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.