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 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. Km, Vmax [forward (Vf) and reverse reactions (Vr)] and the dissociation constants Kd for the binary complexes with NADP and NADPH were as follows. NADP, Km = 35 microns, Kd = 35 microns; cholic acid, Km = 72 microns, deoxycholic acid, Km = 45 microns, Vf = 160 U mg; NAPDH, Kd = 16 microns; 12-oxochenodeoxylic acid, Km = 12 microns, 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 (Km = 4.5 mM) are poorly accepted as coenzyme by HSDH.
Braun
M
M
Department of Enzyme Technology, Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Federal Republic of Germany.
Lünsdorf
H
H
Bückmann
A F
AF
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003013:Clostridium / Q000201:enzymology / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005969:Glutamate Dehydrogenase / Q000138:chemical synthesis* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006913:Hydroxysteroid Dehydrogenases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols / Q000138:chemical synthesis* / Q000378:metabolism*; D013379:Substrate Specificity
D011092:Polyethylene Glycols; D006913:Hydroxysteroid Dehydrogenases; D005969:Glutamate Dehydrogenase