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While for substrates bearing bulky substitutions at C17 position, the activity is essentially lost. Finally, the origin of activity and selectivity for CYP109B1 catalyzed steroid hydroxylation is revealed by computational analysis, thus providing theoretical basis for directed evolution to further improve its catalytic properties.Cyclodextrins (CDs) are a family of α-1,4-linked cyclic oligosaccharides that possess a hydrophobic cavity and a hydrophilic outer surface with abundant hydroxyl groups. This unique structural characteristic all