Fixed trypsinase and its preparation method

A trypsin and immobilized enzyme technology, which is applied in the field of bioengineering to achieve the effects of high catalytic efficiency, good production and application value, and good stability

Inactive Publication Date: 2008-12-03
CHENGDU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims to overcome the shortcomings of existing immobilized trypsin, and provides a novel immobilized trypsin and its preparation method

Method used

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  • Fixed trypsinase and its preparation method
  • Fixed trypsinase and its preparation method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: determination of activity recovery rate

[0026] A. Take 3g of refined dextran derivative powder, place in 40ml of absolute ethanol solution, stir and heat for 5min, then add 30% acetic anhydride polar organic solution, and heat at 65°C for 6h under reflux. After heating, the precipitate was collected by centrifugation, washed with 50% ethanol until the pH was near neutral, and dried at 60°C to obtain the carrier.

[0027] B. Take 1g carrier, dissolve it in 20ml of phosphate buffer solution with a concentration of 200mmol / L and a pH of 6-9, stir at 10°C for 10min, add 10ml of a cross-linking agent solution with a concentration of 10% and 10ml of a cross-linking agent solution with a concentration of 100mg / ml trypsin solution (enzyme activity: 3000U), stirred at 10°C for 4h. After the reaction, adjust the pH of the solution until the immobilized enzyme is completely precipitated, and collect the precipitate by centrifugation to obtain the immobilized tryps...

Embodiment 2

[0030] Embodiment 2: optimum pH and optimum temperature determination

[0031] A. Take 4 g of refined dextran derivative powder, put it in 50 ml of acetone, stir and heat for 5 min, then add a polar organic solution of malonic anhydride with a concentration of 20%, and heat under reflux at 70° C. for 4 h. After heating, the precipitate was collected by centrifugation, washed with 30% ethanol until the pH was near neutral, and dried at 70°C to obtain the carrier.

[0032] B, get 1g carrier, be dissolved in the aforementioned phosphate buffer of 20ml, after stirring 10min at 15 DEG C, add 10ml concentration to the solution and be the trypsin solution that 10% cross-linking agent solution and 10ml concentration are 100mg / ml (enzyme activity is 3000U), stirred at 15°C for 6h. After the reaction is completed, the pH of the solution is adjusted until the immobilized enzyme is completely precipitated, and the precipitate is collected by centrifugation to obtain the immobilized tryps...

Embodiment 3

[0035] Embodiment 3: Determination of acid-base solubility

[0036] A. Take 20 g of refined dextran derivative powder, place it in 500 ml of dimethyl sulfoxide solvent, stir and heat for 5 min, then add 10% succinic anhydride polar organic solution, and heat at 65° C. for 8 h under reflux. After heating, the precipitate was collected by centrifugation and washed with 30% ethanol until the pH was near neutral. Dry at 70°C to obtain the carrier.

[0037] B. Get 10g of carrier, dissolve it in 200ml of buffer, stir at 13°C for 10min, add 100ml of cross-linking agent solution with a concentration of 10% and 100ml of trypsin solution with a concentration of 100mg / ml (enzyme activity is 3000U) to the solution , Stir at 13°C for 4h. After the reaction, adjust the pH of the solution until the immobilized enzyme is completely precipitated, and collect the precipitate by centrifugation to obtain the immobilized trypsin.

[0038] C. Take 10 g of immobilized trypsin, add it to 300 ml of...

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Abstract

This invention discloses new immobilized trypsin and its producing method. Compatible immobilized enzyme carrier that synthesized by dextran derivative is used, enzyme and carrier are used to produce the fixed trypsin by crosslinking agent, the enzyme activity of immobilized trypsin is 60 to 85 percent of non immobilized, activity recovery is 60 to 85 percent. The material is easy to gain, operation is simple, the immobilized trypsin has good stability, it can be repeated used, it can be dissolved in substrate solution and react with solid substrate, its catalysis efficiency is high, and has good application value.

Description

technical field [0001] The invention relates to a trypsin, in particular to a novel immobilized trypsin and a preparation method thereof, belonging to the technical field of bioengineering. Background technique [0002] With the continuous expansion of the application field of biotechnology, trypsin as a catalyst is more and more widely used in industry. Trypsin is a water-soluble protein, which exhibits high catalytic activity in a homogeneous reaction system formed with a substrate solution in a dissolved state. However, the enzyme has poor stability in solution and is prone to self-digestion reaction, and it can only be used once in a solution state, resulting in increased production costs, so its application is limited to a certain extent. In order to solve the difficult problem in the industrial application of trypsin, it can be prepared as immobilized enzyme for use. After the trypsin is prepared as an immobilized enzyme, it has the following advantages: (1) after th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/10C12N9/76
Inventor 张涛王玉明刘桦
Owner CHENGDU MEDICAL COLLEGE
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