纖維素水解主要需要三種纖維水解酵素的協同作用：外切型纖維水解酵素、內切型纖維水解酵素和β-葡萄糖苷水解酵素。土芽孢桿菌G. 70PC53從台灣南部的稻草堆肥中所分離出來。GsCelA是一種由此嗜熱菌土芽孢桿菌(Geobacillus sp. 70PC53, G. 70PC53)分泌，蛋白全長為368個胺基酸的內切型纖維水解酵素。GsCelA歸類於醣苷水解酵素家族5 (glycoside hydrolase family 5, GH5)，其蛋白質結構是典型的TIM-barrel結構。GsCelA酵素作用最適溫度為60℃，pH為5.0。純化後的GsCelA在高於10 ℃的溫度時會進行獨特的蛋白自我截斷作用，從GsCelA的C端移除53個胺基酸的胜肽，而此作用的最佳條件為pH 6-7。這種自我截斷作用並非由蛋白水解酵素的作用而截斷，此截斷作用會受到EDTA和EGTA的抑制，但二價金屬離子可增加自我截斷作用的效率。這種自我截斷作用也發生在原生菌地芽孢桿菌G. 70PC53所分泌的GsCelA，不過它會受到葡萄糖的濃度提高而抑制。在實驗中也發現GsCelA的C端胜肽鍊與非溶性受質蓬鬆化Avicel具有結合能力，並且也與革蘭氏陽性菌的細胞壁肽聚醣具有親和力。原菌自我截斷或者或利用蛋白質工程將GsCelA的C端截斷至60個胺基酸都可提高GsCelA的比活性並且使酵素在高溫更穩定。

SUMMARY
The endoglucanase GsCelA secreted from the thermophilic Geobacillus sp. 70PC53 exhibits excellent lignocellulolytic activity and high thermostability. This enzyme has an optimal temperature at 60℃ and an optimal pH at 5.0. GsCelA belongs to the glycoside hydrolase family 5 with a typical TIM-barrel structure. After the spontaneous self-truncation process, the smaller form GsCelA showed higher activity then that observed in the full-length enzyme. Results of MS-MS analysis indicated that 53 amino acids are removed from the C-terminus of GsCelA via the self-truncation process. This process is not due to the protease contamination, but it can be suppressed by EDTA and EGTA or be recovered by divalent metal ions. The self-truncation process also takes place in vivo in the native Geobacillus sp. 70PC53 strain. The spontaneous or engineered C-terminal truncation up to 60 amino acids from the C-terminus improves GsCelA’s specific activity and renders the enzyme more thermostable. To investigate the importance of specific amino acids on the enzymatic activity of GsCelA, site-directed mutagenesis and protein engineering approach were employed to replace amino acid residues unique to this enzyme. It was demonstrated that single mutations Y195T, D55S, G288T or D289L increase the activity of this enzyme by 30%.
Key words: Endoglucanase, Self-truncation, GH family 5, Geobacillus

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