12-ALPHA-HYDROXYSTEROID DEHYDROGENASE FROM CLOSTRIDIUM GROUP-P, STRAIN-C-48-50 - PRODUCTION, PURIFICATION AND CHARACTERIZATION

NADP(H)-dependent 12-alpha-hydroxysteroid dehydrogenase (HSDH) from Clostridium group P, strain C 48-50, is still expressed at unusual high level (approximately 1% of total protein) under cultivation conditions where the usual expensive brain/heart infusion complex medium is replaced by inexpensive technical grade yeast autolysate. An inexpensive anaerobic bioprocess for the production of HSDH was developed provisionally up to 900-1 scale (9000 U/l, 7 g HSDH, specific activity 1.0 U/mg crude protein, 55 U/g wet cells). By a simple two-step affinity chromatography procedure, easily adaptable to a large-scale operation, using columns of small dimensions of Sephacryl-S-400 - Procion-orange-P-2R (5 cm x 28 cm) and Sephacryl-S-400 - Procion-red-HE-7B (2.6 cm x 14 cm) approximately 140 mg (1.8 x 10(4) U), HSDH was purified to apparent homogeneity and concentrated directly from a crude cell extract (overall yield 53%, specific activity 128 U/mg). As confirmed by fast native and SDS/PAGE, isoelectric focussing and electron microscopy, HSDH has a molecular mass of approximately 105 kDa and consists of four flattened tetrahedrically arranged identical subunits (26 kDa). The enzyme exhibits a rather low isoelectric point of 4.6, a pH optimum of 8.5-9.5 and a temperature optimum of approximately 55-degrees-C for the oxidation of cholic acid. Inhibition by SH reagents and pyridoxal 5'-phosphate has been observed. Chelating agents have no inhibitory effect. The presence of NADP increases considerably the thermostability (t1/2 4-10 d, 25-degrees-C; 2-5 d, 37-degrees-C). Steady-state kinetic analysis for both reaction directions indicated that the reaction proceed through an ordered bi bi mechanism with NADP(H) binding first to the free enzyme. K(m), V(max) [forward (V(f)) and reverse reactions (V(r))] and the dissociation constants K(d) for the binary complexes with NADP and NADPH were as follows. NADP, K(m) = 35-mu-m, K(d) = 35-mu-m; cholic acid, K(m) = 72-mu-m; deoxycholic acid, K(m) = 45-mu-m, V(f) = 160 U/mg; NADPH, K(m) = 8.5-mu-m, K(d) = 16-mu-m; 12-oxochenodeoxycholic acid, K(m) = 12-mu-m, V(r) = 66 U/mg (conditions, 0.1 M potassium phosphate, pH 8.0, 25-degrees-C). N6-functionalized NADP derivatives, e.g. N6-(2-aminoethyl)NADP (K(m) = 4.5 mM) are poorly accepted as coenzyme by HSDH.