我国沿海盐田嗜盐古菌胞外蛋白酶多样性研究

The robustness of extracellular proteases from haloarchaea can expand the application fields of proteases. However, diverse proteases from haloarchaea have not yet been fully explored; only a few of them have been characterized. The numerous proteases from haloarchaea in the various coastal solar salterns of the South and East China are considered worth intensive investigation. In this study, 646 haloarchaeal strains from 12 coastal solar salterns will be screened for extracellular proteases. These proteases will be characterized using the crude enzyme regarding their type and their tolerance to temperature, salinity and pH. The genes of these proteases will be cloned and sequenced. The proteases with good temperature, salinity and pH tolerance will be subjected to heterogeneous expression, purification and characterization. The proteases with desirable properties will be used to accelerate the fish fermentation process. The studies will reveal the diversity of the proteases from haloarchaea in Chinese coastal salterns, as well as the intra- and interspecies diversity of protease genes, thus discovering novel halophilic proteases and providing insights into the molecular evolution of proteases. These studies will provide scientific basis of various halophilic proteases for their catalysis in hypersaline solutions and organic solvents.

嗜盐古菌胞外蛋白酶的鲁棒性能够拓宽蛋白酶的应用范围。然而，嗜盐古菌蛋白酶资源尚未充分挖掘，仅有少数几种蛋白酶得到了鉴定。我国东、南部沿海地区分布着大量的盐田，其嗜盐古菌蛋白酶资源尚未开发。本项目拟从东、南部沿海12个代表性盐田中分离的646株嗜盐古菌出发，筛选胞外蛋白酶产生菌株，表征粗酶液中蛋白酶种类、蛋白酶的催化温度、盐度、pH耐受范围，克隆编码基因；对具有较宽耐受范围的蛋白酶进行异源表达，并对这些蛋白酶进行分离纯化、酶学性质表征；选取具有优良酶学性质的蛋白酶用于缩短鱼露发酵周期研究。项目将揭示我国沿海盐田嗜盐古菌蛋白酶种类的多样性、酶学性质的多样性以及蛋白酶基因的种内和种间多样性，进而鉴定新型嗜盐蛋白酶，并加深对蛋白酶分子进化规律的理解。项目的实施将为高盐体系和有机非水介质中的蛋白酶催化反应提供重要的酶资源。

嗜盐古菌胞外蛋白酶种类单一、纯化困难、未见应用报道。针对上述问题，本项目以阐明嗜盐古菌蛋白酶以及蛋白酶基因的多样性，进而发现新型嗜盐古菌蛋白酶为目标，重点开展了以下研究内容：[1] 新型胞外蛋白酶基因的鉴定；[2] 胞外蛋白酶的异源表达纯化及酶学性质表征；[3] 嗜盐古菌胞外蛋白酶用于鱼露速酿。本项目已在蛋白酶关键残基和结构域、新型蛋白酶的鉴定等方面取得重要研究成果；并且建立了简便、快速、通用的嗜盐古菌胞外蛋白酶异源表达纯化体系，发现嗜盐古菌蛋白酶普遍具有良好的温度、pH和盐度耐受性；最后，探究了嗜盐古菌蛋白酶应用于高盐食品的可行性，发现添加嗜盐古菌蛋白酶可以显著提高鱼露的三氯乙酸可溶性总氮、氨基酸态氮、游离肽、游离氨基酸含量。项目总体目标已经完成。本项目的研究结果不仅丰富了我们对古菌多样化生理代谢的认知，也为嗜盐古菌胞外蛋白酶的适应、进化机制的研究提供了基础，并且为进一步开发极端微生物资源开拓了新的方向。