Identification and Mutation Analysis of Nonconserved Residues on the TIM-Barrel Surface of GH5_5 Cellulases for Catalytic Efficiency and Stability Improvement

文献类型： 外文期刊

第一作者： Zheng, Jie

作者： Zheng, Jie;Qin, Xing;Yang, Kun;Wang, Xiao-Lu;Wang, Ya-Ru;Wang, Yuan;Yao, Bin;Luo, Hui-Ying;Huang, Huo-Qing;Zheng, Jie;Liu, Han-Qing;Tian, Jian

作者机构：

关键词： nonconserved residues; alpha-helices; GH5_5 family cellulases; TIM-barrel fold; catalytic efficiency; lignocellulosic biomass

期刊名称：APPLIED AND ENVIRONMENTAL MICROBIOLOGY （ 影响因子：5.005； 五年影响因子：5.632 ）

ISSN： 0099-2240

年卷期： 2022 年 88 卷 17 期

页码：

收录情况： SCI

摘要： Exploring the potential functions of nonconserved residues on the outer side of alpha-helices and systematically optimizing them are pivotal for their application in protein engineering. Based on the evolutionary structural conservation analysis of GH5_5 cellulases, a practical molecular improvement strategy was developed. Highly variable sites on the outer side of the alpha-helices of the GH5_5 cellulase from Aspergillus niger (AnCel5A) were screened, and 14 out of the 34 highly variable sites were confirmed to exert a positive effect on the activity. After the modular combination of the positive mutations, the catalytic efficiency of the mutants was further improved. By using CMC-Na as the substrate, the catalytic efficiency and specific activity of variant AnCel5A_N193A/T300P/D307P were approximately 2.0-fold that of AnCel5A (227 +/- 21 versus 451 +/- 43 ml/s/mg and 1,726 = 19 versus 3,472 +/- 42 U/mg, respectively). The half-life (t(1/2)) of variant AnCel5A_N193A/T300P/D307P at 75 degrees C was 2.36 times that of AnCel5A. The role of these sites was successfully validated in other GH5_5 cellulases. Computational analyses revealed that the flexibility of the loop 6-loop 7-loop 8 region was responsible for the increased catalytic performance. This work not only illustrated the important role of rapidly evolving positions on the outer side of the a-helices of GH5_5 cellulases but also revealed new insights into engineering the proteins that nature left as clues for us to find. IMPORTANCE A comprehensive understanding of the residues on the alpha-helices of the GH5_5 cellulases is important for catalytic efficiency and stability improvement. The main objective of this study was to use the evolutionary conservation and plasticity of the TIM-barrel fold to probe the relationship between nonconserved residues on the outer side of the alpha-helices and the catalytic efficiency of GH5_5 cellulases by conducting structure-guided protein engineering. By using a four-step nonconserved residue screening strategy, the functional role of nonconserved residues on the outer side of the alpha-helices was effectively identified, and a variant with superior performance and capability was constructed. Hence, this study proved the effectiveness of this strategy in engineering GI-15_5 cellulases and provided a potential competitor for industrial applications. Furthermore, this study sheds new light on engineering TIM-barrel proteins.

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