Foliar application of l-phenylalanine and ferulic acids to pea plants: induced phenylalanine ammonia lyase activity and resistance against Erysiphe pisi

Phenylalanine ammonia lyase (PAL) activity was measured using HPLC in pea leaves following exogenous application of l-phenylalanine and ferulic acid. Treatment with different concentrations (50, 100 and 150 ppm) of l-phenylalanine caused increased activity of PAL in comparison to the control. In pea leaves treated with 50 ppm l-phenylalanine, maximum PAL activity was observed after 72 h of treatment. Application of ferulic acid first reduced PAL activity at lower concentration (50 ppm) but increased at higher concentrations of the compound (100 and 150 ppm) in pea leaves as compared to the control. Maximum PAL activity was 0.19 nM cinnamic acid/min/g fresh wt. after 24 h at 50 ppm and then increased with time. Treatment with both the compounds significantly reduced conidial germination of Erysiphe pisi on pea leaves. They were equally effective at 100 and 150 ppm in reducing conidial germination. The conidial germination on l-phenylalanine-treated leaves was 26% after 24 h and that on ferulic acid-treated leaves was 34% as compared to the control (46%). Foliar application of different concentrations of l-phenylalanine increased the level of ferulic acid in the leaves of pea plants. Maximum accumulation of ferulic acid (79.3 and 83.5 μg/g fresh wt.) was observed following the application of l-phenylalanine after 24 h and 48 h, respectively. At 50 ppm, ferulic acid accumulation in pea leaves was 35.6 and 39.4 μg/g fresh wt. and 74.3 and 86.5 μg/g fresh wt. at 100 ppm.     

Regulatory Properties of Prephenate Dehydrogenase and Prephenate Dehydratase from Corynebacterium glutamicum

Regulatory properties of the enzymes in l-tyrosine and l-phenyalanine terminal pathway in Corynebacterium glutamicum were investigated. Prephenate dehydrogenase was partially feedback inhibited by l-tyrosine. Prephenate dehydratase was strongly inhibited by l-phenylalanine and l-tryptophan and 100% inhibition was attained at the concentrations of 5 × 10^?2mm and 10^?1mm, respectively. l-Tyrosine stimulated prephenate dehydratase activity (6-fold stimulation at 1 mm) and restored the enzyme activity inhibited by l-phenylalanine or l-tryptophan. These regulations seem to give the balanced synthesis of l-tyrosine and l-phenyl-alanine. Prephenate dehydratase from C. glutamicum was stimulated by l-methionine and l-leucine similarly to the enzyme in Bacillus subtilis and moreover by l-isoleucine and l-histidine. C. glutamicum mutant No. 66, an l-phenylalanine producer resistant to p-fluorophenyl-alanine, had a prephenate dehydratase completely resistant to the inhibition by l-phenylalanine and l-tryptophan.     

Regulatory Properties of Chorismate Mutase from Corynebacterium glutamicum

Regulatory properties of chorismate mutase from Corynebacterium glutamicum were studied using the dialyzed cell-free extract. The enzyme activity was strongly feedback inhibited by l-phenylalanine (90% inhibition at 0.1~1 mm) and almost completely by a pair of l-tyrosine and l-phenylalanine (each at 0.1~1 mm). The enzyme from phenylalanine auxotrophs was scarcely inhibited by l-tyrosine alone but the enzyme from a wild-type strain or a tyrosine auxotroph was weakly inhibited by l-tyrosine alone (40~50% inhibition, l-tyrosine at 1 mm). The enzyme activity was stimulated by l-tryptophan and the inhibition by l-phenylalanine alone or in the simultaneous presence of l-tyrosine was reversed by l-tryptophan. The Km value of the reaction for chorismate was 2.9 } 10^?3 m. Formation of chorismate mutase was repressed by l-phenylalanine. A phenylalanine auxotrophic l-tyrosine producer, C. glutamicum 98–Tx–71, which is resistant to 3-amino-tyrosine, p-aminophenylanaine, p-fluorophenylalanine and tyrosine hydroxamate had chorismate mutase derepressed to two-fold level of the parent KY 10233. The enzyme in C. glutamicum seems to have two physiological roles; one is the control of the metabolic flow to l-phenylalanine and l-tyrosine biosynthesis and the other is the balanced partition of chorismate between l-phenylalanine-l-tyrosine biosynthesis and l-tryptophan biosynthesis.     

