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Carbon nano tube immobilized laccase and application thereof

A technology for immobilizing laccase and carbon nanotubes, which is applied in the direction of immobilization on or in inorganic carriers, can solve the problems of leakage of conductive polymers, increased cost, and lack of universality of laccase.

Active Publication Date: 2016-06-08
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved in the present invention is to overcome the limited effect of the existing carbon nanotube-immobilized laccase technology relying solely on carbon nanotubes to increase the electron transfer rate; conductive polymers can accelerate electron transfer, but lead to leakage of conductive polymers Risks, increased costs, unsatisfactory comprehensive effects, and significant modification of enzyme protein molecules, but the process is cumbersome, and it is not universally applicable to laccases from different sources. Provide a method based on direct electron transfer Carbon nanotube immobilized laccase

Method used

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  • Carbon nano tube immobilized laccase and application thereof
  • Carbon nano tube immobilized laccase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] (1) At room temperature (about 25°C), dissolve 2g of halosuccinimide in 20g of acetone, mix 0.4g of potassium carbonate, 1g of potassium metabisulfite, and 2g of carboxylated carbon nanotubes in 20g of water until Completely dissolve the inorganic salt, mix the acetone solution with the aqueous solution, stir at 20°C, 300r / min for 20min, add 160g of chloroform-water mixture (2:1, v / v) for extraction and separation, and take the organic phase after standing for layers , washing the organic phase three times with deionized water whose volume is 1 / 2 of the organic phase layer, and removing chloroform by rotary evaporating the washed organic phase layer at 30° C., to obtain carbon nanotubes grafted with succinimide groups;

[0063] (2) At 30°C and 100r / min, in a reactor equipped with a reflux device, add 2g of N,N-di(carboxymethyl)-L-lysine hydrate, 4g of triethylamine and 40g of diethylamine Methylformamide, heated to reflux for 120min, under the conditions of 30°C and 300...

Embodiment 2

[0070] (1) At room temperature (about 25°C), dissolve 2g of halosuccinimide in 100g of acetone, mix 6g of potassium carbonate, 10g of potassium metabisulfite, and 30g of carboxylated carbon nanotubes in 200g of water until the inorganic Dissolve the salt completely, mix the acetone solution and the aqueous solution, stir at 30°C, 1000r / min for 30min, add 600g of chloroform-water mixture (2:1, v / v) for extraction and separation, and take the organic phase layer after standing and separating. Washing the organic phase 5 times with deionized water whose volume is 1 / 2 of the organic phase layer, and removing the chloroform by rotary evaporating the washed organic phase layer at 45° C., to obtain carbon nanotubes grafted with succinimide groups;

[0071] (2) At 50°C and 300r / min, in a reactor equipped with a reflux device, add 2g of N,N-di(carboxymethyl)-L-lysine hydrate, 12g of triethylamine and 120g of diethylamine Methylformamide, heated to reflux for 80min, under the conditions...

Embodiment 3

[0078] (1) At room temperature (about 25°C), dissolve 2g of halosuccinimide in 30g of acetone, mix 1g of potassium carbonate, 2g of potassium metabisulfite, and 6g of carboxylated carbon nanotubes in 60g of water until the inorganic Dissolve the salt completely, mix the acetone solution with the aqueous solution, stir at 40°C, 550r / min for 40min, add 200g of chloroform-water mixture (2:1, v / v) for extraction and separation, and take the organic phase layer after standing for stratification. The organic phase was washed 4 times with deionized water having a volume of 1 / 2 of the organic phase, and the washed organic phase was evaporated at 40° C. to remove chloroform to obtain succinimide grafted carbon nanotubes;

[0079] (2) Under the conditions of 35°C and 250r / min, in a reactor equipped with a reflux device, add 2g of N,N-di(carboxymethyl)-L-lysine hydrate, 6g of triethylamine and 60g of diethylamine Methylformamide was heated to reflux for 90 minutes. Under the conditions o...

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Abstract

The invention discloses a carbon nano tube immobilized laccase, which is prepared by the following steps of carrying out grafting of succinimido and grafting of a nitrilotriacetic acid functional group on a carbon nano tube; carrying out surface functionalization treatment through copper ion chelating and modifying, and carrying out immobilized reaction with the laccase with copper ion removal. The laccase active center is directly connected with the copper ions of the carbon nano tube surface functional group, so that the real meaning direct electron transfer can be realized, and the shielding effect produced by a laccase protein shell on the direct electron transfer process from the active center to the carbon nano tube is overcome. The carbon nano tube immobilized laccase provided by the invention solves the problem of limitation of the electron transfer rate during the carbon nano tube immobilized laccase catalytic process, the sensitivity of the immobilized laccase in biological detection can be improved, the lowest detection limit during phenol determination is reduced by 85 percent to 96 percent, and the current density in biological fuel cell application is improved by 80 percent to 120 percent.

Description

technical field [0001] The invention relates to the field of immobilization of laccase, in particular to a carbon nanotube immobilized laccase which realizes direct electron transfer by connecting the active center of the laccase with the carbon nanotube and its application. Background technique [0002] Laccase (benzenediol: oxidoreductase, ECl.10.3.2) is a copper-containing polyphenol oxidase, which belongs to the same blue multi-copper oxidase as the ascorbate oxidase in plants and ceruloplasmin in mammals family. Laccase is a copper protein, which generally contains four copper ions. According to the spectral and magnetic characteristics, the copper ions in laccase can be divided into three types: type I Cu, type II Cu, and type III Cu; among them, type I Cu and One type II Cu is a single-electron acceptor, which is paramagnetic, and can be measured by electron paramagnetic resonance (EPR); 2 type III Cu is a two-electron acceptor, which is diamagnetic, and cannot be de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14
CPCC12N11/14
Inventor 王锋徐玲
Owner JIANGSU UNIV
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