宏基因组来源新过水解酶的分子克隆与酶学特性

Molecular cloning and characterization of a novel metagenome-derived bacterial perhydrolase

摘要:

旨在获得具有氯化功能的过水解酶，拓展过水解酶资源，为其工业应用奠定基础。以唐河某造纸厂污泥为材料构建宏基因组文库，通过活性筛选获得一个细菌过水解酶Per822。使用大肠杆菌异源表达Per822，研究纯化后的重组蛋白酶学性质并检测了生成过乙酸的能力。测序结果显示per822编码一个含273个氨基酸的蛋白。Per822是典型的多功能酶代表，分别具有过氧化物酶、卤代酶和酯酶的活性。Per822过水解氯化单氯二甲酮的最适反应pH为4.5，在pH 3.5–8.0范围内酶活性稳定。最适反应温度是55 ℃，在70 ℃以下酶活性稳定且氯化活性能够被Fe2+激活。以乙酸乙酯为共底物Per822显示出较强的产过乙酸能力。重组Per822的高可溶性表达、催化多功能性、较强的产过乙酸能力使得Per822在有机合成、废水处理、杀菌、生物质预处理等方面有着潜在的应用价值。

Abstract:

The aim of this study is to obtain bacterial perhydrolases with chlorination activity, expands the resources of perhydrolases, and lays a foundation for it’s industrial applications. We constructed a metagenomic library using environmental DNA isolated from sludge samples of a paper mill of Tanghe county, and identified a per822 gene encoding a bacteria perhydrolase via activity-based functional screening. Then, we overexpressed Per822 heterologously in Escherichia coli, and characterized the recombinant enzyme after purification. Finally, we further investigated the ability of Per822 to produce peracetic acid (PAA). Sequence analysis revealed that per822 encoded a protein of 273 amino acids. The recombinant Per822 had the activity of peroxidase, esterase and halogenase respectively, and thus was regarded as a typical representative of multifunctional enzymes. The purified Per822 exhibited maximal chlorination activity (hyperhydrolysis) at 55 °C and pH 4.5 with monochlorodimedone as substrate, and the enzyme was stable in the pH range of 3.5–8.0 and below 70 °C. Also, the chlorination activity of this enzyme could be activated by Fe2+. In addition, the enzyme displayed high ability to generate PAA using ethyl acetate as cosubstrate. The highly soluble expression, catalytic versatility and good PAA production capacity of Per822 make it a potential candidate in organic synthesis, wastewater treatment, disinfection and biomass pretreatment, etc.