Phenylalanine Ammonia Lyase Production Research Articles

Potential of Beauveria bassiana formulations to control Thaumastocoris peregrinus (Hemiptera: Thaumastocoridae)

This study evaluated the potential of four formulations of the entomopathogenic fungus Beauveria bassiana for the control of Thaumastocoris peregrinus. For this, the leaves of Eucalyptus dunni were immersed in each delivery of B. bassiana, isolated IBCB 66, and also in sterile distilled water containing Tween 80® (0.01%) (control), totaling five treatments, with five repetitions each. These leaves were placed in germination boxes containing 10 insects in each and, subsequently, kept in an air-conditioned room (27 ± 2 ° C, 60 ± 10% RH, and 12-h photophase). Record longevity for seven days. The experiment was carried out with adults and also with 3rd instar nymphs of T. peregrinus. These formulations were tested for resistance induction, using organic soybean seeds (standard) sown in sand. Ten days after emergence, 40 µL of treatment suspension was competent in 2 mm cuts made on the top of the cotyledon. Phytoalexins and phenylalanine ammonia lyase (PAL) were determined. All formulations caused a reduction in the longevity of T. peregrinus when compared to the control, standing out as formulations T1 and T2, both for adults and for 3rd instar nymphs of T. peregrinus. The T2 expansion also promotes an increase in the production of phytoalexins and PAL. Thus, the IBCB 66 isolate from B. bassiana, supplied T1, has the potential to control nymphs and adults of T. peregrinus and is also a potential resistance inducer.

Pseudomonas taiwanensis (MTCC11631) mediated induction of systemic resistance in Anthurium andreanum L against blight disease and visualisation of defence related secondary metabolites using confocal laser scanning microscopy

Abstract An attempt to evaluate induced systemic resistance in anthurium against blight disease caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad) was studied. Plant associated Pseudomonas taiwanensis and elicitor compounds were used for plant application in greenhouse experiments to evaluate the resistance development against anthurium blight. Application of rhizobacteria P1 and P9 resulted in significant disease reduction to 80.0% and 85.0% respectively. Application of salicylic acid, mancozeb and K2HPO4 also resulted in reduction of disease at 35 days after inoculation. Elevated production of defence related enzymes, proteins and phenols were observed. Application of rhizobacteria P1 resulted in highest production of polyphenol oxidase and phenolic compounds. Production of phenylalanine ammonia lyase was maximum in mancozeb treated plants compared to other treatments. Maximum production of peroxidase enzyme was found in K2HPO4 treated plants whereas application of elicitor compound salicylic acid caused maximum production of defence related proteins compared to other treatments. Confocal microscopic studies revealed that P1 and P9 treated plant petiole showed increased auto fluorescent emissions indicating the distribution of defence related secondary metabolites and phenolic compounds as compared to control plants. Root colonization assay displayed significant results implicating that Rif+ and Cyclo+ resistant rhizobacterial isolates could successfully grow in the root tissue, establishing endophytic association within the host tissue.

Purification and Characterization of Anabaena flos-aquae Phenylalanine Ammonia-Lyase as a Novel Approach for Myristicin Biotransformation.

Phenylalanine ammonia-lyase (PAL) catalyzes the reversible deamination of phenylalanine to cinnamic acid and ammonia. Algae have been considered as biofactories for PAL production, however, biochemical characterization of PAL and its potency for myristicin biotransformation into MMDA (3-methoxy-4, 5-methylenedioxyamphetamine) has not been studied yet. Thus, PAL from Anabaena flos-aquae and Spirulina platensis has been purified, comparatively characterized and its affinity to transform myristicin was assessed. The specific activity of purified PAL from S. platensis (73.9 μmol/mg/min) and A. flos-aquae (30.5 μmol/mg/min) was increased by about 2.9 and 2.4 folds by gel-filtration comparing to their corresponding crude enzymes. Under denaturing-PAGE, a single proteineous band with a molecular mass of 64 kDa appeared for A. flos-aquae and S. platensis PAL. The biochemical properties of the purified PAL from both algal isolates were determined comparatively. The optimum temperature of S. platensis and A. flos-aquae PAL for forward or reverse activity was reported at 30°C, while the optimum pH for PAL enzyme isolated from A. flos-aquae was 8.9 for forward and reverse activities, and S. platensis PAL had maximum activities at pH 8.9 and 8 for forward and reverse reactions, respectively. Luckily, the purified PALs have the affinity to hydroaminate the myristicin to MMDA successfully in one step. Furthermore, a successful method for synthesis of MMDA from myristicin in two steps was also established. Gas chromatography-mass spectrometry (GC-MS) analysis was conducted to track the product formation.

