Synthesis Of Phenylalanine Ammonia-lyase Research Articles

Integrated full-length transcriptome and metabolome analysis of the early defense mechanisms in Paulownia witches’ broom disease

ABSTRACT Paulownia witches’ broom (PaWB) is a devastating disease of Paulownia spp. caused by phytoplasma infection. To reveal the reason for the absence of morphological changes at the early stage of phytoplasma infection, in this study, a combination of second-and third-generation sequencing (single-molecule real-time long-read isoform) and liquid chromatography tandem mass spectrometry were performed to analyse the changes of full-length transcripts and metabolites at the early stage of phytoplasma infection. A total of 140,528 non-redundant full-length transcripts, 597 transcription factors, 1,658 long non-coding RNAs, and 645 metabolites were identified. The third-generation sequencing improved the annotation of the reference Paulownia fortunei (Seem.) Hemsl. genome by 8.86%. Combination analysis of the full-length transcriptome and metabolome revealed that phenolics and phenylalanine ammonia-lyase synthesis related genes were significantly up-regulated in the early stage of phytoplasma-infected P. fortunei. Based on the results of full-length transcriptome and metabolome integrative analysis, we constructed an early defence model diagram of PaWB disease. Our results provide powerful insights into the reason for absence of morphological at the early stage of phytoplasma infection.

A kinetic model for flavonoid production in tea cell culture.

As one of the strategies for efficient production of a metabolite from cell cultures, a kinetic model is very useful tool to predict productivity under various culture conditions. In this study, we propose a kinetic model for flavonoid production in tea cell culture based on the cell life cycle and expression of PAL, the gene encoding phenylalanine ammonia-lyase (PAL)-the key enzyme in flavonoid biosynthesis. The flavonoid production rate was considered to be related to the amount of active PAL. Synthesis of PAL was modelled based on a general gene expression/translation mechanism, including the transcription of DNA encoding PAL into mRNA and the translation of PAL mRNA into the PAL protein. The transcription of DNA was assumed to be promoted at high light intensity and suppressed by a feedback regulatory mechanism at high flavonoid concentrations. In the model, mRNA and PAL were considered to self-decompose and to be lost by cell rupture. The model constants were estimated by fitting the experimental results obtained from tea cell cultures under various light intensities. The model accurately described the kinetic behaviors of dry and fresh cell concentrations, glucose concentration, cell viability, PAL specific activity, and flavonoid content under a wide range of light intensities. The model simulated flavonoid productivity per medium under various culture conditions. Therefore, this model will be useful to predict optimum culture conditions for maximum flavonoid productivity in cultured tea cells.

Salicylic acid and methyl jasmonate improve chilling tolerance in cold-stored lemon fruit (Citrus limon)

Chilling injury (CI) is associated with the degradation of membrane integrity which can be aligned to phenolic oxidation activated by polyphenol oxidase (PPO) and peroxidase (POD), enzymes responsible for tissue browning. Phenylalanine ammonia-lyase (PAL) is a further enzyme prominent in the phenolic metabolism that is involved in acclimation against chilling stress. It was hypothesized that treatment with methyl jasmonate (MJ) and salicylic acid (SA) may enhance chilling tolerance in lemon fruit by increasing the synthesis of total phenolics and PAL by activating the key enzyme regulating the shikimic acid pathway whilst inhibiting the activity of POD and PPO. Lemon fruit were treated with 10μM MJ, 2mM SA or 10μM MJ plus 2mM SA, waxed, stored at −0.5, 2 or 4.5°C for up to 28 days plus 7 days at 23°C. Membrane integrity was studied by investigating membrane permeability and the degree of membrane lipid peroxidation in lemon flavedo following cold storage. The 10μM MJ plus 2mM SA treatment was most effective in enhancing chilling tolerance of lemon fruit, significantly reducing chilling-induced membrane permeability and membrane lipid peroxidation of lemon flavedo tissue. This treatment also increased total phenolics and PAL activity in such tissue while inhibiting POD activity, the latter possibly contributing to the delay of CI manifestation. PPO activity was found to be a poor biochemical marker of CI. Treatment with 10μM MJ plus 2mM SA resulted in an alteration of the phenolic metabolism, enhancing chilling tolerance, possibly through increased production of total phenolics and the activation of PAL and inhibition of POD.

