Identification, Heterologous Expression, and Characterization of a New α‐Galactosidase From <i>Geobacillus kaustophilus</i>

α-Galactosidase enzymes have a variety of applications in numerous areas, including medicine, energy, food, feed, prebiotics, and paper pulp production. A single α-galactosidase gene was identified in the Geobacillus kaustophilus genome through the analysis of the carbohydrate-active enzymes (CAZY) database. The objective of this study was to clone, express heterologously, purify, and biochemically characterize the α-galactosidase enzyme encoded in the G. kaustophilus genome. In order to achieve this, a codon-optimized synthetic gene encoding the α-galactosidase enzyme was cloned into the pQE30 plasmid and expressed in Escherichia coli BL21 DE3 pLysS. The biochemical characterization of G. kaustophilus α-galactosidase revealed that the purified enzyme exhibited optimal activity at 40°C and pH 6.0 in citrate buffer, with demonstrable activity over a broad range of pH and temperature. Furthermore, the GKαGal enzyme exhibited a V_max value of 41.09 U/mg and a 0.248 mM K_m value towards 4-nitrophenyl-α-d-galactopyranoside (pNPGal). In essence, the produced α-galactosidase enzyme has potential applications in the degradation of lignocellulosic biomass, disaccharides production, and in medical contexts.