Enhancement of laccases thermostability in Betaine based Natural Deep Eutectic Solvents

In recent years, natural deep eutectic solvents (NADESs) have gained increasing attention as promising non-toxic solvents for biotechnological applications, due to their compatibility with enzymes and ability to enhance the enzyme activity. Betaine - based NADESs at a concentration of 25 wt% in a buffered aqueous solution [1] were used as media to inhibit thermal inactivation of #rPOXA1b and 5 variants laccases when incubated at 70°C and 90°C for 1 hour. All the tested laccases showed higher residual activity when incubated in NADES solutions compared to the enzymes incubated in the reference solution. Furthermore, the residual activity of laccases in presence of NADESs showed a clear advantage over the use of NADESs’ individual components. Molecular docking simulations were performed to better understand the role of NADESs in the stabilization of laccases towards thermal degradation observed in the experimental work. The 3-dimensional structures of the NADES components and #rPOXA1b and 5 variants laccases were built using YASARA Structure software and the effect of mutations on the on the enzyme stability were evaluated. The binding energies between laccases and NADES components were calculated and compared to the experimental results of the measured residual activities. These findings demonstrate the possibility of pre-incubating enzymes in NADESs as a facile and cost-effective solution to inhibit thermal inactivation of enzymes when exposed to high temperatures for various time.