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Cholesterol oxidase affinity matrix, synthesis and large-scale purification method thereof for cholesterol oxidase

A technology of cholesterol oxidase and oxidase affinity, which is applied in the direction of chemical instruments and methods, oxidoreductase, and other chemical processes, can solve the problems of long purification time, low recovery rate of protein and activity, etc., and achieve increased purification efficiency, large The effect of economic benefits, large industrial application potential

Active Publication Date: 2012-03-21
宁夏妙朗生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Too many purification steps lead to longer purification time and lower recovery of protein and activity

Method used

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  • Cholesterol oxidase affinity matrix, synthesis and large-scale purification method thereof for cholesterol oxidase
  • Cholesterol oxidase affinity matrix, synthesis and large-scale purification method thereof for cholesterol oxidase
  • Cholesterol oxidase affinity matrix, synthesis and large-scale purification method thereof for cholesterol oxidase

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1: Synthesis and application of Sepharose-riboflavin

[0033] Sepharose CL 4B (100g) was washed with 10 times the volume of deionized water, and dried into a wet cake; suspended in 50ml activation buffer (0.8MNaOH, 20% dimethyl sulfoxide, 10% epichlorohydrin) and shaken at 40°C The bed was shaken for 2.5 hours, then poured into a glass frosted funnel, and washed with 10 times the volume of distilled water each time under suction filtration until the pH of the washing liquid reached neutral, and then drained to form a wet cake. The activated Sepharose CL 4B medium was suspended in 500ml of 0.1M NaOH solution, 20ml of ethylenediamine solution was added, and the gel was kept at 30°C for 12h under stirring (200rpm). Rinse with deionized water. NH 2 - Suspend Sepharose CL 4B in 350ml 50% (v / v) ice-bathed acetone solution, then dissolve 4g triazoxide in 80ml-20°C pre-cooled acetone, quickly add the medium suspension, check the pH value in real time, use saturated...

Embodiment 2

[0045] Embodiment 2: Synthesis and application of polychitosan-riboflavin

[0046] Polychitosan (100g) was washed with 10 times the volume of deionized water, and dried into a wet cake; suspended in 50ml activation buffer (0.8MNaOH, 20% dimethyl sulfoxide, 10% epichlorohydrin) at 40 ° C Vibrate on a shaker for 2.5 hours, then pour into a glass frosted funnel, wash with 10 times the volume of distilled water each time under suction filtration, until the pH of the washing solution reaches neutral, and then drain to form a wet cake. The activated Sepharose CL4B medium was suspended in 500ml of 0.1M NaOH solution, 20ml of ethylenediamine solution was added, and the gel was stirred (200rpm) at 30°C for 12h. Rinse with deionized water. NH 2 - Suspend Sepharose CL 4B in 350ml 50% (v / v) ice-bathed acetone solution, then dissolve 4g triazoxide in 80ml-20°C pre-cooled acetone, quickly add the medium suspension, check the pH value in real time, use saturated NaHCO 3 Keep the pH betwee...

Embodiment 3

[0058] Embodiment 3: Synthesis and application of D840-riboflavin

[0059] D840 medium (polystyrene medium, with free amino groups) is suspended in 350ml 50% (v / v) ice bath acetone solution, then dissolve 4g triazoxide in 80ml-20°C pre-cooled acetone, and quickly add the medium suspension , real-time detection of pH value, with saturated NaHCO 3 Keep the pH between 6.5-7.0, keep the reaction temperature at 0-4°C and continue stirring for 2-4h until the white color disappears and the mixture turns transparent to stop the reaction. Use 3 times the medium volume of acetone, acetone: deionized water (1:1), and deionized water to wash the reaction in sequence to obtain dichlorotriazine-amino-Sepharose CL 4B. Weigh 40g of dichlorotriazazine-amino-Sepharose CL 4B, weigh 10g of riboflavin (or photoflavin, photopigment, etc.), dissolve in 80ml of deionized water, shake at 50-60°C for 24h, often during the reaction Check the pH, keep the solution between 11-12 with 1M NaOH. After the...

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Abstract

The invention relates to a cholesterol oxidase affinity matrix formed by linking a cholesterol oxidase affinity ligand and a chromatography media; more preferably, the cholesterol oxidase affinity ligand is FAD (Flavin Adenine Dinucleotide) segmental riboflavin and the chromatography media is Sepharose, chitosan or polyvinyl benzene; the invention further provides a synthesis method for cholesterol oxidase affinity matrix, and a large-scale purification method for cholesterol oxidase by using the cholesterol oxidase affinity matrix; the cholesterol oxidase can be rapidly separated and purified in large scale by using the cholesterol oxidase affinity matrix provided by the invention; on the condition of only using one step of affinity chromatography, the pure cholesterol oxidase is obtained; the consumption time of the entire purification step is short, the active recovery rate of protein is higher and the large-scale popularization and application is applicable.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to the technical field of enzyme production for diagnosis, in particular to a cholesterol oxidase affinity medium and a method for synthesizing and purifying cholesterol oxidase on a large scale. Background technique [0002] Cholesterol oxidase (cholesterol oxidase, EC 11.3.6), referred to as COD, is the first enzyme in the process of cholesterol degradation and metabolism, which can specifically catalyze the substrate cholesterol to generate cholest-4-en-3-one. Microbes are the most important source of cholesterol oxidase, which has a wide range of applications in the fields of clinical diagnosis, food processing, biopharmaceuticals, biochemistry and insect resistance. For example, the use of cholesterol oxidase to measure the content of cholesterol in serum has become an important reference index in clinical diagnosis. Compared with physical and chemical methods to degrad...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/24B01J20/30C12N9/04
Inventor 辛瑜杨海麟张玲夏小乐张玉然仝艳军王武
Owner 宁夏妙朗生物科技有限公司
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