His-tag β-galactosidase supramolecular performance

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

BIOPHYSICAL CHEMISTRY

ELSEVIER SCIENCE BV

Año: 2022 vol. 281

0301-4622

#946;-Galactosidase is an important biotechnological enzyme used in the dairy industry, pharmacology and in molecular biology. 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 the carboxyl terminus in the primary sequence (β-GalHis), which allows its purification by immobilized metal affinity chromatography (IMAC). In this work we compared the functionality and structure of both proteins and evaluated their catalytic behavior on the kinetics of lactose hydrolysis. We observed a significant reduction in the enzymatic activity of β-GalHis with respect to β-GalWT. Although, both enzymes showed a similar catalytic profile as a function of temperature, β-GalHis presented a higher resistance to the thermal inactivation compared to β-GalWT. At room temperature, β-GalHis showed a fluorescence spectrum