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

Author:
Liao, C.H., Sullivan, J., Grady, J., Wong, L.J.C.
Source:
Journal of applied microbiology 1997 v.83 no.1 pp. 10-16
ISSN:
1364-5072
Subject:
Pseudomonas fluorescens, calcium, calcium chloride, cations, cobalt, copper, heparin, iodoacetic acid, isoelectric focusing, lyases, magnesium, pH, purification methods, raw fruit, sodium, spoilage, temperature, thermal stability, vegetables, zinc
Abstract:
An improved method for purification of pectate lyases (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 Km values for sodium polygalacturonate were 1.28 and 1.11 mg ml-1 for PLI and PLII, respectively. Both PLI and PLII were stable at low 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 polylysine 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.
Agid:
2023197