Structural and functional insights about unique extremophilic bacterial lipolytic enzyme from metagenome source

Abstract

In the present investigation, a lipid hydrolyzing gene RPK01 was cloned from metagenome source of hot spring. Expression and purification of recombinant protein revealed single purified protein band of ~24 KDa on 12% SDS-PAGE, and is well corroborated with the deduced molecular weight of protein as calculated from its amino acid sequence. The purified protein displayed high activity towards short chain fatty acids and was found to be completely stable at 30°C till 3h, it further retained ~40% activity at 50°C and 60°C temperature till 3h. Additionally, the pH stability assay showed its functionality in broad range of pH, with maximum stability observed at pH 2.0, it decreases from pH 4.0 to pH 12.0, and nearly showed 40% activity in these pH values. Both circular dichroism and intrinsic Trp fluorescence studies revealed conformational stability of protein structure at wide range of temperature and pH. Enzyme activity enhances in presence of non-ionic surfactants like Tween 20 and TritonX-100. Further, inhibitors of the active site residues including PMSF and DEPC alone were unable to inhibit enzyme activity, while cumulative presence of calcium and inhibitors reduces enzyme activity to 90%, indicating conformational changes in the protein. Molecular simulation dynamics analysis revealed a calcium binding site near the lid helix of this protein(Asn75-Ile80).