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.
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ABSTRACT: Abstract Phenylalanine ammonia lyase (PAL) catalyzes the nonoxidative deamination of L-phenylalanine to form trans-cinnamic acid and a free ammonium ion. It plays a major role in the catabolism of L-phenylalanine. The presence of PAL has been reported in diverse plants, some fungi, Streptomyces and few Cyanobacteria. In the past two decades, PAL has gained considerable significance in several clinical, industrial and biotechnological applications. Since its discovery, much knowledge has been gathered with reference to the enzyme's importance in phenyl propanoid pathway of plants. In contrast, there is little knowledge about microbial PAL. Furthermore, the commercial source of the enzyme has been mainly obtained from the fungi. This study focuses on the recent advances on the physiological role of microbial PAL and the improvements of PAL biotechnological production both from our laboratory and many others as well as the latest advances on the new applications of microbial PAL.Critical Reviews in Biotechnology 05/2013; 34(3). DOI:10.3109/07388551.2013.791660 · 7.84 Impact Factor
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ABSTRACT: Phenylalanine ammonia-lyase (PAL, EC 18.104.22.168) from green bamboo was isolated and cloned from the shell of Bambusa oldhamii. The K(m) of bamboo shell PAL for L-Phe was 476 μM, and the molecular mass of native PAL was estimated as 275 kDa and the molecular mass of a subunit was about 76 kDa, indicating that PAL from bamboo also exists as a tetramer. The optimum temperature for PAL activity was 50°C and the optimal pH 9.0. The identity of the purified bamboo shell PAL was confirmed using Q-TOF tandem MS/MS de novo sequencing. Four PAL genes, designated as BoPAL1 to BoPAL4, were cloned from B. oldhamii. The open reading frames of BoPAL3 and BoPAL4 were 2142 and 2106 bp in size, respectively: BoPAL2-4 contained one intron and two exons, but no intron was found in BoPAL1. BoPAL4 expressed in Escherichia coli possessed both PAL and tyrosine ammonia-lyase activities. While recombinant wild-type PAL proteins had similar biochemical properties to the native bamboo shell PAL, both site-directed mutagenesis of BoPAL1 F133H and BoPAL2 F134H, respectively, showed decreased k(cat)/K(m) values toward L-Phe, whereas BoPAL2 F134H showed a slightly increased k(cat)/K(m) value toward L-Tyr. These data suggest other residues largely control Phe/Tyr substrate specificity. An antibody raised against the purified shell PAL was generated for histochemical studies. In bamboo shell and branch shoots, PAL was localized primarily in sclerenchyma cells.Phytochemistry 10/2010; 71(17-18):1999-2009. DOI:10.1016/j.phytochem.2010.09.019 · 3.35 Impact Factor
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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.