Li Hui-Juan, Tang Bai-Lu, Shao Xuan, Liu Bai-Xue, Zheng Xiao-Yu, Han Xiao-Xu, Li Ping-Yi, Zhang Xi-Ying, Song Xiao-Yan, Chen Xiu-Lan
State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Institute of Marine Science and Technology, Shandong UniversityJinan, China; College of Chemical and Environmental Engineering, Shandong University of Science and TechnologyQingdao, China.
State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Institute of Marine Science and Technology, Shandong University Jinan, China.
Front Microbiol. 2016 Dec 22;7:2016. doi: 10.3389/fmicb.2016.02016. eCollection 2016.
Bacterial extracellular proteases are important for bacterial nutrition and marine sedimentary organic nitrogen degradation. However, only a few proteases from marine sedimentary bacteria have been characterized. Some subtilases have a protease-associated (PA) domain inserted in the catalytic domain. Although structural analysis and deletion mutation suggests that the PA domain in subtilases is involved in substrate binding, direct evidence to support this function is still absent. Here, a protease, P57, secreted by sp. A5-7 isolated from marine sediment was characterized. P57 could hydrolyze casein, gelatin and collagen. It showed the highest activity at 40°C and pH 8.0. P57 is a new subtilase, with 63% sequence identity to the closest characterized protease. Mature P57 contains a catalytic domain and an inserted PA domain. The recombinant PA domain from P57 was shown to have collagen-binding ability, and Phe349 and Tyr432 were revealed to be key residues for collagen binding in the PA domain. This study first shows direct evidence that the PA domain of a subtilase can bind substrate, which provides a better understanding of the function of the PA domain of subtilases and bacterial extracellular proteases from marine sediment.
细菌胞外蛋白酶对细菌营养和海洋沉积有机氮降解至关重要。然而，只有少数来自海洋沉积细菌的蛋白酶得到了表征。一些枯草杆菌蛋白酶在催化结构域中插入了一个蛋白酶相关（PA）结构域。尽管结构分析和缺失突变表明枯草杆菌蛋白酶中的PA结构域参与底物结合，但仍缺乏支持该功能的直接证据。在此，对从海洋沉积物中分离的A5-7菌株分泌的一种蛋白酶P57进行了表征。P57能够水解酪蛋白、明胶和胶原蛋白。它在40°C和pH 8.0时表现出最高活性。P57是一种新的枯草杆菌蛋白酶，与最接近的已表征蛋白酶具有63%的序列同一性。成熟的P57包含一个催化结构域和一个插入的PA结构域。来自P57的重组PA结构域显示具有胶原蛋白结合能力，并且Phe349和Tyr432被揭示为PA结构域中胶原蛋白结合的关键残基。这项研究首次展示了枯草杆菌蛋白酶的PA结构域可以结合底物的直接证据，这为更好地理解枯草杆菌蛋白酶和海洋沉积物中细菌胞外蛋白酶的PA结构域功能提供了依据。
