Thermal Hyperactivation and Stabilization of β-Galactosidase from Bacillus circulans through a Silica Sol-Gel Process...

The research of simple and fast enzyme immobilization methods, preserving the enzyme activity and improving the thermal stability, is in the spotlight. The objective of this work is to develop a β-galactosidase immobilization one-pot route, combining the silica sol-gel encapsulation (SSGE) process with a metal chelation strategy by using chitosan and Ca²⁺, Zn²⁺, or Cu²⁺ cations. The results show that the presence of cations does not affect the encapsulation efficiency (81%) and has positive effects on the maximum catalytic potential, especially at 60 °C and in the presence of Ca²⁺ ions (MPC = 2203). They enhance the biocatalyst thermal stability and promote hyperactivation with respect to the soluble enzyme at 60 °C (1.6 times higher MPC). The biocatalyst prepared with Zn²⁺ ions exhibits also thermal hyperactivation in the first 30 min of heating (1.3 times more residual activity), but the enzyme is not stabilized (0.9 times lower MPC); also, the presence of Cu²⁺ ions does not promote hyperactivation or stabilization of the enzyme (0.3 times lower MPC) at this high temperature. These facts are reflected in the hydrolytic and transgalactosylation activities of the enzyme (33.6-57.4% total lactose conversion), higher than that reported with analogue biocatalysts. The physicochemical characterization of the obtained solid biocatalysts by SEM, TEM, XRF, and XPS indicates that chitosan-metal chelation has an important role in the encapsulation process and that a low metal degree incorporation (8.85 ppm of Ca²⁺) on the solid biocatalyst favors the thermal hyperactivation and stabilization of the evaluated β-galactosidase. This work contributes to the understanding of the SSGE process mediated by chitosan-metal chelates, which is a simple and fast one-pot immobilization strategy.