Graduate Thesis Or Dissertation
 

Pectolytic enzyme production by Fusarium oxysporum f. sp. lycopersici

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  • This investigation was undertaken to determine 1) the effects of various carbon sources in defined media on pectolytic enzyme production, 2) the effect of the incubation period on pectolytic enzyme activity, and 3) the development of improved methods for the purification and characterization of the polygalacturonases (PG) produced by Fusarium oxysporum f. sp. lycopersici. Optimum PG and pectinesterase (PE) production on pectin medium occurred in nine days while 11 days were required for optimum pectin methyl trans-eliminase (PMTE) synthesis. PMTE was the predominant enzyme synthesized on polygalacturonic acid (PGA) with trace amounts of PG and PE. Optimum PMTE synthesis occurred in five days, while PG and PE activity decreased after three days of incubation. Only trace amounts of PG and PMTE were detected in cultures in which glucose was the sole carbon source, and PE production was greater on glucose than on PGA. Polygalacturonate trans-eliminase was not detected in any of the cultures. The action of these enzymes was determined on purified pectic substrates, using improved viscosity reduction and reducing group assays, as well as thin layer chromatography (TLC) procedures for the detection of hydrolysis products. Only endo-PG and endo-PMTE activity was present in the pectin, PGA, and glucose culture filtrates. The results obtained from the TLC of the PG-reaction-mixtures indicated a preferential release of mono-galacturonic acid from PGA, which suggest exo-PG activity. However, this enzyme probably was not an exo-PG because only a small number of the total α-1, 4 linkages were hydrolyzed at relatively high levels of viscosity reduction. About 90% of the PG activity in a pectin medium culture filtrate was adsorbed on CM-cellulose resulting in a 24-34 fold increase in specific activity. Most of the PE activity was similarly adsorbed on CM-cellulose, but the endo-PMTE was not adsorbed. All the pectolytic enzyme activity in the PGA culture filtrates was lost following dialysis against 0. 01M acetate buffer (pH 4. 0). Most of the endo-PG in the filtrates from glucose cultures was not adsorbed on CM-cellulose. An enzyme isolated from the glucose cultures hydrolyzed sodium polypectate reducing its viscosity but yielding an unidentified aldohexose as the major hydrolytic product. Thus, suggesting that this enzyme may have been misclassified in the past as a PG, but it is probably more closely related to the hemicellulases. The chromatographic patterns obtained by gel filtration and ion-exchange chromatography of the PG produced on pectin, PGA, and glucose were correlated with the amount of carbohydrate present in the enzyme fractions. The size of the enzyme units and the number of peaks produced on Sephadex G-75 and CM- and DEAE-cellulose columns increased with increasing carbohydrate concentration. Dialyzed culture filtrate and the fraction not adsorbed on CM-cellulose produced more peaks on the Sephadex G-75 and ion-exchange columns than did the fraction adsorbed on CM-cellulose.
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