Purification and characterization of phenylalanine ammonia-lyase from Ustilago maydis
ABSTRACT Phenylalanine ammonia-lyase (PAL; EC. 220.127.116.11) has been purified to homogeneity from liquid-cultured cells of the phytopathogenic fungus Ustilago maydis by use of heat treatment, protamine and ammonium sulphate precipitation, ion-exchange and gel filtration chromatography, and preparative PAGE. Its native molecular mass was estimated as 320±20 kDa and its subunit molecular mass as 80 kDa. No isoforms of the enzyme were detected, and there was no evidence of glycosylation of the protein. Ustilago PAL was most active at pH 8.8–9.2 and 30° and had a Km for l-phenylalanine of 1.05 mM. The enzyme did not deaminate l-tyrosine. The synthetic inhibitor 2-aminoindan-2-phosphonic acid (AIP) strongly inhibited the enzyme, as did sulphhydryl reagents and carbonyl reagents, whereas t-cinnamate was only moderately inhibitory. Ustilago PAL activity had no requirement for metal ion cofactors, but was inhibited by heavy metal ions (Ag+, Cu2+, and Hg2+). Polyclonal antibodies raised against the purified enzyme readily recognized U. maydis PAL in solution and on Western blots, but only weakly cross-reacted with higher plant PAL.
SourceAvailable from: Lily Novak Frazer
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ABSTRACT: The vase life of cut flowers and foliage is often shortened by vascular occlusions that constrict vase solution supply. Reduction in stem conductivity is typically caused by blockage of cut stem ends and xylem conduits by microbes, physiological plugging, and disruption of water columns in xylem vessels by cavitations and air emboli. Cut flower and foliage longevity can be greatly affected by the chemical composition of the vase solution. A broad range of biocides has been suggested to prevent the proliferation of microorganisms in vase solutions; however, their assumed antimicrobial action may be confounded by their other physicochemical effects. the effect of some chemicals on postharvest longevity and microorganisms in solution of cut carnation 'Delphi' evaluated in a randomized complete block design with three replications. Flowers harvested in paint brush stage and recutted to 60 cm stem length. Vase life evaluated in 20±2 o C temperature, relative humidity 60% and 1800 lux light intensity. The results showed that flowers longevity has significant different (P ≤0.01) and copper sulfate and Halamid ® (Sodium N-Chloro-para-Toluenesulfonamide) were the best treatments. Population (P≤0.001) and relative water content (P≤0.05) were significantly affected by treatments and Halamid ® was the best treatment to microorganisms control and water content. Highly significant negative correlation of relative water content and the bacterial population in solution indicate that the main effect of bacteria in reducing the water uptake.
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ABSTRACT: Cetecol has been known as a component of melanin in teliospores of the corn smut fungus Ustilago maydis. Its metabolic precursor has been assumed to be benzoic acid but it has not been proven yet. This study was carried out to verify the synthesis of benzoic acid and to chase its metabolic origin in U. maydis. For this aim, the catabolic process of phenylalanine was investigated by culturing the fungus in the complete medium containing L--phenylalanine and -trans-cinnamic acid. We detected trans-cinnamic acid, benzoic acid, 4-hydroxybenzoic acid and hydroxybenzoic acid derivatives from the extracts of the fungus cells and cultural filtrates by thin layered chromatography analysis. We also observed that the fungus could completely catabolize L--phenylalanine and produce in the air. Conclusively, this study provided an evidence that U. maydis could produce benzoic acid through catabolic process of phenylalanine.12/2011; 39(3). DOI:10.4489/KJM.2010.39.3.249