Heterologous expression of polygalacturonase genes isolated from Galactomyces citri-aurantii IJ-1 in Pichia pastoris |
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Authors: | Cho Il Jae Yeo In-Cheol Lee Nam Keun Jung Suk Hee Hahm Young Tae |
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Institution: | Department of Biotechnology (BK21 Program), Chung-Ang University, Anseong, 456-756, Republic of Korea. |
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Abstract: | ABSTACT: 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, K (m), were confirmed to be 0.94 and 0.84 mM, respectively. In the presence of Mn(2+), the activity of PG1 and PG2 were increased to 160.8 and 146.4% of normal levels, respectively. In contrast, Cu(2+) and Fe(3+) acted as strong inhibitors to the PGs. |
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