一种来源于Leifsonia shinshuensis DICP 16菌体的b-D-木糖苷酶的分离纯化及其性质

Purification and Characterization of a b-D-xylosidase from Leifsonia shinshuensis DICP 16

摘要:

采用盐析、DE 52、Q-Sepharose Fast Flow阴离子交换层析、Toyopearl Butyl 650C疏水层析以及Sephacryl S-300 HR凝胶过滤层析联用的方法, 从Leifsonia shinshuensis DICP 16菌体中纯化出一种b-木糖苷酶。分离后该酶在SDS-PAGE 上呈单一蛋白质条带, 通过SDS-PAGE和凝胶过滤层析法, 测得该酶是一个由两个分子量约为91 kD的相同亚基组成的同源二聚体。其水解对硝基苯酚木糖苷(pNPX)的最适反应温度为55°C, pH值为7.0。该木糖苷酶在45°C以下, pH 6.0~11.0之间具有很好的稳定性。在45°C, pH值为7.0的条件下, 水解pNPX的Km, Vmax分别为1.04 mmol/L, 0.095 mmol/(min·mg)。研究不同的金属离子对该酶的活性影响, 发现Fe2+和Cu2+是很强的抑制剂。通过对天然木糖苷化合物的水解测试, 发现该酶可以水解人参皂苷Rb3的木糖基, 产生人参皂苷Rd, 却不能水解紫杉烷木糖苷的木糖基。

Abstract:

A b-D-xylosidase from Leifsonia shinshuensis DICP 16 was purified to apparent homogeneity using a combination of ammonium sulfate precipitation, DE 52 anion-exchange, Q-Sepharose Fast Flow anion-exchange, Toyopearl Butyl 650C hydrophobic- interaction and Sephacryl S-300 HR gel-permeation chromatography. The purified xylosidase consisted of two same subunits and had the relative molecular weight of 180 kD as determined by SDS-PAGE and gel-permeation chromatography. The maximal b- D-xylosidase activity occurred at 55°C and pH 7.0. It was stable at 45°C and retained its original activity for 60 min. The stability declined rapidly when the temperature rose above 55°C. The xylosidase was stable in the pH range from 6.0 to 11.0 for 20 h. At pH 7.0 and 45°C the Km for p-nitrophenyl-b-D-xylopyranoside (pNPX) was 1.04 mmol/L and the Vmax was 0.095 mmol nitrophenol/ min/mg xylosidase. The enzyme was inhibited strongly by Fe2+ and Cu2+. It exhibited low levels of activity against other artificial substrates, compared to its activity against pNPX. When different natural xylosides were used as the substrates, the xylosidase showed distinct hydrolysis ability. It could hydrolyze 20-C, b-(1→6)-xyloside of ginsenoside Rb3 (G-Rb3) into ginsenoside Rd, but did not hydrolyze the other b-D-glucosidic bonds of G-Rb3. Additionally, the xylosidase could not hydrolyze C-7 xylosyl-bearing taxanes.