海棲熱袍菌纖維素酶TmCel5A之結晶結構研究與大腸桿菌植酸酶EcAppA之基因改造

Abstract

酵素有提高反應速率且具有受質專一的特性，因此外源性酵素的添加在工業上廣泛地被應用於:提高產品效率如洗衣精、飼料添加物；提高製程效力如生質能源製造、食品加工；增加製程產品專一性如製藥。然而，將來自於自然界菌株的酵素直接應用在工業上仍然有差距，酵素作用的最適溫度、酸鹼度、耐熱性、產量對於不同產業應用、製程仍須優化，以降低產品生產成本、增加應用性。X-光晶體繞射法測定蛋白質三度結構，能提供蛋白質分子間或蛋白質分子與作用物的相互作用;胺基酸的位置則可提供許多序列外的資訊，可做為理性設計法改造酵素的依據，幫助縮短改造酵素所需的時間。此論文分為兩部分，第一個章節，解出蛋白質與受質的複合體結構，以分析其分子間的交互作用:第二個章節，利用已知的結構，進行蛋白質的理性設計改造，有效率地提升酵素活性及耐熱性。 近年來替代能源的需求提高，木質纖維素為植物的結構體，來源豐富，為高潛力的替代能源原料，產程中將木質纖維素中複雜的多醣分解成可利用的單醣，以高溫處理及多種酵素協同處理為發展目標。海棲熱袍菌纖維素酶TmCel5A，具有高耐熱性且對葡萄聚糖及甘露聚糖皆有活性，另外對於有支鏈的葡萄聚糖也有高接受度，適用於複雜的木質纖維素分解。此論文使用X-光繞射分析蛋白質晶體與配體複合體的結構以了解分子間的交互作用。實驗結果得到與兩種配體高解析度(1.29-2.40A)的結構，分析TmCel5A與雙重受質結合位置，發現葡萄糖及甘露糖在C2的氫氧基雖具有不同的方向，但在TmCel5A中皆能不受阻擋地結合;進一步使用一個有木聚糖支鏈的葡萄糖也能疊印上TmCel5A的受質結合區。證實蛋白質與配體複合體的結構能符合酵素活性結果，高解析度結構能細部分析不同受質的作用位置。 植酸酶為飼料添加酶的大宗。植酸為豆科及種子植物儲存磷酸的形式，但單胃動物不具有植酸酶無法利用植酸中的磷酸，飼料中添加植酸酶，可降低植酸酶的抗營養作用，提高磷酸吸收，減少無機磷添加，且增加對環境的友善性。植酸酶作為飼料添加，除了要具蛋白酶耐受性，腸胃道的酸鹼適用性，還要能有對抗飼料製粒時的短暫高熱。大腸桿菌植酸酶EcAppA、布氏檸檬酸桿菌植酸酶CbAppA 、無丙二酸檸檬酸桿菌植酸酶CaAppA，三者皆有高植酸酶活性，其中CbAppA在畢赤巴斯德酵母菌做異源表達時能增加耐熱性，而EcAppA具有蛋白酶耐受性及可成功被畢赤巴斯德酵母菌大量表達且有已知的蛋白質及受質複合體結構。在此論文，以EcAppA做為目標植酸酶，利用序列比對及已知蛋白質結構，使用兩種策略增加活性及提高耐熱性，策略一是比對三種高活性的植酸酶，挑選出在兩個檸檬酸桿菌植酸酶相同但在EcAppA不同的胺基酸，其中再用已知結構選出活性區附近的胺基酸做活性篩選，在11個突變中，V89T能提高17.5%的活性，推測此突變可增加胺基酸的交互作用進而增加活性區的穩定性；策略二是做醣基化格式的改變，由於結構觀察到有一醣基化阻擋在活性區周圍，去除此醣基可提升9.6%的活性，另外將CbAppA獨有的三個醣基化位置分別添加到EcAppA上可觀察到在80℃下處理後，耐熱度由野生型EcAppA的1.8%，增加醣基化的突變增加為5.6-9.5%，且此耐熱有相加的作用，具有多三個醣基化的EcAppA耐熱可增加為33.1%。 在此論文中，提供了具有葡萄聚糖及甘露聚糖雙重受質功能性的纖維素酶TmCel5A蛋白質與配體複合體的結構，說明了在結構上觀察到的相互作用與酵素活性相符合，而高解析度的結構可做為後續改造的依據;而利用植酸酶的結構及其它已知的植酸酶特性及序列，使用不同策略進行改造，可有效率的增加活性及耐熱性，可做為其他酵素改造的參考，改造後的植酸酶有較佳的特性可增進工業應用性。

Exogenous enzymes have been produced and applied to many industrial fields for their abilities of accelerating the rates of the chemical reactions in a substrate specific manner. However, for the industrial applications, the traits of a natural derived enzyme need to be amended, such as the optimal conditions and the stability. Protein structures can be visualized by X-ray diffraction crystallography at atomic level. Thus, the interactions between the residues within protein structures and between substrates can be revealed for the rational design enzyme engineering. In our laboratory, we are devoted to utilize the information from protein structures for further protein engineering. In the thesis, two enzymes were studied. First, the structures of apo form and complex form of a thermostable cellulase were solved to give the whole picture of how the diverse substrates can be accommodated to the active site. Second, by structure-based ration design, the specific activity and thermostability of a phytase were improved. Lignocellulosic bioethanol is a highly potential alternative energy. Enzymes with thermostablility and multiple substrate specificities are preferred to cooperate synergistically, owing to the complexity of the lignocellulosic biomass composition and the production process. A thermostable cellulase Cel5A from Thermotoga maritima, characterized with activity toward both glucan- and mannan-based mainchain and simple and branched chains, is an excellent candidate for industrial application. Here, we report the crystal structures in apo-form and in complex with three ligands, cellotetraose, cellobiose and mannotriose, at 1.29 A to 2.40 A resolution. The open carbohydrate-binding cavity which can accommodate oligosaccharide substrates with extensively branched chains explained the dual specificity of the enzyme. Moreover, our results also suggest that the wide spectrum of TmCel5A substrates might be due to the lack of steric hindrance around the C2-hydroxyl group of glucose or mannose unit from the active-site residues. Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6 to 9.5% residual activity after treatment at 80°C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 33.1% residual activity. In conclusion, the structural information of TmCel5A in complex with its ligands was provided and open up a sight for future dual substrate activity engineering of enzymes in the large glycosyl hydrolase family 5 (GH5) for applications. On the other hand, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications.