Characterization of a glucose-, xylose-, sucrose-, and D-galactose-stimulated β-glucosidase from the alkalophilic bacterium Bacillus halodurans C-125.
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 Zn(2+), Fe(3+), or Cd(2+), but was enhanced by 1 mM Mg(2+) 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.
College of Bioscience and Biotechnology, Yangzhou University, Jiangsu, China.
Hu
H
Xu
Ai-Sheng
AS
Xiong
Wei
W
Zhao
Yong-Sheng
YS
Tian
Ri-He
RH
Peng
Jian-Min
JM
Chen
Quan-Hong
QH
Yao
D001407:Bacillus / Q000201:enzymology*; D050260:Carbohydrate Metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005960:Glucosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D005960:Glucosides; D008670:Metals; D011994:Recombinant Proteins; C025193:4-nitrophenyl beta-D-glucoside; D001617:beta-Glucosidase