Site-directed mutagenesis of recombinant penicillin-G acylase from Escherichia coli and effect on the hydrolytic activity

Abstract

Penicillin-derived antibiotics such as amoxicillin are one of the antibiotics that are widely used. Amoxicillin is synthesized through the availability of raw materials, 6-aminopenicillanic acid (6-APA), with penicillin-G acylase (PGA) as the catalyst. Previous studies revealed a recombinant PGA through the utilization of expression hosts. However, the low activity of the expressed enzyme is still an obstacle to the optimum utilization of its enzyme. In this study, we applied site-directed mutagenesis computationally for initial prediction by creating 36 PGA mutants through homology modeling and predicted their binding affinity as well as bounding interaction mode to penicillin-G through molecular docking. Our research found that four mutants had better potential activity than wild-type PGA. In the first experiment, only the βThr68Tyr PGA mutant was selected to be explored further. The mutant PGA gene inserted into pET-22b plasmid was transformed to Escherichia coli BL21(DE3) by heat shock method. For enzyme expression, IPTG and arabinose were used as inducers. As a result, the specific enzyme activity of mutant PGA showed insignificantly lower values in comparison to wild-type PGA. This activity appeared to be influenced by the formation of inclusion bodies during post-translational protein maturation, as described in this paper.