His-tag β-galactosidase supramolecular performance

Biophys Chem. 2021 Dec 10;281:106739. doi: 10.1016/j.bpc.2021.106739. Online ahead of print.ABSTRACTβ-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 compatible with a partially unstructured protein, however, it exhibited a lower tendency to the thermal-induced unfolding with respect to β-GalWT. The distinctively supramolecular arranges of the proteins would explain the effect of the presence of His-tag on the enzymatic activity and thermal stability.PMID:34923392 | DOI:10.1016/j.bpc.2021.106739

Source: Biophysical Chemistry - December 19, 2021 Category: Chemistry Authors: Sandra S Flores Pedro D Clop Jos é L Barra Carlos E Argara ña Mar ía A Perillo Ver ónica Nolan Julieta M S ánchez Source Type: research