The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing beta-lactam antibiotics, such as cephalexin and ampicillin. N-terminal amino acid sequencing of the purified alpha-amino acid ester hydrolase allowed cloning and genetic characterization of the corresponding gene from an A. turbidans genomic library. The gene, designated aehA, en...

A new penicillin acylase was isolated by cloning and functional screening of DNA isolated from a sand soil enrichment culture. Sequence analysis of this enzyme, PAS2, revealed homology to a group of prominent penicillin G acylases, including the intensively studied enzyme of Escherichia coli ATCC 11105. Accordingly, PAS2 was found to be an Ntn-hydrolase with an N-terminal serine as the catalyti...

Penicillin G acylase (PGA) is a heterodimeric enzyme synthesized as a single-polypeptide precursor that undergoes an autocatalytic processing to remove an internal spacer peptide to produce the active enzyme. We constructed a single-chain PGA not dependent on autoproteolytic processing. The mature sequence of the beta-domain was expressed as the N terminus of a new polypeptide, connected by a r...

Particle size and enzyme protein loading are design parameters of enzyme immobilization affecting biocatalyst performance that can be varied within broad margins. Their effect on mass transfer limitations at different bulk penicillin G concentrations has been studied with glyoxyl agarose immobilized penicillin G acylase biocatalysts of average particle size of 5·10m and 10·10m at protein loadin...

Three mutations on the penicillin acylase surface (increasing the number of Lys in a defined area) were performed. They did not alter the enzyme's stability and kinetic properties; however, after immobilization on glyoxyl-agarose, the mutant enzyme showed improved stability under all tested conditions (e.g., pH 2.5 at 4 degrees C, pH 5 at 60 degrees C, pH 7 at 55 degrees C, or 60% dimethylforma...

A new crystal form of penicillin acylase (penicillin amidohydrolase, E.C. 3.5.1.11) from Escherichia coli W (ATCC 11105) is reported. The crystals were grown using a combination of hanging-drop and streak-seeding methods. The crystals are in the monoclinic space group P2(1) with cell dimensions a = 51.52, b = 131.95, c = 64.43 A, beta = 106.12 degrees. There is one heterodimer in the asymmetric...