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Method or purifying cephalosporin C acyltransferase

An acyltransferase and cephalosporin technology, applied in the field of biochemistry, can solve problems such as long time-consuming, cumbersome elution process, and complicated operation, and achieve the effects of reducing production costs, enhancing curing effect, and extending half-life

Active Publication Date: 2015-11-04
焦作健康元生物制品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purification method of cephalosporin C acylase with column chromatography as the main method needs to use a large amount of eluent, and the operation is complicated; the elution process is cumbersome and time-consuming; limit the increase in production
Therefore, the application of column chromatography in industrial production is limited

Method used

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  • Method or purifying cephalosporin C acyltransferase
  • Method or purifying cephalosporin C acyltransferase
  • Method or purifying cephalosporin C acyltransferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Screening of Ammonium Sulfate Addition in Salting Out Method

[0047] Because the purity of cephalosporin C acylase will affect the purity of 7-ACA, the yield of cephalosporin C acylase and the purity of catalyzed 7-ACA were used as indicators to investigate the addition of ammonium sulfate in the salting-out method. The influence of amount on the purification method of the present invention.

[0048] 1. Preparation of cephalosporin C acylase under different ammonium sulfate addition conditions

[0049] 1) Cool 600 g of cephalosporin C acyltransferase fermentation broth with a solid content of 220 g / L to 4°C, and centrifuge to obtain cell sediment;

[0050] 2) Add 0.02mol / L dipotassium hydrogen phosphate solution to the cell sediment to resuspend the cell sediment, stir evenly, break the cells with an 8MPa high-pressure homogenizer, and cool down to 10°C; Dipotassium hydrogen phosphate solution The weight ratio to the cell pellet is 5:1;

[0051] 3...

Embodiment 2

[0061] Example 2 Screening of Addition Amount of Potassium Dihydrogen Phosphate Solution in Purification of Salting-out Precipitate

[0062] Taking the yield of cephalosporin C acylase and the purity of 7-ACA synthesized by catalysis as indicators, the influence of the amount of ammonium sulfate added in the purification of the salting-out precipitate on the purification method of the present invention was investigated.

[0063] 1. Preparation of cephalosporin C acylase under different additions of potassium dihydrogen phosphate solution in the purification of salting-out precipitate

[0064] 1) Cool 600g of fermentation broth with a solid content of 240g / L to 4°C, and centrifuge to obtain cell sediment;

[0065] 2) Add 0.02mol / L, pH=8 dipotassium hydrogen phosphate solution to the cell sediment to resuspend the cell sediment, stir evenly, break the cells with an 8MPa high-pressure homogenizer, and cool down to 10°C; , the weight ratio of dipotassium hydrogen phosphat...

Embodiment 3

[0082] Example 3 Purification method of cephalosporin C acyltransferase

[0083] 1) Cool 600 g of cephalosporin C acylase fermentation broth with a solid content of 198 g / L to 4°C, and centrifuge to obtain cell sediment;

[0084] 2) Add 0.02mol / L dipotassium hydrogen phosphate solution with pH=8 to the cell sediment to resuspend the cell sediment, stir evenly, break the cells with an 8MPa high-pressure homogenizer, and cool down to 10°C; hydrogen phosphate The weight ratio of dipotassium solution to cell sediment is 5:1;

[0085] 3) Centrifuge to obtain the crude cephalosporin C acyltransferase solution and the bacteria residue;

[0086] 4) Control the temperature of the crude cephalosporin C acyltransferase solution obtained in step 3) at 4°C, add ammonium sulfate to make the concentration of ammonium sulfate 2.0mol / L, stir for 1 hour, carry out salting out, and centrifuge to obtain salting out Precipitate;

[0087]5) Add 0.02mol / L, pH=8 dipotassium hydrogen phosphate so...

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Abstract

The invention provides a method or purifying cephalosporin C acyltransferase. A salt fractionation method is adopted in the method for replacing column chromatography, operation is easy, the production period is short, and cost is low. The purity of the cephalosporin C acyltransferase is high. Therefore, the productivity of the high-quality cephalosporin C acyltransferase in China can be greatly improved, and the needs of domestic and foreign markets can be met.

Description

technical field [0001] The invention belongs to the field of biochemistry, in particular to a method for purifying cephalosporin C acyltransferase from fermentation broth. Background technique [0002] 7-Aminocephalosporanic acid (7-ACA for short) is the most commonly used and important mother nucleus in the synthesis of cephalosporins. The 7-ACA production process mainly includes chemical method and enzymatic method. Chemical methods mainly include acid chloride method and mixed anhydride method, which need to go through tedious processes such as activation, condensation, protection, and deprotection. There are many disadvantages such as long synthesis route, many steps, harsh reaction conditions, and a large amount of three wastes. Compared with the chemical method, the enzymatic synthesis process has many advantages, such as: simple production process, short cycle, mild reaction conditions, and close to neutral pH. In addition, due to the high regio and stereoselectivity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80
CPCC12N9/80C12Y305/01093
Inventor 唐阳刚林楠棋张书汁刘佳幸志伟张欣王悦璋
Owner 焦作健康元生物制品有限公司