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KMID : 0880220120500020332
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Journal of Microbiology
2012 Volume.50 No. 2 p.332 ~ p.340
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Heterologous expression of polygalacturonase genes isolated from Galactomyces citri-aurantii IJ-1 in Pichia pastoris
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Cho Il-Jae
Yeo In-Cheol
Lee Nam-Keun
Jung Suk-Hee
Hahm Young-Tae
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Abstract
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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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KEYWORD
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polygalacturonase, Galactomyces citri-aurantii, Pichia pastoris, heterologous expression
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