Purification and characterization of bile salt hydrolase from Clostridium perfringens.

Abstract

Bile salt hydrolase (cholylglycine hydrolase, EC 3.5.1.24) has been purified to homogeneity (792-fold) from Clostridium perfringens using high performance DEAE-chromatography. The purified enzyme showed a single detectable protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a relative molecular weight ca. 56,000. The intact enzyme had a relative molecular weight (Mr) of ca. 250,000 as determined by nondenaturing PAGE. The NH2-terminal sequence of bile salt hydrolase was determined to be Met-(Ser/Cys)-Arg-Thr-Lys-Leu-Val-Ileu-Thr-Ileu-Gly-Ala-Ser. The purified enzyme was active towards both glycine and taurine conjugates of cholate. The apparent Km and Vmax of the enzyme for glycocholate was estimated to be 0.5 mM and 107 nmol/min.mg protein, respectively. The pH optimum was in the range of 5.8 to 6.4. The enzyme was inhibited 85%, 81%, and 83% by 2 mM iodoacetate, p-chloromercuribenzoate, and phenylmethanesulfonylfluoride, respectively. Rabbit polyclonal antibody was prepared and used to demonstrate a single form of the enzyme in crude cell extracts.