KMID : 0880220090470050542
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Journal of Microbiology 2009 Volume.47 No. 5 p.542 ~ p.548
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Characterization of a Novel ¥â-Glucosidase-Like Activity from a Soil Metagenome
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Jiang Cheng-Jian
Ma Gefei Li Shuangxi Hu Tingting Che Zhiqun Shen Peihong Wu Bo Yan Bing
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Abstract
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We report the cloning of a novel ¥â-glucosidase-like gene by function-based screening of a metagenomic library from uncultured soil microorganisms. The gene was named bgl1C and has an open reading frame of 1,443 base pairs. It encodes a 481 amino acid polypeptide with a predicted molecular mass of about 57.8 kDa. The deduced amino acid sequence did not show any homology with known ¥â-glucosidases. The putative ¥â-glucosidase gene was subcloned into the pETBlue-2 vector and overexpressed in E. coli Tuner (DE3) pLac?; the recombinant protein was purified to homogeneity. Functional characterization with a high performance liquid chromatography method demonstrated that the recombinant Bgl1C protein hydrolyzed D-glucosyl-¥â-(1-4)-D-glucose to glucose. The maximum activity for Bgl1C protein occurred at pH 8.0 and 42¡ÆC using p-nitrophenyl-¥â-D-glucoside as the substrate. A CaCl2 concentration of 1 mM was required for optimal activity. The putative ¥â-glucosidase had an apparent Km value of 0.19 mM, a Vmax value of 4.75 U/mg and a kcat value of 316.7/min under the optimal reaction conditions. The biochemical characterization of Bgl1C has enlarged our understanding of the novel enzymes that can be isolated from the soil metagenome.
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KEYWORD
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uncultured soil microorganisms, function-based screening strategy, ¥â-glucosidase
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