Phenylalanine ammonia‐lyase and phenolic compounds in leaves and fruits of Olea europaea L. cv. Picual during ripening

BACKGROUND: The kinetic and molecular properties of phenylalanine ammonia‐lyase (PAL) in leaves and fruit of the olive tree (Picual variety) have been studied during the seasonal process of fruit maturation. The concentrations of total phenolic compounds, oleuropein, hydroxytyrosol and tyrosol, have also been determined. This study has been made in rainfed 30‐year‐old olive trees in Jaén, Spain, cultivated by the traditional method. RESULTS: PAL specific activity was assayed and hyperbolic kinetics were observed in both organs. The K_m value for L ‐Phe was 0.22 mmol L^?1 in leaf and 0.26 mmol L^?1 in fruit. In leaf, the highest PAL specific activity was found in the stage prior to veraison. By immunoblot, a PAL‐immunoreactive 75 kDa polypeptide was detected in leaf and fruit. In leaf, the level of this protein progressively rose until the last stages of ripening at the same time that total phenols increased. In fruit, PAL activity and protein change as in two series coinciding with different fruit‐maturation period. By immunohistochemistry under light microscopy, PAL was located in the epidermis and parenchyma cells of leaf and fruit. CONCLUSION: These results demonstrate the involvement and regulation of PAL during fruit ripening of olive, cv. Picual. Copyright © 2008 Society of Chemical Industry     

Inhibition of polyphenol oxidase obtained from various sources by 2,3‐diaminopropionic acid

This paper reports for the first time the inhibition of the catecholase activities of mushroom, artichoke (Cynara scolymus L) and Ocimum basilicum L polyphenol oxidase by 2,3‐diaminopropionic acid. Polyphenol oxidases from artichoke and O basilicum L were purified by ammonium sulfate precipitation, dialysis and a Sepharose 4B‐L ‐tyrosine‐p‐aminobenzoic acid‐affinity column. In inhibition studies, 2,3‐diaminopropionic acid showed uncompetitive inhibition for mushroom PPO using catechol and pyrogallol as substrates, competitive inhibition for O basilicum L PPO using catechol as a substrate, and uncompetitive inhibition for artichoke PPO using catechol as a substrate. Furthermore, sodium azide, which is an inhibitor of PPO, was used as an inhibitor for comparison with the inhibition potency of 2,3‐diaminopropionic acid. The highest 2,3‐diaminopropionic acid inhibition observed with O basilicum L (K_i = 0.89 mM ), followed by artichoke (K_i = 1.42 mM ) and mushroom (K_i = 2.47 mM ), respectively. Copyright © 2005 Society of Chemical Industry     

Prevention of Enzymatic Browning of Chinese Yam (Dioscorea spp.) Using Electrolyzed Oxidizing Water

In this study, the effects of electrolyzed oxidizing water (EOW) on the prevention of enzymatic browning of fresh‐cut “Jiu Jinhuang” Chinese yam were investigated. The yams were immersed in the inhibitors for 25 min at 20 °C. Compared with the tap water (TW) treatment, the chromatic attributes were significantly different after 72 h of storage (P < 0.05). The activities of polyphenol oxidase (PPO, EC 1.10.3.1), peroxidase (POD, EC 1.11.1.7), and _L‐phenylalanine ammonia lyase (PAL, EC 4.3.1.5) were inhibited when measured at 24 h. The contents of phenolic acids, including gallic and chlorogenic acid, in the group treated with the slightly acidic electrolyzed water (SAEW) were higher than those treated with TW and neutral electrolyzed water (NEW). The group treated with NEW had the highest total phenol content (P < 0.05, at 24 h), while the group treated with SAEW had the highest flavonoid content (P < 0.05) during storage. Without being treated with inhibitors, the K_m and V_max values of yam PPO were 0.0044 mol/L and 0.02627 U/min, respectively, and the K_i of samples treated with SAEW and citric acid (CA) were 15.6607 and 2.3969 μmol/L, respectively. These results indicate that EOW is beneficial as a browning inhibitor.     

