【作者】 王宇凡；

【导师】 李明春；

【作者基本信息】 南开大学 ， 微生物， 2012， 博士

【摘要】 海藻糖是一种应用广泛的非还原性双糖，在医学、化妆品行业、制药学、食品加工业以及农业等领域都有十分诱人的应用前景。而利用酶法制备海藻糖是现今生产海藻糖的主要手段。在可制备海藻糖的各种酶类中，海藻糖合酶可以利用麦芽糖为底物通过一步反应生产海藻糖，整个过程简单可控，原料成本低廉，因此引起了人们越来越多的关注。然而目前天然的海藻糖合酶其结构与功能之间的关系还未被人们所了解，并且存在着催化效率较低，反应产生的副产物葡萄糖较多等问题，所以急需对该酶的结构功能进行研究，并且建立适合于海藻糖合酶定向进化的方法，对该酶进行改造。我们前期已经克隆得到来源于红色亚栖热菌的海藻糖合酶基因，并成功地将其在大肠杆菌中进行异源表达。该酶具有良好的嗜热性以及稳定性，因此具有工业应用潜力。但是如果将该酶直接用于工业生产，那么它的活性还不是很高，有进一步提升的空间。同时对该酶的结构、作用机理以及热稳定性机制也还不清楚，有必要进行研究，为进一步提升酶的催化效率或热稳定性提供理论依据。在本研究中，首先构建了红色亚栖热菌N端缺失突变子与C端缺失突变子两种缺失突变蛋白。通过对二者活性及二级结构分析发现，仅含C端结构域的蛋白与全长蛋白的二级结构相似，均为α/β型，这也是α淀粉酶家族的典型结构。、缺失C端而仅含有N端结构域的蛋白二级结构发生了变化，说明红色亚栖热菌海藻糖合酶的C端区域对维持蛋白结构稳定性有重要作用。通过构建一系列长度递减的C端片段缺失蛋白，我们发现C端缺失44个氨基酸后，蛋白依然具有活性，而且最适反应温度依然保持在50℃，但在60℃保温1h后残余酶活低于全长蛋白，说明红色亚栖热菌海藻糖合酶的C端能够影响蛋白的热稳定性。而C端缺失44个氨基酸的截短蛋白对底物选择性也发生了改变，它对麦芽糖的亲和力更高，说明红色亚栖热菌海藻糖合酶C端结构域可能对酶与底物的结合有影响。而当C端缺失68个或者更多氨基酸残基时，异源表达的蛋白无法正确折叠为可溶性蛋白，即使经过蛋白质复性后依然没有活性，说明从C末端第68个氨基酸开始的C端片段能够影响蛋白结构与功能。其次，构建了红色亚栖热菌海藻糖合酶与嗜热栖热菌海藻糖合酶C端与N端互换的杂合蛋白TSTtMr与TSMrTt。通过对两种杂合蛋白的表达纯化，测定酶学动力学常数以及温度耐受性等酶学性质，发现拥有相同N端结构域的蛋白，其酶学动力学常数也大致相似，而且有相同的最适反应温度与类似的温度耐受性，说明海藻糖合酶的N端结构域与催化活性密切相关，同时影响酶的嗜热性和热稳定性。而杂合蛋白TSTtMr与麦芽糖的亲和能力较红色亚栖热菌海藻糖合酶有所提高，而能够反映催化效率的kcat/Km值是红色亚栖热菌海藻糖合酶的2倍，说明该杂合蛋白催化效率更高。第三，为了研究影响红色亚栖热菌海藻糖合酶活性与热稳定性的具体区域或位点，本研究对该酶的三维结构进行模拟，并对预测得到的关键位点进行定点突变研究。通过Swiss-Model同源建模，发现红色亚栖热菌海藻糖合酶N端第3位-第543位氨基酸残基属于α淀粉酶家族成员，拥有类似于β/α的结构，该模拟结构中含有一个“口袋”区域，其中含有4个α淀粉酶家族保守区及关键氨基酸残基。通过定点突变，能够确定位于保守区中的H104、D200以及第三保守区对海藻糖合酶的活性起至关重要的作用，当将这些关键位点突变后，蛋白彻底失去活性。此外，位于口袋结构中的Y135、R388、R392位点也影响蛋白的功能，将它们突变为丙氨酸后，蛋白发生了活性丧失或下降。R392A的突变还会影响蛋白的嗜热性，使其在50℃失活，而在30℃具有活性，但与野生型蛋白相比，催化能力大大降低。说明R392位点或附近相关区域能够影响蛋白活性及嗜热性。第四，对红色亚栖热菌海藻糖合酶的定向进化进行了研究。确立了利用甲苯透性化细胞制备粗酶，经过DNS反应后在570nm处测定吸光度，以检验红色亚栖热菌海藻糖合酶活性高低的高通量筛选方法。运用易错PCR以及分段DNA改组法对红色亚栖热菌海藻糖合酶进行定向进化的初步研究，经过一轮易错PCR和一轮分段DNA改组筛选得到一株粗酶活力为野生型红色亚栖热菌海藻糖合酶1.6倍的突变株，该突变子共发生了6个氨基酸位点的突变。将突变蛋白纯化后，研究该酶的催化动力学常数，发现其对麦芽糖为底物时的Km值约是野生型的一半，表示突变子对底物的亲和能力提高了一倍，而突变子的kcat/Km值是野生型的2倍，说明该突变子的催化效率则是野生型的2倍。更多还原

