Optimization of reaction conditions and stabilization of phenylalanine ammonia lyase-containing Rhodotorula glutinis cells during bioconversion of trans-cinnamic acid to L-phenylalanine

Studies were performed to elucidate the optimal reaction conditions (pH, temperature, ammonia concentration and biocatalyst loading) for bioconversion of trans-cinnamic acid (t-CA) to L-phenylalanine (L-Phe) by L-phenylalanine ammonia lyase (PAL) containing Rhodotorula glutinis cells. All treatments with permeabilizing agents stimulated L-Phe production and also enhanced instability of the catalyst, except Triton X-100 which gave a superior (56%) increase in conversion as compared to the control and a significant stabilization of PAL enzyme. Inclusion of several activity modifiers and stabilizer additives in reaction mixtures were shown to enhance the yield of L-Phe and maintained PAL stability over several successive incubations during the bioconversion process. Maximum stabilization of PAL and enhancement of L-Phe production was achieved with addition of 20% polyhydric alcohol (glycerol). The production of L-Phe continued to the fifth cycle and the total yield increased 2.3 times compared to the yield produced by the control (without glycerol addition) during the repeated batch process. Reducing agents such as 2-mercaptoethanol and thioglycolic acid were added to the bioconversion mixture in order to reduce the effects of oxygen on PAL catalyst life. Production of L-Phe by addition of 400 mgL(-1) of thioglycolic acid was maximized over the control by 55%. When both 20% glycerol and 400 mgL(-1) thioglycolic acid were simultaneously present in the reaction mixture, reuseability and stability of biocatalyst (PAL) were extended to eight consecutive cycles and conversion rate and overall productivity of L-Phe were higher than that of the control. These results may lead to improvements in the production of the essential amino acid L-Phe.     

Novel photoreactive cinnamic acid analogues to elucidate phenylalanine ammonia-lyase

4-[3-(Trifluoromethyl) diazirinyl] cinnamic acid derivatives were synthesized to elucidate properties of phenylalanine ammonia-lyase (PAL). 2-Methoxy and 2-biotinylated alkoxy compounds have inhibitory activity on the formation of phenylalanine from cinnamic acid. Specific photolabeling of the enzyme was detected using biotinylated derivatives without the use of radioisotopes. The results indicated that the 4-[3-(trifluoromethyl) diazirinyl] skeleton will be a suitable photoreactive compound to elucidate regulation of phenylpropanoid biosynthesis.     

Production of l-phenylalanine from trans-cinnamic acids by high-level expression of phenylalanine ammonia lyase gene from Rhodosporidium toruloides in Escherichia coli

A combined promoter expression vector pBV–PAL for high-level expression of phenylalanine ammonia lyase gene of Rhodosporidium toruloides was constructed. Pal gene was cloned and inserted into the region between SalI and PstI restriction sites of expression vector pBV220 (containing P_LP_R promoter) to obtain recombinant expression vector pBV220–PAL. The tac promoter obtained from the plasmid pKtac was inserted into the expression vector pBV220–PAL to construct expression vector pBV–PAL. The recombinant plasmid pBV220–PAL and pBV–PAL were introduced into Escherichia coli JM109 by transformation. The result showed that the transformant E. coli JM109 (pBV–PAL) gave a much higher PAL activity than that transformant E. coli JM109 (pBV220–PAL). Recombinant PAL expression level of the transformant JM109 (pBV–PAL) was about 9.6% of total cellular protein, specific enzyme activity was 2.3-fold higher than that of the transformant JM109 (pBV220–PAL), reached 35 U/g (dry cells weight, DCW). PAL specific activity of 123 U/g (DCW) could be achieved in a 5-l fermentor. 80.5% conversion rate of trans-cinnamic acid to l-phenylalanine and 5.12 g/l l-phenylalanine were obtained after 3 h bioconversion using the transformant JM109 (pBV–PAL). The recombinant strain JM109 containing the combined promoter expression vector pBV–PAL was shown to be effective and practical to product l-phenylalanine.     

