Journal of Microbiology

Research Support, Non-U.S. Gov't

Heterologous Expression of Polygalacturonase Genes Isolated from Galactomyces citri-aurantii IJ-1 in Pichia pastoris: Il Jae Cho , In-Cheol Yeo , Nam Keun Lee , Suk Hee Jung , Young Tae Hahm; J. Microbiol. 2012;50(2):332-340. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1290-7

Abstract: The objective of this work was to isolate the polygalacturonase genes of Galactomyces citri-aurantii IJ-1 harvested from rotten citrus peels and to heterologously express these genes in Pichia pastoris. Two polygalacturonase (PG) genes from G. citri-aurantii IJ-1 were obtained and tentatively named PG1 and PG2. The genes were cloned into pPICZαC, and expressed in Pichia pastoris strain GS115 with a native signal peptide or the α-factor secretion signal peptide of Saccharomyces cerevisiae. All of the recombinant proteins were successfully secreted into the culture media and confirmed as a single band with a molecular weight of 35 to 38 kDa by SDS-PAGE. The specific enzyme activities of recombinant PG1 and PG2 purified by His-tag affinity resin were 4,749 and 6,719 U/mg, respectively, with an optimal pH and temperature of pH 4.0 and 50°C. The Michaelis- Menten kinetic constants for PG1 and PG2, Km, were confirmed to be 0.94 and 0.84 mM, respectively. In the presence of Mn2+, the activity of PG1 and PG2 were increased to 160.8 and 146.4% of normal levels, respectively. In contrast, Cu2+ and Fe3+ acted as strong inhibitors to the PGs.

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