嗜碱芽孢杆菌GH13新亚家族碱性淀粉酶N端结构域的功能和进化机制研究

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.

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。

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。前期从Bacillus pseudofirmus 703中得到一个新型碱性淀粉酶Amy703，该酶有一个新的未知功能的N端结构域，该结构域缺失后得到的突变体N-Amy相对野生型Amy703比酶活提高了35倍；最适温度提高了10 ℃；不需要额外添加钙离子维持稳定和活性，但是N-Amy的底物谱变窄。本项目以Amy703和N-Amy为研究对象，主要研究结果如下：. 1. 通过结构域重组研究了N端结构域的底物结合能力。N端结构域是由两个CBM48结构域组成的底物结合结构域，与催化结构域之间形成相互作用界面，参与底物的结合，不能单独与底物结合。. 2. 研究了703菌株体内其他淀粉相关水解酶，发现703菌株不仅可以利用可溶性淀粉，还具有利用支链淀粉、普鲁兰糖和环糊精的多种具特殊性质的淀粉酶类。. 3. 研究了N端结构域与钙离子的构效关系：Ca2+可以促进蛋白质聚集，通过改变蛋白的聚集状态影响酶的活性，过量聚集导致酶活性降低。. 4．结构分析显示N-Amy以二聚体形式进行催化反应，对称分子的N端参与了活性口袋处底物的结合，而Amy703以单体形式存在，这种二聚体状态可能是N-Amy活性大幅提高的原因之一。随后的研究通过亮氨酸拉链人工形成N-Amy的二聚体，通过spy-catcher-tag将N-Amy进行环化，酶活测定结果均显示和Amy703类似的低活性，进一步证实N-Amy的天然二聚体状态是其活性大幅提高的原因。. 5.同源比对显示与N端结构域同源的序列主要来自盐碱菌。因此合成了三个与Amy703分别具有50%，60%，70%同源的淀粉酶，发现三个酶在NaCl存在时活性会显著提高，其中HaAmy为嗜盐淀粉酶，在2.5M NaCl时活性最大，进一步证实N端结构域与嗜盐进化相关。通过比较分析不同pH和盐浓度对703菌株和其同源菌株OF4的生长差异，并结合比较基因组分析了嗜盐嗜碱性差异相关基因，推测703菌株有不同的嗜盐嗜碱机制，为下一步盐碱微生物的适应性改造提供了良好的实验材料。