The estimation of l-phenylalanine ammonia-lyase shows phenylpropanoid biosynthesis to be regulated by l-phenylalanine supply and availability

l-Phenylalanine ammonia-lyase (PAL, E.C. 4.3.1.5) is the first committed enzyme in the pathway leading to phenylpropanoid biosynthesis in higher plants. PAL catalyses the conversion of l-phenylalanine to t-cinnamic acid with the elimination of ammonia. Standard methods for determination of PAL activity in both green and non-green tissues were found to lead to measurements of both l-phenylalanine amino-transferase (PAT, E.C. 2.6.1.1) and PAL activities together. The accurate estimation of PAL activity alone, necessitated the inhibition of PAT by a specific inhibitor of PAT activity, l-aspartic acid. The influence of PAT on the kinetics of PAL activity may explain (i) the diverse properties that have been attributed to PAL and (ii) the controversies regarding the control mechanism underlying the regulation of PAL activity. Evidence is presented for the regulation of phenylpropanoid biosynthesis via substrate supply and availability as opposed to feedback inhibition, during phaseollin production and hypersensitive necrosis in Phaseolus vulgaris.     

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

Summary 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.     

Enzymatic production of the lignin precursor trans-[U-14C]cinnamic acid from l-[U-14C]phenylalanine using l-phenylalanine ammonia-lyase

An enzymatic method using phenylalanine ammonia-lyase (l-phenylalanine ammonia-lyase, EC 4.3.1.5) for the rapid conversion of l-[U-¹⁴C]phenylalanine to the deaminated lignin precursor trans-[U-¹⁴C]cinnamic acid is described. The method produces an experimentally useful ¹⁴C-labelled deaminated lignin precursor unavailable from radiochemical supply companies.     

Inhibition of phenylalanine ammonia-lyase by cinnamic acid derivatives and related compounds

Thirty-five derivatives of cinnamic acid and related compounds were tested for inhibition against phenylalanine ammonia-lyase (PAL) derived from sweet potato, pea and yeast. Caffeic and gallic acids showed inhibition against PAL originating from higher plants, but not against yeast PAL. In contrast, yeast PAL was specifically inhibited by p-hydroxycinnamic and p-hydroxybenzoic acids. The results suggest that caffeic and gallic acids may act as regulatory substances in phenylpropanoid metabolism in higher plants. Inhibition experiments with synthetic cinnamic acid derivatives have revealed that the presence of a hydrophobic aromatic ring, α,β-double bond and carboxyl group is essential for inhibitory activity. 2-Naphthoic acid which fulfills these structural requirements showed a strong inhibition. The size and shape of the active site is discussed from structure-activity relationships of cinnamic acid derivatives. o-Chlorocinnamic acid, one of the strongest inhibitors found in this study showed an inhibitory effect on the growth of the roots of rice seedlings.     

Changes in phenylalanine ammonia-lyase activity in Satsuraa mandarin fruit during ontogeny

PAL activity in young fruits of Citrus unshiu was initiallyvery high. It decreased gradually as the fruit grew and afterthe "June drop", declined markedly. PAL behavior appears tobe closely related to the tissue differentiation of fruit. Proteincontent of the fruit showed a pattern of change similar to thatof PAL activity. Also, the amount of hesperidine was high inimmature fruit. Accumulation of hesperidine per fruit was greatlyenhanced as growth of the fruit progressed, but after PAL activityhad almost disappeared, the accumulation ceased. ¹Present address: Kozo Keikaku Engineering Incorporation, Nakano,Tokyo, Japan. (Received March 16, 1973; )     

Control of phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase in gherkin tissues

Blue light mediates a transient increase in the extractable activity of phenylalanine ammonia-lyase from both cotyledons and hypocotyls of etiolated gherkin seedlings, but concurrent changes in extractable cinnamic acid 4-hydroxylase activity only occur in cotyledons. Excision, followed by incubation in the dark, also results in stimulation of the lyase activity in both tissues, but the hydroxylase activity is only stimulated in cotyledons, again concurrently with the lyase. Stimulated levels of hydroxycinnamic acid esters are, however, only formed following light treatment, indicating the presence of another light-sensitive step in their biosynthesis. Treatment of gherkin tissues with 2-aminooxyacetic acid or α-aminooxy-β-phenylpropionic acid inhibits phenylalanine ammonia-lyase activity in situ, reduces the accumulation of hydroxycinnamic acid esters and presumably reduces the endogenous cinnamic acid pool. This treatment increases extractable lyase activity and delays its peak in activity. In cotyledons, these changes in the lyase are associated with concurrent and similar changes in extractable hydroxylase activity, whilst in hypocotyls the hydroxylase is relatively unaffacted. The increase in phenylalanine ammonia-lyase activity following excision of cotyledons and hypocotyls is prevented by cinnamic acid; in cotyledons the hydroxylase is similarly affected, but after a longer lag. Thus whilst cinnamic acid can modify the extractable activity of the lyase, it cannot itself mediate changes in the extractable activity of the hydroxylase.     

