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標題: 探討十字花科黑腐病菌纖維分解酵素的生化功能
Biochemical function analysis of Xanthomonas campestris cellulase genes
作者: 陳國洲
Chen, Kuo-Chou
關鍵字: 纖維分解酵素;cellulase
出版社: 生命科學院碩士在職專班
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摘 要

纖維分解酵素被廣泛應用在工業上,也與植物致病機制有關。屬革蘭氏陰性菌的 Xanthomonas campestris pv. campestris (簡稱 Xcc) 為專一感染十字花科植物,造成黑腐病 (black rot disease) 的植物病原菌。將台灣分離之菌株 Xc17 與已知全基因序列的ATCC 33913 種源 Xcc 菌序列比對結果,發現 9 個極為近似的纖維分解酵素基因,本研究旨在探討 Xc17 中近似纖維分解酵素基因是否具有生化功能。將先前已由 Xc17 所選殖的 5 個纖維分解酵素基因 Xcc0027、Xcc0028、Xcc1752、Xcc3381、Xcc3521-以 pBBad22k 載體分別轉殖入Xc-HC120 (Xcc3521-cellulase-minus mutant) 與 E. coli JM103y 後,分析菌落分解纖維素的活性結果顯示前者在 LB 中,Xcc3521、Xcc0028 的酵素活性增加 3.9 與 10 倍,而在 1% CMC-LB 中的酵素活性則增加 3.4 與 8 倍。此外,以螢光報導基因載體 pYFL1,試驗各菌株在 LB 與 1% CMC-LB 中分解纖維素的活性,發現 Xcc0028 在 JM103y 菌株中的酵素活性增加 2 與 3.6 倍;在 DH5α菌株的酵素活性增加 3.3 與 3.5 倍,兩者的螢光值均增加 3.3 倍;而在 LE392 菌株的酵素活性與螢光值卻無明顯增加,推測轉殖之酵素基因在該菌株仍有 basal 表現量。酵素反應特性分析顯示 Xcc3521 與 Xcc0028 同為不耐熱、偏酸性酵素,最適作用溫度及 pH 值分別為 30~50˚C 及 4~7 。以 SDS-PAGE 膠體電泳回收 Xc17 胞外主要蛋白後,由質譜定序部份裂解蛋白質片段與 Xcc3521 序列相同,涵蓋率為 23%。本研究初步結果顯示表達之 Xcc3521、Xcc0028 基因具有分解纖維素之酵素活性。

Cellulases were extensively applied in industry and were also involved in pathogenecity of plant pathogens. The gram-negative bacterium Xanthomonas campestris pv. campestris is the causal agent of the black rot disease in crucifers. Alignment of the partial sequence of the Taiwan native Xc17 strain with full genomic sequence of the ATCC 33913 strain revealed nine homologous cellulase genes. Here, we aim to study whether these cellulase genes in Xc17 strain have similar biochemical function. Previously cloned cellulase genes (Xcc0027、Xcc0028、Xcc1752、Xcc3381、Xcc3521) in pBBad22k vectors were transformed into Xc-HC120 ( an Xcc3521-cellulase-minus mutant ) and E. coli JM103y strains for cellulolytic activity assay. The results showed that in LB medium, the enzymatic activity of the former transformant with Xcc0028 or Xcc3521 gene increased by 3.9 and 10 fold and in 1% CMC-LB medium, the activities increased by 3.4 and 8 fold respectively. Furthermore, we used luciferase reporter gene vector pYFL1 to monitor protein expression level and cellulolytic activities both in LB and in 1% CMC-LB medium. The activity of Xcc0028 transformant increased by 2 and 3.6 fold for JM103y strain, and increased by 3.3 and 3.5 fold for DH5α strain respectively. In both strains, the bioluminescence increased by 3.5 fold. There were no significant changes observed for LE392 strain which may indicate basal expression. Both Xcc3521, Xcc0028 are acidic and thermo-labile enzymes. The optimal enzymatic activities were observed in 30-50 ˚C and in the pH range of 4.0-7.0. The LC/MS/MS sequencing of the partial digested peptide fragments of the extracellular protein of Xc17 matched the sequence of Xcc3521 with 23% sequence coverage. Our preliminary study of five putative cellulase genes in Xc17 showed that the Xcc0028 and Xcc3521 have cellulolytic enzyme activities.
其他識別: U0005-0704200810381200
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