Penicillin G acylase (PAC; EC 3.5.1.11) is the key enzyme used in the industrial production of beta-lactam antibiotics. This enzyme hydrolyzes the side chain of penicillin G and related beta-lactam antibiotics releasing 6-amino penicillanic acid (6-APA), which is the building block in the manufacture of semisynthetic penicillins. PAC from Escherichia coli strain ATCC 11105, Bacillus megaterium strain ATCC 14945 and mutants of these two strains is currently used in industry. Genes encoding for PAC from various bacterial sources have been cloned and overexpressed with significant improvements in transcription, translation and post-translational processing. Recent developments in enzyme engineering have shown that PAC can be modified to gain conformational stability and desired functionality. This review provides an overview of recent advances in the production, stabilization and application of PAC, highlighting the recent biotechnological approaches for the improved catalysis of PAC.