorbed and crosslinked on the porous sintering glass ring. The invention combines a physical adsorption methodand a covalence-crosslinking method, draws the strong points to offset the weakness and optimizes coordination. The prepared immobilized laccase is suitable for synthesis of a resveratrol dimer and solves the post-turbidity problem caused by slow juice clarification and long-time storage in beverage processing.

Description

technical field [0001] The invention belongs to the technical field of immobilized enzyme preparation, in particular to a preparation method of immobilized laccase. Background technique [0002] Japanese scholar Tianyan Rokuro discovered an enzyme that can oxidize and harden "tree lacquer" in the sap of the sumac tree. Later, Bertrand (G.E.Bertrand, 1894) studied the enzyme in the lacquer produced in Southeast Asia in detail and named it laccase. Laccases are a type of phenol oxidase that oxidizes hydroquinone (hydroquinone) to p-benzoquinone with oxygen, also known as hydroquinone oxidase. Laccase is a protein that binds multiple copper ions, belongs to cerulocyanin oxidase, has a molecular weight of about 120,000, contains 4 atoms of copper, and can be inhibited by CN-. Laccase survives in the air, and the only product after the reaction is water, which is essentially an environmentally friendly enzyme. In recent years, with the gradual strengthening of environmental awa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/08
Inventor 尉亚辉马丁程亚刚郭斌
Owner 尉亚辉
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