黑曲霉胞外耐高糖β-葡萄糖苷酶的分离纯化及部分特性研究  

Study on Purification and Some Properties of A Highly Glucose-tolerant Extracellular β-Glucosidase from Aspergillus niger

作　　者：赵林果[1] 游丽金[1] 孟鹏[1] 余世袁[1] 

机构地区：[1]南京林业大学化学工程学院,江苏南京210037

出　　处：《林产化学与工业》2007年第6期41-46,共6页Chemistry and Industry of Forest Products

摘　　要：采用硫酸铵盐析、Phenyl Sepharose CL-4B疏水层析、DEAE-Sephamse阴离子交换层析、Sephacryl S-200凝胶过滤层析等步骤，从黑曲霉的发酵液中获得了凝胶电泳均一的两种β-葡萄糖苷酶。其中一种为耐高糖的β-葡萄糖苷酶，葡萄糖抑制系数（Ki）为41．01mmol／L，纯化倍数和得率分别为56．7倍和22．66％。耐高糖β-葡萄糖苷酶单亚基分子质量为114．6ku，以对硝基苯酚-β-肛葡萄糖苷（pNPG）为底物时，米氏常数（Km）值和酶促反应的最大速率（vmax）分别为0．904mmol／L和1．08μmol／min。该酶最适反应温度60℃，最适反应pH值为4．0；在60℃以下及pH值3—7范围内均能保持稳定。所测定的化学试剂中，Ag^＋对酶具有较强的抑制作用，其它金属离子及乙二胺四乙酸对酶的活性影响不大。甲醇、正丁醇有明显激活酶活性的作用，但乙腈、丙酮对酶的抑制作用明显。Two kind of extracellularβ-glucosidases were purified to homogeneity from an Aspergillus niger by ammonium sulfate precipitation, Phenyl Sepharose CL-4B hydrophobic interaction chromatography, DEAE-Sepharose ion exchange chromatography and Sephacryl S-200 HR gel layer chromatography. One of them was a highly glucose-tolerant novelβ-glucosidase with a Ki of 41.01 mmol/L, and the final purification factor 56.7 times and 22.66 % yield were obtained. The enzyme single subunit molecular weight was about 114.6 ku which was identified by SDS-PAGE. The Km and Vmax values of the enzyme were 0.904 mmol/L and 1.08 μmol/min, respectively, using p-nitrophenyl-β-D-glucopyranoside （pNPG） as a substrate. The opti- mum reaction temperature and pH value forβ-glucosidase were 60 ℃ and pH value 4.0, respectively. The enzyme was stable in the pH value range of 3.0-7.0 and up to 60 ℃. The enzyme was greatly inhibited by Ag ^＋ Other metal ions tested and EDTA hand no effects on the activity ofβ-glucosidase. Different organic solvent had different effects on the activity ofβ-glucosidase, methanol and 1-butanol enhanced the activity, whereas acetone and acetonitrile inhibited the activity obviously.

关 键 词：Β-葡萄糖苷酶 耐 南糖 提纯 黑曲霉 

分 类 号：TQ92[轻工技术与工程—发酵工程]