Biochemical characterization of pectate lyases produced by fluorescent pseudomonads associated with spoilage of fresh fruits and vegetables |
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Authors: | C-H Liao J Sullivan J Grady & L-JC Wong |
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Institution: | US Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA, USA,;Department of Biological Sciences, University of Massachusetts, Lowell, MA, USA,;Children's Hospital of Los Angeles, Department of Pathology, University of Southern California, Los Angeles, CA, USA |
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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 K m 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°C or below) for at least 1 month. However, at 37°C, the activity decreased 50% in 36 h. Optimal temperatures for activity were estimated to be 46° and 52°C for PLI and PLII, respectively. Thermal stability of both enzymes at elevated temperatures (48°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. |
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