Design, expression and functional characterization of a thermostable xylanase from Trichoderma reesei
Design, expression and functional characterization of a thermostable xylanase from Trichoderma reesei
复制标题
DOI：
10.1371/journal.pone.0210548
复制
发表时间：
2019-01-16
期刊：
影响因子：
3.7
通讯作者：
Yu, Feng
中科院分区：
文献类型：
--
作者：
He, Jun;Tang, Feng;Yu, Feng
从微生物如里氏木霉中分离的木聚糖酶由于其在各种工业应用中的潜力而吸引了相当大的研究兴趣。然而，天然分离的木聚糖酶不能承受苛刻的条件，如高温和碱性pH。reesei木聚糖酶（Xyn 2），和新的灵活的区域的酶的基础上确定的B因子，分子动力学（MD）参数。为了提高Xyn 2的热稳定性，采用定点突变的方法在Xyn 2的不稳定柔性区引入二硫键，并在毕赤酵母中成功表达了两种重组木聚糖酶XM 1（Xyn 2（Cys 12 -52））和XM 2（Xyn 2（Cys 59 -149））。通过SDS-PAGE估计分泌的重组Xyn 2为24 kDa。有趣的是，XM 1和XM 2在60 ℃下的半衰期分别比天然Xyn 2高2.5倍和1.8倍。XM 1还表现出改善的pH稳定性，并在2.0至10.0的pH值范围内保持超过60%的活性。然而，与XM 2和天然Xyn 2相比，XM 1的比活性和催化效率降低。我们的研究结果不仅有助于阐明蛋白质结构与功能之间的相互作用，而且有助于合理选择靶点以提高酶的热稳定性。
Xylanases isolated from microorganisms such as the Trichoderma reesei have attracted considerable research interest because of their potential in various industrial applications. However, naturally isolated xylanases cannot withstand harsh conditions such as high temperature and basic pH. In this study, we performed structural analysis of the major T. reesei xylanase (Xyn2), and novel flexible regions of the enzyme were identified based on B-factor, a molecular dynamics (MD) parameter. To improve thermostability of the Xyn2, disulfide bonds were introduced into the unstable flexible region by using site-directed mutagenesis and two recombinant xylanases, XM1 (Xyn2(Cys12-52)) and XM2 (Xyn2(Cys59-149)) were successfully expressed in Pichia pastoris. Secreted recombinant Xyn2 was estimated by SDS-PAGE to be 24 kDa. Interestingly, the half-lives of XM1 and XM2 at 60 degrees C were 2.5- and 1.8-fold higher, respectively than those of native Xyn2. The XM1 also exhibited improved pH stability and maintained more than 60% activity over pH values ranging from 2.0 to 10.0. However, the specific activity and catalytic efficiency of XM1 was decreased as compared to those of XM2 and native Xyn2. Our results will assist not only in elucidating of the interactions between protein structure and function, but also in rational target selection for improving the thermostability of enzymes.