Effect of l-phenylalanine on PAL activity and production of naphthoquinone pigments in suspension cultures of Arnebia euchroma (Royle) Johnst

The effects of l-phenylalanine (PHE) on cell growth and production of shikonin and its derivatives, acetylshikonin (ACS) and isobutyrylshikonin (IBS), in suspension cultures of Arnebia euchroma were examined. Supplementing media using PHE have been successfully utilized to enhance shikonin production in cell cultures of other species of Boraginaceae. l-Phenylalanine, the key compound in the phenylpropanoid pathway, is converted by phenylalanine ammonia lyase (PAL) to trans-cinnamic acid, which is the precursor of p-hydroxybenzoic acid (PHB). Coupling of PHB and geranyl pyrophosphate (derived from mevalonate pathway) by p-hydroxybenzoate-m-geranyltransferase leads later to biosynthesis of shikonins. The addition of 0.01 or 0.1 mM PHE to the culture medium stimulated cell proliferation, where the highest observed increase in fresh cell biomass (measured as a ratio of final weight to initial weight) was 12-fold, in contrast to an eightfold increase in control cultures. Whereas, growth media supplemented with 1 mM PHE markedly reduced the rate of cell growth (to only twofold). Precursor feeding had detrimental effects on both ACS and IBS production in all PHE-supplemented media. The highest total content (intracellular + extracellular) of the investigated red pigments (9.5 mg per flask) was detected in the control culture without PHE. ACS was the major component of the naphthoquinone fraction determined in cells and post-culture media. Shikonin itself was found only in the post-culture media from cultures supplemented with 0.01 or 0.1 mM PHE. Increases in PAL activity corresponded well with the accumulation of investigated naphthoquinones in control culture. However, peak PAL activity did not directly correlate with maximum production of shikonin derivatives. Cytotoxicity of extracts, prepared from the cells cultivated in the presence of PHE or in control cultures, was tested on three cancer cell lines: HL-60, HeLa, and MCF-7. The extracts prepared from the untreated control cultures proved to be the most potent against the examined cancer cell lines. The mean inhibitory concentration values were 0.3, 13, and 8 μg ml−1 for the HL-60, HeLa, and MCF-7 cells, respectively.

Impact of soil field water capacity on secondary metabolites, phenylalanine ammonia-lyase (PAL), maliondialdehyde (MDA) and photosynthetic responses of Malaysian kacip fatimah (Labisia pumila Benth).

A randomized complete block design 2 × 4 experiment was designed and conducted for 15 weeks to characterize the relationships between production of total phenolics, flavonoid, anthocyanin, leaf gas exchange, total chlorophyll, phenylalanine ammonia-lyase (PAL) and malondialdehyde (MDA) activity in two varieties of Labisia pumila Benth, namely the var. alata and pumila, under four levels of evapotranspiration replacement (ER) (100%; well watered), (75%, moderate water stress), (50%; high water stress) and (25%; severe water stress). The production of total phenolics, flavonoids, anthocyanin, soluble sugar and relative leaf water content was affected by the interaction between varieties and SWC. As the ER levels decreased from 100% to 25%, the production of PAL and MDA activity increased steadily. At the highest (100%) ER L. pumila exhibited significantly higher net photosynthesis, apparent quantum yield, maximum efficiency of photosystem II (fv/fm) and lower dark respiration rates compared to the other treatment. The production of total phenolics, flavonoids and anthocyanin was also found to be higher under high water stress (50% ER replacement) compared to severe water stress (25% ER). From this study, it was observed that as net photosynthesis, apparent quantum yield and chlorophyll content were downregulated under high water stress the production of total phenolics, flavonoids and anthocyanin were upregulated implying that the imposition of high water stress can enhance the medicinal properties of L. pumila Benth.

