Identification, Heterologous Expression, and Characterization of a New α‐Galactosidase From Geobacillus kaustophilus

ABSTRACT 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 ( p NPGal). In essence, the produced α‐galactosidase enzyme has potential applications in the degradation of lignocellulosic biomass, disaccharides production, and in medical contexts.