Bile acids are associated with gastrointestinal cancers and the enzymes that act on the bile acids are hydroxysteroid dehydrogenases produced by the microbiota in the gut. One such microbe, Methanosphaera stadtmanae, is an archaeon that has recently been detected to have an effect on the gut’s immune and epithelium systems. Using recombinant plasmid from M. stadtmanae, we have overexpressed the 12α-hydroxysteroid dehydrogenase (12α-HSDH) in aerobic conditions by IPTG induction. The 12α-HSDH is responsible for the conversion of toxic bile acids into less toxic bile acids—that are not linked to cancer. After optimization, we found that using ¼ of the flask volume for growing cultures generated 2x bigger cell pellets as compared to half of the flask volume determines the importance of aerobic conditions in cell growth. The lyzed pellets were confirmed for the activity and the protein was purified using Immobilized Metal Affinity Chromatography. Purification was confirmed using SDS-PAGE gel. 12α-HSDH converts the hydroxyl group at the 12-C position of the bile acid to a carbonyl group in the presence of a pyridine nucleotide. We have discovered that 12α-HSDH is NADP-dependent in bile acid reactions and does not show any activity in the presence of NAD. The phylogenetic analysis has placed the enzyme in the short-chain dehydrogenase/reductase (SDR) family. We have focused on the kinetics of the conversion of deoxycholic acid into 12-oxo lithocholic acid using 12α-HSDH and NADP. This was done spectrophotoscopically at A₃₄₀. The steady-state kinetic parameters for the NADP reduction are determined by the method of initial rates. Initial velocities for the enzyme are measured as a function of DCA concentration at fixed NADP concentration. The rates are fit to the Michaelis-Menten equation and the K_m for this reaction is 4.816 ± 3.849 and the K_cat for this reaction is 337.785 s^-1. The enzyme's activity with DCA as a substrate is optimal at a pH of 8. The ideal concentration for crystallization of the enzyme, using the hanging-drop diffusion method, was found to be 1.66 mg/mL, and the preferred conditions for crystallization were found to be 8 days of incubation in 0.1 M HEPES sodium with a pH of 7.5 with 2% v/v PEG 400 and 2.0 ammonium sulfate.

Chloe Caplinger

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