title: Purification, substrate specificity, and N-terminal amino acid sequence analysis of a beta-lactamase-free penicillin amidase from Alcaligenes sp.
abstract: A beta-lactamase-free penicillin amidase from Alcaligenes sp. active against various beta-lactams was purified to homogeneity. The enzyme can hydrolyze penicillin G to 6-amino penicillanic acid (6-APA) and furnish penicillin G from 6-APA and phenyl acetic acid by condensation. The penicillin amidase is a heterodimer of subunit masses of 63 kDa and 22 kDa, respectively. Its isoelectric point is at pH 8.5. Cephalothin was found to be the best substrate. This is a novel type II penicillin amidase which shares the properties of both type II and type III enzymes. It is thermostable and, unlike penicillin amidase from A. faecalis, its stability remains unperturbed even in presence of reductant. An inhibition study by 2-hydroxy-5-nitro benzylbromide indicated the involvement of tryptophan in catalysis by the enzyme.
authors.affiliation: Department of Microbiology, Bose Institute, P1/12 C.I.T. Scheme VII M, Kolkata, 700054, India.
authors.forename: S
authors.initials: S
authors.lastname: Das
authors.forename: J R
authors.initials: JR
authors.lastname: Gayen
authors.forename: A
authors.initials: A
authors.lastname: Pal
authors.forename: K
authors.initials: K
authors.lastname: Ghosh
authors.forename: J P N
authors.initials: JP
authors.lastname: Rosazza
authors.forename: T B
authors.initials: TB
authors.lastname: Samanta
mesh_terms: D000421:Alcaligenes / Q000201:enzymology* / Q000254:growth & development; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism; D002512:Cephalothin / Q000378:metabolism; D002942:Circular Dichroism; D008969:Molecular Sequence Data; D010399:Penicillin Amidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D047090:beta-Lactams; D010399:Penicillin Amidase; D001618:beta-Lactamases; D002512:Cephalothin