云南黑井盐矿土中微生物耐盐内切木聚糖酶的克隆及耐盐机理研究

Hypersaline environments are extreme environments. Microorganisms and enzymes in hypersaline environments are forced to be salt-tolerant. Heijing, having a recorded around 2,000 years of salt production, is known as an ancient salt capital on the famous "Silk Route of the South". Bacteria, actinomycetes and archaebacteria from saline soil of salt mines located in Heijing are diverse and novel, but enzymes with salt tolerance from these microorganisms have not been reported. Salt-tolerant endoxylanases have potential in the processing of hypersaline food (e.g. Chinese cheese and soy sauce) and sea food (e.g. seaweed), and other biotechnological processes where hypersaline conditions are required. Basic research on the mechanism of enzymatic adaptation for salt tolerance is also significant to improve endoxylanases based on molecular strategies. In this study, both the classical culture-dependent and the newly developed culture-independent methods, called m-TAIL-PCR to isolate functional genes directly from metagenome, will be used to clone endoxylanases with salt tolerance. Further site-directed mutagenesis will be employed to reveal the salt-tolerant mechanism of endoxylanases. In a word, this study will contribute greatly to the development of salt-tolerant endoxylanase in the following two aspects: screening novel endoxylanases with salt tolerance from saline soil of a salt mine located in Heijing and revealing salt-tolerant mechanism of endoxylanases.

作为一种极端环境，高盐环境孕育着地球上耐盐的微生物和耐盐酶类。黑井产盐距今已有2000多年的历史，是"南方丝绸之路"上著名的盐都。黑井盐矿土中的耐盐细菌、放线菌及古菌具多样性及新颖性，但其耐盐酶却未被报导。耐盐内切木聚糖酶在高盐食品（如腐乳和酱油）加工、海产品加工（如海藻）及高盐环境生物技术领域具有应用价值，在耐盐机理研究以指导酶的分子改良上具有理论意义。本项目以黑井盐矿土为研究对象，结合微生物的可培养法和最新的直接克隆宏基因组中目的基因的方法（m-TAIL-PCR），克隆耐盐的内切木聚糖酶，进而再通过点突变的方法分析其耐盐机理。该项目对挖掘黑井盐矿土内蕴藏的具有应用潜力的新型耐盐内切木聚糖酶和揭示耐盐内切木聚糖酶的耐盐机理具有重要意义。

木聚糖酶在多个领域都具有应用价值。耐盐木聚糖酶在高盐生物技术领域具有应用优势，对耐盐机理的研究有助于丰富我们对酶在极端环境下的催化适应性的认识，也为相关酶的分子改良提供理论依据。. 本课题分离了云南黑井盐矿土中的微生物，经基因克隆、异源表达和酶学性质研究，获得了耐盐的糖苷水解酶第10、11和30家族内切木聚糖酶各一个。利用研究基础获得的内切木聚糖酶XynAHJ3和其它来源内切木聚糖酶XynAGN16L，对糖苷水解酶第10家族耐盐内切木聚糖酶基因进行DNA家族改组，获得了热盐性改良的突变酶S27B05，该突变酶是野生酶XynAGN16L和XynAHJ3的杂交体，其耐盐适应性主要是通过将野生酶XynAGN16L和XynAHJ3蛋白表面的负电荷区域进行整合，以提高自身的蛋白表面负电荷，进而提高其耐盐性。. 本课题从云南黑井盐矿土微生物中同时获得了糖苷水解酶第43家族木糖苷酶，该酶在NaCl中具有高活性但是稳定性很差，基于上述内切木聚糖酶的耐盐机理分析，通过理性设计和氨基酸位点V322的点突变，增加了该酶在结构域连接区的酸性氨基酸和蛋白表面负电荷，最终使该酶的突变体V322D在NaCl中的稳定性显著提高。. 本课题还从云南黑井盐矿土和云南其它环境微生物中筛选到耐盐的木聚糖酶和其它糖苷水解酶，并揭示了其中5个酶的潜在耐盐分子特性。项目主持人周峻沛以第一作者或通讯作者身份，在Food Chemistry及Journal of Agricultural and Food Chemistry等杂志共发表SCI收录论文9篇。.