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Method for modifying gamma-glutamyl transpeptidase immobilized enzyme by using carrier ampholyte

A technology of glutamyl transpeptidase and ampholyte, which is applied in the direction of immobilization on or in the inorganic carrier, can solve the problems of low success rate, high cost, and long cycle, and achieve improved pH stability and strong buffering The effect of large capacity and specific surface area

Inactive Publication Date: 2014-07-23
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the method of improving the pH tolerance of enzyme preparations is mainly through molecular biology methods such as site-directed mutation or molecular evolution, which not only has a long cycle and high cost, but also has a high degree of uncertainty in the results and a low success rate.

Method used

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  • Method for modifying gamma-glutamyl transpeptidase immobilized enzyme by using carrier ampholyte
  • Method for modifying gamma-glutamyl transpeptidase immobilized enzyme by using carrier ampholyte

Examples

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Embodiment 1

[0031]After fermenting and culturing the recombinant Escherichia coli for 12 hours, add the inducer lactose 2g / L, induce for 20 hours at 37°C, centrifuge, wash and ultrasonically break at a power of 400w, and centrifuge to obtain the supernatant to obtain a crude enzyme solution at 0% to 50%, 50% to 80%, 80% to 100% saturation ammonium sulfate gradient precipitation. Take 80%~100% active protein precipitate and redissolve it with Tri-HCl buffer (pH8.0, 50mmol / L). After dialysis, use DEAE Sepharose Fast Flow ion exchange chromatography and Source15Q ion exchange chromatography to obtain pure enzyme solution . Mesoporous titanium oxide (the specific surface area of ​​mesoporous titanium oxide is 96.539m 2 / g) placed in an oven at 120°C for 10 hours in vacuum to constant weight. Take 1g and put it into a 250mL round bottom flask, then add 100mL toluene and 1.47mL APTES, and react in an oil bath at 100°C under reflux for 6h. After the reaction is over, wash the powder with 250 ...

Embodiment 2

[0034] After fermenting and culturing the recombinant Escherichia coli for 12 hours, add the inducer lactose 2g / L, induce for 16 hours at 23°C, centrifuge, wash, and ultrasonically break at 400w power, centrifuge to get the supernatant to obtain a crude enzyme solution at 0% to 50%, 50% to 80%, 80% to 100% saturation ammonium sulfate gradient precipitation. Take 80%~100% active protein precipitate and redissolve it with Tri-HCl buffer (pH8.0, 50mmol / L). After dialysis, use DEAE Sepharose Fast Flow ion exchange chromatography and Source15Q ion exchange chromatography to obtain pure enzyme solution . Mesoporous titanium oxide (the specific surface area of ​​mesoporous titanium oxide is 96.539m 2 / g) placed in an oven at 100°C for 10 hours in vacuum to constant weight. Take 1g and put it into a 250mL round bottom flask, then add 80mL toluene and 1mL APTES, and react in an oil bath at 60°C under reflux for 6h. After the reaction is over, wash the powder with 250 mL of toluene, ...

Embodiment 3

[0039] After fermenting and culturing the recombinant Escherichia coli for 10 hours, add the inducer lactose 3g / L, induce for 15 hours at 25°C, centrifuge, wash and ultrasonically break at 400w power, and centrifuge to obtain the supernatant to obtain a crude enzyme solution at 0% to 50%, 50% to 80%, 80% to 100% saturation ammonium sulfate gradient precipitation. Take 80%~100% active protein precipitate and redissolve it with Tri-HCl buffer (pH8.0, 50mmol / L). After dialysis, use DEAE Sepharose Fast Flow ion exchange chromatography and Source15Q ion exchange chromatography to obtain pure enzyme solution . Mesoporous titanium oxide (the specific surface area of ​​mesoporous titanium oxide is 96.539m 2 / g) placed in a 100°C oven for 8 hours to a constant weight. Take 1g and add it to a 250mL round bottom flask, then add 80mL toluene and 1mL APTES, and react in an oil bath at 50°C under reflux for 5h. After the reaction is over, wash the powder with 250 mL of toluene, acetone, ...

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Abstract

The invention discloses a method for modifying gamma-glutamyl transpeptidase immobilized enzyme by using carrier ampholyte. The method comprises the following steps: in the environment of a toluene solvent, chemically modifying the surface of mesoporous titanium oxide by 3-aminopropyl triethoxysilane to obtain modified mesoporous titanium dioxide; mixing a gamma-glutamyl transpeptidase liquid with mesoporous titanium dioxide to obtain a mixture and oscillating the mixture to obtain immobilized enzyme; mixing the immobilized enzyme with Pharmalyte (CA) and oscillating for reaction; and filtering out raffinate, washing with a buffer solution and performing filtering-drying. According to the method, gamma-glutamyl transpeptidase (GGT) is immobilized with high-specific-surface-area mesoporous titanium oxide as a carrier and the carrying capacity of the carrier is high; the immobilized enzyme is modified by carrier pharmalyte with isoelectric point approximating the optimum pH of GGT; the pH tolerance of the immobilized gamma-glutamyl transpeptidase is improved effectively simultaneously when the stability of the immobilized enzyme is enhanced, and high activity is maintained in the pH range of 6-11.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for modifying immobilized enzymes. Background technique [0002] In recent years, mesoporous titanium oxide (MTiO 2 ) as a new type of high-performance material that has attracted much attention, its application research in the fields of photocatalysis, adsorption, and separation is very active. At the same time, due to its advantages such as high specific surface area, highly ordered structure, continuously adjustable hollow pore size, high biocompatibility, high mechanical strength, and high hydrothermal stability, it can be used as a carrier of organic or biomolecules in the slow control of drugs. It has broad application prospects in the field of releasing and immobilizing enzymes. [0003] γ-glutamyltranspeptidase (γ-glutamyltranspeptidase, GGT, EC2.3.2.2)) is a key enzyme in the glutamyl cycle in organisms. Potential value in the field of biosynthesis. ...

Claims

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Application Information

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IPC IPC(8): C12N11/14
Inventor 姚忠韩少雯孙芸仲兆祥叶丽静
Owner NANJING UNIV OF TECH
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