纖維素是D-葡萄糖以β-1,4-糖苷鍵組成的長鏈聚合物，分子式（C6H10O5）n，是維管束植物細胞壁的主要成分。臺灣每年達200萬公噸的農業廢棄物，其主要成份為木質纖維素，若能有效轉化木質纖維素為生質能源，不但能解決農業廢棄物的污染問題、日趨減少的能源問題，亦不與人類搶地爭糧。纖維素水解酵素分為三種類型，內切型纖維素水解酵素（Endo-1,4-β-D-glucanase ,EC 3.2.1.4）、外切型纖維素水解酵素（Exo-1,4-β-D-glucanase ,EC 3.2.1.91）與β-葡萄糖苷酵素（β-glucosidase ,EC 3.2.1.21）；此三種酵素是將纖維素水解為葡萄糖發酵反應之速率決定步驟。本研究主要探討從稻稈堆肥中篩選出一株具有纖維水解酵素的細菌Geobacillus sp.。其中此菌具有一段基因長1104 bp，368個胺基酸所組成的endo-glucanase，命名為GsCelA，為glycosyl hydrolase family 5 (GHF5)。GsCelA能作用廣泛的溫度介於45 ℃~80 ℃，且具有極佳的熱穩定度。為了解蛋白質結構上胺基酸對於GsCelA的活性及熱穩定性的影響，因此將GsCelA胺基酸序列與其它glycosyl hydrolase family 5 (GHF5)酵素的胺基酸做序列比對，發現有七個位置的胺基酸是GsCelA特有的。將這七個位置做指定點突變，發現突變E128及P243會降低GsCelA活性，而突變Y195會增加GsCelA活性，但都不影響熱穩定性。在K57、E128、I168、A223及P243位置突變會降低酵素對於pH 5的耐受性，其中K57G降低22 %、E128L降低55 %、I168K降低28 %、A223R降低45 %及P243V降低35 %。有趣的是突變株T51W，其蛋白質降解速率要比GsCelA來的慢。由研究成果希望經由指定點突變(置換)來探討，突變位置對於酵素活性及其熱穩定度等之影響。對於不同要求的發酵特性，因應出相對條件，提高酵素的經濟效益。

Cellulose is a polysaccharide consisting of a linear chain of several hundreds to over ten thousands β-1,4 linked D-glucose units. The formula of cellulouse is (C6H10O5)n, which is an important structural component of cell walls of vascular plants. There are two millions tons of agriculture wastes produced in Taiwan annually, and the main component is lignocellulose, a mixture of lignin- and cellulose-related molecules. Developing an efficient method to convert lignocellulose into biomass energy is can reduce the pollution generated by agriculture wastes and also to address the energy shortage problem. There are three types of cellulases: endo-1,4-β-D-glucanase (EC 3.2.1.4), exo-1,4-β-D-glucanase (EC 3.2.1.91), and β-glucosidase (EC 3.2.1.21), which can break down the polymer of cellulose into glucose. The soil bacterium Geobacillus sp.70PC53, originally isolated from a rice straw compost, synthesizes a glycosyl hydrolase family 5 (GHF5) endo-glucanase, GsCelA, which contains 368 amino acids. GsCelA has high level of thermostability and it can be used from 45 ℃ to 80 ℃. It retains 90 % of its original activity after incubation at 65C for 4 hrs. In order to investigate the structure/function relationship of GsCelA, the amino acid sequences of GsCelA were compared with other GHF5 glycosyl hydrolases. Seven amino acids of GsCelA appear to be unique because they do not exist in other GHF5 enzymes. These amino acids were mutated and impacts of these mutations on enzyme activity and thermostability were investigated. It was discovered that mutations of E128 and P243 reduced, but mutation of Y195 increased enyzme activity, yet all of them had no significant effect on thermostability. In contrast, mutations in K57, E128, I168, A223 and P243 reduced thermostability at pH 5. However, for the mutants at K57G, E128L, I168K, A223R and P243V reduced GsCelA activity by about 22 %, 55 %, 28 %, 45 % and 35 %, respectively. Another interesting observation was that the T51W mutant has a slower rate of self-truncation of C-terminal region than wild-type GsCelA. In conclusion, following the site-directed mutagenesis approach to investigating the activity and thermostability of GsCelA, I have revealed new information for improving GsCelA under suitable conditions, thus enhancing the economical benefit of using this enzyme for hydrolysis of cellulose.

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