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).

Differential proteomic analysis of developmental stages of Acca sellowiana somatic embryos

Feijoa (Acca sellowiana, Myrtaceae), a native fruit species from southern Brazil and northern Uruguay, is considered to constitute a reference system for somatic embryogenesis in woody dicots. This in vitro regenerative pathway is an efficient micropropagation method, and a suitable model system for studies in plant developmental physiology. This study attempts to detect and identify proteins that are expressed during the different developmental stages of somatic embryos of A. sellowiana. Using high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE), a high degree of similarity between protein profiles of the assayed somatic embryos was observed. Of the 74 different protein spots extracted for analysis, 60 were identified by means of 2-DE/MALDI-TOF/MS. Twelve proteins were expressed in all the assayed stages. Ten proteins were expressed in the initial stages and 22 proteins were expressed in the mature developmental stages of somatic embryos. Only one protein was expressed exclusively in the torpedo stage, whereas four were expressed in the pre-cotyledonary, and none in the cotyledonary stage. The proteins identified were involved in the synthesis of phenylalanine ammonia-lyase, a conspicuous polyphenol present in the induction of feijoa embryogenic cultures. The presence of essential proteins of nitrogen metabolism, such as the cytosolic glutamine synthetase protein, was also observed. The physiological implications of these findings are discussed.

Controlled atmosphere and heat shock affect PAL1 and HSP90 mRNA accumulation in fresh-cut endive (Cichorium intybus L.)

Stored fresh-cut endive gradually turns red, since tissue wounding induces de novo synthesis of phenylalanine ammonia-lyase (PAL), which activates the phenylpropanoid pathway. Once the pool of phenolic substrates is increased, polyphenols are oxidized and cause discoloration. Modified atmosphere packaging (MAP) and controlled atmosphere storage (CA) have been used to control browning. This paper describes the combined effects of heat shock (46 °C/120 s) and CA on the quality of cut endive stored at 10 °C. Heat shock protein (HSP90) and PAL expression patterns were analysed in endive sliced tissues. Real-time quantitative PCR (qPCR) was performed for PAL1 and PAL2 as well as for degenerated HSP90 primers. PAL1 and PAL2 were strongly expressed in unheated cut tissues, in which maximum accumulation of PAL1 mRNA occurred 12 h after cutting. Heat shock significantly reduced the level of PAL1 transcripts in sliced endive, which was related to the prevention of discoloration. Controlled atmosphere storage had an effect on PAL1 but not on PAL2 transcription. Peak levels of HSP90 transcript accumulation were found after 6 h, with an increasing and apparently cumulative effect of wounding and CA. Image analysis of the slices after 48 h clearly showed that heat-treated samples were similar to the control, with no significant difference between air and CA storage. These results show that there could be inhibition of PAL1 isoform expression in endive slices after heating.

Differential methods of inoculation of plant growth-promoting rhizobacteria induce synthesis of phenylalanine ammonia-lyase and phenolic compounds differentially in chickpca

Foliar spray and micro-injection of plant growth-promoting rhizobacterial species, viz. Pseudomonas fluorescens and P. aeruginosa on chickpea induced synthesis of phenylalanine ammonia-lyase (PAL) when tested against Sclerotinia sclerotiorum. Induction of PAL was also associated with increased synthesis of phenolic compounds such as tannic, gallic, caffeic, chlorogenic and cinnamic acids. Treatment with P. fluorescens was found to be more effective in inducing phenolic compounds as compared to P. aeruginosa. However, persistence of PAL activity was observed more with P. aeruginosa. Although both the inoculation methods were effective, foliar application was found to be superior to micro-injection in terms of rapid PAL activity leading to the synthesis of phenolic compounds.

