碱性淀粉酶用于纺织退浆及洗涤添加剂，是微生物酶工程领域的研究热点。已从嗜碱芽孢杆菌703中得到一个碱性淀粉酶Amy703，属于糖基水解酶13家族的新亚家族，与其序列同源性在50%以上的酶蛋白均来源于盐碱微生物。Amy703含有一个新的功能不详的N端结构域，将其缺失后的突变体N-Amy比酶活提高了35倍，不需要额外添加钙离子维持稳定和活性，但底物谱变窄。本项目拟以Amy703和N-Amy为研究对象，通过结构域重组研究N端结构域的底物结合能力；通过分析菌株703在不同底物诱导下分泌表达酶谱的变化研究N端结构域对淀粉酶的适配调节作用；通过结构解析和突变分析研究N端结构域与钙离子的构效关系；为淀粉酶的结构域改造和钙离子不依赖性改造提供理论基础。在阐明N端结构域功能的基础上，结合进化分析，研究N端结构域与盐碱微生物适应盐碱环境的关系，为盐碱微生物的适应性改造提供理论指导。

As being widely used for textile desizing and detergent additive, the study on alkaline amylase is always a hot research topic in the field of microbial enzyme engineering. Recently, we isolated an alkaline amylase, Amy703, from Bacillus pseudofirmus 703, which belongs to a new clade of glycosyl hydrolase family 13. Sequence blast analysis indicated that enzymes sharing more than 50% sequence homology with Amy703 were all originated from saline or alkaline microbes, indicating its conserved functionality. Amy703 contains a function unidentified N-terminal domain. Our preliminary studies indicated that the variant N-Amy without N-terminal domain exhibited a 35-fold increase of the enzyme specific activity compared to the wild-type Amy703, and extra calcium was not required anymore to maintain the enzyme stability and activity. It was interesting to note that although the stability and activity were significantly enhanced in the N-Amy variant, its substrate spectrum was narrowed to barely recognize amylopectin and amylose. In this proposal, we plan to characterize the N-terminal domain of Amy703 through various biochemical analysis against wild-type Amy703 and its variant N-Amy. Inhere, the substrate binding ability of N-terminal domain will be studied by domain re-arrangement. Besides, the influence of N-terminal domain on the adaptive adjustment ability of Amy703 will be evaluated through culturing the strain 703 in various media with different substrates, differentiating the enzyme expression spectrum under these conditions. Furthermore, the interaction between the N-terminal domain and calcium ions will also be studied through 3-D structure and mutation analysis. These studies will provide theoretical basis of the Amy703 enzyme engineering for higher activity, broad substrate spectrum, and calcium independence. In addition, the interpretation of the functional roles of N-terminal domain in Amy703 combining with its evolutionary analysis will reveal the mechanism of how the N-terminal domain facilitating the environmental adaptability of the saline or alkaline bacteria, providing guidance for their further engineering against extreme environment.