A putative recombinant beta-galactosidase from Deinococcus geothermalis was purified as a single 79 kDa band of 42 U activity/mg using His-Trap affinity chromatography. The molecular mass of the native enzyme was a 158 kDa dimer. The catalytic residues E151 and E325 of beta-galactosidase from D. geothermalis were conserved in all aligned GH family 42 beta-galactosidases, indicating that this enzyme is also a GH family 42 beta-galactosidase. Maximal activity of the enzyme was at pH 6.5 and 60A degrees C. It has a unique hydrolytic activity for p-nitrophenyl(pNP)-beta-d-galactopyranoside (k (cat)/K (m) = 69 s(-1) mM(-1)), pNP-beta-d-fucopyranoside (13), oNP-beta-d-galactopyranoside (9.5), oNP-beta-d-fucopyranoside (2.6), lactose (0.97), and pNP-alpha-l-arabinopyranoside (0.78), whereas no activity, or less than 2% of the pNP-beta-d-galactopyranoside activity, for other pNP- and oNP- glycosides.