Wound-induced Phenolic Accumulation and Browning in Lettuce (Lactucasativa L.) Leaf Tissue is Reduced by Exposure to n-alcohols

A wound signal originates at the site of injury in lettuce [Lactucasativa (L.)] leaf tissue and propagates into adjacent tissue where it induces a number of physiological responses that include increased phenolic metabolism with the de novo synthesis of phenylalanine ammonia lyase (PAL, EC 4.3.1.5), the synthesis and accumulation of soluble phenolic compounds (e.g., chlorogenic acid), and subsequent tissue browning. Exposing excised mid-rib leaf tissue to vapors (20 μmol·g-1 FW) or aqueous solutions (100 mm) of n-alcohols inhibited this wound-induced tissue browning by 40% and 60%, respectively. Effectiveness of the alcohol increased linearly from ethanol to the seven-carbon heptanol, and then was lost for the longer n-alcohols 1-octanol and 1-nonanol. The 2- and 3-isomers of the effective alcohols did not significantly reduce wound-induced phenolic accumulation at optimal 1-alcohol concentrations, but significant reductions did occur at much higher concentrations (100 μmol·g-1 FW) of the 2-, and 3-isomers. The active n-alcohols were maximally effective when applied during the first 2 h after excision, and were ineffective if applied 12 h after excision. Phospholipase D (PLD) and its product phosphatidic acid (PA) are thought to initiate the oxylipin pathway that culminates in the production of jasmonic acid, and PLD is specifically inhibited by 1-butanol, but not by 2- or 3-butanol. These results suggest that PLD, PA, and the oxylipin pathway may be involved in producing the wound signal responsible for increased wound-induced PAL activity, phenolic accumulation, and browning in fresh-cut lettuce leaf tissue.

Wound-induced phenolic accumulation and browning in lettuce ( Lactuca sativa L.) leaf tissue is reduced by exposure to n-alcohols

A wound signal originates at the site of injury in lettuce ( Lactuca sativa L.) leaf tissue and propagates into adjacent tissue where it induces a number of physiological responses which include increased phenolic metabolism with the de novo synthesis of phenylalanine ammonia lyase (PAL, EC 4.3.1.5), the synthesis and accumulation of soluble phenolic compounds (e.g., chlorogenic acid), and subsequent tissue browning. Exposing excised mid-rib leaf tissue to vapors (20 μmol/(g FW)) or aqueous solutions (100 mM) of n-alcohols inhibited this wound-induced tissue browning by 40 and 60%, respectively. Effectiveness of the alcohol increased linearly from ethanol to the 7-carbon heptanol, and then was lost for the longer n-alcohols 1-octanol and 1-nonanol. The 2- and 3-isomers of the effective alcohols did not significantly reduce wound-induced phenolic accumulation at optimal 1-alcohol concentrations, but significant reductions did occur at much higher concentrations (100 μmol/(g FW)) of the 2-, and 3-isomers. The active n-alcohols were maximally effective when applied during the first 2 h after excision, and were ineffective if applied 6 h after excision. Phospholipase D (PLD) and its products linolenic acid (LA) and phosphatidic acid (PA) are thought to initiate the oxylipin pathway that culminates in the production of jasmonic acid, and PLD is specifically inhibited by 1-butanol, but not by 2-, or 3-butanol. These results suggest that PLD, LA, PA, and the oxylipin pathway may be involved in producing the wound signal responsible for increased wound-induced PAL activity, phenolic accumulation and browning in fresh-cut lettuce leaf tissue.

