黑麴菌β-葡萄醣苷酶II之結構基因分析

STRUCTRURE GENE ANALYSIS OF ASPERGILLUS NIGER β-GLUCOSIDASE II

周積孚(Chi-Fu Chou)

指導教授 ： 顏聰榮

大同大學/工程學院/生物工程學系所/碩士(2004年)

摘要

β-葡萄糖苷酶為催化醣基與氧親核基間反應屬於醣苷酶家族之酵素，由於作用機制為保留式反應，因此可能因其他非水配基作用釋放被酵素所包圍之醣基而達成糖轉移之效果，先前研究中，自黑麴菌Aspergillus niger BCRC 31494中純化之β-葡萄糖苷酶II，除水解活性外，亦具有特定之糖轉移活性，以纖維二醣為基質可合成纖維寡糖，若添加醇類為基質，則可合成相對應之烷基糖苷化合物，產物可成為機能性食品或利用於製藥原料之發展。為達到利用遺傳工程技術將其基因選殖並改良其調控系統以提高酵素表現功能之目的，本研究利用已知β-葡萄糖苷酶II核苷酸序列設計適當引子，利用聚合酶連鎖反應技術，成功對A. niger BCRC 31494 β-葡萄糖苷酶II基因體DNA與RNA反轉譯之cDNA進行定序，基因體序列開放讀架區域核苷酸序列長度為2,948 bp，包括7個外顯子與6個內含子，cDNA核苷酸序列長度為2,583 bp，可轉譯成具860個胺基酸之酵素，根據轉譯之蛋白質序列，酵素屬於醣苷酶家族3，並且為β-葡萄糖苷酶II亞科B之成員。

關鍵字

調控 ； 序列 ； 基因 ； 葡萄醣苷酶 ； 黑麴菌 ； 醣轉移

並列摘要

β-Glucosidase is a member of glycosidase family that catalyzes the transfer of glycosyl group between oxygen nucleophiles. Because of its retaining mechanism, transglycosylation may occurs when the retained glucose released by aglycone other than water. In the previous study, β-glucosidase II was isolated from Aspergillus niger. Besides the typical hydrolysis activity, it was found that processes transglycosylation activity toward specific substrates. Cello-oligosaccharide was synthesized from cellobiose, and alkyl-glucosides were synthesized when supplied with alcohol. The products may apply to functional food and pharmaceutical industrial. For the purpose of enzyme cloning, properties enhancement, and regulation comprehension; we designed primers from known b-glucosidase sequences and succeed in obtaining the DNA and RNA-reversed-transcribed cDNA sequence of A. niger β-glucosidase II. The cloned genomic sequence revealed a 2,948 bp open reading frame with 6 introns and 7 exons. The cDNA showed a 2,583 sequence encoding an 860-amono-acid protein that belongs to glycosyl hydrolase family 3 as well as identified as a member of β-glucosidase subfamily B.

並列關鍵字

aspergillus niger ； glucosidase ； regularion ； sequence ； transglycosylation

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