Enhancement of Phenylalanine Ammonia Lyase Production from Rhodotorula Mucilaginosa by Optimization of Culture Conditions in Batch and Fed-Batch

ABSTRACTThe fermentation of phenylalanine ammonia lyase (PAL) by Rhodotorula mucilaginosa (R. mucilaginosa) was optimized. PAL production was enhanced 8.0-fold from an initial activity of 5 U/g (DCW) with the incorporation of optimization of medium composition, controlled-pH operation, the strategy of batch supplementation with substrate induction and other culture conditions, and the implementation of a fed-batch process. A significant influence of peptone and inoculum size on PAL production was evaluated by Plackett—Burman design and the path of steepest ascent experiment. Among the various amino acids used, it was found that L-Phenylalanine could efficiently induce the biosynthesis of PAL. PAL activity was increased by 18.5% in comparison with the control. Glucose concentration significantly affected PAL synthesis. When the glucose concentration was 1 g/L, PAL activity was enhanced by 1.82- and 6-fold in comparison with those at 5 g/L and 10 g/L, respectively. To further increase PAL production, controlled-pH operation and the strategy of batch supplementation with the inducer was performed in a 5 L stirred tank reactor. The maximum production of PAL reached 41 U/g (DCW).

Defence responses in leaves of resistant and susceptible pepper ( Capsicum annuum L.) cultivars infected with different inoculum concentrations of Phytophthora capsici Leon

Three pepper cultivars (PM-702: resistant, Demre-8 and KM-hot: susceptible) with different resistances to Phytophthora capsici-22 ( P. capsici-22) were inoculated with different concentrations of zoospores to analyze the time course of phenylalanine ammonia lyase (PAL) activity, malondialdehyde (MDA), hydrogen peroxide (H 2O 2), proline and total protein. Samples were collected from the leaves of three pepper cultivars on the 2nd, 4th, and 6th days after infection; important differences were observed in PAL activity, MDA amount, H 2O 2, proline, and total protein with respect to zoospore concentration, infection time, and type of pepper cultivars. Activity of phenylalanine ammonia-lyase (PAL), the first enzyme in the phenylpropanoid biosynthetic pathway, was increased in inoculated leaves of all three cultivars. The increase in production of PAL upon challenge was higher in the infected plants compared to the non-infected (control) plants. An early induction of PAL was observed in the leaves of all peppers infected with P. capsici within two days, making it one of the detected earlier responses to infection. Moreover, pathogen stress increased the rate of lipid peroxidation (which is indicated by increasing MDA content). Production of reactive oxygen species, H 2O 2, and lipid peroxidation increased in pepper leaves by increasing time of infection and inoculum concentration. The findings suggested that PM-702 pepper cultivar was inherently better protected against lipid peroxidation under P. capsici stress than KM-hot and Demre-8. In addition, it was observed that proline content in the leaves of infected susceptible cultivars increased with time, but highest proline and total protein in non-infected (control) leaves were recorded in resistant cultivar.

The Ve-mediated resistance response of the tomato to Verticillium dahliae involves H2O2, peroxidase and lignins and drives PAL gene expression.

