Immobilization method of chitosanase

A technology of chitosanase and chitosan, applied in the field of enzyme engineering, can solve the problems of low stability, affecting substrate contact, low yield, etc., and achieves mild immobilization reaction conditions, long operating life and storage period, The effect of simple process flow

Pending Publication Date: 2021-12-31
CHAMBROAD CHEM IND RES INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many manufacturers use cellulase, protease, lipase and other non-specific enzymes to degrade chitosan, but there are disadvantages such as long production cycle and low yield; chitosanase can specifically hydrolyze chitosan, and can specifically Actively acts on GlcN-GlcN, GlcNAc-GlcN, GlcNAc-GlcNAc glycosidic bonds to hydrolyze chitosan into low molecular weight chitosan oligosaccharides, with mild reaction conditions, no side reactions, high yield of chitosan oligosaccharides, and no environmental pollution Etc
[0004] The use of free chitosan enzymes in practical applications has the following disadvantages: the enzyme has poor stability and is easily denatured and inactivated by the influence of pH and temperature; the enzyme is water-soluble and cannot be separated and recycled after the reaction. In the process, there is often a step of raising the temperature to inactivate the enzyme, which not only increases the cost and wastes energy, but also increases the chance of polluting the product, thus limiting the application of the product
[0005] At present, the immobilization methods of chitosanase are more widely used, including embedding method, adsorption method, cross-linking method, etc. However, the amount of these adsorption, cross-linking or embedding immobilized enzymes is very small, and the loss of enzyme activity is all More serious, even if the recovery rate of some enzyme activities reaches 80%, the cost of consumption is too high. In addition, after embedding, the contact between the substrate and the active site of the enzyme is affected, which increases the steric hindrance; the half-life and survival time of the enzyme are relatively Short, low stability, making it difficult to apply in industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] A kind of immobilization method of chitosanase, concrete steps are as follows:

[0053] Activation of chitosanase carrier

[0054] Weigh 5g carrier resin ESR-3 in a conical flask, add NaH 2 PO 4 -Na 2 HPO 4 100 mL of buffer solution (50 mM, pH 8.0) was added, and 2 mL of 25% glutaraldehyde solution was added, and placed on a shaker at 25° C. for 2 h at 150 rpm. Filter out the carrier, wash with the same buffer solution 5 times, 30 mL each time, subject to the residual glutaraldehyde after washing, filter and dry for later use.

[0055] Immobilization of Chitosanase

[0056] Take 1 g of the above-mentioned activated carrier, add NaH 2 PO 4 -Na 2 HPO 4 Buffer (50mM, pH8.0) 5mL, then add 5ml of chitosan enzyme solution containing 300U (enzyme activity of the enzyme solution is 60U / mL), mix well, seal the bottle mouth, place on a shaker at 25°C at 150rpm Oscillation 20h;

[0057] The resin carrier was filtered out, washed with pure water until the ninhydrin test ...

Embodiment 2

[0060] A kind of immobilization method of chitosanase, concrete steps are as follows:

[0061] Weigh 5g carrier resin ESR-3 in a conical flask, add NaH 2 PO 4 -Na 2 HPO 4 100 mL of buffer solution (50 mM, pH 8.0) was added, and 2 mL of 25% glutaraldehyde solution was added, and placed on a shaker at 25° C. for 3 h at 150 rpm. Filter out the carrier, wash with the same buffer solution 5 times, 30 mL each time, subject to the residual glutaraldehyde after washing, filter and dry for later use.

[0062] Immobilization of Chitosanase

[0063] Take 1 g of the above-mentioned activated carrier, add NaH 2 PO 4 -Na 2 HPO 4 Buffer (50mM, pH8.0) 5mL, then add 10mL of 486U chitosan enzyme solution (enzyme activity of the enzyme solution is 48.6U / mL), mix well, seal the bottle mouth, place on a shaker at 25°C at 150rpm Shake down for 21h;

[0064] The resin carrier was filtered out, washed with pure water until the ninhydrin test effluent was negative, then washed with 1mol / L Na...

Embodiment 3

[0067] Hydrolysis of Chitosan Using Immobilized Chitosanase

[0068] Prepare a chitosan substrate with a concentration of 2%, add 1.5% acetic acid solution to fully swell in a 55°C water bath, adjust the pH value to 5.5, add 0.2g of the immobilized chitosanase in Example 1, and carry out enzymatic hydrolysis at a temperature of 55°C for 20min , take the reaction solution and drop it into 2% NaOH solution until no white flocculent precipitate is produced to stop the reaction, filter out the immobilized chitosan enzyme for next use; use the acetylacetone method to detect the reaction solution, and the chitosan oligosaccharide produced by hydrolysis The viscosity-average molecular weight is 473.23 Da, the color is 429.65 Zeng, and the water solubility is good.

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PUM

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Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to an immobilization method of chitosanase, which comprises the following steps: selecting ESR-3 as carrier resin, activating the carrier resin ESR-3 by using a cross-linking agent glutaraldehyde, then adding chitosanase liquid, carrying out covalent binding reaction on the carrier resin ESR-3 and the chitosanase liquid, finally adding a reducing agent sodium/potassium borohydride for treatment, and obtaining the immobilized chitosanase; and using the obtained immobilized chitosanase for hydrolyzing a chitosan solution to produce water-soluble chitosan oligosaccharide with low molecular weight. By adopting the method, the immobilized chitosanase has the advantages of higher storage and operation stability, easiness in separation of the chitosanase from a substrate and a product, facilitation of recovery and reutilization of the chitosanase and the like, and is suitable for industrial application.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering and provides a chitosan enzyme immobilization method. Background technique [0002] Chitosanase (EC.3.2.1.132) is a specific enzyme for degrading chitosan, acting on the β-1,4-glucosidic bond of chitosan to decompose into low molecular weight chitosan oligosaccharides. Due to its unique biological functions, chitosan oligosaccharide has a wide range of uses in the fields of medicine, agriculture, food, etc., and is a hot spot in research and development at home and abroad. [0003] Compared with the production of chitosan oligosaccharides by traditional acid degradation or oxidative degradation methods, the production of chitosan oligosaccharides by bio-enzyme has the advantages of simple process, low environmental pollution, small molecular weight, good water solubility and high biological activity, so it is becoming more and more popular. It has been paid attention to and has also be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/08C12N9/42C12P19/26C12P19/14
CPCC12N11/08C12N9/2434C12P19/26C12P19/14C12Y302/01132
Inventor 司更花马韵升杨传伦孙建忠王红霞王秀芝梁敬敬
Owner CHAMBROAD CHEM IND RES INST CO LTD
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