嗜鹽菌Bacillus licheniformis NTU-01纖維素水解酶基因之選殖與表現

Molecular cloning and expression of the cellulase gene from halophiles Bacillus licheniformis NTU-01

林培茵(Pei-Yin Lin)

指導教授 ： 徐源泰

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.00121

摘要

內切型纖維素水解酶在纖維廢棄物之再利用上扮演重要角色，它可用以切除纖維素的β-1,4-糖苷鍵而釋放出葡萄糖，在生質能源之水解程序中扮演重要角色。本研究是從台灣南部鹽田分離出之嗜鹽菌株Bacillus licheniformis NTU-01中選殖出一個β-1,4-內切型纖維素水解酶(EC 3.2.1.4)基因，其隸屬於醣苷水解酶第五家族。此目標基因命名為bglC6B，包含一完整之開放式閱讀框架，由1,548個核苷酸所組成，可轉譯出515個胺基酸殘基，與Bacillus sp. NBL420 celA基因(AAK73277.1)有99 %同源性。將bglC6B基因次選殖到表現載體pET-28b vector，並轉形至Escherichia coli BL21 (DE3)進行異源表現。重組後的纖維素水解酶BglC6B以快速蛋白質層析系統進行純化，以SDS-PAGE分析後發現，其蛋白分子量約為50 kDa左右。純化後之重組酵素以CMC當基質，測得其活性高達14. 97 U/ml，而其催化之最適pH和溫度為pH 5和30℃，且溫度可作用範圍極廣，在20-60℃之間皆維持高活性，熱穩定性方面則於40℃下放置4小時，仍有高達72 %的相對活性。在0-15%氯化鈉濃度環境下，皆不至於使酵素失活，顯示酵素可耐高鹽份。上述結果凸顯此酵素表現系統的確可適用於高鹽份之農業纖維廢棄物，在轉化成纖維酒精過程中深具開發潛力。

關鍵字

Bacillus licheniformis NTU-01 ； 內切型纖維素水解酶bglC6B基因 ； 耐鹽性 ； 異源表現

並列摘要

Endocellulase plays an important role in the degradation of fiber wastes, it can cleave the β-1,4-glycosidic bond of cellulose to release glucose, which has great potential in applications such as biomass energy. A β-1,4-endoglucanase (EC 3.2.1.4) gene belonging to the glycosyl hydrolase family 5 was cloned from the halophilic bacteria Bacillus licheniformis NTU-01, isolated from saline area in southern Taiwan. The target gene named bglC6B contains a complete ORF of 1,548 nucleotides that translates 515 amino acid residues, it showed 99% homology to Bacillus sp. NBL420 celA gene(AAK73277.1). The bglC6B gene was subcloned into pET-28b vector and heterogenously expressed by E. coli BL21 (DE3). Recombinant BglC6B, which was purified by fast protein liquid chromatography (FPLC) and based on SDS-PAGE analysis, the recombinant BglC6B showed a molecular mass of 50 kDa. The CMCase activity of purified, recombinant BglC6B is 14.97 U/ml. The enzyme showed an optimal pH and temperature of pH5 and 30℃, it was very stable at the broad temperature range from 20 to 60℃, and it retained 72% relative activity after 4 h incubation at 40℃. The effect of salinity was indicated that the enzyme was active over a range of 0-15% sodium chloride. These results indicate this recombinant BglC6B is active and stable in some extreme environment stress such as high salt concentration, which is maybe suitable for cellulose biodegration of saline agricultural wastes to cellulolytic ethanol.

並列關鍵字

Bacillus licheniformis NTU-01 ； endoglucanase ； bglC6B gene ； halostable ； heterologous expression

參考文獻

簡廷因，2008。利用序列特徵探勘預測酵素催化部位。國立台灣大學資訊工程學系碩士論文。

謝易儒，2008。嗜鹽菌Vibrionaceae sp. NTU 05纖維素水解酶基因之選殖與表現。國立台灣大學園藝學研究所碩士論文。

A Oren, 2002. Diversity of halophilic microorganisms: Environments, phylogeny, physiology, and applications. Journal of Industrial Microbiology & Biotechnology 28, 56-63.

A. Singh and K. Hayashi, 1995. Microbial cellulases: protein architecture, molecular properties, and biosynthesis. Advances in Applied Microbiology 40, 1-44.

Buchanan, Gruissem, Jones, 2000. Biochemistry & molecular biology of plants (1st ed.) American society of plant physiology.