The response of phenylalanine ammonia‐lyase,polyphenol oxidase and phenols to cold stress in the olive tree (Olea europaea L. cv. Picual)

BACKGROUND: In this study the activities of phenylalanine ammonia‐lyase (PAL) and polyphenol oxidase (PPO) and the concentrations of hydroxytyrosol, tyrosol and oleuropein in olive tree (Olea europaea L. cv. Picual) leaves were investigated before and after cold stress by freezing. The air temperature fell to below ?7 °C and, according to the specific field conditions, four categories of orchard were selected: not cold stressed (NS), lightly cold stressed (LS), moderately cold stressed (MS) and heavily cold stressed (HS). RESULTS: In LS and MS samples the PAL specific activity at saturated substrate concentration rose 4.8‐ and 1.9‐fold respectively compared with NS samples. In HS samples the PAL activity declined by 47% compared with NS samples. A low level of PAL protein was detected in all samples affected by cold stress. In LS, MS and HS samples the PPO specific activity at saturated substrate concentration was 1.9‐, 4.4‐ and 9.8‐fold higher respectively than in NS samples. K_m values also increased after cold stress. In MS and HS samples the concentration of oleuropein was 69 and 82% higher respectively than in NS samples, while the concentrations of hydroxytyrosol and tyrosol decreased. CONCLUSION: The response of PAL activity may be part of a recovery process of the olive leaf against cold stress, while the response of PPO and oleuropein may be part of an antioxidant protection mechanism. Copyright © 2009 Society of Chemical Industry     

Characterization of the recombinant succinic semi‐aldehyde dehydrogenase from Saccharomyces cerevisiae

The yeast succinic semi‐aldehyde dehydrogenase gene (SSADH; EC 1.2.1.16) was cloned and overexpressed in Escherichia coli. Based on SDS–PAGE, the molecular mass of the subunit was around 54 kDa, and the purified recombinant enzyme had a tetrameric molecular mass of ca. 200 kDa. The specific activity of the recombinant enzyme was 1.90 µm NADH formed/min/mg, and showed maximal activity at pH 8.4. The recombinant protein was highly specific for succinate semi‐aldehyde (K_m = 15.48 ± 0.14 µm ) and could use both NAD⁺ and NADP⁺ as co‐factors, with K_m values of 579.06 ± 30.1 µm and 1.017 ± 0.46 mm, respectively. Initial velocity studies showed that NADH was a competitive inhibitor with respect to NAD⁺ (K_i = 129.5 µm ) but a non‐competitive inhibitor with respect to succinate semi‐aldehyde. Adenine nucleotides of AMP, ADP and ATP inhibited yeast SSADH activity with K_i = 1.13–10.2 mm, and showed competitive inhibition with respect to NAD⁺ and mixed‐competitive, non‐competitive and non‐competitive inhibition, respectively, with respect to succinate semi‐aldehyde. The kinetic data suggest a 'ping‐pong' mechanism. Copyright © 2014 John Wiley & Sons, Ltd.     

Purification and Biochemical Characterization of Peroxidase Isolated from White Cabbage (Brassica Oleracea var. capitata f. alba)

Peroxidase enzyme was purified for the first time from white cabbage (Brassica oleracea var. capitata f. alba) in a single step using affinity chromatography and some biochemical characteristics of the purified enzyme were determined. The peroxidase was purified 24.7-fold with an overall recovery of 4.3% and a specific activity of 964.5. The molecular weight of the purified peroxidase was approximately 73.2 kDa as calculated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and it showed maximum activity at pH 6.5 and 30°C. For the guaiacol substrate, the K_M and V_max values were found as 3.19 mM and 0.2 EU/mL, respectively. Additionally, the IC₅₀ and K_i values were determined as 0.517 and 0.994 ± 0.453 mM, respectively, for 4-aminobenzohydrazide. 4-amino benzohydrazide showed non-competitive inhibition.     

