Effects of kosmotropic, chaotropic, and neutral salts on Candida antarctica B lipase: An analysis of the secondary structure and its hydrolytic activity on triglycerides

The effects of different concentrations of Hofmeister salts on the hydrolytic activity on triglycerides and the secondary structure of lipase B from Candida antarctica (CALB) were investigated. Structural changes after short- and long-time incubation at high salt concentrations were determined using circular dichroism (CD), fluorescence, and RMSD-RMSF simulations. At 5.2 M NaCl, the hydrolytic activity of CALB on tributyrin (TC4) and trioctanoin (TC8) was enhanced by 1.5 (from 817 ± 3.9 to 1228 ± 4.3 U/mg)- and 8.7 (from 25 ± 0.3 to 218 ± 2.3 U/mg)-folds compared with 0.15 M NaCl, respectively at pH 7.0 and 40 °C. An activity activation was seen with other salts tested; however, long-time incubation (24 h) did not result in retention of the activation effect for any of the salts tested. Secondary structure CD and fluorescence spectra showed that long-time incubation with NaCl, KCl, and CsCl provokes a compact structure without loss of native conformation, whereas chaotropic LiCl and CaCl₂ induced an increase in the α-helical content, and kosmotropic Na₂SO₄ provoked a molten globule state with rich β-sheet content. The RMSD-RMSF simulation agreed with the CD analysis, highlighting a principal salt-induced effect at the α-helix 5 region, promoting two different conformational states (open and closed) depending on the type and concentration of salt. Lastly, an increase in the interfacial tension occurred when high salt concentrations were added to the reaction media, affecting the catalytic properties. The results indicate that high-salt environments, such as 2-5.2 M NaCl, can be used to increase the lipolytic activity of CALB on TC4 and TC8.