Cloning, purification and characterization of a thermostable β-galactosidase from Thermotoga naphthophila RUK-10

Abstract

A novel β-galactosidase gene (Tnap1577) from the hyperthermophilic bacterium Thermotoga naphthophila RUK-10 was cloned and expressed in Escherichia coli BL21 (DE3) cells to produce β-galactosidase. The recombinant β-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-β-d-galactoside (o-NPG) and lactose with the recombinant β-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 70kDa 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 0.3h, respectively. Kinetic studies on the recombinant β-galactosidase revealed Km values for the hydrolysis of o-NPG and lactose of 1.31mM and 1.43mM, respectively. These values are considerably lower than those reported for other hyperthermophilic β-galactosidases, indicating high intrinsic affinity for these substrates. The recombinant β-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.