Simultaneously optimizing multiple properties of β-glucosidase Bgl6 using combined (semi-)rational design strategies and investigation of the underlying mechanisms

• Published in: Bioresource technology 2023-04, Vol.374, p.128792-128792, Article 128792
• Main Authors: Li, Shuifeng, Cao, Lichuang, Yang, Xiangpeng, Wu, Xiangrui, Xu, Shujing, Liu, Yuhuan
• Format: Article
• Language: English
• Subjects: beta-Glucosidase - genetics; beta-Glucosidase - metabolism; Catalytic Domain; Cellulose - chemistry; Enzyme Stability; Glucose - metabolism; Molecular dynamics simulations; Multi-property optimization; Pocket engineering; Semi-rational design; β-Glucosidase
• ISSN: 0960-8524; 1873-2976
• DOI: 10.1016/j.biortech.2023.128792

[Display omitted] •Half-life of M12 was extended from 2 to 40 days using computer-aided strategies.•The activity of M12 was increased by 5.6-fold via pocket engineering.•Conflict between activity and glucose tolerance was overcome by region redesign.•Supplementation of M12 to Celluclast 1.5L increased glucose yield from SCB by 40%.•Mechanisms for property improvement were revealed by structure and dynamics analysis. The performance of β-glucosidase during cellulose saccharification is determined by thermostability, activity and glucose tolerance. However, conflicts between them make it challenging to simultaneously optimize three properties. In this work, such a case was reported using Bgl6-M3 as a starting point. Firstly, four thermostability-enhancing mutations were obtained using computer-aided engineering strategies (mutant M7). Secondly, substrate binding pocket of M7 was reshaped, generating two mutations that increased activity but decreased glucose tolerance (mutant M9). Then a key region lining active site cavity was redesigned, resulting in three mutations that boosted glucose tolerance and activity. Finally, mutant M12 with simultaneously improved thermostability (half-life of 20-fold), activity (kcat/Km of 5.6-fold) and glucose tolerance (ΔIC50 of 200 mM) was obtained. Mechanisms for property improvement were elucidated by structural analysis and molecular dynamics simulations. Overall, the strategies used here and new insights into the underlying mechanisms may provide guidance for multi-property engineering of other enzymes.