Penicillin acylase is commonly used to produce the medical intermediates of 6-Aminopenicillanic acid (6-APA) and 7-Aminodesacetoxycephalosporanic acid (7-ADCA) in industrial process. Nowadays, Penicillin G acylase (PGA) has been widely applied for making pharmaceutical intermediates, while penicillin V acylase (PVA) has been less used for that due to its low activity and poor conversion. In this study, a PVA from Bacillus sphaericus (BspPVA) was employed for directed evolution study with hoping to increase its catalytic efficiency. Finally, a triple mutant BspPVA-3 (T63S/N198Y/S110C) was obtai...