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, BmPGA alpha 144R, BmPGA beta 24F, and BmPGA beta 24F+alpha 144R, were up to 1.3-3.0 times higher values. Compared to the wild-type BmPGA, BmPGA beta 24F+alpha 144R showed superior potential of the synthetic performance, allowing the accumulation of up to twofold more cephalexin at significantly higher conversion rates.