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Three-dimensional melamine sponge complex fe 3+ Methods for Immobilization of Enzymes

A technology of three-dimensional melamine and sponge, applied in the direction of immobilized on or in the inorganic carrier, biochemical equipment and methods, enzymes, etc., can solve the problem of high cost, affecting product quality, free enzyme survival temperature, and narrow pH range and other problems, to achieve the effect of simple recycling and complete three-dimensional structure

Active Publication Date: 2021-07-16
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The temperature and pH ranges in which free enzymes survive are relatively narrow, and enzymes are easily denatured and inactivated
Unstable to heat, acid, alkali, and organic solvents, there are insurmountable shortcomings in catalytic reactions: (1) prone to microbial degradation or aggregation and inactivation; (2) in a homogeneous catalytic system, it is difficult to separate the enzyme from the product , leading to product impurity and affecting product quality; (3) Continuous operation cannot be realized
(4) Free enzymes are not easy to recycle, difficult to reuse, and expensive

Method used

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  • Three-dimensional melamine sponge complex fe <sup>3+</sup> Methods for Immobilization of Enzymes
  • Three-dimensional melamine sponge complex fe <sup>3+</sup> Methods for Immobilization of Enzymes
  • Three-dimensional melamine sponge complex fe <sup>3+</sup> Methods for Immobilization of Enzymes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The melamine sponge purchased in the market is directly punched into a cylindrical shape with a hole diameter of d=1.2cm and a thickness of h=1cm without any treatment. The quality of the sponge is guaranteed to be 10mg (±0.5mg), and the weighed sponge is placed in the 80mM FeCl 3 After soaking in the solution for 10 minutes, take it out with tweezers and squeeze it 5-6 times, blot the water in the sponge with filter paper, and dry it in a vacuum oven at 60°C for 12 hours to obtain the complexed Fe 3+ Materials (MS@Fe 3+ ). Then purify laccase, weigh 300 mg laccase and dissolve in water, centrifuge at 12000 rpm for 15 min in a refrigerated centrifuge at 4°C, take the supernatant to freeze-dry, and store at 4°C for later use. Weigh 100 mg of lyophilized purified laccase and dissolve it in 100 ml, pH=4, 50 mM phosphoric acid buffer, take 20 ml of buffer, add 100 mg of material (MS@Fe 3+ ), and overnight at 25 °C in a shaker. The loading amount of laccase was detected ...

experiment example 1

[0029] Study on Kinetic Equation of Immobilized Enzyme

[0030] By measuring the concentration of different substrates 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) under the condition of 25°C and pH=4 For the reaction rate, calculate the Michaelis-Menten equation parameters of the immobilized enzyme and the free enzyme, including the Michaelis constant Km and the maximum reaction rate Vmax of the immobilized enzyme and the free enzyme. Among them, the Michaelis constant Km represents the affinity between the enzyme and the substrate. The smaller the Km, the stronger the affinity. Vmax represents the maximum reaction rate of the enzyme-catalyzed reaction. The larger the Vmax, the stronger the activity of the enzyme. Such as Figure 4 As shown, the immobilized enzyme and the free enzyme are two straight lines with different slopes. The Vmax and Km values ​​of the immobilized enzyme and the free enzyme are relatively close, and the Vmax of the im...

experiment example 2

[0032] Study on Stability of Immobilized Enzyme

[0033] a.pH stability

[0034] The immobilized enzyme and the free enzyme were respectively placed in the buffer solution of different phosphates with a pH of 2-9, at room temperature, after standing for 12 hours, by measuring 0.5mM substrate 2,2'-azinobis-(3-ethane Oxidation product of diammonium salt ABTS-ABTS +· To determine the activity of free enzyme and immobilized enzyme at different pH. ABTS +· There is a maximum absorption at the ultraviolet wavelength of 415nm. Depend on Figure 5 It can be seen that the immobilized enzyme still retains the characteristics of the free enzyme, and the optimum pH value of it and the free enzyme is around 3, but the immobilized enzyme in MS@Fe 3+ Immobilized enzymes are more resistant to alkaline conditions than free enzymes after the material has been attached.

[0035] b. Thermal stability

[0036] In the environment of pH=4 and temperature 60°C, as the incubation time increases, ...

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Abstract

The invention relates to a three-dimensional melamine sponge complexed with Fe 3+ Methods for immobilization of enzymes. In this method, a cheap commercially available melamine sponge is used as a carrier, and the primary amines on the sponge directly interact with Fe 3+ complexes to immobilize the enzyme. The advantage of this new type of enzyme carrier is that it is safe, simple, fast and non-polluting. After the enzyme is immobilized, the enzyme activity and stability are greatly improved. The sponge is elastic and can be separated and reused by squeezing with tweezers. The immobilized enzyme is used in the dye industry to decolorize and remove dye wastewater. It is an environmentally friendly and efficient catalyst, and has broad application prospects in the pretreatment of complex biological samples and in the medical field.

Description

technical field [0001] The invention relates to a novel overall material for immobilizing enzymes, which belongs to the technical field of functional material preparation. Background technique [0002] Free enzymes are not ideal catalysts for modern industry. The temperature and pH ranges in which the free enzyme survives are relatively narrow, and the enzyme is easily denatured and inactivated. Unstable to heat, acid, alkali, and organic solvents, there are insurmountable shortcomings in catalytic reactions: (1) prone to microbial degradation or aggregation and inactivation; (2) in a homogeneous catalytic system, it is difficult to separate the enzyme from the product , leading to product impurity and affecting product quality; (3) Continuous operation cannot be realized. (4) Free enzymes are not easy to recover, difficult to reuse, and expensive. In order to overcome the shortcomings of free enzymes, people have explored the immobilization of water-soluble enzymes to re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/14C12N9/02C12N9/20C12N9/76
CPCC12N9/0061C12N9/20C12N9/6427C12N11/14C12Y110/03002C12Y301/01003C12Y304/21004
Inventor 张海霞邹玉琳
Owner LANZHOU UNIVERSITY
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