The Biosynthetic Control in Aromatic Amino Acid Producing Mutants of Corynebacterium glutamicum

Regulatory properties of the enzymes involved in aromatic amino acid biosynthesis in the mutant of Corynebacterium glutamicum which produces a large amount of aromatic amino acids were examined. A phenylalanine auxotrophic l-tyrosine producer, pr-20, had a 3-deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthetase released from the feedback inhibition by l-phenylalanine, l-tyrosine and l-tryptophan and had a two-fold derepressed chorismate mutase. A pair of l-phenylalanine and l-tyrosine still strongly inhibited the chorismate mutase activity, though the enzyme was partially released from the inhibition by l-phenylalanine alone. A tyrosine auxotrophic l-phenylalanine producer, PFP-19-31, had a DAHP synthetase sensitive to the feedback inhibition by l-phenylalanine, l-tyrosine and l-tryptophan and had a prephenate dehydratase and a chorismate mutase both partially released from the feedback inhibition by l-phenylalanine. The mutant produced a large amount of prephenate as well as l-phenylalanine. A phenylalanine and tyrosine double auxotrophic l-tryptophan producer, Px-115-97, had an anthranilate synthetase partially released from the feedback inhibition by l-tryptophan and had a DAHP synthetase sensitive to the feedback inhibition. These data explained the mechanism of the production of aromatic amino acids by these mutants and supported the in vivo functioning of the control mechanisms of aromatic amino acid biosynthesis in C. glutamicum previously elucidated in vitro experiments.     

DAHP Synthetase and Its Control in Corynebacterium glutamicum

Properties of 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthetase from Corynebacterium glutamicum were examined using the cell free extract. The optimum pH for the reaction was broad ranging from 5.5 to 7.0 and the optimum temperature was 37°C. Co²⁺ inhibited the enzyme activity at 20°C, whereas Co²⁺ apparently stimulated the enzyme activity at 37°C because the ion protected the enzyme from inactivation at 37°C. Co²⁺ reversed the inhibition of the enzyme activity by EDTA. The activity of DAHP synthetase was feedback inhibited only weakly by l-phenylalanine, l-tyrosine or l-tryptophan alone, but was strongly inhibited synergistically by l-phenylalanine and l-tyrosine. l-Tryptophan enhanced the inhibition by the pair of l-tyrosine and l-phenylalanine. Maximal inhibition was near 90 % in the simultaneous presence of the three amino acids. Sensitivity of the enzyme to the inhibitors was lost during the purification process of the enzyme or during the reaction at 37°C. Especially sensitivity to l-tryptophan was easily lost. Co²⁺ protected the enzyme from the desensitization. Mutants resistant to p-fluorophenylalanine plus l-tyrosine (or 3-aminotyrosine) had DAHP synthetase which was released from the feedback inhibition by the three amino acids. The formation of the enzyme was not affected by aromatic amino acids.     

Microbial Production of l-Phenylalanine from n-Alkanes

A tyrosine auxotroph derived from a hydrocarbon utilizing bacterium, Corynebacterium sp. KY 4309, was found to accumulate a large amount of l-phenylalanine in the broth. The cultural conditions for l-phenylalanine production were studied. The pH value during cultivations exhibited a remakable effect on l-phenylalanine production. The addition of l-tryptophan enhanced the l-phenylalanine accumulation. Shikimic acid and phenylpyruvic acid are possible precursors of phenylalanine biosynthesis in this bacterium. Production of l-phenylalanine attained to a level of 10 mg per ml for 68 hr under optimal conditions.     

