Journal of Biotechnology & Biomaterials
Open Access

Abstract

Among lignocellulolytic enzymes, laccases (EC 1.10.3.2) are the most versatile, broadly specific and well-studied enzyme with wide range of biotechnological potential. Putative laccase (BpCotA) from Bacillus pumilus MK001 was cloned and expressed in E. coli BL21 (DE3) which displayed molecular weight of ~64 kDa, analyzed by SDS-PAGE and western blotting. In addition to soluble bioactive fraction, inactive inclusion body (IB) fraction was also harvested and refolded under optimized conditions resulting in 65% refolding efficiency. As predicted by far UV thermal CD spectra, increase in �²-sheets of the protein structure was recorded after thermal induction, investigated as one of the potent reason for higher thermostability of the protein. Multiple sequence alignment with other similar proteins revealed the occurrence of highly conserved catalytic residues. The 3D structure of BpCotA was constructed through homology modeling (Modeler 9.11). The structure was refined and validated for stereochemical quality by using PROCHECK, ERRAT, Verify 3D and PROSA servers. The best selected model with minimized energy was examined for superimposition with the crystal structure of CotA from B. subtilis which showed 0.33 RMSD value. The potential binding affinities of BpCotA was observed with ferulic acid, vanillic acid, vanillin and caffeic acid involving flexible docking studies, AUTODOCK 4.2 and Hex showed that both ferulic acid and vanillin docked well in the vicinity of predictive active site of recombinant protein. Further, in vitro action of enzyme was analyzed with respective substrates and predicted interactions were confirmed by HPLC generated degradation spectra. Elucidation of thermostability, interactions with phenolics and refolding of IB fraction will deduce the biotechnological applicability of BpCotA.

Biography

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