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Method for separating and purifying transglutaminase

A technique for separation and purification of transglutaminase, applied in the field of separation and purification of transglutaminase, capable of solving difficult production and application problems, achieving high load, simple equipment, and good separation effect

Active Publication Date: 2011-08-10
TAIXING YIMING BIOLOGICAL PRODS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The yield of enzyme activity after CM-cellulose column is generally 55-65%, and the yield of enzyme activity after gel column chromatography is only 20-40%, which is difficult to meet the needs of production and application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] (1) At 0-10°C, equilibrate Amberlite IR-120 strongly acidic cation exchange resin with a phosphate buffer solution with a concentration of 0.02mol / L and pH 5.0, which is 5 times the volume of the resin (v / v), and the flow rate is 3 column volumes / hour;

[0027] (2) Use a volume of 1 / 50 times the amount of resin (v / v) of the transglutaminase activity of 1000U / ml feed solution to feed the balanced cation exchange at a flow rate of 1 / 2 column volume / hour column;

[0028] (3) Use a phosphate buffer solution with a volume of 5 times the amount of resin (v / v) at a concentration of 0.02mol / L and a pH of 5.0 to elute some impurities at a flow rate of 2 column volumes / hour;

[0029] (4) Reuse 3 times the volume of resin (v / v) containing 1mol / L K 2 SO 4 The 0.02mol / L, pH5.0 phosphate eluent elutes transglutaminase at a flow rate of 1 / 2 column volume / hour;

[0030] (5) Collect the obtained eluate and concentrate it through an ultrafiltration device with a molecular cut-off of ...

Embodiment 2

[0035] (1) At 0-10°C, equilibrate the ion exchanger IV weakly acidic cation exchange resin with a Tris-HCl buffer solution with a concentration of 0.1mol / L and pH 7.0 whose volume is twice the amount of resin (v / v), and the flow rate 1 column volume / hour;

[0036] (2) Use a transglutaminase with a volume of 1 / 8 times the amount of resin (v / v) and an activity of 100 U / ml to feed the balanced cation exchange column at a flow rate of 2 column volumes / hour;

[0037] (3) Use Tris-HCl buffer solution with a volume of 3 times the resin volume (v / v) and a concentration of 0.1mol / L, pH 7.0 to elute some impurities at a flow rate of 1.5 times the column volume / hour;

[0038] (4) Reuse 0.1mol / L H 2 SO 4 Tris-HCl eluent of 0.1mol / L, pH7.0 elutes transglutaminase at a flow rate of 1 column volume / hour;

[0039] (5) Collect the obtained eluate and concentrate it through an ultrafiltration device with a molecular cut-off of 10,000 Daltons until the volume of the concentrated enzyme concen...

Embodiment 3

[0044] (1) At 0-10°C, equilibrate Dowex 50 strongly acidic cation exchange resin with a boric acid-borax buffer solution with a volume of 1 times the amount of resin (v / v) and a concentration of 0.15mol / L, pH8.5, with a flow rate of 1 / 2 column volume / hour;

[0045] (2) Use a transglutaminase with a volume of 1 / 20 times the amount of resin (v / v) and an activity of 500 U / ml to feed the well-balanced cation exchange column at a flow rate of 1 column volume / hour;

[0046] (3) Use a boric acid-borax buffer solution with a volume of 2 times the volume of resin (v / v) at a concentration of 0.15mol / L and a pH of 8.5 to elute some impurities at a flow rate of 1 / 2 column volume / hour;

[0047](4) Use 0.15 mol / L, pH 8.5 boric acid-borax eluent containing 0.5 mol / L NaCl with a volume of 1 / 2 times the amount of resin (v / v) at 1 / 5 column volume / hour Flow rate elution transglutaminase;

[0048] (5) Collect the obtained eluate and concentrate it through an ultrafiltration device with a molecu...

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PUM

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Abstract

The invention provides a method for separating and purifying transglutaminase, which effectively increases the specific enzyme activity of the transglutaminase and solves the problems like influence on application due to deep color of the enzyme. In the invention, the transglutaminase with high purity is separated and purified with only one cation exchange resin; and the method has the advantagesof simplicity, low time consumption and high yield, and can be applied to inductiral production for large-scale preparation.

Description

technical field [0001] The invention relates to a method for separating and purifying transglutaminase, in particular to a method for separating and purifying transglutaminase by using cationic resin. Background technique [0002] Transglutaminase, also known as transglutaminase, is a transferase that catalyzes acyl transfer reactions. It can catalyze the intramolecular and intermolecular crosslinking of proteins, the connection between proteins and amino acids, and the hydrolysis of glutamine side chain amide groups in protein molecules. It is considered to be an important enzyme for the production of various new protein processed products It has broad application prospects in food, biopharmaceutical, textile and other fields. [0003] In developed countries, transglutaminase has begun to be widely used, and it has become the second largest enzyme in the food industry after amylase in Japan. The production of transglutaminase by microbial fermentation is a method to obtai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10
Inventor 王戈莎郭宏明
Owner TAIXING YIMING BIOLOGICAL PRODS
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