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Preparation method of wool immobilized enzyme and wool immobilized enzyme column reactor

A technology of immobilized enzymes and reactors, which is applied to specific-purpose bioreactors/fermenters, biochemical equipment and methods, enzyme production/bioreactors, etc., and can solve the problem of poor stability and low reusability of immobilized enzymes and other problems, to achieve the effect of pH stability and repeated use stability enhancement, excellent mass transfer performance and enhanced effect

Pending Publication Date: 2022-03-01
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current immobilization technology of amino acid dehydrogenase and other oxidoreductases still has problems such as poor stability of immobilized enzymes and low recycling activity. More importantly, immobilized enzymes are rarely used in continuous reactors.

Method used

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  • Preparation method of wool immobilized enzyme and wool immobilized enzyme column reactor
  • Preparation method of wool immobilized enzyme and wool immobilized enzyme column reactor
  • Preparation method of wool immobilized enzyme and wool immobilized enzyme column reactor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] (1) Preparation of crude enzyme solution:

[0053] Construction of recombinant expression strain capable of expressing amino acid dehydrogenase with His-tag tag: the gene sequence of amino acid dehydrogenase (AaDH) is shown in SEQ ID NO.1. After the PCR amplification product was identified by 1% agarose gel electrophoresis, the AaDH gene fragment was recovered from the gel, and NdeI and XhoI enzymes were used to carry out double digestion, and the digestion product was recovered, and the same double digestion pET-28a plasmid (with with His-tag tag) for ligation, and the ligated plasmid was transformed into Escherichia coli BL21 (DE3) to obtain the pET28a-AaDH plasmid. The above plasmid was transformed into E.coli BL21(DE3) to obtain a recombinant expression strain E.coli BL21(DE3) / pET28a capable of expressing amino acid dehydrogenase with His-tag tag.

[0054]Cultivation of recombinant Escherichia coli E. coli BL21(DE3) / pET28a: Inoculate the strain into 200 mL of LB me...

Embodiment 2

[0063] (1)-(4) The experimental procedures are as the steps (1)-(4) of Example 1, wherein the wool is treated in the sodium sulfide / urea / sodium lauryl sulfate system for 1 h.

[0064] (5) Detection method of enzyme activity: the activity determination reaction system of amino acid dehydrogenase comprises 500 μ L, 4 mM NADH, 8000 μ L, pH 9.5 concentration is 200 mM ammonium chloride-ammonia water as amino donor, 500 μ L, 40 mM substrate solution, The substrate is α-ketoglutaric acid, and samples are taken every 1 min to measure the absorbance of the reaction solution at a wavelength of 340 nm. Enzyme activity is defined as the amount of enzyme required to consume (or generate) 1 μmol NADH per minute under the above conditions, which is an enzyme activity unit.

[0065] The amino acid dehydrogenase enzyme activity assay system is a buffer solution at different temperatures (buffer temperature includes 30°C, 40°C, 50°C, 60°C, 70°C, 80°C), to investigate the reaction system to GA ...

Embodiment 3

[0067] (1)-(4) The experimental procedures are as the steps (1)-(4) of Example 1, wherein the wool is treated in the sodium sulfide / urea / sodium lauryl sulfate system for 1 h.

[0068] (5) Detection method of enzyme activity: The reaction system for the determination of amino acid dehydrogenase activity includes 500 μL, 4mM NADH, 8000 μL, pH 9.5, ammonium chloride-ammonium water with a concentration range of 200 mM as amino donor, 500 μL, 40 mM substrate solution , the substrate is phenylpyruvate, and samples are taken every 1 min to measure the absorbance of the reaction solution at a wavelength of 340 nm. Enzyme activity is defined as the amount of enzyme required to consume (or generate) 1 μmol NADH per minute under the above conditions, which is an enzyme activity unit.

[0069] (6) Determination of temperature stability of immobilized enzyme: the wool immobilized enzyme cross-linked with GA and metal ion Cu 2+ After the coordination, the wool-immobilized enzyme was kept i...

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Abstract

The invention discloses a preparation method of a wool immobilized enzyme and a wool immobilized enzyme column reactor, and belongs to the technical field of immobilized enzymes. Wool is a natural flexible material, the keratin content accounts for 80% or above of the wool, many effective functional groups such as amino-terminated groups, carboxyl-terminated groups and sulfydryl groups can be exposed on the wool subjected to treatment and surface modification, and the wool has good biocompatibility and mechanical strength and is beneficial to enzyme immobilization. The column reactor is widely applied to analysis and production, has excellent mass transfer performance, is simple in preparation method, can be repeatedly used, and is suitable for separation and purification of various components and industrial production application. The obtained wool immobilized enzyme and the wool immobilized enzyme column reactor have good catalytic ability and stability. The method has the advantages of simple process, mild preparation conditions and convenience in continuous production.

Description

technical field [0001] The invention relates to a method for immobilizing redox enzymes, belonging to the field of enzyme immobilization. Background technique [0002] Oxidoreductase is a general term for a class of enzymes that can catalyze the redox between two molecules, including a variety of important enzymes. Among them, amino acid dehydrogenase (AaDH) can catalyze reversible amino acid oxidative deamination and ketoacid reductive amination reactions. For the reductive amination reaction of unnatural substrates, the substrate 2-oxo-4-phenylbutyric acid ethyl ester (EOPB) was selected in the ammonium-containing salt system (providing NH 4 + acceptor) for reductive amination to generate L-homophenylalanine (LHPA) with high selectivity. The unnatural amino acid L-homophenylalanine (LHPA) can be synthesized by using AaDH, so that it can be more widely used in the fields of medicine, new biomaterials and cosmetics. [0003] Due to the poor stability of free dehydrogenas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/02C12N9/06C12N9/04C12M1/40C12M1/00
CPCC12N11/02C12N9/0014C12N9/0006C12Y101/01037C12M21/18
Inventor 王世珍江亮雷航彬
Owner XIAMEN UNIV
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