A novel thermostable β -galactosidase from Geobacillus kaustophilus HTA42

A novel thermostable β -galactosidase gene, designated as GkGal1A , from the thermophilic bacterium Geobacillus kaustophilus HTA426 was cloned and heterologously overexpressed in Escherichia coli ( E. coli ). Based on the sequence analysis, GkGal1A belongs to the glycosyl hydrolase family 1 that was the first β -galactosidase of bacterial origins expressed by us in this family. The apparent molecular weight of GkGal1A determined by sodium deodecyl sulfate-polyacrylamide gel electrophoresis is 52000. It exhibited the highest activity toward p -nitrophenyl- β - D -galactopyranoside at pH 7.8 and 70 °C and displayed high thermal stability. Divalent cations are prerequisite for the activity of GKGal1A, with the highest activity in the presence of Mn 2+ . Moreover, the three-dimensional structure of GkGal1A was modeled to speculate the structure of the catalytic residues and the reaction mechanism. The catalytic residues consisting of Glu166 and Glu355 were verified by site-directed mutagenesis.