Thermal Hyperactivation and Stabilization of β-Galactosidase from Bacillus circulans through a Silica Sol–Gel Process Mediated by Chitosan–Metal Chelates

Abstract

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.

Optimization of the synthesis process of β‐galactosidase/carboxymethylchitosan‐silica biocatalyst powders, stabilized with lyoprotectants for potential dietary applications

“…One International unit of β‐gal activity (IU) was defined as the amount of enzyme producing 1 μmol of o‐NP per minute under the described conditions. The values were reported as IU/ml or IU/mg sample, according to the concentration factor in Equation (1) and o‐NP molar absorptivity, e = 0.5601 cm/M (Bultema et al, 2014; Ospina et al, 2019). …”

“…Approximately 22 mg of solid biocatalyst were resuspended in 4 mL of 100 mM acetate buffer pH 6.0. The value obtained by the activity assay of a 100 μL aliquot was multiplied by this factor in Equation (2) (Ospina et al, 2019). …”

“…The supernantant was analyzed by UV–vis spectroscopy at 280 nm at zero time ( P i ) and at the end ( P f ) of the immobilization process (Ospina et al, 2019). The IY P was calculated by Equation (5) …”

“…The silica gel sol route involves the polycondensation of siliceous species around the enzyme, which allows the synthesis of a biocatalyst with the enzyme homogeneously distributed in the solid material obtained (Ospina et al, 2019). Even, though the enzyme entrapment causes diffusion restrictions, which affect the kinetic biochemical parameters, but it does not modify the acid/base mechanism for substrate hydrolysis (Escobar et al, 2020; Soto et al, 2017).…”

Multipoint Immobilization at Inert Center of Papain on Homo-Functional Diazo-Activated Silica Support: A Way of Restoring “Above Room-Temperature” Bio-Catalytic Sustainability

“…At present, enzyme immobilization via multipoint covalent attachment is a big issue: not only solving the problem of separation, it recovers the catalytic activity, specificity, or selectivity, , explicitly the enantio-selectivity over the native enzymes, , but advances the enzyme stability against the fluctuation of temperature, , pH, solvent parameters, and reaction conditions. Immobilized enzymes get hold of a suitable enzymatic microenvironment in porous media; in such microenvironments, intermolecular-inactivation processes have never been experienced, which come into view either for precipitation or proteolysis or interaction with external hydrophobic interfaces. , Second, increasing the enzyme rigidity via covalent binding, particularly multipoint attachment, − prevents any conformational change or the subunit dissociation of multimeric proteins ,,, that brings the stability of the immobilized proteins, sometimes even better over their native form.…”