Effect of temperature and chlorination of pre-washing water on shelf-life and physiological properties of ready-to-use iceberg lettuce

The effects of cold and warm, chlorinated water as well as warm water without chlorination for pre-washing trimmed, cored iceberg lettuce heads was assessed regarding the shelf-life and physiological properties of the resulting ready-to-use (RTU) produce. On a pilot-plant scale, lettuce heads were shredded with or without pre-washing (50 °C, no chlorine; 4 and 50 °C, 200 mg/l free chlorine; 60 s). After shredding, lettuce was washed for 90 s (tap water, 4 °C), spin-dried, packaged in 200 g consumer-sized bags, and stored at 4 °C for ≤ 9 days. Samples were analyzed for their headspace O 2 and CO 2 levels, sensory properties, microbiological status, and phenylalanine ammonia-lyase (PAL) activity at regular intervals throughout processing and storage. Parallelly, instrumental color and texture measurements were performed. All pre-washing procedures of lettuce heads lowered the initial counts of total aerobic bacteria, pseudomonades, and Enterobacteriaceae by ≥ 1.63, ≥ 1.55, and ≥ 1.85 log 10 cfu/g, respectively, while the single-wash of shredded lettuce in tap water without any pre-washing resulted in significantly smaller reduction rates (0.23, 0.11, and 0.71 log 10 cfu/g, respectively). During storage, all pre-washing procedures had a persistent effect on bacterial counts of the RTU lettuce. Furthermore, all pre-washing procedures delayed vascular tissue browning at cut edges and retarded the decline in the overall visual quality of the samples. Whereas wound-induced PAL synthesis was reduced by the use of warm water (± chlorine), O 2 depletion and CO 2 evolution in the corresponding bags were slightly enhanced, thus proving that processing kept the food as a living tissue. Industrial relevance Ready to use fresh like produce is getting increasing attention and industrial relevance. The aim of this study was to specifically overcome problems associated with product heterogeneity. Interestingly, the use of warm tap water without chlorination as a pre-washing step proved as effective alternative to the application of cold chlorinated water without substantially compromising vitality and freshness of the product.

Wound-induced increases in phenolic content of fresh-cut lettuce is reduced by a short immersion in aqueous hypertonic solutions

Wounding during the preparation of fresh-cut lettuce induces the synthesis of phenylalanine ammonia lyase (PAL, EC 4.3.1.5), and the synthesis and accumulation of specific phenolic compounds (e.g., chlorogenic acid) that contribute to subsequent tissue browning. Increased PAL activity and phenolic content were not induced in non-wounded lettuce that was kept in close proximity to wounded tissue through either a vapor or aqueous signal. The phenolic content of fresh-cut Iceberg and Romaine lettuce was reduced in tissue pieces immediately immersed for 2 h in hypertonic aqueous mannitol solutions (0.3–0.9 M) after cutting. Re-wounding tissue segments 1 day after the initial excision did not produce a second rise in PAL activity in those pieces in which wound-induced increases in PAL activity had been suppressed by a previous soak in hypertonic aqueous mannitol solutions. It appears that the hypertonic solution did not cause the loss of a portion of the wound signal through efflux of water from the tissue, but rather induced a general stress-related resistance to further abiotic stresses.

Involvement of putative chemical wound signals in the induction of phenolic metabolism in wounded lettuce.

Cutting leaves of Romaine lettuce (Lactuca sativa L. cv. Longifolia) produces a wound signal that induces the synthesis of phenylalanine ammonia lyase (PAL, EC 4.3.1.5) and the accumulation of phenolic compounds in cells up to 2 cm from the site of injury, and tissue browning near the site of injury. The response of leaves within a head of Romaine lettuce to putative chemical wound signals [abscisic acid (ABA), jasmonate (JA) and methyl jasmonate (MeJA)] differed significantly with leaf age. Exposure of harvested heads of lettuce to ABA, JA, MeJA, or salicylic acid (SA) did not induce changes in PAL activity, the concentration of phenolic compounds or browning in mature leaf tissue that was similar to the level induced by wounding. Methyl jasmonate applied as vapour (10, 100 or 1000 &mgr;l kg-1 FW), or as an aqueous spray or dip (0.01-100 &mgr;M) at 5 or 10 degrees C did not produce an effect on PAL activity or browning that differed significantly from the untreated controls. In contrast, JA, MeJA and SA did induce elevated levels of PAL activity in younger leaves. However, the levels induced were far lower than those induced by wounding. Wound induced phenolic metabolism in mature leaves appears to be induced by different signals than those functioning in young leaves.

