Investigation into the structure and activity of conjugated bile salt hydrolase and related penicillin acylase

Abstract

The Penicillin acylases are used in the commercial production of 6-amino penicillanic acid (6-APA), the starting compound for the synthesis of semi-synthetic penicillins. The enzyme bile salt hydrolase (BSH) is related to penicillin V acylase and has potential application in lowering the serum cholesterol level in hypercholestremic humans or to prevent hypercholesterolemia in individuals with normal cholesterol. We report in this thesis the extensive studies carried out on a bile salt hydrolase (Cholylglycin hydrolase, EC 3.5.1.24) from Bifidobacterium longum ( BlBSH) and two penicillin G acylases (PGAs) (penicillin amidohydrolase, EC 3.5.1.11) from Kluyvera citrophila (KcPGA) and Alcaligence faecalis (AfPGA). These enzymes could be placed in N-terminal nuleophile (Ntn) hydrolase superfamily. Ntn hydrolases are a class of enzymes that share a common fold of áââá- core structure possessing a N-terminal catalytic nucleophile residue. In BSH the N-terminal nucleophile is cysteine while in PGAs it is serine. The Ntn hydrolases invariably produce their active form from the corresponding precursor by an intra-molecular autocatalytic cleavage to create a free amino group at the nucleophile residue. Bile salt hydrolase (BSH) catalyses the hydrolysis of glycine or taurine conjugated bile salt into the corresponding amino acid residues and the deconjugated bile salt. The ability to deconjugate bile salts is widespread among the members of the genus Bifidobacterium. The deconjugation of six different salts by all bifidobacteria strains that inhabit animal gut is one way of transforming the bile salt in intestine. In recent years therapeutic use of bile salt deconjugation by lactic acid bacteria to lower serum cholesterol level is being tried. Penicillin acylases, a subclass of -lactam antibiotic acylase superfamily, catalyze the selective hydrolysis of relatively stable amide bond in penicillins and some cephalosporins while leaving the labile -lactam ring intact. The capacity of penicillin G acylases to catalyze the acylation of amino group of key intermediates can be used in the environment-friendly synthesize of semi-synthetic -lactam antibiotics. PGAs are also useful for other applications such as peptide synthesis, removal of protecting groups and separation of racemic mixtures of certain compounds.