BackgroundVerticillium dahliae is a fungal pathogen that infects a wide range of hosts. The only known genes for resistance to Verticillium in the Solanaceae are found in the tomato (Solanum lycopersicum) Ve locus, formed by two linked genes, Ve1 and Ve2. To characterize the resistance response mediated by the tomato Ve gene, we inoculated two nearly isogenic tomato lines, LA3030 (ve/ve) and LA3038 (Ve/Ve), with V. dahliae.ResultsWe found induction of H2O2 production in roots of inoculated plants, followed by an increase in peroxidase activity only in roots of inoculated resistant plants. Phenylalanine-ammonia lyase (PAL) activity was also increased in resistant roots 2 hours after inoculation, while induction of PAL activity in susceptible roots was not seen until 48 hours after inoculation. Phenylpropanoid metabolism was also affected, with increases in ferulic acid, p-coumaric acid, vanillin and p-hydroxybenzaldehyde contents in resistant roots after inoculation. Six tomato PAL cDNA sequences (PAL1 - PAL6) were found in the SolGenes tomato EST database. RT-PCR analysis showed that these genes were expressed in all organs of the plant, albeit at different levels. Real-time RT-PCR indicated distinct patterns of expression of the different PAL genes in V. dahliae-inoculated roots. Phylogenetic analysis of 48 partial PAL cDNAs corresponding to 19 plant species grouped angiosperm PAL sequences into four clusters, suggesting functional differences among the six tomato genes, with PAL2 and PAL6 presumably involved in lignification, and the remaining PAL genes implicated in other biological processes.An increase in the synthesis of lignins was found 16 and 28 days after inoculation in both lines; this increase was greater and faster to develop in the resistant line. In both resistant and susceptible inoculated plants, an increase in the ratio of guaiacyl/syringyl units was detected 16 days after inoculation, resulting from the lowered amount of syringyl units in the lignins of inoculated plants.ConclusionsThe interaction between the tomato and V. dahliae triggered a number of short- and long-term defensive mechanisms. Differences were found between compatible and incompatible interactions, including onset of H2O2 production and activities of peroxidase and PAL, and phenylpropanoid metabolism and synthesis of lignins.

Optimization of medium for phenylalanine ammonia lyase production in E. coli using response surface methodology

A culture medium for phenylalanine ammonia lyase (PAL) production in E. coli was developed following preliminary studies by means of response surface methodology (RSM). The medium components having significant effect on the production were first identified by using a fractional factorial design. Then, central composite design (CCD) was used to optimize the medium constituents and explain the combined effects of four medium constituents: glucose, yeast extract, (NH4)2HPO4 and MgSO4. A quadratic model was found to fit the PAL production. CCD revealed that the optimum values of the test variables for PAL production were glucose 28.2 g/L, yeast extract 5.01 g/L, (NH4)2HPO4 7.02 g/L and MgSO4 1.5 g/L. PAL production of 62.85 U/g, which was in agreement with the prediction, was observed in the verification experiment. In comparison to the production of basal medium, 1.8-fold increase was obtained.

Optimal culture condition for the production of phenyalanine ammonia lyase from E. coli

The effects of culturing conditions on phenylalanine ammonia lyase production by a recombinant E. coli strain were investigated by using a controlled fed-batch fermentation system. In a 5 L fermentor, the optimal com- position of the batch medium was 2% glucose, 1% yeast extract, 0.7% K2HPO4, 0.8% KH2PO4, 0.5% (NH4)2SO4, 0.1% MgSO4·7H2O. The optimal feed glucose solution was 50%. Glucose concentration and pH of the culture broth were maintained at about 2.0 g/L and 7.0 during the fed-batch phase, respectively. Following 24-h cultivation, 0.2 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG) was added and temperature was shifted from 37 o C to 42 o C to induce pal gene expression. Under optimal conditions, a high productivity of 300 U/g could be achieved after 48 h culture, and a cell density of OD600 about 82 was obtained at 52 h culture at 500 r/m stirrer speed and 1 vvm, respectively.

A horizontal gene transfer at the origin of phenylpropanoid metabolism: a key adaptation of plants to land.

BackgroundThe pioneering ancestor of land plants that conquered terrestrial habitats around 500 million years ago had to face dramatic stresses including UV radiation, desiccation, and microbial attack. This drove a number of adaptations, among which the emergence of the phenylpropanoid pathway was crucial, leading to essential compounds such as flavonoids and lignin. However, the origin of this specific land plant secondary metabolism has not been clarified.ResultsWe have performed an extensive analysis of the taxonomic distribution and phylogeny of Phenylalanine Ammonia Lyase (PAL), which catalyses the first and essential step of the general phenylpropanoid pathway, leading from phenylalanine to p-Coumaric acid and p-Coumaroyl-CoA, the entry points of the flavonoids and lignin routes. We obtained robust evidence that the ancestor of land plants acquired a PAL via horizontal gene transfer (HGT) during symbioses with soil bacteria and fungi that are known to have established very early during the first steps of land colonization. This horizontally acquired PAL represented then the basis for further development of the phenylpropanoid pathway and plant radiation on terrestrial environments.ConclusionOur results highlight a possible crucial role of HGT from soil bacteria in the path leading to land colonization by plants and their subsequent evolution. The few functional characterizations of sediment/soil bacterial PAL (production of secondary metabolites with powerful antimicrobial activity or production of pigments) suggest that the initial advantage of this horizontally acquired PAL in the ancestor of land plants might have been either defense against an already developed microbial community and/or protection against UV.ReviewersThis article was reviewed by Purificación López-García, Janet Siefert, and Eugene Koonin.

