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A preparation method of affinity chromatography medium for separating and purifying hexokinase

A technology for separation and purification of hexokinase, which is applied in the field of preparation of affinity chromatography media, and achieves the effects of low price, easy acquisition and good application prospects

Active Publication Date: 2022-01-25
JIANGXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Glucosamine belongs to hexose, which can be used as the substrate of hexokinase and has a certain affinity for hexokinase. So far, there has been no report on using glucosamine as an affinity ligand to separate hexokinase by affinity chromatography

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The invention provides a method for preparing an affinity chromatography medium for separating and purifying chitosanase, comprising the following steps:

[0033] S1. First, take 5 g of washed and drained agarose 6FF gel and place it in a 250 ml Erlenmeyer flask, add 50 ml of sodium periodate solution with a concentration of 0.01 g / mL, place it in a constant temperature shaking incubator, and control the temperature at 25 ℃, and the rotation speed was 150rpm to carry out the aldylation reaction for 1h, and the aldylation agarose 6FF gel was prepared;

[0034] S2. Take 1 g of the aldylated agarose gel prepared in step S1, put it in a sand core funnel, wash it with distilled water, drain it, transfer it to a 250 mL conical flask, add 50 mL of glucosamine solution with a concentration of 0.01 g / mL, Place in a constant temperature shaking incubator, control the temperature at 37°C, and carry out the coupling reaction at 150rpm for 12h;

[0035] S3, transfer the agarose gel...

Embodiment 2

[0047] The invention provides a method for preparing an affinity chromatography medium for separating and purifying hexokinase, comprising the following steps:

[0048] S1. First, take 2 g of washed and drained agarose 6FF gel, 1 g of agarose CL-6B gel, and 2 g of agarose 4B gel, put them into a 250 ml Erlenmeyer flask, and add 50 ml of high concentration of 0.03 g / mL The sodium iodate solution was placed in a constant temperature shaking incubator, the temperature was controlled at 25°C, and the rotation speed was 150rpm, and the aldylation reaction was carried out for 1 hour to prepare the aldylated agarose 6FF gel;

[0049] S2. Take 1 g of the aldylated agarose gel prepared in step S1, place it in a sand core funnel, wash it with distilled water, drain it, transfer it to a 250 mL Erlenmeyer flask, add 50 mL of glucosamine solution with a concentration of 1 g / mL, Place in a constant temperature shaking incubator, control the temperature at 37°C, and carry out the coupling re...

Embodiment 3

[0061] The invention provides a method for preparing an affinity chromatography medium for separating and purifying hexokinase, comprising the following steps:

[0062] S1. First, take 2 g of washed and drained agarose 6FF gel, 1 g of agarose CL-6B gel, and 2 g of agarose 4B gel, put them into a 250 ml Erlenmeyer flask, and add 50 ml of high concentration of 0.03 g / mL The sodium iodate solution was placed in a constant temperature shaking incubator, the temperature was controlled at 20°C, and the rotation speed was 250rpm, and the aldylation reaction was carried out for 2 hours to prepare the aldylated agarose 6FF gel;

[0063] S2. Take 1 g of the aldylated agarose gel prepared in step S1, place it in a sand core funnel, wash it with distilled water, drain it, transfer it to a 250 mL Erlenmeyer flask, add 50 mL of glucosamine solution with a concentration of 1 g / mL, Place in a constant temperature shaking incubator, control the temperature at 30°C, and carry out the coupling r...

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Abstract

The invention provides a method for preparing an affinity chromatography medium for separating and purifying hexokinase, which comprises the following steps: S1. Firstly, agarose gel is used as a substrate, and the sodium periodate solution is used for aldylation reaction to prepare Obtaining an aldylated agarose gel; S2, followed by using glucosamine as an affinity ligand, and coupling on the aldylated agarose gel prepared in step S1; S3, and then preparing by using sodium borohydride solution to reduce step S2 The obtained coupling product is prepared to obtain a stable affinity chromatography medium; S4, finally, the affinity chromatography medium prepared in step S3 is washed with distilled water and dried, and stored in ethanol solution for later use. The preparation method provided by the invention has the advantages of low raw material price, easy acquisition, simple preparation process, stable and reliable purification method, easy control, low equipment requirements, good application prospects and good economic and social benefits.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing an affinity chromatography medium for separating and purifying hexokinase. Background technique [0002] Hexokinase is a six-carbon sugar phosphorylase with hexose as the specific substrate, and plays an important role in the glycolysis process of organisms. Hexokinase is an allosteric enzyme that catalyzes glucose from a stable state to an active state. It has a small Km value, strong affinity for substrates, and low specificity. It can act on a variety of six-carbon sugars. Utilizing its action principle and characteristics, the enzyme is mainly used in clinical diagnostic reagents, especially in the clinical detection of glucose in blood. Hexokinase is a key enzyme in the preparation of diagnostic kits, and its stability directly affects the stability of diagnostic kits. Therefore, preparing hexokinase with high stability is the key to formulating a diagno...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/24B01J20/28B01D15/38B01J20/30
CPCB01J20/24B01J20/28047B01D15/3814
Inventor 阳文静游清徽蔡险峰徐贤柱王曼莹
Owner JIANGXI NORMAL UNIV