乳白耙齿菌染料脱色过氧化物酶的催化特性研究

乳白耙齿菌F17（Irpex lacteus F17）是项目组早期选育对多种染料具有良好脱色和降解性能的白腐担子真菌。基因组数据表明该菌株不仅具有分泌型的ClassⅡ过氧化物酶（PODs）种类，且含有5个编码染料脱色过氧化物酶（DyPs）的基因。前期研究发现，该菌株DyPs具有不同于真菌ClassⅡ类过氧化物酶的独特氨基酸序列和蛋白结构特征，且异源表达的重组酶（Il-DyP4）表现出宽泛的底物范围以及对蒽醌和偶氮染料高效的催化效率。因此，本项目系统分析该菌株5个DyPs蛋白的序列特征和其在系统进化过程中的地位；研究DyPs的生化和理化性质及其对典型底物的催化氧化途径和机理；通过蛋白晶体生长和结构解析，结合定点突变、等温滴定量热法和电子顺磁共振技术，分析酶与底物的相互作用及其酶蛋白自由基的产生，阐明DyPs的底物氧化位点及其催化特性，为该新型功能酶对环境异生物质的降解和去除提供理论指导。

Irpex lacteus F17 is an indigenous white-rot basidiomycete fungus which has already been isolated by our group. Research on genome sequencing of I. lacteus F17 helped to reveal the species of secreted Class II peroxidases (PODs), and found 5 DyPs coding genes. Based on our previous study, this kind of enzymes from I. lacteus F17 show a unique protein structure made from a special amino-acid sequence distinguished from fungal class II peroxidases (PODs). Besides, the heterologous expressed DyP (Il-DyP4) in Escherichia coli show a broad substrate specificity and a high catalytic efficiency to anthraquinone as well as azo dyes. Therefore, to find the phylogenetic position of DyPs, our project will start from analyzing the specific pattern of its amino acid sequence, especially those 5 DyPs coding region, align the sequence to other homologous enzymes and identify the specialty in its protein structure. We will also study the biochemical, physical and chemical properties of the recombinant DyPs, including their abilities to degrade a wide range of substrates. Besides, an enzymatic degradation metabolites analysis and degradation mechanism study will be performed on typical substrates. Furthermore, aiming to detect the interactions between DyP and its substrates and the formation of radicals on DyP proteins, several technologies will be applied, including site-directed mutations, isothermal titration calorimetry and electron paramagnetic resonance technologies, together with the crystal structure analysis of DyP from I. lacteus F17, all of which will help us to identify DyP substrate oxidation sites and understand the catalytic properties. The purpose of this project is to provide theoretical guidance to the degradation and even removal of xenobiotics in the environment using the new functional enzyme.