The effect of ethylene on phenylalanine ammonia lyase (PAL) induction by a fungal elicitor in Phaseolus vulgaris

The effects of ethylene on the induction of phenylalanine ammonia-lyase (PAL) by cell wall elicitors of Colletotrichum lindemuthianum was investigated in leaves of susceptible and resistant cultivars of Phaseolus vulgaris (cultivars Pinto and Kievit respectively). Induction of PAL by exogenously supplied ethylene and associated test substances alone, without elicitor, was negligible. With elicitor alone, cv. Kievit responded to lower concentrations than cv Pinto. Exogenously supplied ethylene augmented the response of cv. Kievit only in the presence of limiting concentrations of elicitor. When concentrations of elicitor giving maximum PAL induction were used, ethylene was without effect in cv. Kievit also. Aminoethoxyvinylglycine (AVG) partially inhibited the induction of PAL in cv. Kievit and this inhibition could be reversed by aminocyclopropane-1-carboxylic acid (ACC) and ethylene. However, since cv. Pinto did not respond to ethylene, the application of AVG in the presence of elicitor was, as expected, without effect. Unless introduced within 2 h of elicitor application, ethylene was without effect on PAL production in cv. Kievit also; likewise, the inhibitory effect of AVG was minimal if not injected within the same time period following elicitor. Elicitor rapidly induced the synthesis of ACC during this 2 h period leading to the production of stress ethylene and this was completely inhibited by AVG and completely reversed by ACC. The effect of the elicitor, after injection into the leaf of cv. Kievit, was to change both the distribution and relative concentrations of the soluble proteins. Ethylene enhanced this effect and the 40 % increase in PAL activity observed in the presence of the hormone can largely be accounted for by the increase in the concentration of the enzyme. Changes brought about by the elicitor were eliminated by inhibitors of protein and nucleic acid synthesis, the effects of which could not be reversed by ethylene. These results are discussed in relation to other studies. It has been proposed that elicitors induce the biosynthesis of ethylene and PAL independently. Ethylene is involved in the activation of the induced response leading to the synthesis of PAL, and thereby phytoalexins, particularly when the response to elicitor is sub-optimal.

Interaction of cowpea with Phytophthora vignae: inheritance of resistance and production of phenylalanine ammonia-lyase as a resistance response

The interactions between Phytophthora vignae and two cowpea cultivars are described. The cultivars are near-isogenic but differ in their resistance to P. vignae race 2. Cultivar Caloona is resistant, cv. Poona is susceptible. Resistance of cv. Caloona to race 2 is controlled by a single dominant gene and is expressed throughout the life of the plant in all tissues at temperatures up to 35°C. Inoculation of cut cowpea epicotyls with a plug of agar containing race 2 hyphae provides a reliable method of obtaining host tissue which responds in a predictable way to the pathogen. Phenylalanine ammonia-lyase (PAL) activity increases rapidly in invaded epicotyl tissue of cv. Caloona in response to inoculation with race 2. Increased PAL activity in advance of the hyphae is not detected. Low levels of PAL are produced by cv. Poona and cv. Caloona in response to inoculation with a race virulent on both cultivars (race 3). Application of l -2-aminooxy-3-phenylpropionic acid (1 m m ) or aminooxy acetic acid (10 m m ) through the cut bases of hypocotyls of cv. Caloona seedlings results in this cultivar, normally resistant to P. vignae race 2, becoming susceptible.

