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KMID : 1234420090370020099
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Korean Journal of Microbiololgy and Biotechnology
2009 Volume.37 No. 2 p.99 ~ p.104
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Cloning of β-Glucosidase Gene from Streptomyces coelicolor A3(2) and Characterization of the Recombinant β-Glucosidase Expressed in Escherichia coli
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Kim Jae-Young
Kim Bong-Kyu
Yi Yong-Sub
Kang Chang-Soo
Ahn Joong-Hoon
Lim Yoong-Ho
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Abstract
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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 20oC to 70oC, and increased activity using β-pNPGA from 30oC to 50oC, 1.8 times higher activity at 50oC than at 30oC. 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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KEYWORD
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β-glucosidase, deglycosylation, Streptomyces coelicolor A3(2), substrate specificity
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