Cloning of β-Glucosidase Gene from Streptomyces coelicolor A3(2) and Characterization of the Recombinant β-Glucosidase Expressed in Escherichia coli
2009
Kim, J.Y., Hoseo University, Asan, Republic of Korea | Kim, B.K., Konkuk University, Seoul, Republic of Korea | Kang, C.S., Hoseo University, Asan, Republic of Korea | Yi, Y.S., Hoseo University, Asan, Republic of Korea | Ahn, J.H., Konkuk University, Seoul, Republic of Korea | Lim, Y.H., Konkuk University, Seoul, Republic of Korea
The β-glucosidase gene from Streptomyces coelicolor A3(2) was cloned and expressed in Escherichia coli. The ORF consisted of 1377 nucleotides encoding 51 kDa in a predicted molecular weight. Effects of pH indicated that the β-glucosidase showed similar activity using α-pNPG(ρ-nitrophenyl-α-D-glucopyranoside), β-pNPG(ρ-nitrophenyl-β-D-glucopyranoside), and β-pNPF(ρ-nitrophenyl-β-D-fucopyranoside) at range of pH 3 to 10, and high activity using β-pNPGA (ρ-nitrophenyl-β-D-galactopyranoside) from pH 5 to 10, especially, 3.3 times higher activity at pH 9. Effects of temperature indicated that the β-glucosidase showed low activity using α-pNPG, β-pNPG, and β-pNPF from 20℃ to 70℃, and increased activity using β-pNPGA from 30℃ to 50℃, 1.8 times higher activity at 50℃ than at 30℃. According to activity determination of other substrates, the enzyme was active on daidzin, genistin, and glycitin, inactive on esculin and apigenin-7-glucose. The EDTA and DTT as reducing agents inhibited β-glucosidase activity, but SDS and mercaptoethanol did not inhibit. Monovalent or divalent metal ions such as MnSO₄, CaCl₂, KCl, and MgSO₄ did not inhibited β-glucosidase activity. CuSO₄ and NaCl showed low inhibition, and ZnSO₄ inhibited 3.3 times higher than control.
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