Trichoderma reesei 所產生xylanase之純化與生化特性探討

Purification and biochemical Properties of xylanase from Trichoderma reesei

王柏堯(Po-Yao Wang)

指導教授 ： 許成光 ； 鍾雲琴

亞洲大學/健康學院/生物科技學系碩士班/碩士(2005年)

摘要

寡木醣為深具潛力的機能性食品素材，可藉由聚木醣酵素(xylanase)水解聚木糖而得到具生理活性之木二糖、木三糖等不同鏈長之寡醣。本研究目的為純化Trichoderma reesei聚木醣酵素並探討其生化特性。實驗將T. reesei液體培養5天所得的粗酵素液，利用超過濾濃縮(5k pellicon XL filter)、陰離子交換層析(DEAD Sepharose)和膠體過濾層析(Sephacryl S-200HR)等步驟純化後，可獲得比活性為4433 U/mg之聚木醣酵素。利用 SDS-polyacrylamide gel electrophoresis得知此酵素分子量為20.25 kDa。以等電焦集電泳測得此酵素pI值為9.3，為一鹼性蛋白質。此聚木醣酵素水解聚木醣的最適反應溫度與pH值分別為50℃和4.0。且於pH 2~4較穩定，溫度方面則在30~50℃反應30分鐘仍有95%以上的殘餘活性。以樺木聚木醣為基值時，Km值為8.85 mg/ml，Vmax為5.89 μmole/min/mg，活化能則為7.8 kcal/mole。金屬離子Ca2+存在下，酵素活性會被活化，而Hg2+抑制酵素活性最為明顯。化學修飾劑N-bromosuccinimide (NBS)會完全抑制酵素活性，而N-acetylimidazole (NAI)則會促進酵素活性。此酵素水解樺木聚木醣的主要產物為木二榶與木三榶，為一內切型聚木醣酶。

關鍵字

聚木醣酵素 ； 純化 ； 寡木醣

並列摘要

Xylooligosaccharides, such as xylobiose and xylotriose, have been considered as a potential health food. They can be produced by digesting hemicellulose with xylanase. The aim of this study was to purify xylanase from Trichoderma reesei and determine the biochemical properties of the enzyme. Crude xylanase was obtained by incubating T. reesei for 5 days, and then the crude enzyme was concentrated with a 5k-cut off fitter membrane, purified with a DEAD-Sepharose ion exchange chromatography followed by a Sephacryl S-200 HR gel filtration chromatography. The molecular weight of the purified xylanase measured by SDS-PAGE was approximately 20.25 kDa. It’s pI and specific activity were 9.3 and 4433 U/mg, respectively. Optimal reaction temperature and pH for the xylanase were 50℃ and 4.0, respectively. The xylanase was more stable at pH range of 2-4 and maintained 95% residual activity after incubating at 30~50℃ for 30 min. When using birchwood xylan as the substrate, the Km and Vmax of the xylanase were 8.85 mg/ml and 5.89 μmole/min/mg, respectively. Activation energy of the xylanase was 7.8 Kcal/mole. The xylanase activity was inhibited by either the presence of Hg2+ or N- bromosuccinimide (NBS) but enhanced by Ca2+ and N-acetylimidazole (NAI). The hydrolysates obtained by digesting birchwood xylan with the xylanase contained mainly xylobiose and xylotriose.

並列關鍵字

xylanase ； Trichoderma reesei ； Purification

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