南海沉积物细菌M4家族蛋白酶MP4301酶原成熟过程及其对颗粒有机氮（PON）的降解机制

海洋沉积物中颗粒有机氮的有效水解是其回到海洋生态系统氮循环的关键，微生物分泌的蛋白酶在这一过程中发挥重要作用，研究这些蛋白酶的表达加工过程及其对颗粒态有机氮的水解效应对于深入认识微生物在海洋中氮的生物地球化学循环中的作用机制具有重要意义。本项目以来源于南海沉积物细菌的M4家族蛋白酶MP4301为研究对象，开展该酶成熟过程及其对颗粒有机氮水解机制的研究。从转录、蛋白加工和酶活性三个水平分别考察该酶的成熟过程及其对环境因子的响应，对环境因素在该酶成熟过程中的调控作用进行全面阐释，并应用前导肽置换和定点突变技术研究前导肽辅助酶原成熟的分子机制，同时用MP4301对天然颗粒态蛋白进行水解，观测其吸附水解过程，以期对MP4301的成熟过程、生态学效应机制及其对环境因子的响应有全面的了解。本项目的实施将为阐释微生物生物催化剂参与生物地球化学循环过程的分子机制提供理论依据。

Effective hydrolysis of marine sedimentary particulate organic nitrogen is the key step for their returning to the nitrogen cycle, and microbial extracellular proteases play a significant role in this process. Research on expression, processing of these enzymes, as well as hydrolysis effects on particulate nitrogen, will offer a deeper understanding of the mechanism of microbial biogeochemical cycle on the earth. This project will focus on maturing process and hydrolysis mechanism of a marine sedimentary bacterial protease MP4301. Maturing process and its response for environmental factors of the protease will be investigated in transcriptional, protein processing, and enzymatic aspect, respectively. At the same time, site directed mutagenesis and homologous propeptide swapping will be used to construct a series of propeptide mutants to illustrate molecular mechanism of the propeptide assisting zymogen maturing. Furthermore, we will apply this protease MP4301 on hydrolyzing natural particulate proteins to observe the absorption and hydrolysis pattern. This research can give a comprehensive understanding on the maturing processing, ecological effects, and responses for environmental factors of the protease MP4301. The project will provide theoretical basis on molecular mechanism of biocatalysts driving biogeochemical cycle.