Gene clone and characterization of a novel thermostable β-galactosidase with transglycosylation activity from Thermotoga naphthophila RUK-10

Abstract

We cloned and expressed a new recombinant β-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 β-D-galactopyranoside at the optimum pH and temperature of 6.5 and 80 °C, respectively. TN0949 can also hydrolyze lactose at the optimum pH and temperature of 5.2 and 80 °C, respectively. The K _m values for the hydrolyses of o-nitrophenyl β-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 °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.

Keywords

β-Galactosidase / Thermostable / Transglycosylation

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