Abstract
β-Glucosidase (BGL) plays a key role in cellulose hydrolysis. However, it is still a great challenge to enhance product tolerance and enzyme activity of BGL simultaneously. Here, we utilized one round error-prone PCR to engineer the Penicillium oxalicum 16 BGL (16BGL) for improving the cellulosic ethanol yield. We identified a new variant (L-6C), a triple mutant (M280T/V484L/D589E), with enhanced catalytic efficiency (\({k}_{cat}/{K}_{m}\)) for hydrolyzing pNPG and reduced strength of inhibition (\({K}_{m}^{app}/{K}_{I}\)) by glucose. To be specific, L-6C achieved a \({K}_{m}^{app}/{K}_{I}\) of 0.35 at a glucose concentration of 20 mM, which was 3.63 times lower than that attained by 16BGL. The catalytic efficiency for L-6C to hydrolyze pNPG was determined to be 983.68 mM−1 s−1, which was 22% higher than that for 16BGL. However, experiments showed that L-6C had reduced binding affinity (2.88 mM) to pNGP compared with 16BGL (1.69 mM). L-6C produced 6.15 g/L ethanol whose yield increased by about 10% than 16BGL. We performed molecular docking and molecular dynamics (MD) simulation, and binding free energy calculation using the Molecular Mechanics/Poisson Boltzmann surface area (MM/PBSA) method. MD simulation together with the MM/PBSA calculation suggested that L-6C had reduced binding free energy to pNPG, which was consistent with the experimental data.
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Acknowledgements
We thank Dr. Xiaoqiang Huang from Department of Computational Medicine and Bioinformatics, University of Michigan, for the guidance on molecular docking, MD simulation, and binding free energy calculation. This work was supported by the National Natural Science Foundation of China (21666010, 31360217), and the Doctoral Starting up Foundation of Jiangxi Normal University (5451).
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Wang, K., Huang, Q., Li, H. et al. Co-evolution of β-glucosidase activity and product tolerance for increasing cellulosic ethanol yield. Biotechnol Lett 42, 2239–2250 (2020). https://doi.org/10.1007/s10529-020-02935-9
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DOI: https://doi.org/10.1007/s10529-020-02935-9