title: Conformational flexibility of lipase Lip1 from Candida rugosa studied by electronic spectroscopies and thermodynamic approaches.
abstract: We have used second-order orthogonal designs to obtain empirical models that describe the combined effect of pH and temperature on the secondary structure of a lipase (Lip1) from Candida rusosa. The equations that describe lipase conformational flexibility were derivated from the enzyme alpha helix fraction obtained from the experimental matrix. The thermal unfolding of lipase at different pH values was followed by measuring the circular dichroism signal as a function of temperature over a temperature range of 20-80 °C. The results showed a melting temperature of 58.9 °C at pH 5.5, while at pHs 7.0 and 8.6, the temperature values were 50.2 °C and 36.1 °C respectively. The optimum experimental conditions of conformations with high content of alpha helix were found at high temperature and pH, both at zero time and at one-hour incubation time of enzyme. Important variations in the enzyme secondary structure were induced for the pH and temperature. In contrast, minor changes were observed during the incubation time. This behaviour suggests that the medium pH induces a modification in the enzyme secondary structure and not due to a result of a progressive denaturation process. From the values of thermodynamic functions at different pHs, the system at initial state of unfolding process is previously disordered by the pH effect.
authors.affiliation: Facultade de Ciencias de Ourense, Universidade de Vigo, As Lagoas s/n E-32004 Ourense, Spain.
authors.forename: J P
authors.initials: JP
authors.lastname: Fuciños González
authors.forename: G
authors.initials: G
authors.lastname: Bassani
authors.forename: B
authors.initials: B
authors.lastname: Farruggia
authors.forename: G A
authors.initials: GA
authors.lastname: Picó
authors.forename: L
authors.initials: L
authors.lastname: Pastrana Castro
authors.forename: M L
authors.initials: ML
authors.lastname: Rua
mesh_terms: D002175:Candida / Q000201:enzymology*; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis; D013696:Temperature; D013816:Thermodynamics
chemical_list: D008049:Lipase