Inhibition of mRNA levels and activities by trans-cinnamic acid in elicitor-induced bean cells

Addition of trans-cinnamic acid to bean cell suspension cultures after their treatment with fungal elicitor resulted in the loss of induced phenylalanine ammonia-lyase (PAL) enzyme activity. In contrast elicitor-induced chalcone synthase (CHS) activity was arrested but did not decline, whereas chalcone isomerase (CHI) activity was relatively unaffected. However, translational activities of extracted polysomal mRNAs encoding these three enzymes were depressed by cinnamate treatments as were levels of PAL and CHS polysomal mRNAs and rates of transcription of these genes measured in isolated nuclei. Treatment of elicitor-induced cultures with l-α-aminooxy-phenylpropionic acid (AOPP), a potent inhibitor of PAL activity (and therefore cinnamate production) in vivo, resulted in increased production of PAL and CHS mRNAs. Addition of cinnamate to elicitor-treated cultures inhibited the appearance of a number of polypeptides translated in vitro from polysomal mRNA, although at least nine polypeptides were specifically induced by cinnamate treatment. We conclude that cinnamic acid potentially could act as an in vivo modulator of the synthesis of phenylpropanoid pathway enzymes although it is not yet fully possible to rule out less specific inhibitory effects.

Production of phenylalanine ammonia-lyase (PAL): isolation and evaluation of yeast strains suitable for commercial production of l-phenylalanine

Phenylalanine Ammonia-Lyase (PAL) containing microorganisms were isolated from a wide variety of natural habitats. The best 21 strains to emerge from the primary screen were screened for PAL activities in both directions using l-phenylalanine and t-cinnamate substrates. Twelve of the latter strains were compared for total cell production and PAL activity and 7 isolates were chosen for examination of the extent of PAL induction in various media. On the basis of these screens, isolate SPA 10 (identified as Rhodotorula rubra) was selected for further optimization. Growth was optimal at 28° C and pH 5.0, although cellular PAL activity was shown to be higher at sub-optimal temperatures (36° C) and pH (8.0) for growth. Synthesis of PAL was repressed when grown in the presence of various sugars and NH 4 + ions. Manipulation of fermentation conditions enabled PAL synthesis to occur at maximum biomass levels, upon glucose exhaustion. PAL was rapidly inactivated within cells shortly after maximum synthesis was attained: feeding of d,l-isoleucine and low concentrations of d,l-phenylalanine, and shifting of fermentation temperature conferred catalyst stability for fermentations over 100 h. These results demonstrate the suitability and superiority of isolate SPA 10 for the commercial production of l-phenylalanine from trans-cinnamic acid.

The biosynthesis of benzoic acid in Bramley's Seedling apples infected by Nectria galligena Bres

Radioactive benzoic acid was synthesized from glucose- 14C and l-phenylalanine- 14C in apples infected with Nectria galligena and in apples infused with protease extracted from tissue rotted by N. galligena. Phenylalanine ammonia-lyase (PAL) activity was rapidly induced in apples infused with the fungal protease but a lag phase occurred following inoculation with the pathogen. Production of benzoic acid was maximal approximately 3 days after maximum PAL production. Enzymes extracted from necrotic apple tissue converted phenylalanine to cinnamic and benzoic acids. P. expansum, a non-protease producing apple pathogen, did not induce either PAL activity or elicit benzoic acid accumulation in apples.

The significance of carbohydrate metabolism in flavonoid synthesis in strawberry leaf disks

The production of phenylalanine ammonia-lyase and flavonoids in illuminated strawberry leaf disks was dependent on a supply of carbon dioxide, while in darkness carbon dioxide had no effect on the small rates of production. An external supply of sucrose could remove the carbon dioxide dependency of flavonoid and phenylalanine ammonia-lyase production and a supply of carbon dioxide could remove the stimulation of phenylalanione ammonia-lyase and flavonoid production brought about by external sucrose. The uptake and metabolism of external sucrose by leaf disks was stimulated by light and the light-stimulated uptake was inhibited by DCMU. The synthesis of flavonoids and phenylalanine ammonia-lyase were inhibited by DCMU probably through its inhibition of CO 2 fixation or sucrose uptake. Photosynthesis and carbohydrate metabolism were found to be of extreme importance in controlling the synthesis of flavonoids in leaf disks.