Abstract
本論文之目的在探討木腐黴纖維素外雙醣水解脢Ｉ酵素之功能與性質，及其基因之選殖、定序、異體表現與基因座定位。當木腐黴受到纖維素等因子誘導後，會製造大量纖維素水解脢，其中纖維素外雙醣水解脢Ｉ佔胞外蛋白質總量的百分之六十以上。經由蛋白純化及電泳分析顯示，此酵素之分子量約為 68,000，等電位值約為4.0。採用對硝基苯-β-纖維雙醣甘為反應基質測得此酵素之比活性為每毫克1.4國際單位。相對於其他纖維素水解脢，纖維素外雙醣水解脢Ｉ對於結晶形纖維素有較佳之作用能力。這是因為它對纖維素具有高度結合能力。比較此酵素的纖維素外雙醣水解能力及纖維素結合能力發現，兩者之物理與化學性質不盡相同，由此研判，這兩種作用能力應分屬於酵素之不同部份。 經由分子選殖法得到之纖維素外雙醣水解脢Ｉ基因（包括其互補DNA及基因座DNA）進行核酸定序分析及比對發現，此基因可轉譯一含513胺基酸之蛋白質，其中並含有兩段插入子。此一來自koningii菌株的基因和早先已知來自reesei菌株者之間只有六個核酸次序不同:一個在5'端未轉譯位置，二個在轉譯部位造成無意義突變，另三個在第一插入子中。在此基因之上游，發現有五個部位其序列與其他真菌發現的碳代謝抑制子結合部位相同。在此數個部位之下游與轉譯起始點之間，亦有數個類似真核生物轉錄因子結合部位之序列存在。 利用脈衝場凝膠電泳技術，我們進一步探討纖維素外雙醣水解脢Ｉ基因及內木聚糖脢Ⅱ基因之染色體座落。首先經由電泳分析，將木腐黴基因座分出五條帶，其大小分別為4.4、4.7、 5.7、6.7及7.2百萬鹼基對。其中最小者具有極明亮之螢光強度，顯示可能有兩條以上同樣大小的染色體在此位置，由此得到木腐黴基因座大小至少有28.7百萬鹼基對。經南方氏墨漬法分析得知纖維素外雙醣水解脢Ｉ基因及內木聚糖脢Ⅱ基因均和 4.4百萬鹼基對大小之染色體帶雜合，但此帶可能含兩條以上之染色體，故有待進一步研究以確定此二基因是否在同一染色體上。Cellobiohydrolase I (CBH I) from a filamentous fungusTrichoderma koningii G-39 has been purified by SP- SephadexC-50 and DEAE-Sephadex A-50 ion-exchange chromatographies. Itshowed a molecular weight of 68 k and pI of 4.0 upon sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)and isoelectric focusing. This enzyme has a specific activityof 1.4 unit/mg using p-nitrophenyl-b-D-cellobioside as asubstrate and prefers to interact toward crystallinesubstrates. It exhibited both exocellobiohydrolytic andcellulose binding activities, with different chemical andphysical characteristics. CBH I encoding gene cbh1 has beencloned, including a 1.4 kb cDNA and 7.0 kb genomic DNA. Thesequence analysis reveals that part of the cloned sequenceencodes a 513 amino acids-protein. The coding region is splitby two introns with lengths of 67 and 63 base pairs.Nucleotide sequence alignment to the putative promoter regionof cbh1 reveals significant homology to the carbon cataboliterepressor binding sites of Aspergillus nidulans CREA andSaccharomyces cerevisiae MIG1 promoters which respond to a wide-domain regulatory system of glucose repression. In addition,several transcription factor binding site-like sequences areidentified in 5' flanking region of cbh1 as well. Theelectrokaryotype of T. koningii is obtained by pulsed field gelelectrophoresis (PFGE). The fungal chromosomes were separatedto five bands. The lowest band showed non-proportionalfluorescent intensity, it is possible that at least two similarsize chromosomes comigrated on this band. Accordingly, thefungal genome is thought to be constituted of five or morechromosomes. The sizes of chromosomes are estimated as 4.4,4.7, 5.7, 6.7 and 7.2 Mb, respectively, and the genome size iscalculated to be about 28.7 Mb. The results of PFGE followedby Southern hybridization indicates that cbh1 and xyl2, a genecoding for an endoxylanase, are both mapped at the lowest band.