文献类型： 外文期刊

第一作者： Zheng, Fei

作者： Zheng, Fei;Zheng, Jie;Wang, Yuan;Tu, Tao;Wang, Xiaoyu;Yao, Bin;Luo, Huiying;Zheng, Fei;Wang, Xiaoyu;Xie, Xiangming;Vermaas, Josh V.;Beckham, Gregg T.

作者机构：

关键词： (beta alpha)(8)-barrel structure; GH5 endoglucanase; hybrid enzymes; structure-based recombination; thermostability

期刊名称：APPLIED AND ENVIRONMENTAL MICROBIOLOGY （ 影响因子：4.792； 五年影响因子：5.26 ）

ISSN： 0099-2240

年卷期： 2019 年 85 卷 5 期

页码：

收录情况： SCI

摘要： Cellulases from glycoside hydrolase family 5 (GH5) are key endoglucanase enzymes in the degradation of diverse polysaccharide substrates and are used in industrial enzyme cocktails to break down biomass. The GH5 family shares a canonical (beta alpha)(8)-barrel structure, where each (beta alpha) module is essential for the enzyme's stability and activity. Despite their shared topology, the thermostability of GH5 endoglucanase enzymes can vary significantly, and highly thermostable variants are often sought for industrial applications. Based on the previously characterized thermophilic GH5 endoglucanase Egl5A from Talaromyces emersonii (TeEgl5A), which has an optimal temperature of 90 degrees C, we created 10 hybrid enzymes with elements of the mesophilic endoglucanase Cel5 from Stegonsporium opalus (SoCel5) to determine which elements are responsible for enhanced thermostability. Five of the expressed hybrid enzymes exhibit enzyme activity. Two of these hybrids exhibited pronounced increases in the temperature optimum (10 and 20 degrees C), the temperature at which the protein lost 50% of its activity (T-50) (15 and 19 degrees C), and the melting temperature (T-m) (16.5 and 22.9 degrees C) and extended half-lives (1(1/2)) (similar to 240- and 650-fold at 55 degrees C) relative to the values for the mesophilic parent enzyme and demonstrated improved catalytic efficiency on selected substrates. The successful hybridization strategies were validated experimentally in another GH5 endoglucanase, Cel5 from Aspergillus niger (AnCel5), which demonstrated a similar increase in thermostability. Based on molecular dynamics (MD) simulations of both the SoCelS and TeEgl5A parent enzymes and their hybrids, we hypothesize that improved hydrophobic packing of the interface between alpha(1) and alpha(2) is the primary mechanism by which the hybrid enzymes increase their thermostability relative to that of the mesophilic parent SoCel5. IMPORTANCE Thermal stability is an essential property of enzymes in many industrial biotechnological applications, as high temperatures improve bioreactor throughput. Many protein engineering approaches, such as rational design and directed evolution, have been employed to improve the thermal properties of mesophilic enzymes. Structure-based recombination has also been used to fuse TIM barrel fragments, and even fragments from unrelated folds, to generate new structures. However, little research has been done on GH5 endoglucanases. In this study, two GH5 endoglucanases exhibiting TIM barrel structure, SoCel5 and TeEgl5A, with different thermal properties, were hybridized to study the roles of different (beta alpha) motifs. This work illustrates the role that structure-guided recombination can play in helping to identify sequence function relationships within GH5 enzymes by supplementing natural diversity with synthetic diversity.

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