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.

Purification and characterization of an exo-polygalacturonase from botrytis cinerea: Lee, Tae Ho , Kim, Byung Young , Chung, Young Ryun , Lee, Sang Yeol , Lee, Chang Won , Kim, Jae Won; J. Microbiol. 1997;35(2):134-140.

Abstract: Botrytis cinerea T81-1 has been shown to produce at least four different polygalacturonases into a liquid medium containing citrus pectin, a carbon sousrce. One of the enzymes, which had an apparent molecular weight of 66 kDa estimated by denatured polyacrylamide gel electrophoresis, was purified to electrophoretic homogeneity by a series of procedures including a cetone precipitation, ion exchange, heparin affinity, and reverse phase column chromatographies. The molecular weight of native enzyme was determined to be 64 kDa by gel permeation chromatography indicating the enzyme to be a single polypeptide chain. By viscometric analysis, the enzyme was revealed as exo-polygalacturonase. The enzyme activity was inhibited by divalent cations such as Ca^2+, Mg^2+, and Cu^2+. Km and Vmax for polygalacturonic acid hydrolysis were 0.33 mg/ml and 28.6 nM/min, respectively. The optimum temperature for enzymatic activity was 50℃. And the enzyme showed optimal pH values between 4.0 and 5.0. The enzyme was stable upto 12 hours in the range of pH 3 to 8 and at temperature below 30℃.

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