探討海棲熱袍菌Cel5A 的生化鑑定及定點突變

Abstract

植物生物質中的纖維素和半纖維素，經由水解酶處理後，可用於生產生物燃料。在我們先前的研究發現熱纖維梭菌的纖維素酶/木聚醣酶雙功能酵素CtCel5E和極端嗜熱海棲熱袍菌的纖維素酶/甘露聚醣酶雙功能酵素TmCel5A，兩者有高度的序列同源性，但卻擁有不同的雙功能活性。其中一部分序列具有顯著差異，稱之為flexible loop，當把CtCel5E的flexible loop置換為TmCel5A的序列後，得到一具有三功能酵素，稱為CtCel5E-Tmloop，具有纖維素酶/木聚醣酶/甘露聚醣酶活性。 於本論文中，經由表現、純化和定性在大腸桿菌中表現的重組蛋白TmCel5A，發現不只具有原本報告中提到的纖維素酶/甘露聚醣酶雙功能活性，亦同時具有木聚醣酶的活性。並藉由定點突變確認位於flexible loop區域的三個胺基酸H205、 W210、Y198，將三者突變為丙氨酸(alanine)後會顯著減少活性，說明三者對於催化作用的重要性，其中尤以H205和W210對甘露聚醣酶影響更為劇烈。 此外，藉由保留性的Tmloop結構比對發現4個纖維素酶，並表現熱纖維梭菌的纖維素酶CtCelC，發現不同於原本報告提到的單一活性，而同樣是一個三功能酵素具有纖維素酶/木聚醣酶/甘露聚醣酶的活性，說明了flexible loop此序列對多功能活性是必要的。

Cellulose and hemicelluloses from plant biomass can be used to produce biofuel after enzymatic process. We have engineered Clostridium thermocellum Cel5E (CtCel5E), a bifunctional cellulase/xylanase, into a tri-functional cellulase/xylanase/mannanase, named CtCel5E-Tmloop, by replacing its flexible loop region with that in Thermotoga maritima Cel5A (TmCel5A), a homologous bifunctional cellulase/mannanase, suggesting that the Tmloop might be essential for multi-functionality. In this thesis, the recombinant TmCel5A expressed in E. coli was purified and characterized. Surprisingly, TmCel5A also showed low xylanase activity, in addition to the known cellulase and mannanase activities. By site-directed mutagenesis, H205A, W210A, and Y198A in the loop region showed significantly reduced activities, indicating their importance in catalysis. In particular, H205 and W210 are essential for mannanase activity. Moreover, by searching the conserved Tmloop structures in other cellulases, we identified Clostridium thermocellum CelC (CtCelC) also with tri-functional cellulase/xylanase/mannanase activity.