Bitterness of Diastereomers of a Hexapeptide (Arg-Arg-Pro-Pro-Phe-Phe) Containing d-Phenylalanine in Place of l-Phenylalanine

In order to investigate the steric requirement of the C-terminal hydrophobic amino acid of the bitter peptide, Arg-Arg-Pro-Pro-Phe-Phe, for the production of bitterness, we prepared analogs of it containing d-phenylalanine. in place of l-phenylalanine. The analogs with l-phenylalanine at the C-terminal exhibited stronger bitterness than those with d-phenylalanine at the C-terminal. We confirmed that the configuration of the C-terminal hydrophobic amino acid is important for the increase in bitterness.     

Micropolyamide Thin-layer Chromatography of Fluorescein-thiohydantoin-Amino Acids at Picomole Level

The formation of aromatic l-amino acid decarboxylase in bacteria was studied with intact cells in a reaction mixture containing the aromatic l-amino acids, 3,4-dihydroxy-l-phenyl-alanine, l-tyrosine, l-phenylalanine, l-tryptophan and 5-hydroxy-l-tryptophan. Activity was widely distributed in such genera as Achromobacter, Micrococcus, Staphylococcus and Sarcina. Bacterial strains belonging to the Micrococcaceae showed especially high decarboxylase activity toward l-tryptophan, 5-hydroxy-l-tryptophan and l-phenylalanine. M. percitreus AJ 1065 was selected as a promising source of aromatic l-amino acid decarboxylase. Results of experiments with this bacterium showed that the aromatic amine formed from l-tryptophan by the enzymatic method was identical with tryptamine. M. percitreus constitutively produced an enzyme which exhibited decarboxylase activity toward l-tryptophan. However, when large amounts of the aromatic l-amino acids listed above or the tryptamine formed from l-tryptophan were added, enzyme formation was repressed.

Cells with high enzyme activity were prepared by cultivating this bacterium at 30°C for 24 hr in a medium containing 0.5% glycerol, 0.5% yeast extract, 0.5% Polypepton, 3.0 vol % soybean protein hydrolyzate, 0.1% KH₂PO₄, 0.1% MgSO₄ · 7H₂O, 0.001% FeSO₄ · 7H₂O and 0.001% MnSO₄ · 5H₂O in tap water (pH 8.0).     

Liver-specific Induction of Ribosomal Protein Gene Expression by Amino Acid Starvation in Rats

An aminoacylase, inducibly formed in Bacillus thermoglucosidius grown with a synthetic compound, acetamidocinnamate, was used for enzymatic synthesis of l-phenylalanine from chloroacetamido-cinnamate. The reaction system consisted of the hydrolysis of chloroacetamidocinnamate to phenylpyruvate by aminoacylase and the reductive amination of phenylpyruvate to l-phenylalanine by phenylalanine dehydrogenase. The coenzyme NADH consumed was regenerated by a coupled reaction with formate dehydrogenase. Under optimum conditions for l-phenylalanine production, more than 98% of the initially added chloroacetamidocinnamate was converted effectively to l-phenylalanine without appreciable decomposition or racemization.     

Purification,Crystallization and Properties of Tryptophanase from Proteus rettgeri

An inducible tryptophanase was crystallized from the cell extract of Proteus rettgeri grown in a medium containing l-tryptophan. The purification procedure included ammonium sulfate fractionation, heat treatment, DEAE-Sephadex and hydroxylapatite column chromatographies. Crystals were obtained from solutions of the purified enzyme by the addition of ammonium sulfate.

The crystalline enzyme preparation was homogeneous by the criteria of ultracentrifugation and zone electrophoresis. The molecular weight was determined to be approximately 210,000.

The crystalline enzyme catalyzed the degradation of l-tryptophan into indole, pyruvate and ammonia in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from 5-hydroxy-l-tryptophan, 5-methyl-l-tryptophan, S-methyl-l-cysteine and l- cysteine. l-, d-Alanine, l-phenylalanine and indole inhibited pyruvate formation from these substrates.     

