Engineering of multiple arginines into the Ser/Thr surface of Trichoderma reesei endo-1,4-β-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH
Engineering of multiple arginines into the Ser/Thr surface of Trichoderma reesei endo-1,4-β-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH
复制标题
DOI：
10.1093/protein/15.2.141
复制
发表时间：
2002-02-01
期刊：
影响因子：
--
通讯作者：
Leisola, M
中科院分区：
文献类型：
--
作者：
Turunen, O;Vuorio, M;Leisola, M
研究了表面酶数目的增加对里氏木霉内切-1，4-β-木聚糖酶II的酶活和稳定性的影响。在两个突变体系列中，突变体的数量增加。第一组在蛋白质表面的不同侧上含有六个组氨酸。这些生物碱对热稳定性没有显著影响。但最适pH范围变窄。另一系列的五个木聚糖酶被工程化到'Ser/Thr表面'，由位于'右手样'木聚糖酶一侧的双层β-折叠的一部分形成。这些突变使酶的活性向碱性区域移动了大约0.5 -1.0个pH单位。此外，在50- 55 ℃，酶在无底物存在下的稳定性显著下降，但在高温下，Ser/Thr表面的丝氨酸能提高酶的活性。在底物存在下，在60- 65 ℃下热稳定性增加4-5倍。因此，底物中和了Ser/Thr表面丝氨酸的去稳定作用，并揭示了相同突变的稳定作用。只有在Ser/Thr表面引入5个甘氨酸后，才能清楚地看到甘氨酸在高温下的稳定作用。
We studied the effects of increase in the number of surface arginines on the enzyme activity and stability of Trichoderma reesei endo-1,4-beta-xylanase II. The number of arginines was increased in two mutant series. The first set contained six arginines on different sides of the protein surface. These arginines had no significant effect on the thermostability. However, the optimal pH region became narrower. Another series of five arginines was engineered into the 'Ser/Thr surface', formed of part of the double-layered beta-sheet located on one side of the 'right-hand-like' xylanase. These mutations shifted the activity profile to the alkaline region by similar to0.5-1.0 pH units. In addition, the arginines on the Ser/Thr surface increased the enzyme activity at high temperature, although the enzyme stability in the absence of substrate decreased significantly at 50-55degreesC. In the presence of the substrate, the thermostability increased 4-5-fold at 60-65degreesC. Thus, the substrate neutralized the destabilizing effect of Ser/Thr surface arginines and revealed a stabilizing effect of the same mutations. The stabilizing effect of arginines at high temperatures was seen clearly only when five arginines were introduced into the Ser/Thr surface.