AUTHOR=Huang Jia-Jun , Hu Hao-Xuan , Lu Yu-Jing , Bao Ya-Dan , Zhou Jin-Lin , Huang Mingtao 

TITLE=Computer-Aided Design of α-L-Rhamnosidase to Increase the Synthesis Efficiency of Icariside I

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=10

YEAR=2022

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2022.926829

DOI=10.3389/fbioe.2022.926829

ISSN=2296-4185

ABSTRACT=<p>Icariside I, the glycosylation product of icaritin, is a novel effective anti-cancer agent with immunological anti-tumor activity. However, very limited natural icariside I content hinders its direct extraction from plants. Therefore, we employed a computer-aided protein design strategy to improve the catalytic efficiency and substrate specificity of the α-L-rhamnosidase from <italic>Thermotoga petrophila</italic> DSM 13995, to provide a highly-efficient preparation method. Several beneficial mutants were obtained by expanding the active cavity. The catalytic efficiencies of all mutants were improved 16–200-fold compared with the wild-type TpeRha. The double-point mutant DH was the best mutant and showed the highest catalytic efficiency (<italic>k</italic><sub><italic>cat</italic></sub>/<italic>K</italic><sub><italic>M</italic></sub>: 193.52 s<sup>−1</sup> M<sup>−1</sup>) against icariin, which was a 209.76-fold increase compared with the wild-type TpeRha. Besides, the single-point mutant H570A showed higher substrate specificity than that of the wild-type TpeRha in hydrolysis of different substrates. This study provides enzyme design strategies and principles for the hydrolysis of rhamnosyl natural products.</p>