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 KM and Vmax 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 Ki of 365 mM.
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Acknowledgments
This work was supported by grants from; the Chulalongkorn University Dutsadi Phiphat Scholarship, a Chulalongkorn University Graduate Scholarship to Commemorate the 72nd Anniversary of His Majesty King Bhumibol Adulyadej, and Mrs. Ratanavalee In-ochanon from the PTT Public Company Limited.
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Boonvitthya, N., Tanapong, P., Kanngan, P. et al. Cloning and expression of the Aspergillus oryzae glucan 1,3-beta-glucosidase A (exgA) in Pichia pastoris . Biotechnol Lett 34, 1937–1943 (2012). https://doi.org/10.1007/s10529-012-1001-9
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DOI: https://doi.org/10.1007/s10529-012-1001-9