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Immobilized carboxylesterase and preparation method and application thereof

A technology for immobilizing carboxylate and carboxylesterase, applied in the direction of immobilized enzymes, biochemical equipment and methods, enzymes, etc. Loading efficiency and other issues, to achieve good chemical and thermal stability, good reusability, and simple synthesis process

Pending Publication Date: 2021-01-05
ANHUI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these traditional enzyme carriers do not have good tunability and crystallinity, which may lead to low protein loading efficiency, instability, and enzyme leaching, thereby affecting the activity of immobilized enzymes

Method used

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  • Immobilized carboxylesterase and preparation method and application thereof
  • Immobilized carboxylesterase and preparation method and application thereof
  • Immobilized carboxylesterase and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Preparation of EstC@ZIF

[0044] Preparation of EstC protein solution: According to the steps of Example 1-4 in the patent application CN201910137250.2, EstC was expressed and purified.

[0045] Then, the purified protein solution was put into a Millipore ultrafiltration concentration tube (MW 10,000Da), centrifuged at 5,000 rpm for 30 min at 4°C, and the protein solution was concentrated to a concentration of 1.0 mg / mL.

[0046] Preparation of 2-methylimidazole solution: Weigh 2-methylimidazole and dissolve it in a certain volume of deionized water to prepare a 2-methylimidazole solution with a concentration of 2M.

[0047] Preparation of zinc acetate solution: Weigh zinc acetate and dissolve it in a certain volume of deionized water to prepare a zinc acetate solution with a concentration of 1M.

[0048] Add 3.5mL of the above-prepared 2-methylimidazole solution, 4.0mL of the concentrated EstC protein solution, and 2.1mL of deionized water into the Ep tube,...

Embodiment 2

[0059] Embodiment 2: the preparation of immobilized carboxylesterase

[0060] Add 4.25 mL of the 2-methylimidazole solution with a concentration of 2M prepared in Example 1, 3 mL of the concentrated EstC protein solution with a concentration of 1.0 mg / mL, and 2.55 mL of deionized water into the Ep tube together, and vortex Shake on the instrument for 30s, then add 0.2mL of the 1M zinc acetate solution prepared in Example 1, shake again for 30s, mix well, fix the Ep tube on a horizontal shaker, and react at 37°C for 30min, the reaction solution turns from clear to turbid , a large amount of precipitate was produced, and the precipitate was obtained by centrifugation at 6,000 rpm for 10 min. The precipitate was washed three times with ultrapure water and dried at 37°C to obtain the immobilized carboxylesterase.

Embodiment 3

[0061] Embodiment 3: the preparation of immobilized carboxylesterase

[0062] Add 2.75 mL of the 2-methylimidazole solution with a concentration of 2M prepared in Example 1, 4 mL of the concentrated EstC protein solution with a concentration of 1.0 mg / mL, and 3.05 mL of deionized water into the Ep tube together, and vortex Shake on the instrument for 30s, then add 0.2mL of the 1M zinc acetate solution prepared in Example 1, shake again for 30s, mix well, fix the Ep tube on a horizontal shaker, and react at 37°C for 30min, the reaction solution turns from clear to turbid , a large amount of precipitate was produced, and the precipitate was obtained by centrifugation at 6,000 rpm for 10 min. The precipitate was washed three times with ultrapure water and dried at 37°C to obtain the immobilized carboxylesterase.

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Abstract

The invention discloses immobilized carboxylesterase and a preparation method and application thereof. The carboxylesterase EstC is apoenzyme, and ZIF-8 is an immobilized carrier. The preparation method comprises the following steps of: (1) after carboxylesterase solution is mixed with 2-methylimidazole solution, adding zinc acetate solution, carrying out oscillation again, and after even mixing is carried out, reacting in a table concentrator at a temperature of 20-40 DEG C for 15-60 min; (2) carrying out centrifugation on reactant to obtain centrifugation supernatant and a centrifugation product; and (3) washing and drying the centrifugation product for multiple times to obtain the immobilized carboxylesterase. The immobilized carboxylesterase has high immobilization efficiency and goodactivity. Compared with free enzyme, the immobilized carboxylesterase further improves tolerance for reaction environment and parts of organic solvents and detergents, and shows good recycling performance. The immobilized carboxylesterase is applied to an aspect of bioremediation pesticide pollution, can generate an obvious hydrolysis function for chlorpyrifos, and has the good recycling performance.

Description

technical field [0001] The invention belongs to the technical field of immobilized enzyme preparation, and in particular relates to an immobilized carboxylesterase and its preparation method and application. Background technique [0002] Esterase (EC 3.1.1.x, esterase) is a general term for a large class of hydrolytic enzymes that catalyze the cleavage and formation of ester bonds. Carboxylesterase (EC 3.1.1.3, carboxyl ester hydrolase) that hydrolyzes short-chain fatty acids (≤10) with smaller chain lengths and has strong selectivity for water-insoluble long-chain fatty acids (≥10) Lipase (EC 3.1.1.1, triacylglycerol hydrolase). As a very important class of biocatalysts in organic chemistry, esterase has excellent chemoselectivity, regioselectivity and stereoselectivity, and the reaction process mediated by it usually does not require specific cofactors and no side reactions. occur. Because of these advantages, esterase has gradually become one of the most important indu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/089C12N9/18A62D3/02A62D101/04A62D101/26
CPCC12N11/089C12N9/18A62D3/02A62D2101/04A62D2101/26
Inventor 王宝娟吴爽安丽华刘珠琴王敖侯焕焕徐永祥施金叶蒋荣立吴甘霖
Owner ANHUI NORMAL UNIV
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