Graduate Thesis Or Dissertation
 

Production of pectic enzymes by Verticillium albo-atrum

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/ht24wm83w

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  • The main objectives of this study were to determine the effect of certain environmental conditions on the quantity of poly-galacturonase (PG) produced by V. albo-atrum, and to partially purify and characterize the PGs produced by this fungus in culture and in infected tomato plants. V. albo-atrum produced twenty times as much PG when sodium polypectate was the carbon source as when grown on pectin. Optimum PG production occurred 6-8 days after inoculation of the medium. Less pectinmethyesterase (PME) was produced when V. albo-atrum was grown on polypectate as the carbon source than when grown on pectin. PME production was observed early in the growth of the fungus. No PME activity could be detected from culture filtrates after eight days when either pectin or sodium polypectate was the carbon source. Three elution schedules were used to purify PGs from a DEAE-cellulose column. Step-wise elution produced residual peaks of PG activity, and gradient elution gave poor separation of the PGs. Flushing the DEAE-cellulose column with distilled water and NaC1 gave the best separation of the two endo-PGs. PME was associated with all fractions containing PG activity. Assays of PG activity in fractions separated by column chromatography suggested that V. albo-atrum produced at least two endo-PGs in culture. One endo-PG preferentially hydrolyzed sodium polypectate to large fragments; however, the fragments were eventually hydrolyzed to monogalacturonic acid. The second endo-PG preferentially hydrolyzed small fragments from the substrate. Fractions containing the endo-PG that preferentially hydrolyzed the substrate to large fragments gave ratios of viscosity reduction to reducing group liberation at least five times as high as those obtained for fractions containing the endo-PG that preferentially hydrolyzed the substrate to small fragments. Culture filtrates from cultures incubated at room temperature but not on a shaker produced less PG than cultures grown on a shaker at room temperature. When culture filtrates of the still cultures were eluted from the DEAE-cellulose column, fractions from the first PG peak eluted from the column hydrolyzed trigalacturonic acid slowly to monogalacturonic acid. Cultures grown at 30°C produced only trace amounts of PG activity. Growth of the fungus at 30°C was similar to cultures grown at lower temperatures. However, the cultures grown at lower temperatures produced relatively high PG titers. The crude enzyme preparation of V. albo-atrum from infected tomato plants yielded pectic fragments as well as monogalacturonic acid. This indicated the presence of an endo-PG that hydrolyzed small fragments from the substrate. Column chromatography of this enzyme on DEAE-cellulose using a step-wise elution yielded a PG fraction that preferentially hydrolyzed the substrate into small fragments similar to fractions obtained by a step-wise elution of culture filtrates. Evidence was obtained that V. albo-atrum produced trace amounts of pectintranseliminase in culture and in infected plants. Additional studies will be required to understand the significance of this enzyme in the Verticillium wilt syndrome.
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