His-tag β-galactosidase supramolecular performance

FLORES, SANDRA S.; CLOP, PEDRO D.; BARRA, JOSÉ L.; ARGARAÑA, CARLOS E.; PERILLO, MARÍA A.; NOLAN, VERÓNICA (AUTOR CORRESPONDIENTE); SÁNCHEZ, JULIETA M. (AUTOR CORRESPONDIENTE)

BIOPHYSICAL CHEMISTRY

ELSEVIER SCIENCE BV

Año: 2022 vol. 281

0301-4622

β-Galactosidase is an important biotechnological enzyme used in the dairy industry, pharmacology and in molecularbiology. In our laboratory we have overexpressed a recombinant β-galactosidase in Escherichia coli(E. coli). This enzyme differs from its native version (β-GalWT) in that 6 histidine residues have been added to thecarboxyl terminus in the primary sequence (β-GalHis), which allows its purification by immobilized metal affinitychromatography (IMAC). In this work we compared the functionality and structure of both proteins and evaluatedtheir catalytic behavior on the kinetics of lactose hydrolysis. We observed a significant reduction in theenzymatic activity of β-GalHis with respect to β-GalWT. Although, both enzymes showed a similar catalytic profileas a function of temperature, β-GalHis presented a higher resistance to the thermal inactivation compared toβ-GalWT. At room temperature, β-GalHis showed a fluorescence spectrum compatible with a partially unstructuredprotein, however, it exhibited a lower tendency to the thermal-induced unfolding with respect to β-GalWT. Thedistinctively supramolecular arranges of the proteins would explain the effect of the presence of His-tag on theenzymatic activity and thermal stability.