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Novel fructosyltransferase immobilization method

A new technology of fructosyltransferase, which is applied in the direction of immobilization on/in the organic carrier, can solve the problems of low utilization rate of immobilized enzyme, irregular shape of carrier, limitation of immobilized enzyme, etc., and achieve production equipment investment less, increase the enzyme load, the effect of uniform particle shape

Inactive Publication Date: 2009-09-23
云南健生生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, most of our country adopts the one-step cross-linking method to immobilize enzymes, that is, the cross-linking agent reacts with the enzyme and the carrier in the same system. In addition, the shape of the carrier is irregular, which will lead to: organic macromolecular substances as the mechanical Low strength, fragile reaction process, less enzyme loading, low utilization of immobilized enzymes, etc., which limit the use of immobilized enzymes

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Add the 2% chitosan acetic acid solution containing 0.01% binder polyvinylpyrrolidone dropwise into the 1M sodium hydroxide solution regulated by ethanol with a dropping pill machine, filter the obtained chitosan spherical particles and rinse thoroughly with deionized water. Wash until neutral.

[0027] The immobilized enzyme carrier was resuspended with a phosphate buffer solution with a pH of 7.0, wherein the ratio of the wet carrier to the buffer solution was 1:8 by mass. The carrier was activated by adding 0.1% cross-linking agent, shaking gently for 2 hours, and then washing away the excess cross-linking agent with deionized water.

[0028] Spherical chitosan particles were resuspended in 12 U / ml fructosyltransferase solution at a mass ratio of 1:10, fixed at 16°C for 10 hours to obtain immobilized enzyme particles, washed with deionized water to remove free enzymes, and obtained Immobilized fructosyltransferase. Measure its activity to be 40U / g, carry out enzyma...

Embodiment 2

[0030] A 2% chitosan acetic acid solution containing 0.03% polyvinylpyrrolidone was added dropwise into a 1M sodium hydroxide solution adjusted by ethanol with a dropping pill machine, and the obtained chitosan spherical particles were filtered and fully washed with deionized water until neutralized. sex.

[0031] The immobilized enzyme carrier was resuspended with a phosphate buffer solution with a pH of 7.0, wherein the ratio of the wet carrier to the buffer solution was 1:8 by mass. The carrier was activated by adding 0.15% cross-linking agent, shaking gently for 2 hours, and then washing off the excess cross-linking agent with deionized water.

[0032] Spherical chitosan particles were resuspended in 30 U / ml fructosyltransferase solution at a mass ratio of 1:10, fixed at 16°C for 10 hours to obtain immobilized enzyme particles, washed with deionized water to remove free enzyme, and immobilized Fructosyltransferase. Measure its activity to be 80U / g, carry out enzymatic re...

Embodiment 3

[0034] A 2% chitosan acetic acid solution containing 0.05% polyvinylpyrrolidone was added dropwise into a 1M sodium hydroxide solution adjusted by ethanol with a dropping pill machine, and the obtained chitosan spherical particles were filtered and fully washed with deionized water until neutralized. sex.

[0035] The immobilized enzyme carrier was resuspended with a phosphate buffer solution with a pH of 7.0, wherein the ratio of the wet carrier to the buffer solution was 1:8 by mass. The carrier was activated by adding 0.3% cross-linking agent, shaking gently for 2 hours, and then washing away the excess cross-linking agent with deionized water.

[0036] Spherical chitosan particles were resuspended in fructosyltransferase solution with a concentration of 50 U / ml at a mass ratio of 1:10, fixed at 16°C for 10 hours to obtain immobilized enzyme particles, washed with deionized water to remove free enzymes, and obtained Immobilized fructosyltransferase. Measure its activity t...

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Abstract

The invention relates to a novel fructosyltransferase immobilization method, comprising the following steps: (1) adding 0.01 percent-0.05 percent adhesive polyvinyl pyrrolidone into chitosan acetic acid solution; (2) dripping the chitosan acetic acid solution to NaOH solution regulated by ethanol for preparing chitosan spherical particles and washing the chitosan spherical particles to be neutral by using distilled water; (3) placing the neutral chitosan spherical particles in phosphate buffer solution with pH of 7, adding glutaraldehyde cross-linker to lead the glutaraldehyde cross-linker to be bonded on the chitosan spherical particles and fully washing to remove the glutaraldehyde which is not bonded; (4) placing the chitosan spherical particles which are bonded with the glutaraldehyde in the phosphate buffer solution with pH of 7 and then adding fructosyltransferase therein for immobilization. The novel fructosyltransferase immobilization method immobilizes the fructosyltransferase, improves the mechanical strength of chitosan as an immobilized enzyme carrier and improves the enzyme loading capacity; and the immobilized enzyme prepared by the method can be used repeatedly for a plurality of times.

Description

technical field [0001] The invention relates to a novel immobilization method of fructosyltransferase. technical background [0002] The application of immobilized enzyme technology in biochemical industry can make enzymes reusable, easy to separate from substrates and products, reduce process links, and also help continuous and automatic operation of enzymatic reactions, thereby reducing production costs. Therefore, this technology It plays a very important role in actual production. [0003] Fructose-oligosaccharides, such as kestose, kestose, and kestose pentaose, have the function of regulating blood lipids and intestinal beneficial bacteria, and are a class of functional sugar compounds that are very beneficial to human health. At present, the production of fructo-oligosaccharide is mainly realized by the catalysis of liquid fructosyltransferase. In order to reduce its production cost and promote the production of fructo-oligosaccharide, the research on its immobilized...

Claims

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Application Information

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IPC IPC(8): C12N11/10C12N11/06
Inventor 李世平段进军唐石荣牟云青路福平毛淑红别松涛
Owner 云南健生生物科技有限公司
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