Biochemical characterization of pectate lyases produced by fluorescent pseudomonads associated with spoilage of fresh fruits and vegetables

An improved method for purification of pectate Iyases (PLI and PLII) from culture fluids of Pseudomonas fluorescens CY091 and Ps. viridiflava PJ-08-6 by using a phosphocellulose cation exchanger was described. Analysis of purified PLI and PLII by sodium dodecyl sulphate-polyacrylamide and isoelectric focusing gel electrophoresis revealed that both enzymes had been purified to near homogeneity. Optimal Ca2+ concentration required for PLI and PLII activity was determined to be 0.5 mmol l(-1), The Ca2+ requirement could not be replaced by other metal cations such as Mg2+, Cu2+, Zn2+, Fe3+ and Co2+ Optimal pH for activity was determined to be between 8.5 and 9.0. The k(m) values for sodium polygalacturonate were 1.28 and 1.11 and ml(-1) for PLI and PLII, respectively. Both PLI and PLII were stable at ion temperatures (25 degrees C or below) for at least 1 month. However, at 37 degrees C, the activity decreased 50% in 36 h, Optimal temperatures for activity were estimated to be 46 degrees and 52 degrees C for PLI and PLII, respectively. Thermal stability of both enzymes at elevated temperatures (48 degrees C or higher) increased when CaCl2 or a positively charged molecule such as poly lysine was present, but decreased when polygalacturonate or a negatively charged molecule such as heparin was present. PLI and PLII exhibit differential degrees of sensitivity to group-specific inhibitors, including iodoacetic acid and diethylpyrocarbonate. This result suggests that both sulphydryl and imidazole groups are important for the catalytic function of PLI and PLII.