Effect of low organic solvents concentration on the stability and catalytic activity of HSL-like: Analisys from psychrophiles to (hyper thermophiles)

A comparative study aiming at analysing the catalytic activity and stability of four related enzymes belonging to the HSL family in the presence of several water-miscible organic solvents was performed. The studied enzymes were four carboxylesterases: PsyHSL (psychrophilic), Aes (mesophilic), EST2 (thermophilic) and AFEST (hyperthermophilic). The effect of the solvents on catalytic activity was measured by adding solvents up to 20% (v/v) to the assay mixture. For all enzymes at low solvent concentrations (4-5%) the catalytic activity (U/mg) increased up to 3-4-fold; only diethyl ether exerted a negative effect when compared to the reference value without solvent. EST2 and AFEST retained more than 100% of activity for concentrations of solvent >10% (v/v). Similar results were obtained for the stability analysis; in fact these two enzymes appeared more stable than the mesophilic and psychrophilic counterparts. The kinetic parameters of PsyHSL, EST2 and AFEST in all the tested solvents showed an increase in kcat but a negative effect on KM values. In the case of PsyHSL the KM increase was very low resulting in a substantial increase of the specific constant (S); in particular, the addition of dimethyl sulfoxide increased the kcat and the s values about 4- and 3-fold, respectively. We tested the ability of the enzymes to synthesize esters with the aim to discriminate if the decrease of activity in the presence of organic solvents was due to protein denaturation or an inversion of the hydrolytic reaction toward synthesis. While for PsyHSL and Aes the synthesis was very low and completely abolished in presence of solvents, instead for EST2 and AFEST the esters synthesis increased up to 10-fold in the presence of solvents, although the hydrolytic activity dropped about 300-fold.