以大腸桿菌表現β-glucosidase及熱穩定性改良之研究

Abstract

生質酒精是一個極具潛力的能源之ㄧ。目前生質酒精的製程把纖維素水解及酒精發酵分成兩個部份，成兩個部份，若把兩個步驟結合成單一步驟將能降低40 %的生產成本。纖維素水解酶分成三種，分別為endoglucanase、exoglucanase及β-glucosidase，本實驗分別把五個β-glucosidase表現在E. coli表面以期能應用在水解發酵同步化。從醣類水解資料庫中，根據發酵的環境（37 °C, pH 7.0）選出五個細菌來源的β-glucosidases ( E.C. 3. 2. 1. 21 )，分別為BglA、BglB（Bacillus sp.）、BgxA（Erwinia sp.）、CelA、SalB（Azospirillum sp.）。BglA利用表面系統表現，活性為0.3 μmole/min/100 ml（OD600 = 1.0的菌液），BgxA則可達3.5 μmole/min/100 ml，均超越原本菌株的活性。 BglA，根據文獻，比活性為2417 units/mg同時具有3D結構，此部分利用生物資訊方法，藉由尋找BglA內能形成離子鍵的位置，利用點突變方式增加離子鍵的數目進而提高熱穩定性。BglA野生株粗萃取在55 ℃、pH 7之半衰期為15 min，Q216K則約為20 min，預期結合所有提高熱穩定性的突變點可得到耐高熱的BglA。根據實驗結果，利用離子鍵增加BglA熱穩定性的方法除電荷平衡外，須同時考慮B-fator，形成離子鍵的距離及改變胺基酸conserved與否。

Bioehtanol is an important energy to substitute gasoline. In this study, the β-glucosdiase expressed on the surface of E. coli which can be used for cellulose degradation and ethanol fermentation, synchronously. From the glycoside hydrolase database, five bacterial β-glucosidases ( E.C. 3. 2. 1. 21 ), BglA、BglB、BgxA、CelA and SalB with the optimum activity ( 37 °C, pH 7.0 ), were selected to express in E. coli. The activity of BglA was 0.3 μmole/min/100 ml which was expressed on the surface with surface display system 1（SDV1）. Furthermore, the activity of BgxA reached 3.5 μmole/min/100 ml which was expressed on the surface with SDV2. BglA from Bacillus sp. containing three dimensional structure was used to improve thermostability by site-directed mutagenesis. Ten possible sites for increasing ionic pairs of BglA were found. Half life of wild type BglA was 15 min at pH 7 and 55 ℃. The thermostability of Q216K was improved for half life of 20 min at pH 7 and 55 ℃. Therefore, the high themoresistance enzyme can be obtained by combination of single substitution for increasing salt bridges. In conclusion, charge balance, B-factor, ionic bonds distance and the conserved region of BglA need to be considered for rational design of thermostable BglA.