title: Heterologous expression of polygalacturonase genes isolated from Galactomyces citri-aurantii IJ-1 in Pichia pastoris.
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.
authors.affiliation: Department of Biotechnology (BK21 Program), Chung-Ang University, Anseong, 456-756, Republic of Korea.
authors.forename: Il Jae
authors.initials: IJ
authors.lastname: Cho
authors.forename: In-Cheol
authors.initials: IC
authors.lastname: Yeo
authors.forename: Nam Keun
authors.initials: NK
authors.lastname: Lee
authors.forename: Suk Hee
authors.initials: SH
authors.lastname: Jung
authors.forename: Young Tae
authors.initials: YT
authors.lastname: Hahm
mesh_terms: D000595:Amino Acid Sequence; D002957:Citrus / Q000382:microbiology*; D003001:Cloning, Molecular; D005638:Fruit / Q000382:microbiology; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010935:Plant Diseases / Q000382:microbiology; D011096:Polygalacturonase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004718:Saccharomycetales / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D011096:Polygalacturonase