Cloning and functional characterization of AhyAP-T65Lig, an ATP-dependent DNA ligase from Trabzonvirus AP-T65
VII. International Enzyme and Bioprocess Days, Trabzon, Türkiye, 26 - 28 Ağustos 2026, ss.45, (Özet Bildiri)
- Yayın Türü: Bildiri / Özet Bildiri
- Basıldığı Şehir: Trabzon
- Basıldığı Ülke: Türkiye
- Sayfa Sayıları: ss.45
- Karadeniz Teknik Üniversitesi Adresli: Evet
Özet
DNA ligases are essential enzymes in all living organisms and viruses. They play critical roles in genome maintenance and
recombination by sealing single-strand nicks and repairing damaged DNA. Due to these features, DNA ligases are widely
used in gene cloning applications and in vitro processing of DNA. In this study, the DNA ligase–encoding gene from the
Aeromonas hydrophila phage Trabzonvirus AP-T65 was cloned and characterized.
DNA ligase encoding gene from AP-T65 was identified by whole genome analysis, PCR amplified and sequenced. After
verification, AP-T65 DNA ligase (AhyAP-T65Lig) was cloned into the pET28(a)+ expression vector, overexpressed and
purified. The ligation efficiency of AhyAP-T65Lig was evaluated using ligation-mediated PCR and qPCR. Biochemical
characterization and structural analyses were performed for evaluation of the enzyme. AhyAP-T65Lig shared 51.81%
sequence similarity with the DNA ligase of Aeromonas phage BUCT695, and 53.35% with that of Salmonella phage
PKM.Hi.22.6. Molecular weight of the enzyme was determined as 41.1 kDa. AhyAP-T65Lig was active between 4 - 37 °C,
with an optimum temperature of 4 °C and an optimum pH of 8.0. Maximum activity was observed in the presence of 1
mM ATP, 10 mM DTT, 10 mM MgCl2, and 1 mM NaCl. Sticky-ended, linearized pGEM-T Easy and pET28(a)+ vectors were
circularized by AhyAP-T65Lig. The enzyme retained its activity for an extended period at both 4 °C and -20 °C.
The low-temperature optimum, high in vitro yield, and long shelf-life of AhyAP-T65Lig make it a promising enzyme for
biotechnological applications