Magnetic cross-linked enzyme aggregate, preparation method and application thereof

A technology of enzyme aggregates and cross-linking enzymes, applied in directions such as immobilization on or in inorganic carriers, immobilization on/in organic carriers, etc., can solve problems such as instability of Schiff bases and cannot be effectively solved, and achieve Good cross-linking effect, improved stability, high catalytic efficiency

Inactive Publication Date: 2014-07-23
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The magnetically cross-linked enzyme aggregates prepared by the above method can be magnetically separated, which not only effectively solves the problem of agglomeration caused by separation from the reaction system by means of centrifugation and filtration, but also ensures that the enzyme can be fully cross-linked and improves its stability. , so that it can be reused many times, but the unstable problem of the generated Schiff base cannot be effectively solved

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] (1) Weigh 200mg of lipase (based on protein content) and place it in 40mL of 100mM phosphate buffer, stir magnetically at room temperature for 20min to fully dissolve the lipase and form a homogeneous solution, then place it at room temperature at 150rpm Oscillate in a shaker at a high speed for 5 hours;

[0047] (2) Weigh 20 mg self-made amine functionalized magnetic nanoparticles (Fe 3 o 4 -PEI) (Refer to the published literature Hu, Y.R., Guo, C., Wang, F., Wang, S.K., Pan, F., Liu, C.Z., 2013. Improvement of microalgae harvesting by magnetic nanocomposites coated with polyethyleneimine for the preparation method .Chem.Eng.J.242,341-347.) placed in a conical flask containing 5mL of 100mM phosphate buffer solution with pH=8, ultrasonicated in an ice bath for 25min under ultrasonic conditions with a frequency of 40KHz and a power of 240w, the dispersion was even ;

[0048] (3) Pour (1) into the dispersed magnetic particle system prepared in (2), shake at room temper...

Embodiment 2

[0053] (1) Weigh 200 mg of lipase (based on protein content) and place it in 40 mL of 100 mM phosphate buffer, stir magnetically for 20 min at room temperature to fully dissolve the lipase, add 1.5 g of 1-hydroxyethyl-3-methylimidazole tetra Fluoroborate, low-temperature ultrasonic to make it evenly dispersed, after forming a homogeneous solution, place it in a shaker at room temperature and 150 rpm for 4 hours;

[0054] (2) Weigh 20 mg self-made amine functionalized magnetic nanoparticles (Fe 3 o 4 -PEI) (Refer to the published literature Hu, Y.R., Guo, C., Wang, F., Wang, S.K., Pan, F., Liu, C.Z., 2013. Improvement of microalgae harvesting by magnetic nanocomposites coated with polyethyleneimine for the preparation method .Chem.Eng.J.242,341-347.) placed in a conical flask filled with 5mL of 100mM phosphate buffer solution with pH=8, ultrasonicated in an ice bath for 20min under ultrasonic conditions with a frequency of 40KHz and a power of 240w, the dispersion was even ; ...

Embodiment 3

[0062] (1) Weigh 200 mg of lipase (based on protein content) and place it in 40 mL of 100 mM phosphate buffer, stir magnetically at room temperature for 20 min to fully dissolve the lipase, add 1.5 g of 1-carboxymethyl-3-methylimidazolium Fluorosulfonimide salt, low-temperature ultrasonic to make it uniformly dispersed, after forming a homogeneous solution, place it in a shaker at room temperature and 150 rpm for 4 hours;

[0063] (2) Weigh 20 mg self-made amine functionalized magnetic nanoparticles (with dendritic NH 2 Fe 3 o 4 -PMMA) (Refer to the published literature Liu, X., Guan, Y.P., Wang, Q., Ren, X.F., Yang, M.Z., 2012. The Surface Modification of Magnetic Poly(methyl acrylate) Microspheres with Dendron and Application for the preparation method in Au(III)Adsorption.J.Appl.Polym.Sci.126,1956-1964.) was placed in a conical flask containing 5mL of 100mM phosphate buffer solution with pH=8, at a frequency of 40KHz and a power of 240w Ultrasound in an ice bath for 30 m...

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Abstract

The present invention relates to a magnetic cross-linked enzyme aggregate, a preparation method and an application thereof. The magnetic cross-linked enzyme aggregate comprises an enzyme aggregate and surface-functionalized magnetic particles, wherein the enzyme aggregate and the surface-functionalized magnetic particles are integratedly cross-linked through p-benzoquinone. The preparation method comprises: uniformly mixing an enzyme solution and surface-functionalized magnetic particles, adding a precipitating agent of the enzyme to make the enzyme form an enzyme aggregate, and adding p-benzoquinone as a cross-linking agent to integratedly cross-link the enzyme aggregate and the surface-functionalized magnetic particles so as to form the magnetic cross-linked enzyme aggregate. The magnetic cross-linked enzyme aggregate has characteristics of high immobilization efficiency, high enzyme activity recovery rate and good operation stability, and can be reused as a biocatalyst.

Description

technical field [0001] The invention relates to the technical field of enzyme immobilization, in particular to a magnetic cross-linked enzyme aggregate prepared by using p-benzoquinone as a cross-linking agent to cross-link enzyme aggregates and surface functionalized magnetic particles, as well as its preparation method and application. Background technique [0002] Laccase is a multi-copper oxidase that has been widely used as a green catalyst in material synthesis, degradation of environmental pollutants and dye decolorization, biological detection and electrochemical analysis, pulp bleaching and wastewater treatment in the paper industry, degradation of fiber raw materials etc., have broad application prospects (Jin Rong, Zhang Feilong, 2013. Application technology of laccase. China Raw Lacquer 32,35–42). However, laccase has the problems of low expression level, complicated separation and purification operation, and the free enzyme is directly used for catalysis, which ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12N11/08
Inventor 刘春朝刘英郭晨
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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