Bile acids are essential to maintain gut health, but at high concentrations they have been linked to diseases such as cancers in the different parts of the gastrointestinal tract. 12α-Hydroxysteroid Dehydrogenases are enzymes produced by gut bacteria known for oxidizing bile acids and transforming them from the toxic to the nontoxic forms. The focus of the research is to determine the kinetic parameters of the conversion of cholic acid to deoxycholic acid, catalyzed by 12α-HSDH in the presence of NADP⁺ by replacing the hydroxyl group at 12-C position with keto group. Using recombinant plasmid from Methanosphaera stadtmanae, 12α-HSDH was overexpressed in aerobic conditions by IPTG induction. It was found that growing cultures in flask with one fourth of its volume generated twice the amount of cell pellets compared to half of flask volume, confirming the oxygen requirement for the cell growth. The crude extract from pellets was tested to confirm the activity of the enzyme spectrophotometrically at 340 nm by measuring the reduction of NADP⁺ to NADPH. The His-tagged protein was purified using Immobilized Metal Affinity Chromatography and confirmed for MW 29 kDa using SDS-PAGE. Results from the spectrophotometer scans with NAD⁺ confirmed that 12 α-HSDH is strictly NADP⁺ dependent with bile acid substrates and does not show any activity with NAD⁺. The enzyme was confirmed to have optimal activity at pH 7.9. The phylogenetic analysis of the enzyme suggested that it belongs to the short-chain dehydrogenase and reductase (SDR) family. The steady state kinetic parameters for the reduction of NADP⁺ were measured through initial rates. The rates obtained as a function of various CA concentrations and fixed NADP⁺ concentration was fit to the Michaelis-Menten equation and gave the K_mof 0.401 mM. The turnover number of the reaction was determined to be 34.9 s^-1and the catalytic efficiency was calculated to be 87.0 s^-1 mM^-1. To obtain the good quality crystals of the enzyme, crystal screening was conducted to obtain the feasible conditions. The several conditions have been optimized and reproduced using hanging-drop diffusion method with or without ligands.

Larisa Ramos Diaz

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