Optimal Conditions for the Enzymatic Production of l-Aromatic Amino Acids from the Corresponding 5-Substituted Hydantoins

The reaction conditions for the production of l-tryptophan from dl-5-indolyl- methylhydantoin by Flavobacterium sp. AJ-3940, and the cultural conditions for the formation of the enzyme involved by this bacterium were investigated. The optimal pH of this reaction was around 8.5 and the optimal temperature was between 45 to 55°C. The amount of l-tryptophan produced was remarkably increased by the addition of inosine, which formed a water insoluble adduct with l-tryptophan, to the reaction mixture because of the release of end-product inhibition by l-tryptophan. This enzyme was inducibly and intracellularly produced by Flavobacterium sp. AJ-3940 in proportion to the increase in cell growth. Cells showing high activity were obtained using a medium containing 5 g glucose, 5 g (NH₄)₂SO₄, 1 g KH₂PO₄, 3 g K₂HPO₄, 0.1 g MgSO₄ · 7H₂O, 0.01 g CaCl₂ · 2H₂O, 50 ml corn steep liquor and 3.5 g dl-5-indolylmethylhydantoin in a total volume of 1 liter (pH 7.0). Under the best conditions, 43 mg/ml of l-tryptophan was produced from 50 mg/ml of dl-5-indolylmethylhydantoin with a molar yield of 97% in the presence of cells of Flavobacterium sp. AJ-3940. In addition, other l-aromatic amino acids such as l-phenylalanine, l-tyrosine, l-DOPA and related l-amino acids were also produced from the corresponding 5-substituted hydantoins by this bacterium containing the l-tryptophan-producing enzyme induced by dl-5-indolylmethylhydantoin.     

Role of Phenylalanine Deaminase and Tyrase in the Lignification of Bamboo

A pronounced increase of the activity of phenylalanine deaminase and tyrase during the lignification of bamboo shoots was observed. With progressing maturation from the basal part to the upper part of immature bamboos the pattern of the activity of the enzymes moved toward the upper part of the tissues, where the most active lignification was taking place, and the amount of cinnamic acid derivatives in the tissues was found to be in good accordance with the activity of the enzymes. l-Phenylalanine-G-¹⁴C and l-tyrosine-G-¹⁴C were both well incorporated into the lignin of bamboos. These results indicate that phenylalanine deaminase and tyrase are synthesized progressively just before the lignification of the bamboos and by the enzymes l-phenylalanine and l-tyrosine are deaminated to cinnamic acid derivatives and incorporated into lignin.     

Occurrence of 3-Hydroxy-3-methyl-glutaryl Coenzyme A Reductase in Sweet Potato

Illumination of the dark-grown Euglena gracilis, both the wild-green type and a permanently bleached mutant, for 4 hr at 2,000 lux caused about 6-fold increase of the cellular content of total l-ascorbic acid. The increase was mainly due to an increase of reduced-form l-ascorbic acid. From the action spectrum only blue light was found to be effective for the increase. Darkening stopped the increase and reillumination started a renewed increase. The activity of l-gulono-γ-lactone dehydrogenase, catalyzing the last step of l-ascorbic acid biosynthesis, was also increased two fold by illumination for 2 hr, and was changed in parallel to that of the cellular content of l-ascorbic acid depending on the presence or absence of illumination. The augmentation of l-ascorbic acid formation was markedly inhibited by various inhibitors and uncouplers, but not by dichlorophenyldimethylurea. The results in sum suggest that the light-dependent increase of l-ascorbic acid formation in E. gracilis is not primarily associated with photosynthesis, but is apparently related to the adaptation of the dark-grown cells to the illuminated state.     

Purification,characterization, and gene cloning of a novel aminoacylase from Burkholderia sp. strain LP5_18B that efficiently catalyzes the synthesis of N-lauroyl-l-amino acids

ABSTRACT

An N-lauroyl-l-phenylalanine-producing bacterium, identified as Burkholderia sp. strain LP5_18B, was isolated from a soil sample. The enzyme was purified from the cell-free extract of the strain and shown to catalyze degradation and synthesis activities toward various N-acyl-amino acids. N-lauroyl-l-phenylalanine and N-lauroyl-l-arginine were obtained with especially high yields (51% and 89%, respectively) from lauric acid and l-phenylalanine or l-arginine by the purified enzyme in an aqueous system. The gene encoding the novel aminoacylase was cloned from Burkholderia sp. strain LP5_18B and expressed in Escherichia coli. The gene contains an open reading frame of 1,323 nucleotides. The deduced protein sequence encoded by the gene has approximately 80% amino acid identity to several hydratase of Burkholderia. The addition of zinc sulfate increased the aminoacylase activity of the recombinant E. coli strain.     