Inhibitory effects of selected pesticides on peroxidases purified by affinity chromatography

The objective of this study was to determine the in vitro inhibition effects of seven commonly used pesticides including 2,4-d-acid dimethylamine, fenoxaprop-p-ethyl, glyphosate isopropylamine, haloxyfop-p-methyl, cypermethrin, λ-cyhalothrin, and dichlorvos on the peroxidase purified from turnip (Brassica rapa L.) and black radish (Raphanus sativus L.) using 4-amino benzohydrazide affinity column chromatography. The purification factors for the turnip and black radish peroxidases were found to be 263.29-fold (with a yield of 12.89%) and 36.20-fold (with a yield of 6.90%), respectively. Among these compounds, λ-cyhalothrin showed the strongest inhibitory effect against turnip peroxidase (K_i: 1.23 × 10^?2 ± 0.21 × 10^?2 mM) as noncompetitive inhibition. On the other hand, cypermethrin demonstrated the highest inhibition effect against black radish peroxidase (K_i: 2.14 × 10^?2 ± 0.08 × 10^?2 mM) as competitive inhibition.     

ENZYMATIC PROPERTIES OF A PROTEASE FROM THE HEPATOPANCREAS OF SHRIMP, PENAEVS OMENTAIM

The protease purified from hepatopancreas of shrimp, Penaeus orientals, had high proteolytic activity in the pH range of 7.0 to 9.5. Temperature optimum for hydrolysis of casein was 70C. The protease was stable at neutral and alkaline pH and unstable at acidic pH. The apparent Michaelis‐Menten constant (K_m) and the turnover number (V_max) of the protease on hydrolysis of N‐CBZ‐L‐tyrosine p‐nitrophenyl ester (CBZ‐Tyr‐NE) and N‐CBZ‐L‐phenylalanine p‐nitrophenyl ester (CBZ‐Phe‐NE) ‐were similar, however, those for N‐CBZ‐L‐cysteine p‐nitropher.yl ester (CBZ‐Cys‐NE) were different. K_m and V_max for hydrolysis of casein by the protease were determined to be 0.31% and 5.21s^‐1, respectively. The N‐terminal sequence of the protease showed higher homology with the collagenase of crab and trypsins from Crustacea. Myosin heavy chain (MHC) was the primary substrate during proteolysis with the protease. Actin/tropomyosin were degraded progressively during 2 h incubation but to a lesser extent than MHC.     

Partial characterization of polyphenoloxidase from a hybridized wheat (Triticum aestivum L.)

Polyphenol oxidase was extracted and partially purified from wheat leaves by a procedure that included ammonium sulfate fractionation followed by dialysis and gel filtration chromatography. These procedures led to 35.21-fold purification with 17.65% recovery. Optimum pH, temperature, and ionic strength were determined with six substrates. Some kinetic properties of the enzyme such as V _max, K _M, and k _cat were calculated for the substrates. The k _cat/K _M values of the PPO for catechol, catechin, pyrogallol, l-dopa, dopamine, and 4-methyl catechol were 31408, 31167, 28404, 15378, 4865, and 4967 mM/min, respectively. The best substrate of wheat PPO was found to be catechol. The native molecular weight of the PPO was estimated to be 243 kDa based on its mobility in gel filtration column. The inhibitory effects of glutathione, sodium azide, ascorbic acid, oxalic acid, l-cysteine, and thiourea on the reaction catalyzed by the enzyme were tested, and I ₅₀ values were estimated to be 8.0 mM, 10.12 mM, 11.18 mM, 77.33 mM, 183 mM, and 413 mM, and K _i constants were also calculated as 0.416 ± 0.244 mM, 0.317 ± 0.208 mM, 0.820 ± 0.111 mM, 13.80 ± 1.179 mM, 14.10 ± 5.069 mM, and 130 ± 62.45 mM, respectively, by means of Lineweaver–Burk graphs. The most effective inhibitor was glutathione. Glutathione, sodium azide, oxalic acid, and thiourea were competitive inhibitors, whereas ascorbic acid and l-cysteine were also noncompetitive inhibitors.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM FRESH-CUT CHINESE WATER CHESTNUT

The characteristics of polyphenol oxidase (PPO) from Chinese water chestnut (CWC) and its potential inhibitors for browning reactions were investigated. PPO was isolated from fresh‐cut CWC and was purified on a Sephadex G‐100 column, with a yield of total activity close to 10%. The molecular weight, Michaelis constant (K_m), substrate specificity, optimal pH and temperature of CWC PPO were examined. Kinetic studies indicated that the K_m and V_max values of CWC PPO for catechol were 10.32 mmol/L and 6.452 × 10⁴ U/min, respectively. The optimal pH and temperature for CWC PPO was 6.5 and 40C, respectively. Among the browning inhibitors tested, 4‐hexylresorcinol, at a concentration of 0.3 mmol/L, showed the strongest inhibition (70%) against the PPO activity of CWC, followed by 3.0 mmol/L N‐acetyl‐L‐cysteine with an inhibition of 53%.     

