Purification and characterization of an endopolygalacturonase from the rough lemon pathotype ofAlternaria alternata,the cause of citrus brown spot disease☆

摘要: Abstract Alternaria alternata rough lemon pathotype (AC325) produced polygalacturonase in liquid culture containing 1% pectin. Synthesis of the was repressed by addition 5% sucrose to culture. The enzyme purified about 31-fold from filtrates four steps: ammonium sulphate precipitation; CM-Sepharose; cation exchange HPLC; and gel filtration HPLC. isolated acted an endo fashion: hydrolysis 1.13% glycosidic bonds polygalacturonic acid resulted a 50% reduction viscosity. endopolygalacturonase glycoprotein, molecular weight approximately 60 kDa as estimated SDS polyacrylamide electrophoresis. enzyme's isoelectric point 8.8, pH optimum for activity 5.0. K m V max values were 0.1 mg ml −1 4.76×10 −3 mmol reducing group min protein, respectively. N-terminal amino sequence identified Gly-Pro-Asp-Asp-Ile-Tyr-Ala. gene ( PGN1 ) Cochliobolus carbonum race 1 detected single band possessing high homology AC325 genome on Southern blot. same probe also hybridized PCR-product genomic DNA, using forward primer designed reverse conserved among several known fungal endopolygalacturonases.