A new role for penicillin acylases: degradation of acyl homoserine lactone quorum sensing signals by Kluyvera citrophila penicillin G acylase.

Use of penicillin acylases for the production of semi-synthetic penicillins is well-known. Escherichia coli penicillin G acylase (EcPGA) has been extensively used for this purpose; however, Kluyvera citrophila penicillin G acylase (KcPGA) is assumed to be a better substitute, owing to its increased resilience to extreme pH conditions and ease of immobilization. In the present article we report a new dimension for the amidase activity of KcPGA by demonstrating its ability to cleave bacterial quorum sensing signal molecules, acyl homoserine lactones (AHL) with acyl chain length of 6-8 with or without oxo-substitution at third carbon position. Initial evidence of AHL degrading capability of KcPGA was obtained using CV026 based bioassay method. Kinetic studies performed at pH 8.0 and 50 °C revealed 3-oxo-C6 HSL to be the best substrate for the enzyme with V(max) and K(m) values of 21.37+0.85 mM/h/mg of protein and 0.1+0.01 mM, respectively. C6 HSL was found to be the second best substrate with V(max) and K(m) value of 10.06+0.27 mM/h/mg of protein and 0.28+0.02 mM, respectively. Molecular modeling and docking studies performed on the active site of the enzyme support these findings by showing the fitting of AHLs perfectly within the hydrophobic pocket of the enzyme active site.

Increasing synthetic performance of penicillin G acylase from Bacillus megaterium by site-directed mutagenesis.

Site-directed mutagenesis based on predicted modeled structure of pencillin G acylase from Bacillus megaterium (BmPGA) was followed to increase its performance in the kinetically controlled synthesis of cephalexin with high reactant concentrations of 133 mM 7-amino-desaceto-xycephalosporanic acid (7-ADCA) and 267 mM D: -phenylglycine amide (D-PGA). We directed changes in amino acid residues to positions close to the active site that were expected to affect the catalytic performance of penicillin acylase: alpha Y144, alpha F145, and beta V24. Alpha F145 was mutated into tyrosine, alanine, and leucine. Alpha Y144 and beta V24 were mutated into arginine and phenylalanine, respectively. The S/H ratios of three mutants, BmPGAalpha144R, BmPGAbeta24F, and BmPGAbeta24F+alpha144R, were up to 1.3-3.0 times higher values. Compared to the wild-type BmPGA, BmPGAbeta24F+alpha144R showed superior potential of the synthetic performance, allowing the accumulation of up to twofold more cephalexin at significantly higher conversion rates.