BROWNING INHIBITION AND QUALITY MAINTENANCE OF FRESH-CUT CHINESE WATER CHESTNUT BY ANOXIA TREATMENT

Experiments were conducted to test the effects of anoxia treatments on the quality of fresh‐cut Chinese water chestnut (CWC). Fresh‐cut CWC was exposed to pure N₂ for 0 (control) or 4 h, then placed in trays overwrapped with plastic film and finally stored at 4C. Changes in surface discoloration, eating quality, disease incidence, concentrations of total soluble solids and titratable acidity, activities of phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO), and membrane permeability were analyzed. The short‐term anoxia treatment significantly inhibited surface discoloration and disease development, and markedly reduced the loss of eating quality in association with a higher concentration of total soluble solids. Exposure of fresh‐cut CWC to pure N₂ gas for 4 h significantly reduced membrane permeability, but it increased PAL and PPO activities. Thus, the browning inhibition of the fresh‐cut CWC by the short‐term anoxia treatment may be because of the maintenance of compartmentation between enzymes and their substrates, preventing enzymatic reaction. The result suggests that short‐term anoxia treatment exhibits potential for extending the shelf life of fresh‐cut CWC.     

Screening of phenylalanine ammonia lyase in plant tissues,and retention of activity during dehydration

BACKGROUND: Oral therapy with phenylalanine ammonia lyase (PAL), naturally encapsulated in plant cells, may provide a potential alternative treatment for hyperphenylalaninemic patients, including those with phenylketonuria. Therefore different sources of plant tissue were investigated for PAL activity. RESULTS: Enzyme activity was highest in grain seedlings, with maximal enzyme activity in 7‐day‐old red spring wheat (Triticum aestivum L.) seedlings. The PAL activities of leaves and roots/endosperm of wheat seedlings were 11.90 ± 2.64 and 6.48 ± 1.59 µmol h^?1 g^?1 dry weight respectively. Three PAL‐related polypeptides with molecular weights of 74, 83 and 103 kDa were identified in wheat seedling leaf tissues, while only the 74 kDa polypeptide was detected in root/endosperm tissues. Dehydration was investigated as a method of concentrating PAL in wheat seedlings. Freeze‐drying was found to retain the most PAL activity (>90% recovery on a dry weight basis) compared with air drying and vacuum microwave drying for both leaf and root/endosperm samples. CONCLUSION: This study has led to a better understanding of PAL activity and stability in plant tissues and provides the basis for developing a natural plant preparation as a dietary supplement for the treatment of hyperphenylalaninemia. Copyright © 2007 Society of Chemical Industry     

PARTIAL PURIFICATION AND CHARACTERIZATION OF NEUTRAL TREHALASE FROM COMMERCIAL BAKER'S YEAST, SACCHAROMYCES CEREVISIAE

The neutral trehalase of a commercial baker's yeast (S. cerevisiae) strain has been partially purified using ammonium sidfate fractionation and DEAE‐cellulose column chromatography techniques. Trehalase was precipitated between 35–50% ammonium sulfate saturation and approximately 5–8 fold purification was achieved. The yeast cAMP‐dependent protein kinase was also precipitated in the same fraction and these two proteins were separated by DEAE‐cellulose column chromatography. Trehalase became totally inactive after ion exchange chromatography, “cryptic trehalase” (tre‐c), but was later activated with the addition of partially purified protem kinase together with cAMP and ATP. A 215 fold purification was obtained after DEAE‐ceUulose column chromatography. One mM EDTA caused complete inhibition of the enzyme in crude extract, however the inhibition levels in ammonium sulfate and DEAE‐cellulose fractions were 73.5% and 50%, respectively. Optimal pH range and temperature of the enzyme were determined as pH 6–6.8 and 30C, respectively. The kinetic parameters, K_m and V_max, were estimated as 11.78 mM trehalose and 12.47 μmole glucose/min‐mg protein, respectively.     