Compartmentation of Phenolic Compounds and Phenylalanine Ammonia-Lyase in Leaves of Phyllanthus tenellus Roxb. and their Induction by Copper Sulphate

The compartmentation of phenolic compounds in mature leaves of Phyllanthus tenellus and their induction by copper sulphate were analysed at histological and subcellular levels. Light and electron microscopy studies demonstrated that the vacuoles of spongy cells were the main sites of phenolic accumulation. Spraying plants with copper sulphate induced punctated lesions formed by groups of necrotic cells which accumulated brownish substances. Histochemical tests and fluorescence microscopy analysis of the sprayed leaves indicated that the phenolic compounds increased in spongy cells within the lesions. Ultrastructural analyses showed that 3h after elicitation, the organelles of the cells within the lesion started to collapse and the content of phenolic substances increased in the vacuole of spongy cells. Antibody against phenylalanine ammonia-lyase (PAL) from parsley cross-reacted with the crude extract of P. tenellus leaves. Two isoforms, one of 65kD and the other of 66kD, were identified. Immunocytochemical studies showed that PAL was synthesized in the palisade and spongy cells, mainly in the cytoplasm and chloroplasts. The phytotoxicity of Cu2+ions induced the accumulation of PAL in sub-cellular compartments of palisade cells. PAL accumulation started to increase 3h after elicitation and reached a maximum after 6h, decreasing 12h post-induction. The increase of PAL was more evident in cells within the necrotic punctated regions than in surrounding cells. Since the vacuole of palisade cells did not accumulate phenolic compounds, the in situ studies suggested that the end products of PAL synthesis play a role in palisade cell wall reinforcement or might accumulate in other tissues. The symptoms induced by copper sulphate suggest that this abiotic elicitor may be a useful tool in the understanding of the regulation of biosynthetic phenolic pathways inP. tenellus .

Synthesis of 3-deoxyanthocyanidin phytoalexins in sorghum occurs independent of light

Aetiolated mesocotyls of sorghum were inoculated with the anthracnose pathogen,Colletotrichum sublineolum,and placed in the light or in the dark for 120 h. A time course study showed that each of the four known 3-deoxyanthocyanidin phytoalexins accumulated regardless of whether seedlings had been incubated in the light or the dark. Analyses of extracts by high performance liquid chromatography and plasma desorption mass spectrometry confirmed the presence of each of the phytoalexins. The phytoalexins were never detected in the control, uninoculated plants, regardless of the light treatment. Total RNA was extracted from the mesocotyls, and a time course of RNA hybridization revealed accumulation of transcripts for phenylalanine ammonia-lyase (PAL) and chalcone synthase at times consistent with those expected to be required for phytoalexin synthesis. PAL transcripts accumulated to higher levels in light-grown than in dark-grown seedlings. Importantly, chalcone synthase transcripts accumulated in response to inoculation and accumulation was independent of light. The accumulation of phytoalexins in the dark and the increase of RNA transcripts question the assumption that light is always required to initiate phenolic compound synthesis at the level of PAL. The results suggest that in sorghum the induction of the synthesis of PAL transcripts and the resultant synthesis of the 3-deoxyanthocyanidin phytoalexins occurs as a response to fungal infection and probably is separate from the induction of PAL and phenolic compound synthesis which occurs as a response to light.

Peroxidase production from carrot hairy root cell culture

Carrot hairy roots have shown vigorous growth and extensive lateral root branching in the absence of phytohormone. The production of peroxidase which was not detected in suspension cells was observed in high amounts in carrot hairy root. High peroxidase activity (19.2 Ug −1 fresh cell weight) was obtained by increasing the total phenolics concentration in the media. Peroxidase synthesis could be increased using elicitors such as yeast extract, V 2O 5, and Hg 2Cl 2, which are known to induce the synthesis of PAL (phenylalanine ammonia lyase). As the initial sucrose concentration in the medium increased, peroxidase activity and total phenolics increased while final cell mass decreased. A high level of sucrose also affected the elevation of the phenolics concentration in the cell. By adopting a two-stage culture method, the activity of peroxidase (55 U g −1 fresh cell weight) was ten times higher than that of a single-stage culture (5 U g −1 fresh cell weight) and about three times higher than that of field-grown horseradish on an activity per weight basis.

