Han Chengye, Yu Shiyuan, Ouyang Jia, Li Xin, Zhou Juan, Xu Yan
Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing 210037, China.
Sheng Wu Gong Cheng Xue Bao. 2010 May;26(5):623-9.
We engineered a disulphide bridge between two adjacent double-layered beta-sheet at the N-terminal region of Trichoderma reesei endo-1,4-beta-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 degrees C to 60 degrees C, relative to XYN-OU. At 70 degrees 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 degrees 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 degrees C in 30 min. The result of the mutated xylanase indicated that constructed disulphide bridge could improve its thermostability at relatively higher temperature.
我们通过定点突变在里氏木霉内切 - 1,4 - β - 木聚糖酶II（XYN II）的N端区域两个相邻的双层β - 折叠之间构建了一个二硫键。天然木聚糖酶XYN - OU和突变木聚糖酶XYN - HA12（T2C、T28C和S156F）分别在毕赤酵母中表达。两种木聚糖酶均被纯化并进行了特性分析。相对于XYN - OU，XYN - HA12的最适温度从50℃提高到了60℃。在70℃时，XYN - OU和XYN - HA12的失活半衰期分别为1分钟和14分钟。XYN - HA12的最适pH为5.0，与XYN - OU相似。然而，XYN - HA12在50℃、pH 3.0至10.0条件下保温30分钟后仍能保留超过50%的活性。对于XYN - OU，在50℃、pH值4.0至9.0条件下保温30分钟后能保留超过50%的活性。突变木聚糖酶的结果表明，构建的二硫键可以在相对较高温度下提高其热稳定性。
