| Summary: | 碩士 === 國立陽明大學 === 生物化學研究所 === 91 === In this study, the xylanase and β- xylosidase secreted by Xylaria regalis were isolated , purified and characterized. We used the avicel and oat spelts xylan as the only carbon source to induce the production of xylan-degrading enzyme by this fungus. The fungus was grown in a rotary shaker at 25℃ for 26 days. And the culture filtrate was collected and further purified by anion exchanger column (QAE Sephadex A 25), hydrophobic interaction column (phenyl 650M) and gel filtration column (Sephadex G 75), respectively. The specific activity of the finally purified xylanase was 1.30x103 unit /mg and its purity was increased 11 fold, and the recovery was 34.6%. The molecular weight of xylanase estimated by SDS-PAGE was approximately 30.8 kDa. The optimal pH and temperature for this enzyme was pH 5.5 and 50℃. The xylanase was stable above pH 5 at low temperature. When the enzyme treated with distinct metal ions, the activity was significantly inhibited by Cu2+and Hg2+. Xylanase activity was also inhibited by EDTA or β-mercaptoethanol. The results show that the active site of xylanase may have disulfide bond which can stabilize the xylanase third structure and certain metal ions may be the cofactor of the enzyme.
We collected the culture filtrate for the purification of β-xylosidase. Then culture filtrate was precipitated by 80% ammonium sulphate and further purified by hydrophobic interaction column (phenyl 650M), anion exchanger column (QAE) and gel filtration column (Sephadex G 75), respectively. The specific activity of the finally purified β-xylosidase was 4.00x10-1 unit /mg. The recovery of the enzyme was 24.2% and the purity was increased 22.97 fold. The molecular weight of β-xylosidase estimated by SDS-PAGE was approximately 45 kDa . The optimal pH and temperature for β-xylosidase was pH 5.5 at 50℃. β-xylosidase was stable above pH 5 at low temperature. Most of the tested metal ions have no remarkable effects on the activity of the β-xylosidase, whereas the Fe2+and Hg2+ significantly inhibit it. In addition, the activity of the β-xylosidase was not affected by EDTA or β-mercaptoethanol.
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