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 ...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-nitrophenylβ-D- galactopyranoside at the optimum pH and temperature of 6.5 and 80 ℃, respectively. TN0949 can also hydrolyze lactose at the optimum pH and temperature of 5.2 and 80 ℃, respectively. The Km values for the hydrolyses of o-nitrophenyl fl-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 ℃ 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.展开更多
文摘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-nitrophenylβ-D- galactopyranoside at the optimum pH and temperature of 6.5 and 80 ℃, respectively. TN0949 can also hydrolyze lactose at the optimum pH and temperature of 5.2 and 80 ℃, respectively. The Km values for the hydrolyses of o-nitrophenyl fl-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 ℃ 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.
