【摘要】 从Aspergillus niger NG1306菌株中扩增得到β-木糖苷酶基因anxyl,与pPICZαA连接构建了表达载体pPICZαA-anxyl,进一步在毕赤酵母KM71H中诱导表达获得了目的酶蛋白重组β-木糖苷酶（Anxyl）;并将其用于人参皂苷Rb₃及C-Mx的生物转化及催化动力学参数表征.研究结果表明,Anxyl可将人参皂苷Rb₃和C-Mx分别转化为人参皂苷Rd和C-K.酶学性质研究表明,该酶最适反应pH=2. 5,最适反应温度为35℃;在pH=2. 0～5. 0,20和30℃条件下其具有较好的稳定性,Mg²⁺对酶活具有促进作用.Anxyl对葡萄糖和乙醇有较好的耐受性,其催化对硝基苯基-β-D-吡喃木糖苷（pNPX）、人参皂苷Rb3和C-Mx水解的米氏常数（Km）值分别为3. 1,1. 55和1. 04 mmol/L,酶的最大反应速率（Vmax）分别为1. 9×10²,0. 8×10³和8×10³mmol/min,酶的转换数（Kcat）值分别为5. 55,0. 17和1. 85 s^-1,表明该酶对天然底物Rb₃和C-Mx具有更高的亲和力.更多还原

【Abstract】 The β-xylosidase gene anxyl which was amplified from Aspergillus niger NG1306 was cloned into pPICZαA to construct the recombinant expression vector（ pPICZαA-anxyl）. The recombinant protein（Anxyl）was further expressed in Pichia pastoris KM71 H for ginsenoside Rb₃,C-Mx biotransformation and catalytic kinetic study. The results showed that anxyl could convert ginsenosides Rb₃ and C-Mx to Rd and C-K,respectively. The results of corresponding enzymatic properties showed that the optimum pH of Anxyl was 2.5,and the optimum reaction temperature was 35 ℃ . It has good stability at pH of 2. 0—5. 0 and 20—30 ℃ . Mg²⁺ could improve the catalytic activity of Anxyl and had good tolerance for glucose and ethanol. The Kmvalues of p-nitrophenyl-β-D-xylopyranoside（pNPX）,ginsenoside Rb₃ and C-Mx were 3.1,1.55 and 1.04 mmol/L,the V_max values of p NPX,Rb₃ and C-Mx were 1.9 102,0.8 103 and 8 103 mmol/min,and the Kcatvalues of pNPX,Rb₃ and C-Mx were 5.55,0.17 and 1.85 s^-1,respectively. The results indicated that the recombinant Anxyl had a higher affinity for the natural substrates Rb₃ and C-Mx.更多还原