熱纖梭菌之纖維水解與半纖維水解雙功能酵素的蛋白質結構分析與工程改造

Abstract

纖維素及半纖維素為生質能源的主要來源；降解這些長碳鏈多醣物質至單醣分子是需要透過纖維水解酶與半纖維水解酶共同協同作用。在本篇論文中所研究熱纖梭菌Clostridium thermocellum純化出來的雙功能水解酵素Cel5E可同時降解木質纖維素的主要組成纖維素及半纖維素中的聚木醣(xylan)。其纖維水解酶降解纖維素最適溫度及酸鹼值分別為50度和pH 5.0；而聚木醣水解酶降解聚木醣最適溫度及酸鹼值分別為60度和pH 6.0。 藉由薄膜層析法與酵素動力學實驗，顯示CtCel5E分解纖維素比分解聚木醣來的有效率，而鈣、鎂及鍶離子和還原劑DTT皆能增進CtCel5E的雙功能水解活性。第209號麩胺酸(Glu209)及第314號麩胺酸(Glu314)為雙功能水解酵素的催化位置。此外，藉由與纖維雙醣水解酶(β-1,4 glucosidase)的協同作用下，可完全將CtCel5E主要水解產物中的雙醣分子降解成葡萄糖，而這些單醣分子後續可被微生物發酵利用做生產生質酒精之用。據我所知，目前並無任何文獻指出為何5號家族的內切纖維水解酶同時也可辨認異聚木醣，為了找尋哪些胺基酸對於酵素受質專一性是重要的，藉由結晶學實驗，CtCel5E的酵素結構已被成功解出，且利用單定點突變實驗去找出哪些胺基酸對於酵素的水解能力是必要的。與TmCel5A酵素(PDB: 3MMU)的立體結構做比較後，觀察到CtCel5E第274號麩胺酸(Glu274)到第290號天門冬胺酸(Asp290)的位置具有段長度較短且易擺動的結構，且第270號酪胺酸(Tyr270)對於酵素催化過程中扮演重要的角色。再者，第128號精胺酸(Arg128)對於CtCel5E在pH 4.0酸性環境下降解纖維素的過程是重要的。 該酵素與受質共結晶的實驗仍在進行中，相信該實驗未來將會提供更完整關於酵素催化反應機制的資訊，也相信該實驗對於未來生質能源工業應用上有很大的助益。

Cellulose and hemicellulose are sources of biofuel production. Their degradation into monosaccharide requires action of cellulase (endoglucanase, exoglucanase, and β-glucosidase) and hemicellulase. Cel5E from Clostridium thermocellum belongs to family 5 of the glycoside hydrolases. It shows activity against both cellulose and hemicellulose (xylan), suggesting it is an unusual bifunctional β-1,4 endoglucanase/β-1,4 xylanase. The optimal conditions for endoglucanase and xylanase activities are 50°C, pH 5.0 and 60°C, pH 6.0, respectively. By thin-layer chromatography (TLC) analysis and kinetic assays, CtCel5E displays a higher catalytic efficiency on hydrolysis of cellulose than on hydrolysis of xylan. In addition, some metal ions and chemical reagents such as Ca2+, Mg2+, Sr2+ as well as Dithiothreitol (DTT) can improve both endoglucanase and xylanase activities of CtCel5E. Glu209 and Glu314 were identified as the catalytic sites for its dual-activities. Besides, CtCel5E displays obvious synergistic effects with β-1,4 glucosidase, which can further degrade cellulolytic biomass into fermentable sugars. To my knowledge, there is no literature explained why family 5 endoglucanase can also hydrolyze heteroxylan. In order to investigate which amino residues are responsible for substrate specificity, CtCel5E was crystallized. Crystals of CtCel5E have been obtained using sitting drop method, and structure has been determined. To distinguish amino acids essential for catalysis, mutagenesis experiments were performed. By structures superimposition with TmCel5A (PDB: 3MMU), residues from Glu274 to Asp290 may be a shorter flexible loop and Tyr270 play crucial role in enzyme catalysis. Moreover, Arg128 is important for CtCel5E cellulase to function at pH 4.0. Crystallization of ligand-bound CtCel5E is underway, and we believe it can provide detailed information for structure-based engineering, leading to potential biofuel industrial applications.