The present invention provides engineered penicillin G acylase (PGA) enzymes, polynucleotides encoding the enzymes, compositions comprising the enzymes, and methods of using the engineered PGA enzymes.

The present disclosure relates to engineered penicillin G acylase (PGA) enzymes having improved properties, polynucleotides encoding such enzymes, compositions including the enzymes, and methods of using the enzymes.

A mutant penicillin acylase and uses thereof are provided. Compared to the amino acid sequence set forth in SEQ ID NO: 1, the mutant includes at least one mutation selected from the group consisting of: F146K, F24R, F71Y, N241K, G385Y, and G385R. By introducing mutations into the penicillin acylase derived from Kluyvera citrophila, the invention obtains a mutant enzyme exhibiting enhanced and well-coordinated hydrolytic and synthetic activities. It can be used for the synthesis of -lactam antibiotics, particularly for the one-step preparation of amoxicillin from penicillin potassium salt, thereby avoiding the isolation of the intermediate 6-APA. The present invention provides a key enzyme for the efficient production of -lactam antibiotics and is poised to significantly advance the innovation of their manufacturing technology.