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A kind of immobilized cephalosporin C acylase and preparation method thereof

A cephalosporin and acylase technology, applied in directions such as being fixed on/in an organic carrier, can solve the problems of increased cost, shortened immobilization time, long immobilization time, etc., and achieves the effect of solving production costs

Active Publication Date: 2019-02-19
SHANGHAI INST OF PHARMA IND CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem solved by the present invention: aiming at the problem that the immobilization time in the existing cephalosporin C acylase immobilization technology is longer, both 20 hours or longer, which increases the production cost, the present invention provides a New immobilized cephalosporin C acylase and its preparation method
Compared with the prior art, this method has the advantages of greatly shortening the immobilization time, and the immobilized enzyme obtained under the same conditions has higher enzyme activity, stability and reusability, overcomes the shortcomings of the existing literature, and solves the problem of The problem of cost increase caused by excessive time in the prior art is more suitable for industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] (1) Put ES-1 resin (purchased from Tianjin Nankai Hecheng Technology Co., Ltd.) in 400ml of 0.1mol / L potassium phosphate buffer solution with pH 8.0, let it stand at 25°C for 15min, wash with water for 3 times, and use Dry it with a vacuum pump and store it at 4°C for later use;

[0022] (2) Take 1 g of pretreated ES-1 resin, add 200 U of enzyme solution, and fix in 1.0 mol / L, pH 9.5 potassium phosphate buffer solution at 25°C and 150 r / min for 12 hours;

[0023] (3) Wash the immobilized enzyme obtained above with 0.1mol / L, pH 8.0 sodium phosphate buffer three times to remove unbound protein on the surface, and filter with a vacuum pump, and store the immobilized enzyme at 4°C spare;

[0024] (4) The obtained immobilized enzyme is carried out the mensuration of apparent enzyme activity recovery rate, the immobilized enzyme that is about to reclaim is added in the 0.1mol / L PBS solution of the pH8.0 that contains 5.0mg / ml CPC, at 37 ℃ , 150r / min shaker reaction for 15mi...

Embodiment 2

[0026](1) With embodiment 1, earlier carrier is carried out pretreatment;

[0027] (2) Take 1 g of pretreated ES-1 resin, add 200 U of enzyme solution, and fix in 0.5 mol / L, pH 8.5 potassium phosphate buffer at 35°C and 150 r / min on a shaker for 8 hours;

[0028] (3) reclaim immobilized enzyme, with the step (3) of embodiment 1;

[0029] (4) Detection of activity, same as step (4) of Example 1, the apparent enzyme activity recovery rate of the immobilized enzyme at this moment is 41%, and the apparent enzyme activity of the immobilized enzyme is 82U / g.

Embodiment 3

[0031] Other operations are as in Example 1, the only difference being that the potassium phosphate buffer concentration is 0.5mol / L during immobilization, the apparent enzyme activity recovery rate of the immobilized enzyme at this moment is 45%, and the apparent enzyme activity of the immobilized enzyme It is 90U / g.

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Abstract

The invention discloses immobilized cephalosporin C acylase and a preparation method thereof. The immobilized cephalosporin C acylase comprises ES-1 resin and cephalosporin C acylase fixed on the ES-1 resin. The preparation method comprises steps as follows: the ES-1 resin and the cephalosporin C acylase are dissolved in a phosphate buffer solution with the concentration being 0.5-1.0 mol / L and the pH being 7.0-10.5 and immobilized at the temperature of 15-35 DEG C for 8-20 h. Compared with the prior art, the provided method for preparing the immobilized cephalosporin C acylase is short in immobilizing time and high in enzyme activity recovery rate, the problem of high production cost in the prior art is solved, and the method is more suitable for industrial production.

Description

technical field [0001] The invention relates to the field of immobilized enzymes, in particular to an immobilized cephalosporin C acylase and a preparation method thereof. Background technique [0002] Cephalosporin C acylase is a kind of N-terminal nucleophilic (Ntn) hydrolase, which can directly catalyze cephalosporin C (CPC) to generate 7-aminocephalosporanic acid (7-aminocephalosporanic acid, 7-ACA), and 7-ACA is an important intermediate for the production of semi-synthetic cephalosporins in the pharmaceutical industry, and is in great demand. Therefore, the immobilization of the enzyme has broad application prospects in industry. [0003] The reported immobilized cephalosporin C acylase mainly adopts the covalent method, such as reference Characteristic of immobilized cephalosporin C acylase and its application in one-step enzymatic conversion of cephalosporin C to 7-aminocephalosporanicacid. Xiangwei Zhu, Hui Luo, Yanhong Chang, Houbo Su, Qiang Li, Huimin Yu, Zhongy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/08
Inventor 胡又佳张伟徐雪丽谢丽萍刘艳龚桂花刘海元
Owner SHANGHAI INST OF PHARMA IND CO LTD
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