Cloning and expression of the Aspergillus oryzae glucan 1,3-beta-glucosidase A (exgA) in Pichia pastoris.

Nassapat Boonvitthya, Phatrapan Tanapong, Patcharaporn Kanngan, Vorakan Burapatana, Warawut Chulalaksananukul

Biological Science Program, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Patumwan, Bangkok 10330, Thailand. nassapat.boonvitthya@hotmail.com

Biotechnology letters 2012 Oct

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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.

Citation

Nassapat Boonvitthya, Phatrapan Tanapong, Patcharaporn Kanngan, Vorakan Burapatana, Warawut Chulalaksananukul. Cloning and expression of the Aspergillus oryzae glucan 1,3-beta-glucosidase A (exgA) in Pichia pastoris. Biotechnology letters. 2012 Oct;34(10):1937-43