甲基黄嘌呤N7-脱甲基酶CdnC的可溶性表达及其酶学性质  

Soluble expression and enzymatic properties of methylxanthine N 7-demethylase CdnC

作　　者：李双岐 燕云 高子晴[1] LI Shuangqi;YAN Yun;GAO Ziqing(School of Biological Engineering,Dalian Polytechnic University,Dalian 116034,China)

机构地区：[1]大连工业大学生物工程学院,辽宁大连116034

出　　处：《大连工业大学学报》2025年第2期103-108,共6页Journal of Dalian Polytechnic University

基　　金：辽宁省民族药功效成分开发与应用重点实验室开放课题(2021JH13/10200038)。

摘　　要：以实验室前期筛选得到的咖啡因降解菌Paraburkholderia caf feinilytica CF1产的甲基黄嘌呤N7-脱甲基酶CdnC为研究对象,通过密码子优化和pMAL-c5X表达载体成功可溶性表达并纯化出N7-脱甲基酶蛋白,并对该酶进行酶学性质表征。CdnC底物专一,与氧化还原酶CdnD及谷胱甘肽硫转移酶CdnE共同作用脱去7-甲基黄嘌呤的N7位甲基,不能作用于咖啡因3,7-二甲基黄嘌呤和1,7-二甲基黄嘌呤的N7位甲基。SDS-PAGE和分子筛层析确定CdnC-MBP是一个分子质量为76.3 ku的单倍体蛋白。对CdnC进行酶学性质表征,其最适反应温度为30℃,最适反应pH为8.0,pH稳定区间为7.0~8.0。对CdnC的最适缓冲体系进行筛选,确定其最适缓冲液为50 mmol/L Tris-HCl+50 mmol/L NaCl(pH 8.0)。本实验成功实现了N7-脱甲基酶的可溶性表达,并确定了CdnC在咖啡因降解中的功能。A methylxanthine N 7-demethylase named as CdnC that produced by the caffeine-degrading bacterium Paraburkholderia caffeinilytica CF1,was successfully expressed and purified by codon optimization and the pMAL-c5X expression vector.CdnC shows substrate specificity and works together with oxidoreductase CdnD and glutathione s-transferase CdnE to remove the N 7 methyl group of 7-methylxanthine,but not 3,7-dimethylxanthine and 1,7-dimethylxanthine.CdnC-MBP was identified by SDS-PAGE and molecular size chromatography as a monomer protein with a molecular weight of 76.3 ku.The enzymatic characterization of CdnC revealed that its optimum reaction temperature was 30℃,and the optimum reaction pH and pH stable interval were 8.0 and 7.0 to 8.0,respectively.The optimal buffer system for CdnC was determined to be 50 mmol/L Tris-HCl+50 mmol/L NaCl(pH 8.0).The soluble expression of N 7-demethylase was successfully achieved,and the function of CdnC in caffeine degradation was determined.

关 键 词：咖啡因 甲基黄嘌呤N7-脱甲基酶 咖啡因降解菌 单倍体蛋白 

分 类 号：Q71[生物学—分子生物学]