Aneurinibacillusthermoaerophilus耐热性聚木糖酶之纯化与生化特性探讨.docVIP

Aneurinibacillus thermoaerophilus耐熱性聚木 糖酶之純化與生化特性探討 Purification and characterization of thermostable xylanase from Aneurinibacillus thermoaerophilus 研究生：李臻育 Li, Chen-Yu 指導教授：許詳純 Sheu, Shyang-Chwen 【摘要】 由於環境污染日益嚴重及能源危機接踵而至，因此如何減少環境污染及尋求替代性再生能源成為一重要課題。本實驗自高溫發酵農業廢棄物中篩選出一株能分泌耐熱性聚木糖酶之菌株，經由其型態分析、菌體脂肪酸組成及16S rDNA等分析，鑑定為Aneurinibacillus thermoaerophilus。利用燕麥聚木糖為唯一碳源的Mandels-Reese培養基誘導後，可分泌聚木糖酶，培養三天後其粗酵素液可達最高活性4.5 U/mL。粗酵素液經過濃縮、硫酸銨鹽沉澱、Q-spharose FF離子交換層析、phenyl spharose FF疏水性管柱層析及sphacryl S-200HR膠體過濾層析等步驟純化後，可成功分離出主要的三型聚木糖酶，分別命名為Xyl A、Xyl B及Xyl C，且經sphacryl S-200HR膠體過濾層析及非還原性SDS電泳判定其分子量，結果顯示其分子量分別為66 kDa、130 ka與14 kDa。Xyl B及Xyl C之pI值分別為5.5及9.0。Xyl C純化比活性可達354.4 U/mg，純化倍率達36.4倍。 進一步以A. thermoaerophilus誘導後分泌之主要聚木糖酶Xyl C為材料，探討其生化特性，其最適作用溫度及pH分別為70℃及pH 6，60?C作用3小時後仍保有66%之活性，於pH 6~10的環境中30分鐘仍未達其半衰期。5 mM 之Na+、Mn+2、Fe+2及Zn+2存在均可顯著提升反應活性，又以Mn+2存在下可提高酵素活性1.4倍為最高；而5 mM Hg+2、Cu+2、Mg+2、β-ME和PMSF的添加對酵素活性均有抑制作用，其中以Hg+2為最強之酵素抑制劑。Xyl C聚木糖酶僅對樺木與燕麥聚木糖基質具有高特異性，並以薄層色層分析確認Xyl C聚木糖酶水解樺木聚木糖的產物型態，比對後發現產物以木二糖、木三糖及木寡糖為主，因此可證實由A. thermoaerophilus分泌之Xyl C聚木糖酶為一內切型聚木糖酶。 關鍵字：Aneurinibacillus thermoaerophilus、聚木糖酶、純化、特性探討 【Abstract】 Because of the increasing environmental pollution and energy crisis, how to reduce the pollution and find a renewable energy source become an important issue. We have screened one thermophilic bacterial strain from compost which had xylanase activity. The bacterial strain was identified as Aneurinibacillus thermoaerophilus by morphological and phenotypic characteristics, cellular fatty acids analysis and 16S rDNA sequence. A. thermoaerophilus could produce extracellular multi-xylanases by using oat spelt xylan as the inducer. After 3 days of incubation, the highest xylanase activity (4.5 U/mL) was reached. Purification of xylanase was performed by ultrafiltration, ammonium sulfate precipitation, Q-sepharose FF anion exchange chromatography, phenyl sepharose FF hydrophobic chromatography and sephacryl S-200HR gel filtration chromatography. Three xylanases named as Xy