The effect of high pressure carbon dioxide on the inactivation kinetics and structural alteration of phenylalanine ammonia-lyase from Chinese water chestnut: An investigation using multi-spectroscopy and molecular docking methods

The effect of high pressure carbon dioxide (HPCD) on the inactivation kinetic and structure of phenylalanine ammonia-lyase (PAL) from Chinese water chestnut (CWC) was studied in this paper. The inactivation kinetic of PAL treated by HPCD (1–4 MPa, 35–55 °C, and 5–60 min) were determined and fitted to the first order kinetics model with calculating kinetic parameters. As revealed by the circular dichroism spectral, the α-helix and β-turn content in secondary structure increased and the β-sheet content decreased. And the intensity of the fluorescence spectra reflecting tertiary structure decreased, together with the λ_max blue-shifted with the increasing pressure. Fluorescence and circular dichroism spectral results indicated that the conformation of PAL was altered by HPCD. The findings of particle size distribution and ζ potential showed that HPCD could cause the aggregates of PAL particles. Moreover, molecular docking indicated the interactions between small molecules (CO₂, H₂CO₃, HCO₃⁻, and CO₃²⁻) and PAL might result in a decrease in PAL activity by forming steric hindrance, preventing substrate from binding. Finally, this paper proposed a potential mechanism for inactivation of PAL by HPCD treatment, where the loss in PAL activity was correlated to changes in secondary and tertiary structure of PAL, which was induced by aggregation effect of HPCD.     

Characterization and inhibition of <Emphasis Type="Italic">Rosmarinus officinalis</Emphasis> L. polyphenoloxidase

Polyphenoloxidase (PPO) from Rosmarinus officinalis L. was fractionated by ammonium sulfate ((NH₄)₂SO₄) precipitation and dialysis, and then some of its kinetic properties such as optimum pH and temperature, substrate specificity, thermal inactivation, and inhibition were investigated using 4-methylcatechol, catechol, and pyrogallol as substrates. The protein content of Rosmarinus officinalis L. extracts was determined according to Bradford’s method. Kinetic parameters, K _m and V _max, were calculated from Lineweaver–Burk plots. According to V _max/K _m ratio, 4-methylcatechol was the most suitable substrate. The optimum temperature and pH values were 20, 30 and 30 °C, and 7, 8 and 8 for 4-methylcatechol, catechol, and pyrogallol substrates, respectively. The thermal inactivation of PPO was investigated at 35, 55, and 75 °C. The enzyme activity decreased with increasing temperature. The effect of different inhibitors on partly purified Rosmarinus officinalis L. PPO was spectrophotometrically investigated. For this purpose, ascorbic acid and l-cysteine were used to inhibit the activity of Rosmarinus officinalis L. PPO at different concentrations. From the experimental results, it was found that l-cysteine is a more effective inhibitor than ascorbic acid due to lower K _i values.     

A preliminary study of monosaccharide composition and α‐glucosidase inhibitory effect of polysaccharides from pumpkin (Cucurbita moschata) fruit

In this work, crude polysaccharide extracts were extracted from pumpkin (Cucurbita moschata) fruit by hot water. After removal of proteins and purification, polysaccharides of pumpkin fruit (PP1‐1) were subjected to structural identification. Gas chromatography analysis indicated that PP1‐1 comprised of galactose (86.4%), and glucose (13.6%). The molecular weight of PP1‐1 was measured to be 0.87 × 10⁴ Da by gel permeation chromatography. The inhibitory kinetic evaluation showed that it was non‐competitive inhibition of PP1‐1 on the α‐glucosidase‐catalysed hydrolysis of PNPG. The Michaelis–Menten constant (K_m) was 0.106 m , and the inhibitory constants (K_i), 0.435 mg.     

