定点突变提高里氏木霉木聚糖酶 (XYN II) 的稳定性

Enhancing stability of Trichoderma reesei xylanase (XYN II) by site-directed mutagenesis

摘要:

通过定点突变的方法，在来源于里氏木霉Trichderma reesei的木聚糖酶XYN II的N-末端两个β折叠片层间添加二硫键，以提高木聚糖酶的稳定性。原酶XYN-OU和突变酶XYN-HA12 (T2C、T28C和S156F) 分别在毕赤酵母中分泌表达，突变酶与原酶纯化后进行酶学性质比较。结果表明：突变酶最适反应温度由50℃提高到60℃；在70℃的半衰期由1 min提高到14 min；最适反应pH为5.0，与原酶保持一致，但是在50℃、30 min条件下的pH稳定范围由4.0~9.0扩展到3.0~10.0。对木聚糖酶分子改良的结果反映出在β片层间添加二硫键可以有效改善酶在较高温度下三维结构的刚性，提高热稳定性。

Abstract:

We engineered a disulphide bridge between two adjacent double-layered β-sheet at the N-terminal region of Trichoderma reesei endo-1,4-β-xylanase II(XYN II) by site-directed mutagenesis. The native xylanase XYN-OU and the mutated xylanase XYN-HA12 (T2C, T28C and S156F) were separately expressed in Pichia pastoris. Both xylanases were purified and characterized. The optimum temperature of XYN-HA12 was increased from 50°C to 60°C, relative to XYN-OU. At 70°C, the halftime of inactivation for XYN-OU and XYN-HA12 were 1 min and 14 min, respectively. The optimum pH of XYN-HA12 was 5.0, similar to XYN-OU. However, XYN-HA12 could retain over 50% activity from pH 3.0 to 10.0 at 50°C for 30 min. As for XYN-OU, it could retain over 50% activity from the pH value 4.0 to 9.0 at 50°C in 30 min. The result of the mutated xylanase indicated that constructed disulphide bridge could improve its thermostability at relatively higher temperature.