transform 4-androstenedione (AD) to1,4-androstadiene-3,17-dione (ADD). in this study, we investigated how the active site of KstD211 affected substrate specificity by protein modeling and site-directed mutagenesis. We found that Tyr116 played an important role in recognizing steroid substrates. The mutation of Tyr116 to Ile enhanced the conversion rate of 4AD from 54% to 87%, compared with KstD211. in a pilot-scale reaction, the productivity of ADD by converting 4-AD reached 3.32 g/L/h. Furthermore, our data also revealed that different F116 mutants exhibited distinct specificity for a variety of steroidal substrates. Therefore, our work has provided the potential application of to KstD211 to dehydrogenize steroids in pharmaceutical industry.
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