一种产碱假单胞菌产生的地塞米松降解酶的提取纯化和鉴定  

Extraction,Purification and Identification of a Dexamethasone-degrading Enzymes Generated by Pseudomonas Alcaligenes

作　　者：朱黎黎[1] 杨致邦[2] 杨茜[1] 石中全[3] 邓西川[2] 

机构地区：[1]重庆医科大学基础医学院病原生物学教研室,重庆400016 [2]重庆医科大学基础医学实验教学中心病原生物学与免疫学实验室,重庆400016 [3]重庆三峡医药高等专科学校免疫学与病原生物学教研室,重庆404120

出　　处：《生物医学工程学杂志》2015年第5期1044-1049,共6页Journal of Biomedical Engineering

基　　金：重庆市教委科学技术研究资助项目(KJ1402506)

摘　　要：将分离到的1株对地塞米松有降解作用的产碱假单胞菌进一步驯化。采用渗透休克法分离该菌各区域蛋白,将分离的具有高酶活性的胞内蛋白用硫酸铵盐析、离子交换层析和葡聚糖凝胶层析提取和纯化。纯化蛋白用HPLC检测其对地塞米松的降解活性,酶切后质谱鉴定。结果表明驯化后的产碱假单胞菌对地塞米松的降解率从23.63%提高到52.84%,降解地塞米松的酶主要存在于该菌胞质内,分子量约为41kD;纯化后的酶蛋白比活力达到1.02U·mg-1,该蛋白的5条肽段的氨基酸序列与异戊酰辅酶A脱氢酶的部分序列匹配,可能是一种新的甾体类化合物降解酶。本文为用微生物修复技术清除水环境的地塞米松污染提供了新策略。In this research a strain of isolated Pseudomonas alcaligenes which causes degradation of dexamethasone was acclimated further and its proteins of every position in the bacterium were separated by the osmotic shock meth od. The separated intracellular proteins which had the highest enzyme activity were extracted by the salting out with ammonium sulfate and were purified with the cation exchange chromatography and gel chromatography. The purified proteins which was active to cause degradation of dexamethasone had been detected were cut with enzyme and were analyzed with mass spectrometry. The results showed that the degradation rate to dexamethasone by acclimated Pseudomonas alcaligenes were increased from 23.63 ~ to 52.84~. The degrading enzymes were located mainly in the intracellular of the bacteria and its molecular weight was about 41 kD. The specific activity of the purified degrad- ing enzymes were achieved to 1.02 U · mg-1. Its 5-peptide amino acid sequences were consistent with some se quences of the isovaleryl-CoA dehydrogenase. The protein enzyme may be a new kind degrading enzyme of steroidal compounds. Our experimental results provided new strategies for cleanup of dexamethasone in water environment with microbial bioremediation technique.

关 键 词：产碱假单胞菌 地塞米松 降解酶 提取 纯化 鉴定 

分 类 号：R378[医药卫生—病原生物学] X172[医药卫生—基础医学]