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Method for preparing agar oligosaccharide from immobilized agarase

A technology of agar oligosaccharides and agarase, which is applied in the direction of immobilization on or in the inorganic carrier, fermentation, etc., can solve the problems that the activity is easily affected by the environment, it is difficult to reuse and separate out, and achieve immobilization The method is simple and effective, the cost is low, and the effect of cost reduction

Inactive Publication Date: 2015-05-27
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although free enzymes can be directly applied to the preparation of agar oligosaccharides, their activity is easily affected by the environment and it is difficult to reuse in the reaction system and the product is difficult to separate from the system

Method used

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  • Method for preparing agar oligosaccharide from immobilized agarase
  • Method for preparing agar oligosaccharide from immobilized agarase
  • Method for preparing agar oligosaccharide from immobilized agarase

Examples

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Embodiment 1

[0034] Example 1 Preparation of carboxyl functionalized magnetic carrier according to the present invention

[0035] Weigh 8.1g FeCl 3 ·6H 2 Dissolve O in a conical flask filled with 142.5 mL of deionized water, stir and heat to 70°C; weigh 4.4g of FeCl 2 4H 2 O was dissolved in 10 mL of deionized water, filtered, and 7.5 mL of the supernatant was added to the Erlenmeyer flask; under vigorous stirring conditions, 18 mL of concentrated ammonia water with a mass fraction of 25% was quickly added, and after 1 min of reaction, gradually Add 4.66 g of oleic acid dropwise, and stir vigorously at 70°C for 1 h; after the reaction, a black sol-like substance is obtained, and the obtained precipitate is separated from the reaction system by applying an external magnetic field; washed 2-3 times with alcohol to remove Excess oleic acid was washed with deionized water to neutral pH; 160 mL of KMnO at a concentration of 10 mg / mL was added 4 solution, ultrasonically oscillated for 8 h ...

Embodiment 2

[0036] Example 2 The carboxyl functionalized magnetic Fe of the present invention 3 O 4 Optimization of Immobilization Conditions for Nanoparticle-immobilized Agarase

[0037] 1) Effect of glutaraldehyde concentration on agarase immobilization

[0038] The amount of immobilization carrier was 20 mg, the crosslinking time of glutaraldehyde was 3 h, the amount of enzyme added was 3 mL, the immobilization time was 3 h, and the immobilization temperature was 4°C. The influence of glutaraldehyde concentration on the immobilization of agarase was investigated. The maximum immobilized enzyme activity and enzyme activity recovery rate were obtained when the concentration of glutaraldehyde was 4%, which were 361.7 U and 14.2% respectively. The results are shown in figure 2 .

[0039] 2) The effect of the amount of enzyme added on the immobilization of agarase

[0040] The amount of immobilization carrier was 20 mg, the crosslinking time of glutaraldehyde was 3 h, the conc...

Embodiment 3

[0047] Example 3 The optimal reaction conditions for preparing agar oligosaccharides by immobilizing agarase on a carboxyl-functionalized magnetic carrier according to the present invention

[0048] 1) Optimum reaction temperature for immobilized agarase

[0049] In parallel, take 6 copies of 20 mg immobilized agarase in 50 mL Erlenmeyer flasks, add 10 mL of pH7.0 agar solution with a concentration of 0.2%, respectively, at 30°C, 35°C, 40°C, 45°C, 50°C , at 55°C for enzymatic hydrolysis. After the reaction, the optimum temperature of the immobilized enzyme was measured to be 40°C, the results are shown in Figure 7 .

[0050] 2) The optimal reaction pH of immobilized agarase

[0051] Take 6 copies of 20 mg immobilized agarase in parallel, and prepare agar solution with a concentration of 0.2% with buffer solutions of pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5 and pH 8.0 respectively. Take out 10 mL of agar solution with different pH and add immobilized agarase for enzymatic ...

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Abstract

The invention discloses a method for preparing an agar oligosaccharide from immobilized agarase. The method comprises the following steps: firstly, preparing magnetic Fe3O4 nanoparticles embedded in oleic acid by virtue of a coprecipitation method, and adding a KMnO4 solution to oxidize the nanoparticles so as to carboxyl-functionalized magnetic Fe3O4 nanoparticles; then, reacting the carboxyl-functionalized magnetic Fe3O4 nanoparticles with a coupling agent, namely glutaraldehyde, for 2h, wherein the concentration of the glutaraldehyde is 4%; after removing a cleaning fluid through magnetic separation, adding an enzyme fluid for immobilizing at 4 DEG C for 1h so as to obtain immobilized agarase, wherein enzyme addition is 1mL (850U); and under an oscillating condition, carrying out an enzymatic hydrolysis reaction on agar dissolved in a reaction medium and the immobilized enzyme, and after the reaction, magnetically separating to obtain a new agar oligosaccharide and recovering the immobilized enzyme. The immobilizing process disclosed by the invention is simple, low in operation cost, good in enzymatic activity recovery rate, good in the stability of the immobilized agarase, repeatable in use, easy in product separation and applicable to large-scale production.

Description

technical field [0001] The invention relates to the technical field of preparation of agar oligosaccharides, in particular to a method for producing agar oligosaccharides with immobilized enzymes. Background technique [0002] Agar oligosaccharides generally refer to oligosaccharides with a degree of polymerization (DP) of 2-10 formed by hydrolysis of agar polysaccharides connected by repeating units of agarose, including agar oligosaccharides and new agar oligosaccharides, agar oligosaccharides Glue oligosaccharides not only have the general properties of functional oligosaccharides, but also have many physiological functional properties that cannot be replaced by ordinary oligosaccharides, such as inducing apoptosis, anti-oxidation, anti-cancer, anti-inflammatory, anti-allergic activity and anti-starch aging It is a kind of oligosaccharide with great development potential. In the research on agar oligosaccharides, acid degradation and enzymatic degradation are commonly us...

Claims

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Application Information

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IPC IPC(8): C12P19/14C12N11/14
Inventor 肖安风倪辉蔡慧农肖琼姜泽东李利君杨秋明黄高凌杨远帆杜希萍陈艳红
Owner JIMEI UNIV
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