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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。
A strain of isolated Pseudomonas alcaligenes degrading dexamethasone was further domesticated. The osmotic shock method was used to separate the proteins of each region of the bacterium. The isolated intracellular protein with high enzymatic activity was extracted and purified by ammonium sulfate salting-out, ion exchange chromatography and Sephadex G-30. The purified protein was assayed for its degradation activity on dexamethasone by HPLC and identified by restriction enzyme digestion. The results showed that the degradation rate of dexamethasone induced by P. domestica increased from 23.63% to 52.84%, and the enzyme degrading dexamethasone mainly existed in the cytoplasm of the bacterium with a molecular weight of about 41 kD. The purified enzyme The specific activity of the protein reached 1.02U · mg-1. The amino acid sequence of the 5 peptides of this protein matched the partial sequence of isoenzyme A coenzyme A dehydrogenase, which may be a new steroid degrading enzyme. This article provides a new strategy for microbial decontamination of dexamethasone in aqueous environments.