Screening for Penicillin G Acylase (PGA)-Producing Bacteria and Gene Cloning Using Degenerate Oligonucleotide Primed-PCR

Authors

Masdalifah Masdalifah, Department of Food and Biotechnology, Faculty of Agricultural Technology, Universitas Brawijaya, Malang 65145, Indonesia
Sri Rezeki Wulandari, Research Center for Genetic Engineering, National Research and Innovation Agency (BRIN), Banten 15314, Indonesia
Gabriela Christy Sabbathini, Research Center for Genetic Engineering, National Research and Innovation Agency (BRIN), Banten 15314, Indonesia
Maria Ulfah, Research Center for Genetic Engineering, National Research and Innovation Agency (BRIN), Banten 15314, Indonesia
Dini Achnafani, Research Center for Pharmaceutical Ingredients and Traditional Medicines, National Research and Innovation Agency (BRIN), Banten 15314, Indonesia
Ahmad Wibisana, Research Center for Pharmaceutical Ingredients and Traditional Medicines, National Research and Innovation Agency (BRIN), Banten 15314, Indonesia
Feronika Heppy Sriherfyna, Department of Food and Biotechnology, Faculty of Agricultural Technology, Universitas Brawijaya, Malang 65145, Indonesia
Is Helianti, Research Center for Genetic Engineering, National Research and Innovation Agency (BRIN), Banten 15314, Indonesia
Niknik Nurhayati, Research Center for Genetic Engineering, National Research and Innovation Agency (BRIN), Banten 15314, IndonesiaFollow

Abstract

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 in-tracellular 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. Sec-ondary 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.

Recommended Citation

Masdalifah, Masdalifah; Wulandari, Sri Rezeki; Sabbathini, Gabriela Christy; Ulfah, Maria; Achnafani, Dini; Wibisana, Ahmad; Sriherfyna, Feronika Heppy; Helianti, Is; and Nurhayati, Niknik (2025) "Screening for Penicillin G Acylase (PGA)-Producing Bacteria and Gene Cloning Using Degenerate Oligonucleotide Primed-PCR," Makara Journal of Science: Vol. 29: Iss. 1, Article 4.
DOI: 10.7454/mss.v29i1.2327
Available at: https://scholarhub.ui.ac.id/science/vol29/iss1/4