【Abstract】 Trehalsoe is a non-reducing disaccharide widely used in pharmaceutical industry,cosmetic industry, food, agriculture, and many other fields. Because of its ability ofprotection, trehalose attracts lots of interest. Nowadays, trehalose is mainlymanufactured through enzymatic pathways. Among the enzymes which couldproduce trehalose, trehalose synthase (TreS) could convert maltose into trehalose inone step reaction, and the raw material is cheap, which makes it get more and moreconcerning. However, the relationship between the structure and function of TreS hasnot been investigated deeply. Moreover, the efficiency of some native TreS is not veryhigh. So it is important to study about the relationship between the structure andfunction and construct the method for directed evolution of TreS.In our previous study, the treS gene had been cloned from a thermophilicMeiothermus ruber strain CBS-01and expressed in Escherichia coli to characterizeits properties. Because of its thermophilicity and thermostability, TreS from M. ruber(TSM) was fit to produce trehalose in industry. However, the activity of TSM was nothigh enough to be applied in industry. It was necessary to improve the efficiency ofTSM for application and investigate the thermoadapatation mechanism of theenzyme.Firstly, the N-and C-terminal domains of TSM were constructed and expressedin E. coli. The C-termianl domain and wild-type TSM shared the similar secondarystructure, both of which were α/β type，a typical structure of α-amylase super family.While the N-terminal domain without C-terminal region underwent the change insecondary structure. It implied that the C-terminal domain was important for themaintenance of the structure of protein.Moreover, a series of proteins with different deletion at C terminus of TSM wereconstructed to find the region which affected the function of TSM. In a result, thetruncated protein ΔC44, which was deleted44amino acid residues from C terminus,changed the affinity to the substrates. It preferred maltose to trehalose as its substrate. Meanwhile, the truncated protein ΔC68and more amino acid residues missed from Cterminus could not fold correctly in solution form. The result showed that the44amino acid residues from C terminus may affect the linkage between the protein andsubstrate, while the region from the residue68from C terminus played a key role inthe protein folding.Secondly, the N-terminal and C-terminal domains of TSM and trehalosesynthase from Thermus thermophilus (TST) were switched to ascertain which of themplayed the important role in the characteristics and function of TreS. Two fusionproteins TSTtMr (N-terminal domain of TST fused with C-terminal domain of TSM)and TSMrTt (N-terminal domain of TSM and C-terminal domain of TST) wereconstructed. The enzymes with the same N-terminal domains shared the similarkinetics parameters and optimum temperature, indicating that the N-terminal domainsthemselves also play a major role in determining the thermostability and activity ofenzymes. Additionally, the fusion protein TSTtMr displayed the higher kcat/Kmvaluethan that of TSM, indicating that it could convert maltose to trehalose moreefficiently from a kinetic point of view.Thirdly, the three-dimensional structure of the N-terminal domain (3-543residues) of TSM was predicted at the tertiary level as a GH13domain of α-amylasesuper family. There was a putative catalytic cleft in the N-terminal domain. Fourconserved region and key sites of α-amylase super family were located in the cleft.Through site-directed mutagenesis, H104, D200, and the third conserved region werefound to play important roles in the activity of TSM. Besides, Y135, R388, and R392,which were also located in the cleft, could affect the function of TSM as well. Themutagenesis at these sites led to the complete loss or sharp decrease in the activity.The mutant R392A was inactive at50℃and kept a little activity at30℃, suggestingthat R392or the region near R392could influence the activity and thermophilicity ofTSM.Fourthly, the method for the directed evolution of TSM was constructed. Aproper process to prepare the crude enzyme was achieved with cell suspension treatedby2%toluene to get permeabilized cells. After DNS analysis, the absorbance at570nm was used to compare the activity of the mutants. Moreover, error-prone PCR and DNA shuffling for the directed evolution of TSM were performed to construct thelibrary of mutants. After screening the library, a mutant with6site substitution wasselected. The activity of the mutant was1.6fold of that of wild-type TSM. The Kmvalue of the mutant was a half of TSM, implying the affinity of mutant was2-foldhigher. And the catalytic efficiency was2-fold of wild-type TSM.更多还原