三相法提取猪肝酯酶及其在制备D -7-ACA中的应用

Extraction of Esterase from Pig Liver by Three-phase Partitioning and Its Application in Preparation of D -7-ACA

酯酶广泛存在于动物、植物和微生物中,具有作用范围广、底物特异性高等特点。该酶能够催化许多含有酯、酰胺、硫酯或乙酰基的化合物的水解与合成,在生物催化领域具有重要的用途。对来源于猪肝中的酯酶采用三相分离的方法进行了提取,并利用猪肝酯酶的水解特性,研究了其催化7-氨基头孢烷酸(7-ACA)脱乙酰基制备去乙酰基-7-氨基头孢烷酸(D-7-ACA)的过程。主要研究结果:确立了三相法分离提取猪肝酯酶的方法,考察了有机溶剂种类、有机溶剂用量、硫酸铵浓度以及体系的pH和提取温度等条件对三相法分离提取猪肝酯酶的影响。实验结果表明在硫酸铵质量分数为45%、异丁醇与水相之比为1∶1、pH7.0、温度为30℃的条件下,酶活回收率为54.84%,纯化倍数为6.32倍。以猪肝酯酶为催化剂,研究了猪肝酯酶催化7-ACA脱乙酰基制备D-7-ACA,在pH7.5、温度35℃、底物浓度2%、酶添加量30 U/mL的反应条件下,7-ACA转化率达到94.33%。

Esterase is widely distributed in animals,plants,and microorganisms.It has wide substrate specificity and can catalyze hydrolysis and synthesis of a variety of compounds containing esters,amides,sulfones,or acetyl groups.Esterase has important applications in the field of biocatalysis.This paper studies the extraction of the enzymology of esterase from pig liver,and its ability to catalyze the deacetylation of 7-aminocephalosporanic acid to produce deacetyl-7-aminocephalosporanic acid.The main results are as follows:Extraction of esterase from pig liver by three-phase partitioning has been established.The effects of different organic solvents,organic solvents dosage,ammonium sulfate concentration,and the pH of the system and extraction temperature on the three-phase extraction of pig liver esterase have been investigated.The experimental results showed that under the conditions of ammonium sulfate mass fraction 45%,isobutanol to water ratio of 1:1,pH 7.0,and temperature 30℃,the enzyme activity recovery rate was 65.79%,and the purification factor was 2.31.With pig liver esterase as a catalyst,the preparation of D-7-ACA by deacetylation of 7-ACA was studied.The conversion rate of 7-ACA reached 94.33%when the preparation conditions were pH 7.5,temperature 35℃,substrate concentration 2%and enzyme addition was 30 U/mL.