Stimulated Synthesis of Phenylalanine Ammonia-lyase in Spirodela oligorrhiza treated with 1-Amino-2-Phenylethylphosphonic Acid

Summary 1-Amino-2-phenylethylphosphonic acid (PheP) stimulated activity of phenylalanine ammonia-lyase (PAL), increased the content of phenylalanine, and reduced the content of anthocyanins and phenols in Spirodela oligorrhiza (Kurz.) Hegelm.PheP enhanced incorporation of L-[U-14C]-leucine into PAL.It is suggested that (1) PheP forms labile complexes with PAL in vivo, which dissociate in vitro releasing the active enzyme, and (2) the plant compensates the inhibition by increased biosynthesis of PAL.

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.

Isolation of various hyperactive mutants of phenylalanine ammonia-lyase containing yeasts

Various strains of phenylalanine ammonia-lyase (PAL) containing yeasts were mutagenised using ultraviolet (UV) irradiation. Two of the yeast, SPA 1 and SPA 17, produced UV kill curves with a distinct shoulder reflecting their diploid nature. Of the analogues used, p-fluoro-D,L-phenylalanine and β-2-thienyl-D,L-alanine selected the greatest frequency of mutants with the highest PAL activities: one such mutant, FP10M6, exhibited five times the PAL activity of the parent SPA 10. Mutants constitutive for PAL synthesis could not be isolated using any selection regime, including resistance to relatively high concentrations of 2-deoxyglucose. Three of the hyperactive PAL mutants examined were not impaired for growth and their PAL-induction profiles were not different from the respective parents. Inactivation of PAL shortly after peak synthesis was found with all mutants examined, although this was not extensively investigated. Of the 21 strains mutagenised, mutants from three of the wild-type yeasts were found to exhibit moderate growth at 37 °C, while growth of the parent strains was greatly impaired. The latter temperature-resistant mutants exhibited a twofold increase in PAL activity as compared with the parent strains at either 30 or 37 °C. Under biotransformation reaction conditions, several of the mutants were capable of producing more than 15 g L-phenylalanine∙L−1. Although the stability of the PAL catalyst varied markedly among different mutants, some of the most productive mutants did exhibit good reuseability under sequentially repeated batch reaction conditions.

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 mode of inhibition of the biosynthesis of phenylalanine ammonia lyase by its product cinnamic acid in aging potato parenchyma tissue

Excised potato parenchyma tissue upon aging in air and light, showed synthesis of RNA, as assessed by the incorporation of [3H]-uridine, reaching a maximum in 18 h. The increase in RNA synthesis was in parallel with the increase in phenylalanine ammonia lyase synthesis. The treatment of the tissue, initially with actinomycin D at 15 μg/g tissue, or with trans-cinnamate at 3·5 mM, caused 65% and 50% inhibition of RNA synthesis, respectively. The inhibition was reduced to 20% by delaying the treatment of trans-cinnamate to the ninth hour. A comparative study on the nuclear fractions, isolated from trans-cinnamate treated and untreated aged parenchyma tissues showed that trans-cinnamate treatment inhibited thein vitro RNA synthesis by 38%. Studies on thein vivo synthesis of PAL and other proteins showed that trans-cinnamate treatment mainly impaired thede novo synthesis of PAL. The total RNA, isolated from trans-cinnamate treated parenchyma, was 66% less efficient in the translation of PAL, in cell free wheat germ protein synthesizing system. The translation product, purified by affinity chroma tography on phenylalanine conjugated sepharose-4B was found to be homogeneous and showed a single radioactive peak corresponding to protein band on polyacrylamide gel electrophoresis.