Masdalifah, Sri Rezeki Wulandari, Gabriela Christy Sabbathini, Maria Ulfah, Dini Achnafani, Ahmad Wibisana, Feronika Heppy Sriherfyna, Is Helianti, Niknik Nurhayati
The growing concern over antibiotic resistance has driven global efforts to explore innovative solutions, including the use of Penicillin G acylase (PGA) to produce semisynthetic β-lactam antibiotics. This study screened four potential intracellular PGA-producing bacteria: Alcaligenes faecalis InaCC B444 (AfPGA), Kluyvera cryocrescens InaCC B850 (KcPGA), Providencia rettgeri InaCC B25 (Pr25PGA), and P. rettgeri InaCC B466 (Pr466PGA). Penicillin G Acylase encoding genes (pgas) were isolated from them using a Degenerate Oligonucleotide Primed-PCR (DOP-PCR) approach and sequenced. Microbiological assays confirmed all tested crude extracts to exhibit inhibitory effects. Penicillin G was used for evaluating hydrolytic activity and 6-Amino Penicillanic Acid (6-APA) coupled with D-p-Hydroxyl-phenylglycine methyl ester hydrochloride (DHPGME) for the synthetic activity. Pr466PGA and Pr25PGA showed the highest synthetic and hydrolytic activities, respectively. DOP-PCR successfully amplified a 2,517 bp pga-encoding Pr25PGA. The deduced amino acid sequence shared 95.1% identity with the known PGA from P. rettgeri PX04. Secondary structure analysis of Pr25PGA revealed 35% α-helices, 16% β-sheets, and 49% coils, suggesting that the enzyme may be flexible and dynamic, with structural stability primarily provided by the α-helices and β-sheets. These findings offer valuable insights for the future design and application of Pr25PGA, particularly in the production of semisynthetic β-lactam antibiotics. © 2025, Universitas Indonesia. All rights reserved.
Department of Food and Biotechnology, Faculty of Agricultural Technology, Universitas Brawijaya, Malang, 65145, Indonesia; Research Center for Genetic Engineering, National Research and Innovation Agency (BRIN), Banten, 15314, Indonesia; Research Center for Pharmaceutical Ingredients and Traditional Medicines, National Research and Innovation Agency (BRIN), Banten, 15314, Indonesia
