Figure 9
4 Conclusions
In summary, biocatalysis in organic solvents is very appealing for the industry in producing many intermediates and fine chemicals, and improving the organic solvents stability of enzyme is urgent for biomanufacturing via protein engineering. The beneficial mutation sites were identified via regional random mutation, and the best mutant T23I/T200K/P260S (M3) was obtained by combinatorial mutation, which not only enhanced the resistance against organic solvents, but also improved the catalytic efficiency of the enzyme. MD results showed that increasing the rigidity of the loop region on the protein surface displayed a positive correlation with organic solvents resistance. And the increase of intra- and intermolecular interactions stabilized the protein structure so that mutant could tolerate high concentration organic solvents. Under 25% DMSO, M3 displayed better catalytic performance toward 1-acetylnaphthalene than WT. In conclusion, M3 is a potential excellent biocatalyst for the synthesis of (R )-NEA due to its high organic solvent stability.
Authors’ contributions The design and conduct of experiments were executed by J. Huang, S. Qiu, C. N. Wang, C. J. Lyu, F. F. Fan, S. Hu and W. R. Zhao. The writing and revising of manuscript were done by S. Qiu, J. Q. Mei, and C. N. Wang. Project administration was in the charge of J. Huang and L. H. Mei. All authors read and approved the manuscript
Funding This research was funded by the National Natural Science Foundation of China (Nos. 32071268, 32201037, 31971372, 22103071), Ningbo “Scientific and Technological Innovation 2025” Key Project (2020Z080).
Data availability All data and materials are available upon reasonable request.