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Method for immobilizing fructosyl transferase by taking mesoporous molecular sieve-chitosan as carrier

A technology of fructosyltransferase and mesoporous molecular sieve, which is applied in the field of food enzyme engineering, can solve the problems of easy leakage and poor stability of enzymes, and achieve the effects of low cost, good stability and many times of reuse

Inactive Publication Date: 2012-01-18
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, SBA-15 is a molecular sieve with a pore structure, its pore diameter is adjustable from 5-30nm, it is non-physiologically toxic, and has good stability. There are a large number of hydroxyl groups on its surface, which can adsorb enzyme proteins and can be applied to the immobilization of enzymes , but the enzyme bound by adsorption alone is easy to leak and has poor stability, so other steps are needed to improve

Method used

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  • Method for immobilizing fructosyl transferase by taking mesoporous molecular sieve-chitosan as carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Accurately weigh 30 mg of mesoporous molecular sieve SBA-15, add 18 mL of fructosyltransferase (total enzyme activity 400 U) diluted with 50 mM pH5. 15°C) shake for 2 hours, add 1 mL of 1.0% chitosan (pre-dissolved in 1.0% glacial acetic acid), then add 1 mL of 2.5% glutaraldehyde aqueous solution, shake in a constant temperature shaker for 4 hours, and centrifuge at 4000 rpm for 5 minutes , transfer the supernatant, wash the precipitate with 50mM pH5.0 citric acid-phosphate buffer to remove residual glutaraldehyde and unfixed enzyme protein, centrifuge again, and repeat the washing 3 times to obtain the immobilized fructosyl Transferase. The supernatant obtained by centrifugation was collected to determine the activity of immobilized enzyme 328U, the recovery rate was 82.0%, and the protein adsorption amount was 382mg protein / g SBA-15.

Embodiment 2

[0027] Accurately weigh 30 mg of mesoporous molecular sieve SBA-15, add 18 mL of fructosyltransferase (total enzyme activity 300 U) diluted with 50 mM pH7.0 citric acid-phosphate buffer, place in a constant temperature shaker (150 rpm) and shake for 2 h, Add 1mL of 2.0% chitosan (dissolved in 1.0% glacial acetic acid in advance), then add 1mL of 1.5% glutaraldehyde aqueous solution, shake in a constant temperature oscillator for 4h, centrifuge at 4000rpm for 5min, and transfer the supernatant , washed the precipitate with 50mM citric acid-phosphate buffer solution of pH 7.0 to remove residual glutaraldehyde and unimmobilized enzyme protein, centrifuged again, and repeated washing 3 times to obtain immobilized fructosyltransferase. The supernatant obtained by centrifugation was collected to determine the enzyme activity of 235 U of the immobilized enzyme, the recovery rate of enzyme activity was 78.3%, and the protein adsorption amount was 356 mg protein / g SBA-15.

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Abstract

The invention discloses a method for immobilizing fructosyl transferase by taking mesoporous molecular sieve-chitosan as a carrier, and belongs to the technical field of food enzyme engineering. The method comprises the following steps of: absorbing zymoprotein on a mesoporous molecular sieve SBA-15 which is taken as a carrier, adding chitosan and a bifunctional reagent glutaraldehyde and furtherabsorbing and cross-linking, and immobilizing the fructosyl transferase by a two-step absorption-crosslinking method so as to firmly immobilize enzyme molecules on the carrier. The recovery rate of enzyme activity of the immobilized enzyme is over 75 percent, and the thermal stability and the pH stability are superior to those of free enzyme. Compared with other immobilization methods, the invention has the advantages that: the method is easy to operate and lower in cost, and has a good immobilization effect, conditions are mild, and the enzyme molecules are difficult to leak; and the product, namely the immobilized fructosyl transferase can be applied to the production of high-purity fructooligosaccharides.

Description

technical field [0001] The invention discloses a method for preparing immobilized fructosyltransferase with mesoporous molecular sieve-chitosan as a carrier, which belongs to the technical field of food enzyme engineering. Background technique [0002] Fructooligosaccharides, referred to as FOS, also known as oligofructose or saccharotriose oligosaccharides, the molecular formula is: G-F-Fn, n=1~3 (G is glucose, F is fructose). It is composed of sucrose and 1 to 3 fructosyl groups combined with fructosyl groups in sucrose through β-2-1 glycosidic bonds to form kestose, kestose, kestose, and kestose pentaose and their mixtures. In industry, fructo-oligosaccharides are generally generated from sucrose through the transglycosylation of fructosyltransferase (Fruetosyltransferase, EC 2.4.1.9, FTS). The enzymatic reaction mechanism is as follows: [0003] [0004] Fructo-oligosaccharide (FOS) is a functional oligosaccharide, which is characterized by low calorie and low sweetn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12N11/10
Inventor 江波吴保承缪铭李晓卉张涛杨春霞
Owner JIANGNAN UNIV
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