초록
The β-glucosidase gene Tt-bgl from Thermotoga thermarum DSM 5069T was cloned and overexpressed in Escherichia coli. A simple strategy, induction at 37<SUP>o</SUP>C with no IPTG, was explored to reduce the inclusion bodies, by which the activity of Tt-BGL was 13U/mL in LB medium. Recombinant Tt-BGL was purified by heat treatment followed by Ni-NTA affinity. The optimal activity was at pH 4.8 and 90<SUP>o</SUP>C. The activity of Tt-BGL was significantly enhanced by methanol and Al<SUP>3+</SUP>. The enzyme was stable over pH range of 4.4-8.0, and had a 2-h half life at 90<SUP>o</SUP>C. The V<SUB>max</SUB> for p-nitrophenyl-β-d-glucopyranoside and ginsenoside Rb1 was 142U/mg and 107U/mg, while the K<SUB>m</SUB> was 0.59mM and 0.15mM, respectively. The activity of the enzyme was not inhibited by ginsenoside Rb1 (36g/L). It was activated by glucose at concentrations lower that 400mM. With glucose further increasing, the activity of Tt-BGL was gradually inhibited, but remained 50% of the original value in even as high as 1500mM glucose. Under the optimal conditions, Tt-BGL transformed ginsenoside Rb1 (36g/L) to Rd by 95% in 1h.