Purification of phenylalanine ammonia lyase from Rhodotorula glutinis

D’Cunha, Godwin B.; Satyanarayan, Vaduvatha; Nair, P.M.

doi:10.1016/0031-9422(95)00914-0

Cited by 60 publications

(25 citation statements)

References 23 publications

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“…The reactions are performed in 150 μl reaction mixtures with 6 μg recombinant NnPAL1, 15 mM L-phenylalanine and 50 mM Tris–HCl (pH 8.5), and are terminated with the addition of concentrated HCl [57]. …”

Section: Methodsmentioning

confidence: 99%

Molecular evolution and functional characterisation of an ancient phenylalanine ammonia-lyase gene (NnPAL1) from Nelumbo nucifera: novel insight into the evolution of the PAL family in angiosperms

Gui

Wang

et al. 2014

BMC Evol Biol

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BackgroundPhenylalanine ammonia-lyase (PAL; E.C.4.3.1.5) is a key enzyme of the phenylpropanoid pathway in plant development, and it catalyses the deamination of phenylalanine to trans-cinnamic acid, leading to the production of secondary metabolites. This enzyme has been identified in many organisms, ranging from prokaryotes to higher plants. Because Nelumbo nucifera is a basal dicot rich in many secondary metabolites, it is a suitable candidate for research on the phenylpropanoid pathway.ResultsThree PAL members, NnPAL1, NnPAL2 and NnPAL3, have been identified in N. nucifera using genome-wide analysis. NnPAL1 contains two introns; however, both NnPAL2 and NnPAL3 have only one intron. Molecular and evolutionary analysis of NnPAL1 confirms that it is an ancient PAL member of the angiosperms and may have a different origin. However, PAL clusters, except NnPAL1, are monophyletic after the split between dicots and monocots. These observations suggest that duplication events remain an important occurrence in the evolution of the PAL gene family. Molecular assays demonstrate that the mRNA of the NnPAL1 gene is 2343 bp in size and encodes a 717 amino acid polypeptide. The optimal pH and temperature of the recombinant NnPAL1 protein are 9.0 and 55°C, respectively. The NnPAL1 protein retains both PAL and weak TAL catalytic activities with Km values of 1.07 mM for L-phenylalanine and 3.43 mM for L-tyrosine, respectively. Cis-elements response to environmental stress are identified and confirmed using real-time PCR for treatments with abscisic acid (ABA), indoleacetic acid (IAA), ultraviolet light, Neurospora crassa (fungi) and drought.ConclusionsWe conclude that the angiosperm PAL genes are not derived from a single gene in an ancestral angiosperm genome; therefore, there may be another ancestral duplication and vertical inheritance from the gymnosperms. The different evolutionary histories for PAL genes in angiosperms suggest different mechanisms of functional regulation. The expression patterns of NnPAL1 in response to stress may be necessary for the survival of N. nucifera since the Cretaceous Period. The discovery and characterisation of the ancient NnPAL1 help to elucidate PAL evolution in angiosperms.

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Section: Methodsmentioning

confidence: 99%

Molecular evolution and functional characterisation of an ancient phenylalanine ammonia-lyase gene (NnPAL1) from Nelumbo nucifera: novel insight into the evolution of the PAL family in angiosperms

Gui

Wang

et al. 2014

BMC Evol Biol

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“…The crude enzyme was obtained by centrifugation. PAL activity was measured by the increase in the absorbance at 290 nm (D'Cunha et al, 1996). The assays were performed in 3 ml reaction solution containing 50 mm Tris-HCl buffer (pH 8.8), 20 mm l-phe and 50 μl enzyme solution.…”

Section: Enzyme Extraction and Pal Activity Assaymentioning

confidence: 99%

Changes of Antioxidant Enzyme and Phenylalanine Ammonia-Lyase Activities during Chimonanthus praecox Seed Maturation

Gao

2008

Zeitschrift Für Naturforschung C

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Changes in peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) and phenylalanine ammonia-lyase (PAL) activities were studied during Chimonanthus praecox seed maturation. According to our findings the protein content increased steadily from 8 to 12 weeks after flowering, and thereafter decreased significantly. Similarly, SOD and POD activities increased gradually up to 12 weeks after flowering and then declined. PAL activity declined gradually during seed maturation. CAT activity, however, showed no changes during seed maturation. By means of polyacrylamide gel electrophoresis (PAGE), SOD and POD isoenzymes were observed during seed maturation. The staining intensities of SOD and POD isoenzymes correlated well with SOD and POD activities as obtained by an assay in solution. These findings suggest that POD, SOD and PAL may be involved in the growth and development during Chimonanthus praecox seed maturation.

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“…The resulted RNA was then separated on denaturing agarose gels and transferred onto nylon membranes (GE Healthcare, Piscataway, NJ, USA) and hybridized to radioactive DNA probes as described previously [19]. Determination of enzyme activities PAL activity was assayed using the method as described previously [21] with slight modification. Briefly, the assay was carried out in a reaction mixture containing 0.1 M Tris-HCl buffer (pH 8.5), 1 mM 2-mercaptoethanol, 50 mM phenylalanine, and 1 ml aliquot of enzyme.…”

Section: Northern Blot Analysismentioning

confidence: 99%

Regulation of polyphenols accumulation by combined overexpression/silencing key enzymes of phyenylpropanoid pathway

Chang¹,

Luo²,

He³

2009

ABBS

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There is a growing interest in the metabolic engineering of plant with increased desirable polyphenols such as chlorogenic acid (CGA) and rutin. In this study, the effects of overexpression of both phenylalanine ammonia lyase (AtPAL2), the first enzyme of the phenylpropanoid pathway, and hydroxycinnamoyl-CoA quinate:hydroxycinnamoyl transferase (NtHQT), the last enzyme of CGA biosynthesis, and the overexpression of AtPAL2 together with silencing of NtHQT were investigated in tobacco. Transgenic tobacco plants overexpressing AtPAL2 showed two and five times increases of CGA and rutin levels than the wild-type (WT) plants, respectively. Overexpression of NtHQT further increases the accumulation of CGA in the AtPAL2 plants to about three times than that of the WT level, while silencing of NtHQT in AtPAL2 plants results in 12 times increase in rutin level than that of the WT plants. Simultaneous overexpression of phenylalanine ammonia lyase (PAL) and overexpression/silencing HQT could be used for the production of functional food with increased polyphenols.

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Purification of phenylalanine ammonia lyase from Rhodotorula glutinis

Cited by 60 publications

References 23 publications

Molecular evolution and functional characterisation of an ancient phenylalanine ammonia-lyase gene (NnPAL1) from Nelumbo nucifera: novel insight into the evolution of the PAL family in angiosperms

Molecular evolution and functional characterisation of an ancient phenylalanine ammonia-lyase gene (NnPAL1) from Nelumbo nucifera: novel insight into the evolution of the PAL family in angiosperms

Changes of Antioxidant Enzyme and Phenylalanine Ammonia-Lyase Activities during Chimonanthus praecox Seed Maturation

Regulation of polyphenols accumulation by combined overexpression/silencing key enzymes of phyenylpropanoid pathway

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