초록
<P><B>Abstract</B></P><P>12α‐Hydroxysteroid dehydrogenase (12α‐HSDH) has the potential to convert cheap and readily available cholic acid (CA) to 12‐oxochenodeoxycholic acid (12‐oxo‐CDCA), a key precursor for chemoenzymatic synthesis of the therapeutic bile acid ursodeoxycholic acid (UDCA). In this work, a native nicotinamide adenine dinucleotide (NAD<SUP>+</SUP>)‐dependent 12α‐hydroxysteroid dehydrogenase (<I>Rr</I>12α‐HSDH) from <I>Rhodococcus ruber</I> was identified using a structure‐guided genome mining (SSGM) approach, which is based on the structure of cofactor pocket and the conserved nicotinamide cofactor binding motif alignment. <I>Rr</I>12α‐HSDH was heterologously overexpressed in <I>Escherichia coli</I> BL21 (DE3), purified and characterized. The purified <I>Rr</I>12α‐HSDH showed a high oxidative activity of 290 U mg<SUP>−1</SUP><SUB>protein</SUB> toward CA, with a catalytic efficiency (<I>k</I><SUB>cat</SUB>/<I>K</I><SUB>M</SUB>) of 5.10×10<SUP>3</SUP> mM<SUP>−1</SUP> s<SUP>−1</SUP>. In a preparative biotransformation (100 mL), CA (200 mM, 80 g L<SUP>−1</SUP>) was efficiently converted to 12‐oxo‐CDCA in 1 h, with a 85% isolated yield and a space‐time yield (STY) of up to 1632 g L<SUP>−1</SUP> d<SUP>−1</SUP>. Furthermore, <I>Rr</I>12α‐HSDH was shown to be able to catalyze the oxidation of other 12α‐hydroxysteroids at high substrate loads (up to 200 mM), giving the corresponding 12‐oxo‐hydroxysteroids in 71%–85% yields, indicating the great potential of <I>Rr</I>12α‐HSDH as a promising biocatalyst for the synthesis of various therapeutic bile acids.</P>