沙雷氏菌(Serratia marcescens)Yj1的分离鉴定及菌体有机磷降解酶的分离纯化

doi:10.13560/j.cnki.biotech.bull.1985.2015.07.027

摘要/Abstract

摘要： 从大豆土壤中分离纯化得到一株具有卵磷脂和乐果降解能力的菌株Yj1，对该菌株进行鉴定、生长条件优化、酶活性鉴定以及有机磷降解酶的分离纯化。结果表明，Yj1与Serratia marcescens WW4(CP003959.1)的16S rDNA相似度为99%。正交试验对所需培养基进行优化，得到该菌株的最佳生长条件为甘露糖、蛋白胨和pH 8的组合。Yj1菌株在两种磷源条件下，菌株生长量均很低，但72 h内以大豆卵磷脂为磷源时的菌体生长情况优于乐果。以大豆卵磷脂为磷源时酸性磷酸酶、碱性磷酸酶与有机磷降解酶活性明显高于以乐果为磷源时的酶活，且72 h内碱性磷酸酶活性一直都高于酸性磷酸酶和有机磷降解酶。硫酸铵沉淀法结合阳离子交换层析成功从Yj1菌体中分离纯化了有机磷降解酶，SDS-PAGE结果显示纯化的蛋白为单一条带。且阳离子交换层析的提纯倍数是硫酸氨沉淀的5.303倍，硫酸氨沉淀为粗酶的1.416倍。

关键词: 沙雷氏菌, Yj1, 乐果, 大豆卵磷脂, 有机磷降解酶

Abstract: The strain Yj1 isolated and purified from the soybean soil could degrade soy lecithin and dimethoate. The 16S rDNA identification of the strain, optimization of growth conditions and examination of enzyme activities were performed and organophosphate degradation enzyme from Yj1 was separated and purified. The results revealed that the similarity of 16S rDNA in Yj1 and Serratia marcescens WW4(CP003959. 1)was 99%. The orthogonal experiment for the culture medium demonstrated that the optimal growth condition of Yj1 was the combination of mannose, peptone and pH8. The growth rate of Yj1 was poor in either of 2 phosphorus sources；however, the growth rate in soy lecithin was better than that in the dimethoate within 72 h. Moreover, the activities of acid phosphatase, alkaline phosphatase and organophosphate degrading enzyme(ODE)were higher when the soybean lecithin as the sole phosphorus source than those of dimethoate, and the activity of alkaline phosphatase was obviously greater than that of acid phosphatase and ODE within 72 h. ODE was successfully purified from Yj1 by combining ammonium sulfate precipitation and SP Sepharose Fast Flow. SDS-PAGE results showed that the purified protein was in a single band. The purified multiple by SP Sepharose Fast Flow was 5. 303 times as many by ammonium sulfate precipitation, and by the latter 1. 416 times as many by crude enzyme.

Key words: Serratia marcescens, Yj1, dimethoate, soy lecithin, organophosphate degrading enzyme

于亭, 王春红, 张婷婷, 汲添, 武志海, 杨美英. 沙雷氏菌(Serratia marcescens)Yj1的分离鉴定及菌体有机磷降解酶的分离纯化[J]. 生物技术通报, 2015, 31(7): 180-187.

Yu Ting, Wang Chunhong, Zhang Tingting, Ji Tian, Wu Zhihai, Yang Meiying. Isolation and Identification of Serratia marcescens Yj1，and Separation and Purification of Organophosphate Degrading Enzymes[J]. Biotechnology Bulletin, 2015, 31(7): 180-187.

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