DNA Ligase From <i>Thermococcus radiotolerans</i>: Recombinant Production, Characterization, Up‐Scale Fermentation, and In Silico Evaluation

The present study describes molecular cloning, Escherichia coli expression, purification, and characterization of a DNA ligase from Thermococcus radiotolerans (TR). Optimal pH, temperature, media composition, and inducer concentrations for the production of wet cellular mass (WCM) with active enzyme have been validated. Recombinant DNA ligase-TR displayed a 44 kDa protein band on SDS-PAGE, and enzyme activity was measured at 50°C in the presence of 50 mM Tris-Cl buffer adjusted at pH 7 and supplemented with 0.4 mM ATP ± 0.1 mM NAD⁺. The enzyme was able to ligate two restriction products of EcoRI, independent of the presence of NAD⁺. The subject enzyme exhibited better activity than T4 DNA ligase. Maximum WCM was obtained at pH 7.3 and 30°C in modified M9NG medium when induced with 0.4 mM isopropyl β-d-1-thiogalactoside (IPTG). Protein sequence alignment studies have shown that DNA ligase-TR exhibits a novel and specific primary structure that is different from DNA ligases isolated from eukaryotic and other thermophilic species reported in the literature. According to in silico studies, Lys²³⁸ interacts with adenosine monophosphate (AMP) and the residues responsible to engage nicked DNA include Arg⁴¹, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>Ar</mml:mi> <mml:msup><mml:mi>g</mml:mi> <mml:mn>43</mml:mn></mml:msup> </mml:mrow> <mml:annotation>${\mathrm{Ar}}{{\mathrm{g}}^{43}}$</mml:annotation></mml:semantics> </mml:math> , <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>Ar</mml:mi> <mml:msup><mml:mi>g</mml:mi> <mml:mn>50</mml:mn></mml:msup> </mml:mrow> <mml:annotation>${\mathrm{Ar}}{{\mathrm{g}}^{50}}$</mml:annotation></mml:semantics> </mml:math> , <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>Ar</mml:mi> <mml:msup><mml:mi>g</mml:mi> <mml:mn>51</mml:mn></mml:msup> </mml:mrow> <mml:annotation>${\mathrm{Ar}}{{\mathrm{g}}^{51}}$</mml:annotation></mml:semantics> </mml:math> , <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>Ar</mml:mi> <mml:msup><mml:mi>g</mml:mi> <mml:mn>267</mml:mn></mml:msup> </mml:mrow> <mml:annotation>${\mathrm{Ar}}{{\mathrm{g}}^{267}}$</mml:annotation></mml:semantics> </mml:math> , <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>Ar</mml:mi> <mml:msup><mml:mi>g</mml:mi> <mml:mn>270</mml:mn></mml:msup> </mml:mrow> <mml:annotation>${\mathrm{Ar}}{{\mathrm{g}}^{270}}$</mml:annotation></mml:semantics> </mml:math> , and Arg³⁶⁷. About 51% secondary structure consists of α-helices and β-pleated sheets. High-temperature stability, better half-life, and high activity of DNA ligase-TR advocate its suitability in the DNA manipulation procedures.