Kaur_2020_Int.J.Biol.Macromol__

In the present investigation, a lipid hydrolyzing gene RPK01was cloned from metagenome source of hot spring. Expression and purification of recombinant protein revealed a protein band of ~24 KDa on 12% SDS-PAGE and is well corroborated with the deduced molecular weight 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 degrees C till 3h, and retained ~40% activity at 50 degrees C and 60 degrees C temperature till 3h. Additionally, the pH stability assay showed its functionality in broad range pH, with maximum stability observed at pH2.0, it decreases from pH4.0 to pH12.0 and has retained ~ 40% activity in these pHs. Both circular dichroism and intrinsic Trp fluorescence studies revealed conformational stability of protein structure in 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 (Asn75-Ile80).