Heterologous expression of polygalacturonase genes isolated from Galactomyces citri-aurantii IJ-1 in Pichia pastoris

摘要

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.