Gene Clone and Characterization of a Novel Thermostable β-Galactosidase with Transglycosylation Activity from Thermotoga naphthophila RUK-10

We cloned and expressed a new recombinant beta-galactosidase(TN0949) from Thermotoga naphthophila RKU-10 with the pET28a(+) vector system in Escherichia coli BL21(DE3), and determined its catalytic capability to synthesize alkyl glucosides. The recombinant enzyme was purified to a single band via heat treatment and Ni2+-NTA affinity chromatography. The molecular mass of the recombinant enzyme was estimated to be 79 kDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). TN0949 can hydrolyze o-nitrophenyl beta-D-galactopyranoside at the optimum pH and temperature of 6.5 and 80 degrees C, respectively. TN0949 can also hydrolyze lactose at the optimum pH and temperature of 5.2 and 80 degrees C, respectively. The Km values for the hydrolyses of o-nitrophenyl beta-D-galactopyranoside and lactose were 0.82 and 83.65 mmol/L, respectively. TN0949 was stable over a wide range of pH(3.0 to 7.0) after 24 h of incubation. The half-lives of TN0949 at 75, 80 and 85 degrees C were 22, 6 and 1.33 h, respectively. The enzyme displayed the capability to use lactose as the transglycosylation substrate to synthesize butyl galactopyranoside and hexyl galactopyranoside, indicating its suitability as a candidate industrial biocatalyst.