Biotechnological production and applications of microbial phenylalanine ammonia lyase: a recent review

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.     

Metabolism of Aromatic Amino Acid in Microorganisms

Phenylalanine ammonia-lyase, which catalyzes the conversion of l-phenylalanine to trans-cinnamic acid and ammonia, has been partially purified from the cells of Rhodotorula. Some of the properties of this phenylalanine ammoyia-lyase were investigated. The enzyme was stable in phosphate buffer of pH over the range of 6.0 to 7.0 On heating, the enzyme was stable up to 50°C, but above 60°C, it was destroyed. The enzyme activity was strongly inhibited by p-chloromercuribenzoate at 10^?5 m and almost recovered by the addition of glutathione or mercaptoethanol at 10^?3 m. The present enzyme preparation of Rhodotorula also catalyzed the deamination of l-tyrosine to trans-p-coumaric acid. trans-p-Coumaric acid was isolated from the reaction mixture and identified by its absorption spectra. The rates of deamination showed optima at pH 9.0 and 9.5 for l-phenylalanine and l-tyrosine, respectively.     

The Production of Phenylacetaldehyde from l-Phenylalanine in Tea Fermentation

Tea leaves macerated with l-phenylalanine generated rose like aroma. The gas chromatogram of the essential oil obtained from these leaves showed extremely large peak of phenylacetaldehyde. The evidence for degradation of l-phenylalanine to phenylacetaldehyde and carbon dioxide was given by the radioactive tracer experiment using l-phenylalanine-U-¹⁴C. The phenylacetaldehyde was presumed to be an intermediate product in tea fermentation from the data on the changes of the compound in tea fermentation process.     

Salt Effects on Plastein Formation by α-Chymotrypsin

The plastein formation by α-chymotrypsin from an ovalbumin hydrolysate was affected in an order of valency of salts when the concentration of each salt was 1 m. Monovalent cations were rather effective at this concentration and enhanced the plastein yield by 10%. In the presence of NaCl, the plastein formation showed two distinct maximal rates at its concentrations of 0.1 m and 0.8 m. The first maximum was considered to be resulted from an increase in enzyme activity, since chymotryptic hydrolysis of both N-acetyl-l-tyrosine ethyl ester and benzyloxycarbonyl-l-phenylalanine p-nitrophenyl ester was activated at an NaCl concentration of 0.1 ~ 0.2 m. The second maximum was ascribed to the salting-out of the product due to the higher concentration of NaCl. A salt-tolerant protease was also used to confirm the above conclusions. It was observed that this enzyme was much effective in producing a plastein at a high NaCl concentration. This may be due to the fact that both the enzyme activation effect and the product salting-out effect participate co-operatively.     

5′-Nucleotidase from Yeast,Having Special Affinity for 5′-AMP

Three kinds of diketopiperazines which have retarditive activity for the growth of plant seedlings and plant roots at concentrations ranging from 1 : 2,500 to 1 : 100,000, were isolated from the neutral fraction by extracting the cultured broth of Rosellinia necatrix. These three diketopiperazines have been proved to be l-prolyl-l-leucine anhydride, l-prolyl-l-valine anhydride and l-prolyl-l-phenylalanine anhydride respectively, and the last one seems to be a new diketo-piperazine.

Furthermore, a crystalline wax having m.p. 52°C, a physiologically inactive substance, was also isolated from the same neutral fraction and presumed to be the saturated hydrocarbon of n-pentacosane C₂₅H₅₂.     

Optical Resolution of dl-Phenylalanine with Acylase

New devices for resolution of DL-phenylalanine by an enzymatic method have been developed by using ammonium N-acetyl-DL-phenylalanate as a substrate. In this procedure, crystals of l-phenylalanine and ammonium N-acetyl-d-phenylalanate are separated alternately or simultaneously from reaction mixtures containing acylase, as first crops. The whole resulting solution including acylase can be reused. Ammonium acetate formed as a by-product was found to inhibit the enzyme.