Selective precipitation of l-phenylalanine using copper ions and its application in the bioconversion of phenylpyruvic acid

l-Phenylalanine can be selectively precipitated from a biotransformation reaction mixture as an insoluble diphenylalanine-copper complex. During the bioconversion of phenylpyruvic acid to l-phenylalanine, none of the substrates or co-products reacted with copper ions to form insoluble salts. Using immobilized cells of Pseudomonas fluorescens, l-phenylalanine produced from phenylpyruvate was precipitated in situ using copper acetate and the reaction continued by supplying fresh substrate. After 30 h, 36.5 grams of partially pure lphenylalanine were recovered from 1 liter by treatment with hydrogen sulphide. A recycle packed bed reactor was set up with the l-phenylalanine (product) in the reactor effluent continuously precipitated with copper ions. After 102h operation, 103.7 g of partially pure l-phenylalanine was recovered from the pooled precipitates. The merits of this efficient recovery method are discussed.     

Kinetic analysis of the inhibition of phenylalanine ammonia-lyase by 2-aminoindan-2-phosphonic acid and other phenylalanine analogues

The conformationally restricted phenylalanine analogue 2-aminoindan-2-phosphonic acid (AIP) inhibits phenylalanine ammonia-lyase (PAL) competitively in a time-dependent manner. This phenomenon was investigated in more detail with the heterologously expressed, highly purified homotetrameric PAL-1 isozyme from parsley. The kinetic analysis revealed that the enzyme-inhibitor complex is formed in a single "slow" step with an association rate of k(2)=2.6+/-0.04 10(4) M(-1) s(-1). The inhibition is reversible with a dissociation rate of k(-2)=1.8+/-0.04 10(-4) s(-1) and an equilibrium constant of K(i)=7+/-2 nM. The previously described PAL inhibitor (S)-2-aminooxy-3-phenylpropanoic acid [(S)-AOPP] was also found to be a slow-binding inhibitor of PAL-1. The carboxyl analogue of AIP, 2-aminoindan-2-carboxylic acid, served as a substrate of PAL-1 and was converted to indene-2-carboxylic acid.     

Changes in phenylalanine ammonia-lyase activity during xylem differentiation in Coleus and soybean

Summary Xylem differentiation was induced in cultured Coleus internode slices when grown in the light on a simple agar/sucrose/IAA medium and in darkgrown soybean callus tissue when cultured on a complex defined medium containing 5×10^-7 M kinetin. In the Coleus system, the activity of phenylalanine ammonialyase followed the same time course as the formation of lignified wound vessel members. The specific activity of PAL was higher in the soybean callus tissues grown on 5×10^-7 M kinetin, which produced tracheary elements, than in the soybean tissue grown on 10^-8 M kinetin, which did not produce tracheids. These observations suggest that PAL is a marker enzyme for xylogenesis and that PAL activity may be a rate limiting step in lignification.Abbreviations IAA indole 3-yl acetic acid - NAA -naphthalene acetic acid - 2,4D 2,4,dichlorophenoxyacetic acid - DNA deoxyribose nucleic acid - TCA trichloracetic acid - PAL phenylalanine ammonia-lyase     

Protection of phenylalanine ammonia-lyase from proteolytic attack

Phenylalanine ammonia-lyase contained within permeabilized cells of Rhodosporidium toruloides was protected from proteolytic attack by trypsin, chymotrypsin and duodenal juice. The inactivation by the proteases was biphasic. The enzyme contained within the yeast cells had a similar Km for phenylalanine and Ki for cinnamic acid to the protein in free solution. Phenylalanine ammonia-lyase present in the yeast depleted duodenal juice of free phenylalanine, while the enzyme in free solution did not. The possibility of using permeabilized cells of R. toruloides as a vehicle for protecting orally ingested therapeutic enzymes from proteolytic inactivation is discussed.     

Changes of antioxidant enzyme and phenylalanine ammonia-lyase activities during Chimonanthus praecox seed maturation

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.