Purification and characterization of phenylalanine ammonia-lyase from Ustilago maydis
ABSTRACT Phenylalanine ammonia-lyase (PAL; EC. 184.108.40.206) 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.
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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.The Korean Journal of Mycology. 01/2011; 39(3).
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ABSTRACT: The aim of this study was to investigate the effect of tryptophan (Trp) on the production of total phenolic compounds (TP) in the mycelial culture of Ganoderma neo-japonicum (G. neo-japonicum). Among various amino acids tested, Trp was best for increasing the TP content by 13.2-fold compared to the control. Trp also increased the phenylalanine content via Trp-specific activation of chorismate mutase. Although 12 mM of Trp was the optimal concentration for TP production, it significantly inhibited mycelial growth. This growth inhibition was reversed by the addition of yeast extract (YE) in a dose-dependent manner. Consequently the combined addition of Trp (12 mM) and YE (8 g/L) stimulated TP production up to 73.5 mg gallic acid equivalent per g dry weight of mycelial culture. Furthermore, the phenolic profiling revealed that quercertin occupied 34.1% of the total amount of phenolic compounds tested and the resveratrol concentration increased at the highest rate (49.1-fold) compared to that of the control. These results provided strong evidence that the accumulation of phenolic compounds in the G. neo-japonicum mycelia was regulated by the availability of substrates involved in the phenylpropanoid pathway, which may be useful in regulation and optimization of G. neo-japonicum culture for efficient production of valuable nutraceuticals.Journal of the Korean Society for Applied Biological Chemistry 53(3). · 0.54 Impact Factor
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ABSTRACT: We have grown both polycrystalline and partially textured cobalt films by magnetron sputter deposition in the range of thickness (50–200nm). Kinetic roughening of the growing film leads to a controlled rms surface roughness values (1–6nm) increasing with the as-grown film thickness. Ion erosion of a low energy 1keV Ar+ beam at glancing incidence (80°) on the cobalt film changes the surface morphology to a ripple pattern of nanometric wavelength. The wavelength evolution at relatively low fluency is strongly dependent on the initial surface topography (a wavelength selection mechanism hereby confirmed in polycrystalline rough surfaces and based on the shadowing instability). At sufficiently large fluency, the ripple wavelength steadily increases on a coarsening regime and does not recall the virgin surface morphology. Remarkably, the use of a rough virgin surface makes the ripple amplitude in the final pattern can be controllably increased without affecting the ripple wavelength.Applied Surface Science 01/2011; 257(9):4432-4438. · 2.54 Impact Factor