棘白菌素B脱酰基酶工程菌的构建及酶学性质研究  

Heterologous expression and enzymatic properties of echinocandins B deacylase in Streptomyces coelicolor

作　　者：邹树平[1] 廖思行 牛坤[1] 郑裕国[1] 沈寅初[1] 

机构地区：[1]浙江工业大学生物与环境工程学院,浙江杭州310014

出　　处：《浙江工业大学学报》2016年第1期99-103,共5页Journal of Zhejiang University of Technology

基　　金：国家自然科学基金资助项目(21176224);教育部博士点新教师基金资助项目(2012331712004)

摘　　要：棘白菌素B(ECB)脱酰基酶能选择性催化水解脱去ECB酰基侧链,得到ECB母核,可用于合成新型抗真菌药物阿尼芬净.从实验室保藏的犹他游动放线菌(Actinoplanes utahensis)ZJB-0801基因组中克隆到ECB脱酰基酶基因,并将其构建入表达质粒pSET152,通过接合转移的方法将表达质粒导入到天蓝色链霉菌(Streptomyces coelicolor)A3中,构建可以高效表达ECB脱酰基酶的工程菌.对重组ECB脱酰基酶的酶学性质进行了考察,结果表明:重组酶的最适反应温度为45℃,最适pH为7.5.Zn^(2+),Mg^(2+),Mn^(2+),Fe^(2+)对重组酶有显著的激活作用,而Cu^(2+),Co^(2+)具有显著地抑制作用.动力学参数分析表明:该酶对底物ECB的K_m和V_(max)分别为0.356mmol/L和397μmol/(min·mg).Echinocandin B（ECB）deacylase can catalyze echinocandin B.The side chain amide bond was removed.In this way,ECB nucleus was acquired.In the new antifungal,the chemicalenzymatic synthesis has important applications in the preparation of anidulafungin.Clone of ECB dealyse gene from Actinoplanes utahensis ZJB-0801,and linked into the overexpression vector pSET152.Then the plasmid was transformed into Streptomyces coelicolorA3.This genetically engineered strain could transform ECB into ECB nucleusand.Investigating its enzymatic properties,the results showed that the optimum temperature for the recombinant enzyme is 45℃,the optimum pH is 7.5.Zn^2＋,Mg^2＋,Mn^2＋,Fe^2＋are activated on the recombinant enzyme.The Cu^2＋and Co^2＋are inhibition on the recombinant enzyme.Enzyme kinetic parameters Kmand Vmax are 0.183mmol/L and 0.085μmol/min·mg,respectively.

关 键 词：棘白菌素B脱酰基酶 天蓝色链霉菌 重组表达 酶学性质 

分 类 号：Q814[生物学—生物工程]