12α‐Hydroxysteroid dehydrogenase from Clostridium group P, strain C 48–50

NADP(H)‐dependent 12α‐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 × 28 cm) and Sephacryl‐S‐400—Procion‐red‐HE‐7B (2.6 cm × 14 cm) approximately 140 mg (1.8 × 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°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 ( t 1/2 4—10 d, 25°C; 2—5 d, 37°C). Steady‐state kinetic analysis for both reaction directions indicated that the reaction proceeds 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 μm, K d = 35 μm; cholic acid, K m = 72 μm; deoxycholic acid, K m = 45 μm, V f = 160 U/mg; NADPH, K m = 8.5 μm, K d = 16 μm; 12‐oxochenodeoxycholic acid, K m = 12 μm, V = r = 66 U/mg (conditions, 0.1 M potassium phosphate, pH 8.0, 25°C). N 6 ‐functionalized NADP derivatives, e.g. N 6 ‐(2‐aminoethyl)NADP ( K m = 4.5 mM) are poorly accepted as coenzyme by HSDH.