Abstract
The glucan 1,3-beta-glucosidase A gene (exgA) from Aspergillus oryzae and fused to the Saccharomyces cerevisiae signal peptide (α-factor) was expressed under the control of either a constitutive (GAP) or an inducible (AOX1) promoter in Pichia pastoris. A 1.4-fold higher extracellular enzyme activity (2 U/ml) was obtained using the AOX1 inducible expression system than with the GAP constitutive promoter (1.4 U/ml). The purified recombinant ExgA enzyme, with a yield of 10 mg protein/l culture supernatant, was about 40 kDa by SDS-PAGE analysis with a specific activity of 289 U/mg protein. The enzyme was optimally active at 35 °C and pH 5.0 and displayed a K(M) and V(max) of 0.56 mM and 10,042 μmol/(min mg protein), respectively, with p-nitrophenyl-β-D-glucopyranoside as the substrate. Moreover, it was tolerant to glucose inhibition with a K(i) of 365 mM.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Aspergillus oryzae / enzymology*
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Aspergillus oryzae / genetics
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Cloning, Molecular
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DNA, Fungal
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Electrophoresis, Polyacrylamide Gel
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Fungal Proteins / biosynthesis*
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Fungal Proteins / chemistry
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Fungal Proteins / genetics
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Glucan 1,3-beta-Glucosidase / biosynthesis*
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Glucan 1,3-beta-Glucosidase / chemistry
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Glucan 1,3-beta-Glucosidase / genetics
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Glucose / metabolism
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Histidine / genetics
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Histidine / metabolism
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Oligopeptides / genetics
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Oligopeptides / metabolism
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Pichia / enzymology
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Pichia / genetics*
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Polymerase Chain Reaction
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Recombinant Fusion Proteins / biosynthesis*
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Temperature
Substances
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DNA, Fungal
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Fungal Proteins
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His-His-His-His-His-His
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Oligopeptides
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Recombinant Fusion Proteins
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Histidine
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Glucan 1,3-beta-Glucosidase
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Glucose