Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis.
A high-molecular-weight (250 000) bile salt hydrolase (cholylglycine hydrolase, EC 3.5.-.-) was isolated and purified 128-fold from the "spheroplast lysate" fraction prepared from Bacteroids fragilis subsp. fragilis ATCC 25285. The intact enzyme had a molecular weight of approx. 250 000 as determined by gel infiltration chromatography. One major protein band, corresponding to a molecular weight of 32 500, was observed on 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis of pooled fractions from DEAE-cellulose column chromatography (128-fold purified). The pH optimum for the 64-fold purified enzyme isolated from Bio-Gel A 1.5 M chromatography was 4.2 and bile salt hydrolase activity measured in intact cell suspensions had a pH optimum of 4.5. Substrate specificity studies indicated that taurine and glycine conjugates of cholic acid, chenodeoxycholic acid and deoxycholic acid were readily hydrolyzed; however, lithocholic acid conjugates were not hydrolyzed. Substrate saturation kinetics were biphasic with an intermediate plateau (0.2--0.3 mM) and a complete loss of enzymatic activity was observed at high concentration for certain substrates. The presence or absence of 7-alpha-hydroxysteroid dehydrogenase was absolutely correlated with that of bile salt hydrolase activity in six to ten strains and subspecies of B. fragilis.
D000581:Amidohydrolases / Q000302:isolation & purification / Q000378:metabolism*; D001441:Bacteroides fragilis / Q000201:enzymology*; D001647:Bile Acids and Salts / Q000494:pharmacology; D006000:Glycocholic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D013104:Spheroplasts / Q000201:enzymology; D013329:Structure-Activity Relationship
D001647:Bile Acids and Salts; D000581:Amidohydrolases; C023154:choloylglycine hydrolase; D006000:Glycocholic Acid