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Method for immobilizing β-fructofuranosidase and glucose oxidase double enzymes by sol-gel method

A technology of fructofuranosidase and glucose oxidase, which is applied in the field of enzyme engineering, can solve the problems of inability to fix other enzymes, application limitations, and reduced enzyme activity, and achieves improved lactulose oligosaccharide yield, low cost and good stability. Effect

Active Publication Date: 2021-11-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ordinary sol-gel method for the immobilization of β-fructofuranosidase will inhibit the release of the enzyme activity embedded in the gel, and the enzyme activity shown will be significantly reduced, and it will not be able to simultaneously with β-fructofuranosidase. Immobilization of other enzymes limits its application

Method used

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  • Method for immobilizing β-fructofuranosidase and glucose oxidase double enzymes by sol-gel method
  • Method for immobilizing β-fructofuranosidase and glucose oxidase double enzymes by sol-gel method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] First activate the Arthrobacter sp.10137 strain, insert the preserved Arthrobacter into the activation medium (glucose 2% (w / v); yeast extract 0.15% (w / v); magnesium sulfate 0.01% (w / v); Potassium dihydrogen phosphate 0.2% (w / v); diammonium hydrogen phosphate 0.6% (w / v); pH7.0-7.2), cultured at 30° C. for 24 hours to obtain activated strains. Carry out seed culture afterwards, draw the bacterial classification after 1ml activation to house 100ml seed culture medium (glucose 2% (w / v); Yeast extract 0.15% (w / v); Peptone 0.3% (w / v); Magnesium sulfate 0.01% (w / v); Potassium dihydrogen phosphate 0.2% (w / v); Diammonium hydrogen phosphate 0.6% (w / v), pH7.0-7.2) in a 250ml Erlenmeyer flask, in a shaker for 30 Cultivate at ℃ for 24h, with a shaking speed of 125r / min. Carry out fermentation culture then, seed culture solution is connected with 100ml fermentation medium (sucrose 4% (w / v); Yeast extract 1.2% (w / v): peptone 0.8% (w / v) by 2% inoculum size access ); Magnesium sulfat...

Embodiment 2

[0050] First activate the Arthrobacter sp.10137 strain, insert the preserved Arthrobacter into the activation medium (glucose 2% (w / v); yeast extract 0.15% (w / v); magnesium sulfate 0.01% (w / v); Potassium dihydrogen phosphate 0.2% (w / v); diammonium hydrogen phosphate 0.6% (w / v); pH7.0-7.2), cultured at 30° C. for 24 hours to obtain activated strains. Carry out seed culture afterwards, draw the bacterial classification after 1ml activation to house 100ml seed culture medium (glucose 2% (w / v); Yeast extract 0.15% (w / v); Peptone 0.3% (w / v); Magnesium sulfate 0.01% (w / v); Potassium dihydrogen phosphate 0.2% (w / v); Diammonium hydrogen phosphate 0.6% (w / v), pH7.0-7.2) in a 250ml Erlenmeyer flask, in a shaker for 30 Cultivate at ℃ for 24h, with a shaking speed of 125r / min. Carry out fermented culture then, seed culture liquid is connected with 100ml fermentation medium (sucrose 4% (w / v); Yeast extract 1.2% (w / v): peptone 0.8% (w / v) by 3% inoculum size access ); Magnesium sulfate 0.2...

Embodiment 3

[0054] First activate the Arthrobacter sp.10137 strain, insert the preserved Arthrobacter into the activation medium (glucose 2% (w / v); yeast extract 0.15% (w / v); magnesium sulfate 0.01% (w / v); Potassium dihydrogen phosphate 0.2% (w / v); diammonium hydrogen phosphate 0.6% (w / v); pH7.0-7.2), cultured at 30° C. for 24 hours to obtain activated strains. Carry out seed culture afterwards, draw the bacterial classification after 1ml activation to house 100ml seed culture medium (glucose 2% (w / v); Yeast extract 0.15% (w / v); Peptone 0.3% (w / v); Magnesium sulfate 0.01% (w / v); Potassium dihydrogen phosphate 0.2% (w / v); Diammonium hydrogen phosphate 0.6% (w / v), pH7.0-7.2) in a 250ml Erlenmeyer flask, in a shaker for 30 Cultivate at ℃ for 24h, with a shaking speed of 125r / min. Carry out fermented culture then, seed culture solution is connected with 100ml fermentation medium (sucrose 4% (w / v); Yeast extract 1.2% (w / v): peptone 0.8% (w / v) by 4% inoculum size access ); Magnesium sulfate 0...

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Abstract

The application provides a method for immobilizing β-fructofuranosidase and glucose oxidase dual enzymes, comprising the steps of: (1) production of β-fructofuranosidase; (2) separation and purification of β-fructofuranosidase (3) Purified β-fructofuranosidase and glucose oxidase solution are fixed on the gel by sol-gel method in the presence of silane coupling agent. Compared with traditional immobilized β-fructofuranosidase to produce lactulose-oligosaccharides, the present invention adopts sol-gel method to immobilize β-fructofuranosidase and glucose oxidase dual enzymes for the first time to improve the stability of enzyme and obtain higher The recovery rate of enzyme activity is high, the yield of lactulose oligosaccharide is improved, and compared with the traditional method, the operation is simple and the conditions are mild. It is an environmentally friendly green method.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to a new method for producing lactulose-oligosaccharides through co-immobilization of β-fructofuranosidase and glucose oxidase by a sol-gel method. Background technique [0002] β-fructofuranosidase (EC 3.2.1.26) is a glycosidase that widely exists in plants and microorganisms. It not only has hydrolysis, can catalyze the hydrolysis of sucrose into glucose and fructose, but also has transglycosylation, and can be widely used Used in the synthesis of functional oligosaccharides, food additives, modification and modification of glycosides and other biologically active substances. However, the properties of β-fructofuranosidases from different sources are different, and the ability of transglycosylation and receptor specificity are also different. As a raw material, the fructosyl produced by the hydrolysis of sucrose is transferred to the C at the reducing end of lactose 1...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/089C12N9/04C12N9/26
CPCC12N9/0006C12N9/2431C12N11/08C12Y101/03004C12Y302/01026
Inventor 金征宇龙杰潘婷徐学明谢正军周星柏玉香焦爱权田耀旗赵建伟王金鹏
Owner JIANGNAN UNIV
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