The interaction of Bovine Serum Albumin (BSA) with limonene has been studied by UV-visible spectroscopy, fluorescence spectroscopy and molecular docking, and its effects on protein conformation, topology and stability were determined by Circular Dichroism (CD), Dynamic Light Scattering (DLS) and Differential Scanning Calorimetry (DSC). A gradual decrease in Stern–Volmer quenching constants with the increase in temperature showed the static mode of fluorescence quenching. The obtained binding constant (K_b) was ∼10⁴ M⁻¹. The temperature dependent K_b, Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes were calculated, which revealed that the reaction is spontaneous and exothermic. The UV-visible spectra showed a change in the peaks within the aromatic region indicating hydrophobic interactions with Trp, Tyr and Phe in the protein. Moreover, limonene induced an increase in α-helical contents probably on the cost of random coils or/and β-sheets of BSA, as observed from the far-UV CD spectra. The topology of BSA in the presence of limonene was slightly altered, as obtained from DLS results. The stability was also enhanced as revealed through thermal denaturation study by DSC and CD. Molecular docking study depicted that limonene fits into the hydrophobic pocket close to Sudlow site I in domain IIA of BSA. The present study will be helpful in understanding the binding mechanism of limonene and associated stability and conformational changes.

The effect of pH on the conformational behavior of Candida antartica lipase B (CaLB) has been monitored by spectroscopic and calorimetric studies. The results obtained from far and near-UV CD, intrinsic fluorescence and ANS binding studies indicate that CaLB exhibits the characteristic properties of a molten globule in acidic (protonated) conditions at pH 1.4. The molten globule state retained about 67% of its secondary structure with a substantial loss of tertiary structure at pH 1.4. Moreover, equilibrium unfolding studies indicated that the ‘molten-globule-like’ state unfolds in a non-cooperative manner and is thermodynamically less stable than that of the native state. The molten globule possessed a slightly higher R_h than its native state. The DSC thermogram shows a high heat signal at pH 7.4, and a low heat signal at pH 2.6, and suggests that CaLB is likely to have undergone structural changes during the thermal unfolding. However partially unfolded CaLB at pH 1.4 does not produce a DSC peak which proves the existence of the molten globule state at pH 1.4 as supported by spectroscopic data. The Stokes radius of the MG state obtained by SEC experiments is found to be 33% larger than the native state, but essentially smaller than the denatured state.

Bacterial quorum sensing (QS) is a density dependent communication system that regulates the expression of certain genes including production of virulence factors in many pathogens. Bioactive plant extract/compounds inhibiting QS regulated gene expression may be a potential candidate as antipathogenic drug. In this study anti-QS activity of peppermint (Mentha piperita) oil was first tested using the Chromobacterium violaceum CVO26 biosensor. Further, the findings of the present investigation revealed that peppermint oil at sub-MICs strongly interfered with acyl homoserine lactone (AHL) regulated virulence factors and biofilm formation in Pseudomonas aeruginosa and Aeromonas hydrophila. The result of molecular docking analysis attributed the quorum sensing inhibitory activity exhibited by peppermint oil to menthol. Assessment of ability of menthol to interfere with quorum sensing systems of various Gram-negative pathogens comprising diverse AHL molecules revealed that it reduced the AHL dependent production of violacein, virulence factors and biofilm formation indicating broad-spectrum anti-QS activity. Using two E. coli biosensors, MG4/pKDT17 and pEAL08-2, we also confirmed that menthol inhibited both the las and pqs quorum sensing systems. Further, findings of the in vivo studies with menthol on nematode model Caenorhabditis elegans showed significantly enhanced survival of the nematode. Our data identified menthol as a novel broad spectrum quorum sensing inhibitor.