葡萄糖透過自我聚合(self-assembly)而組成的纖維素,為大自然界最豐富的天然聚合物,其降解必須透過三種纖維素酶的協同作用(synergistic effect)來完成。絲狀真菌具有纖維素酶的生產力,Aspergillus niger為其中之ㄧ,具有能力生產endoglucanase、cellobiohydrolase、β-glucosidase三種酵素。 本實驗室已將Aspergillus niger BCRC 31494中β-glucosidase基因分析完畢(周積孚,2004),故在此分析另外兩個酵素的基因序列與結構。內切型纖維素纖維分解酵素(endoglucanase B,EGLB)與外切型纖維素纖維分解酵素(cellobiohydrolase A,CBHA)。eglB以直鏈纖維素為基質,在水溶性或非水溶性纖維素中都可以表現,降解後可產生還原端與非還原端葡萄糖,並且減短纖維素長度;cbhA以水溶性纖維素(avicel)為基質,透過avicel誘導後,可將直鏈葡萄糖降解成纖維二糖的雙糖分子。本篇論文主要定序此兩條基因的序列,並分析基因結構與尋找上游可能調控序列。 經由NCBI資料庫搜尋已知的序列設計適當引子,利用聚合酶連鎖反應技術成功的對A. niger BCRC 31494 cbhA與eglB基因體DNA與RNA反轉錄之cDNA進行定序,並利用DNAMAN軟體進行比對與分析。eglB基因體序列全長為1,217 bp,包括5個內含子,cDNA核苷酸序列長度為996 bp,可轉譯成331個胺基酸之酵素;cbhA基因體開放讀架區域核苷酸序列長度為1,512 bp,包括3個內含子,cDNA核苷酸序列長度為1,359 bp,可轉譯成453個胺基酸之酵素,並根據RPS-BLAST分析轉譯蛋白質序列,EGLB為醣苷酶家族5,CBHA為醣苷酶家族7。
Cellulose is the most abundant and renewable biopolymer on Earth. Cellulose has been used for centuries; however, it's enormous potential as a renewable source of energy was recognized only after the cellulose degrading enzymes or "cellulases" had been identified. Microorganisms including fungi, bacteria and actinomycetes produce mainly three types of cellulase: endoglucanase, exoglucanase and β-glucosidase. Aspergillus niger can be used in function food and pharmaceutical industrial. For the purpose of enzyme cloning, propertise enhancement, and regulation comprehension; we designed primers from known endoglucanase and cellobiohydrolase sequence and succeed in obtaining the DNA and RNA-reversed transcribed cDNA sequence of A. niger endoglucanase and cellobiohydrolase. The endoglucanase B cloned genomic sequence revealed a 1,217 bp open reading frame with 5 introns and 6 exons. The cDNA showed a 996 bp sequence encoding an 331 amino acid protein that belongs to glycosyl hydrolase family 5. The full length of cellobiohydrolase A is 1,512 bp. Its cDNA shows 1,359 bp. There are 3 intron in the cbhA gene. The CBHA belongs of glycosyl hydrolase 7.