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