A novel P-galactosidase gene (Tnap1577) from the hyperthermophilic bacterium Thermotoga naphthophila RUK-10 was cloned and expressed in Escherichia coli BL21 (DE3) cells to produce P-galactosidase. The recombinant P-galactosidase was purified in three steps: heat treatment to deactivate E. coli proteins, Ni-NTA affinity chromatography and Q-sepharose chromatography. The optimum temperatures for the hydrolysis of o-nitrophenyl-beta-D-galactoside (o-NPG) and lactose with the recombinant P-galactosidase were found to be 90 C and 70 C, respectively. The corresponding optimum pH values were 6.8 and 5.8, respectively. The molecular mass of the enzyme was estimated to be 70 kDa by SDS-PAGE analysis. Thermostability studies showed that the half-lives of the recombinant enzyme at 75 C, 80 C, 85 C and 90 C were 10.5, 4, 1, and 0.3 h, respectively. Kinetic studies on the recombinant P-galactosidase revealed Km values for the hydrolysis of o-NPG and lactose of 1.31 mM and 1.43 mM, respectively. These values are considerably lower than those reported for other hyperthermophilic beta-galactosidases, indicating high intrinsic affinity for these substrates. The recombinant P-galactosidase from Thermotoga naphthophila RUK-10 also showed transglycosylation activity in the synthesis of alkyl galactopyranoside. This additional activity suggests the enzyme has potential for broader biotechnological applications beyond the degradation of lactose. (C) 2014 Elsevier Ltd. All rights reserved.