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Method for preparing modified alpha-amylase

A amylase modification technology, applied in the field of bioscience enzyme engineering, can solve the problems of poor environmental adaptability, short enzyme activity retention time, poor stability, etc., and achieve the effect of improving physical and chemical properties and thermal stability

Active Publication Date: 2016-07-27
SHENZHEN POLYTECHNIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Although α-amylase has been widely used in various industries, due to the characteristics of protein molecules, there are still some shortcomings in the application process, such as poor environmental adaptability, short enzyme activity retention time and poor stability.

Method used

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  • Method for preparing modified alpha-amylase
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  • Method for preparing modified alpha-amylase

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preparation example Construction

[0034] Such as figure 1 , the embodiment of the present invention provides a preparation method of modified α-amylase, comprising:

[0035] Step S101: using an oxidizing agent to oxidize chitosan to obtain oxidized chitosan;

[0036] Chitosan is formed by condensation of 2-acetylamino-2-deoxy-β-D-glucan and 2-amino-2-deoxy-β-D-glucan through β-1,4 glycosidic bonds. The cationic biopolymer is the product of de-N-acetylation of chitin; the chitosan has good biocompatibility, good antibacterial activity and strong adaptability to the environment. The chitosan according to the embodiment of the present invention can be chitosan with a high deacetylation degree, and its deacetylation degree is above 85%, for example, it can be 85%-95%.

[0037] In the embodiment of the present invention, an oxidizing agent is used to oxidize chitosan to obtain oxidized chitosan. Specifically, the oxidizing agent oxidizes two adjacent carbon atoms respectively connected with hydroxyl groups on the...

Embodiment 1

[0074] Weigh 10 mg of chitosan and dissolve in 10 ml of sodium acetate buffer solution containing a certain concentration of sodium periodate, the pH of the sodium acetate buffer solution is 5.0, and the concentration is 50 mmol / L, and stir for 30 minutes under dark conditions. Add 14.5ml of ethylene glycol to stop the reaction and let stand for 2 hours. Under dark conditions, the above solution was dialyzed for 48 hours with pH 5.0 and 200 mmol / L sodium acetate buffer solution, and the water was changed 3 times in the middle. The oxidized chitosan solution was mixed with 8 mg of α-amylase, redissolved in 2.0 ml of acetic acid, and stirred for 4 hours in the dark. Then, 1.0 mol / L NaBH4 was added dropwise to the above solution with constant stirring and reacted for 4 hours. Finally, the obtained product was dialyzed for 48 hours in a sodium acetate buffer solution with a pH of 5.0 and a concentration of 50 mmol / L to obtain a modified α-amylase. All the above operations were p...

Embodiment 2

[0076] Weigh 10 mg of chitosan and dissolve it in 10 ml of sodium acetate buffer solution containing a certain concentration of sodium periodate, the pH of the sodium acetate buffer solution is 5.0, and the concentration is 50 mmol / L, and stir for 60 minutes under dark conditions. Add 14.5ml of ethylene glycol to stop the reaction and let stand for 2 hours. Under dark conditions, the above solution was dialyzed for 48 hours with pH 5.0 and 200 mmol / L sodium acetate buffer solution, and the water was changed 3 times in the middle. The oxidized chitosan solution was mixed with 10 mg of α-amylase, redissolved in 2.0 ml of acetic acid, and stirred for 4 hours in the dark. Then, 1.0 mol / L NaBH4 was added dropwise to the above solution with constant stirring and reacted for 6 hours. Finally, the obtained product was dialyzed for 48 hours in a sodium acetate buffer solution with a pH of 5.0 and a concentration of 50 mmol / L to obtain a modified α-amylase. All the above operations we...

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Abstract

The invention discloses a method for preparing modified alpha-amylase. The method includes the steps that chitosan is oxidized through an oxidizing agent to obtain oxidized chitosan; the oxidized chitosan and alpha-amylase are reacted to obtain a cross-linking reaction mixture; a reducing agent is added into the cross-linking reaction mixture, reacting is carried out, and the modified alpha-amylase is obtained. By means of the mode, the environmental suitability and the stability of the alpha-amylase can be improved.

Description

technical field [0001] The invention relates to the field of bioscience enzyme engineering, in particular to a preparation method of modified alpha-amylase. Background technique [0002] Biological enzyme is a new type of biocatalyst developed with the progress of biotechnology. Compared with traditional catalysts, it has the advantages of high efficiency, low energy consumption, and environmental friendliness. α-amylase, also known as starch-1,4-dextrinase (α-1,4-glucan glucan hydrolase, E.C.3.2.1.1), is widely distributed in animals, plants, and microorganisms. Alpha-amylases have been extensively studied. The main function of α-amylase is to randomly cut α-1,4 glycosidic bonds from the inside of starch molecules to generate dextrin and reducing sugars. Therefore, it is characterized by a sharp drop in the viscosity of the substrate solution and the disappearance of the iodine reaction, and finally The product is mainly maltose when decomposing amylose. At present, α-am...

Claims

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Application Information

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IPC IPC(8): C12N9/26C12N9/96
CPCC12N9/2414C12N9/96C12Y302/01001
Inventor 唐勇军代建国金刚
Owner SHENZHEN POLYTECHNIC
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