Characterization of a Glucose-, Xylose-, Sucrose-, and d-Galactose-Stimulated β-Glucosidase from the Alkalophilic Bacterium Bacillus halodurans C-125
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The gene (Bhbgl) encoding a β-glucosidase from the alkalophilic bacterium Bacillus halodurans C-125 was synthesized chemically via the PCR-based two-step DNA synthesis (PTDS) method and expressed in Escherichia coli. Bhbgl contained an open reading frame (ORF) of 1359 bp encoding a 453-amino acid protein belonging to glycoside hydrolase family 1 (GHF1), and the deduced molecular mass of recombinant Bhbgl (52,488 Da) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited a high specific activity with o-nitrophenyl-β-d-glucopyranoside (oNPGlu) and an apparent K m value of 0.32 mM. With oNPGlu as the substrate, Bhbgl displayed pH and temperature optima of ~7.0 and 50°C, respectively. The enzyme was relatively stable under alkaline conditions and >50% activity was retained after incubation at pH 9.5 for 24 h at 4°C. Recombinant Bhbgl activity was inhibited by 5 mM Zn2+, Fe3+, or Cd2+, but was enhanced by 1 mM Mg2+ and other metal ions. Enzyme activity was also stimulated by at least four sugars (sucrose, d-galactose, xylose, glucose) at concentrations ranging from 50 to 800 mM.
This research was supported by the Youth Fund of the Shanghai Academy of Agricultural Sciences (Grant No. 2008-6, 2009-19), the Shanghai Basic Research Project (Grant No. 08JC1418000), the Key Project Fund of the Shanghai Municipal Committee of Agriculture (Grant No. 2008-7-5), and the Key Project Fund of the Shanghai Municipal Committee of Agriculture (Grant No. 2009-6-4). We thank Dr John Buswell for linguistic revision of the manuscript.
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