Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36056
標題: 細菌性beta-1,3-葡萄聚醣水解酵素之選殖與應用
The Cloning and Application of beta-1,3-Glucanase from Bacteria
作者: 洪堂耀
Hong, Tang Yao
關鍵字: beta-1;beta-1;3-glucanase;S. sioyaensis;Paenibacillus sp.;antifungal;3-葡萄聚糖水解酵素;Streptomyces sioyaensis;Paenibacillus sp.;抗真菌
出版社: 生物科技學研究所
摘要: 
本論文從本土性菌株中,選殖beta-1,3-葡萄聚糖水解酵素的對應基因,分析此酵素的生化特性及抗真菌活性,評估其發展為生物防治藥劑的潛力。
首先從一株本土菌株Streptomyces sioyaensis選殖到一beta-1,3-葡萄聚糖水解酵素基因。其胺基酸序列N端與其它菌株的葡萄聚糖水解酵素基因具有高度的同源性,是屬於醣基水解酵素第16家族;而C端則和一些蛋白質的碳水化合物結合區第六家族具有同源性。此醣基水解酵素會專一性水解beta-1,3-鍵結的葡萄聚糖,水解方式為內切型。全長蛋白質和C端碳水化合物結合區截斷蛋白質對水溶性beta-1,3-葡萄聚糖有著近似的比活性,但對不溶性聚糖則以全長蛋白質的活性較高,全長蛋白質具有吸附多種不溶性聚糖的功能,顯示S. sioyaensis beta-1,3-葡萄聚糖水解酵素的C端碳水化合物結合區可增加與不溶性聚糖的親和性,推測因而增加了醣基水解酵素區域與聚糖的碰撞機會所致。
另外從土壤樣品中篩選到一株具高beta-1,3-葡萄聚糖水解酵素活性的菌株Paenibacillus sp.,其beta-1,3-葡萄聚糖水解酵素會專一性的水解beta-1,3-鍵結的葡萄聚糖,水解方式為內切型,會吸附多種不溶性聚糖。此菌株或S. sioyaensis beta-1,3-葡萄聚糖水解酵素,都可以破壞立枯絲核菌和腐霉菌菌絲的細胞壁,Paenibacillus sp. beta-1,3-葡萄聚糖水解酵素對此兩株真菌菌絲生長的抑制較為顯著。而從Paenibacillus sp.菌株中也選殖到一段beta-1,3-葡萄聚糖水解酵素基因,此基因所表達的蛋白質,對立枯絲核菌的菌絲生長也有明顯的抑制。
由以上結果可知,所純化或選殖的Paenibacillus sp. beta-1,3-葡萄聚糖水解酵素其抗真菌的效果較為顯著,期將來能夠應用於轉基因作物中,提高轉基因作物防禦病原性真菌的入侵。

A gene encoding beta-1,3-glucanase was isolated from Streptomyces sioyaensis. The N-terminal amino acid sequence shares similarity to bacterial endo-beta-1,3-glucanases classified in glycosyl hydrolases family 16 (GHF 16), while the C-terminus is a putative carbohydrate binding module (CBM) grouped into CBM family 6. The glycosyl hydrolase domain preferentially catalyzes the hydrolysis of glucans with beta-1,3 linkage, and has an endolytic mode of action. Binding assay indicated that the C-terminal CBM binds to various insoluble beta-glucans. The full-length and the CBM-truncated proteins had similar specific activity to soluble beta-1,3-glucan, whereas the former had much stronger specific activity to insoluble beta-1,3-glucans. This result suggests that the C-terminal CBM may enhance the affinity of the Streptomyces beta-1,3-glucanase to insoluble substrates, leading to the increase of the encounter events between the hydrolase domain and substrates.
A 44-kDa beta-1,3-glucanase was purified from the culture medium of a Paenibacillus strain. This purified enzyme preferentially catalyzes the hydrolysis of glucans with beta-1,3-linkage, has an endolytic mode of action, and showed binding activity to various insoluble polysaccharides. The enzymes from Paenibacillus and S. sioyaensis had the ability to damage the cell wall structures of the growing mycelia of phytopathogenic fungi, Pythium aphanidermatum and Rhizoctonic solani AG-4. Nonetheless, the Paenibacillus enzyme had a much stronger effect on inhibiting the growths of the tested fungi. Meanwhile, a beta-1,3-glucanase gene was isolated from the Paenibacillus sp. The recombinant protein expressed from Escherichia coli had effect on inhibiting the growths of R. solani AG-4.
URI: http://hdl.handle.net/11455/36056
Appears in Collections:生物科技學研究所

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