L‐Phenylalanine ammonia‐lyase activity and concentration of phenolics in developing olive (Olea europaea L cv Arbequina) fruit grown under different irrigation regimes

Changes in L ‐phenylalanine ammonia‐lyase (PAL, EC 4.3.1.5) activity and total phenolic, ortho‐diphenolic and fat contents of olive flesh in response to different irrigation treatments applied to olive tree cv Arbequina were studied during fruit ripening. Results indicate that the fat content of olive flesh at harvest was not affected by irrigation, although olives from the most heavily irrigated treatment reached their final fat content (dry weight) earlier than those from other irrigation treatments. PAL activity and phenolic content, expressed on a dry weight basis, decreased during fruit development and were affected by irrigation, being lowered as the water supplied increased. Good correlations were established between PAL enzymatic activity and the polyphenol and ortho‐diphenol contents of olive flesh, indicating that PAL is involved in the phenolic metabolism of olive fruit. The phenolic content of the oil depends on the PAL activity in the fruit, which varies with changes in water status. © 2002 Society of Chemical Industry     

Investigation of a wild pear species (Pyrus elaeagnifolia subsp. Elaeagnifolia Pallas) from Antalya,Turkey: polyphenol oxidase properties and anti-xanthine oxidase,anti-urease,and antioxidant activity

Polyphenol oxidase properties and anti-xanthine oxidase, anti-urease, and antioxidant activity were investigated in Pyrus elaeagnifolia subsp. elaeagnifolia Pallas harvested from Antalya, Turkey. Optimum pH and temperature were 7.0 and 30°C, respectively. Selected kinetic properties of polyphenol oxidase was evaluated.The K_m and V_max-values, using 4-methylcatechol as substrate, were calculated as 3.57 mM and 4781 U/mg protein, respectively. IC₅₀-values ranged from 0.0036 to 4.0231 mM against sodium metabisulphite, ascorbic acid, sodium azide, and benzoic acid, while ascorbic acid was the strongest inhibitor. Aquatic extracts of the sample exhibited strong antioxidant capacity and substantial xanthine oxidase and urease inhibition, with IC₅₀-values of 10.75 ± 0.11 and 0.97 ± 0.03 mg/mL, respectively.     

KINETIC STUDIES AND MECHANISM OF CATALYSIS OF MALIC DEHYDROGENASE FROM DIOSCOREA ROTUNDATA TUBER

ABSTRACT The kinetic mechanism of catalysis by malic dehydrogenase (EC 1.1.1.37) isolated from yam (Dioscorea rotundata) tuber has been delineated. Initial velocity studies with the enzyme in the presence and absence of products of the reaction revealed an ordered sequential mechanism. The K_m values obtained from secondary plots were 0.05, 0.08, 0.48 and 2.56 mM for NADH, OAA, and NAD⁺ and L-malic acid, respectively. Product inhibition studies in both the forward and backward reactions support an ordered Bi-Bi sequential mechanism. This begins with an obligatory binding of NAD⁺ to form the first binary complex and a final release of NADH.     

The role of 1‐methylcyclopropene in lignification and expansin gene expression in peeled water bamboo shoot (Zizania caduciflora L.)

BACKGROUND: Water bamboo shoot (WBS) (Zizania caduciflora L.) is a fleshy aquatic vegetable susceptible to lignification. In this study, effects of 1‐methylcyclopropene (1‐MCP) anti‐ethylene treatment on lignification of harvested peeled WBS were investigated. RESULTS: Peeled shoots were treated with 0.5 µL L^?1 1‐MCP for 20 h at 20 °C and then stored at 20 °C for up to 9 days. Sensory quality, lignin content and activities of the lignification‐associated enzymes peroxidase (POD) and phenylalanine ammonia lyase (PAL) were evaluated. Expression of expansin (ZcExp) was also assessed. 1‐MCP application maintained better sensory quality and inhibited the increase in lignin content. Lessened lignification was associated with reduced activities of POD and PAL. Moreover, 1‐MCP‐treated shoots showed lower expression of the ZcExp gene. CONCLUSION: 1‐MCP pretreatment suppressed the synthesis of lignin and thereby delayed lignification in peeled WBS. Copyright © 2011 Society of Chemical Industry