中文摘要

本實驗的目的在純化從地熱谷溫泉土壤所篩選之煙麴黴 -葡萄糖
苷酶及其基因片段之比對，以期利用在工業上纖維素之分解。-葡萄
糖苷酶粗酵素液之最適溫度及最適 pH 分別為 65℃、5.0 且比活性為
1.95 U/mg protein，活性與發表文獻之-葡萄糖苷酶相當。將粗酵素液
使用超過濾膜進行濃縮約 50 倍後經 Sephacryl-S200 及 Sephacryl-S300
之膠體過濾，則有明顯分離效果。以 SDS-PAGE 確認其分子量為 170
kDa。-葡萄糖苷酶在 pH 4-9 呈現相當之穩定性，由熱穩定性實驗顯示，
75 ℃加熱 1 小時殘餘活性仍將近 65 ﹪，若溫度上升至 85 ℃，活性僅
剩 40 %。除此之外，-葡萄糖苷酶於-mercaptoethanol、Tween-80 及
SDS 存在下活性被些微抑制，這些特性將有利於工業化生產時應用。

實驗共使用六種方法（LiCl 法，Total RNA 試劑，RNAzolTM B 試

劑，ULTRASPECTM RNA 試劑，TriPureTM Kit 試劑和 TRIzol 試劑）進
行 A. fumigatus 之 RNA 抽取，結果以 TRIzol 試劑效果最佳。首先設計
九組-葡萄糖苷酶基因之引子，其中 5’端五組，3’端四組，再加 oligo
（dT）以二十五種組合進行聚合酶連鎖反應，其中僅 F1/oligo （dT）
引子對具增幅效果，其 PCR 產物經定序完成後，利用 NCBI 資料庫中
之 BLAST 軟體進行比對。目前我們所得到的 DNA 產物，尚未在 NCBI
上搜尋到相關保守序列或已註冊之序列。

關鍵字：-葡萄糖苷酶、RNA。

Abstract

The objective of the present study is to particial purify and

characterize the properties of -glucosidase from Aspergillus fumigatus
which screened from Hot Spring of Taipei Valley and comparison of
genetic fragments of -glucosidase for industrial cellulose degradation. The
optimal temperature and pH of -glucosidase were 65℃and 5.0,
respectively. Furthermore, the specific activity of -glucosidase, 1.95 U/mg

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protein, is similar to those of reported literatures. The crude enzyme
solution was concentrated by ultramembrane filtration and then purified by
size exclusion techniques. Significant resolution effect was obtained by
Saphacryl-S200 and Saphacryl-S300. The molecular weight was
determined by SDS-PAGE of 170 kDa. -glucosidase showed broader pH
stability in the range of pH 4-9. The thermostable study demonstrated that
65% residual activity was maintained when the -glucosidase was
incubated at 75℃ for an hour, and only 40% activity was remained if
incubation temperature up to 85℃ under the defined condition. In addition,
-mercaptoethanol, Tween-80 and SDS showed a little inhibition on -
glucosidase activity and it revealed great highlight in industrial enzyme
production. Six common methodologies, LiCl method, Total RNA reagent,
RNAzolTM B reagent, ULTRASPECTM RNA reagent, TriPureTM Kit reagent
and TRIzol reagent, were employed for RNA extraction of Aspergillus
fumigatus. The resulted illustrated that TRIzol reagent was the best kit for
RNA extraction. An oligo（dT）and nine sets of primer of -glucosidase
gene were designed which were 5 sets for 5’ terminal and 4 sets for 3’
terminal. Totally, 25 combinations were used for PCR amplification and
only F1/oligo （ dT ） showed some amplified fragment. The product was
sequenced and compared to the registered sequences of -glucosidase by
BLAST program in NCBI. Conclusively, it hard to identify the amplified
product since no highly conserved region was found between our fragment
and registered ones.

Key words: -glucosidase, RNA.

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