Purification and identification of linoleic acid hydroperoxides generated by soybean seed lipoxygenases 2 and 3

It has been known that lipoxygenase (LOX) isozymes exhibit differences in product formation, but most product information to date is for LOX 1 among soybean (Glycine max) LOX isozymes. In this study, LOXs 2 and 3 were purified and used to generate hydroperoxide (HPOD) products in an in vitro system using linoleic acid as a substrate in the presence of either air or O2. The products were analyzed to determine their stereoisomeric characteristics. The control (no enzyme) showed only low levels of hydroperoxide production and no stereoisomeric specificity. LOX 2 shows high specificity in product formation, producing roughly 4 times more 13-HPOD than 9-HPOD, nearly all of which was 13-S(Z,E)-HPOD. LOX 3 produced more 9-HPOD than 13-HPOD at approximately a 2:1 ratio. No single stereoisomer was predominant among LOX 3 products. These results demonstrate that different isozymes of LOX have characteristic product profiles in in vitro reactions.     

Purification and partial characterization of lipoxygenase from desert truffle (Terfezia claveryi Chatin) ascocarps

A lipoxygenase from Terfezia claveryi Chatin ascocarp, a mycorrhizal hypogeous fungus, is described for the first time. The higher proportion of PUFA in T. claveryi ascocarps makes lipid rancidity the main factor limiting its storage life. Thus, the studies on LOX from T. claveryi are important because this enzyme, among other roles, may be involved in an alteration of lipids leading to consumer rejection. The enzyme has been purified to apparent homogeneity by phase partitioning in the presence of Triton X-114, followed by two steps of cation-exchange chromatography. The purified T. claveryi LOX preparation consisted of a single major band with an apparent molecular mass of 66 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymic activity exhibited a strong specificity toward linoleic and linolenic acids as substrates, while only 32% activity was observed using gamma-linolenic acid. The pH optimum of this enzyme was pH 7.0. When the enzyme reacted with linoleic acid, it produced a single peak, which comigrated with standard 13-hydroperoxy-octadecadienoic acid; 13-hydroperoxy-octadecatrienoic acid was produced during the reaction with linolenic acid.     

桃脂氧合酶(LOX)基因家族cDNA全长克隆与序列分析

根据脂氧合酶保守序列设计的简并引物从桃（瑞蟠5号）果肉总RNA中扩增出795bp的脂氧合酶（lipoxygenase,LOX）基因的保守序列,并结合GeneBank中公开的LOX基因的EST序列经Blast比对分析、序列组装之后设计基因特异引物（GSP）。利用RACE技术共获得3条不同的桃LOX基因cDNA全长序列,分别命名为PpLox1-3。序列分析结果表明,这3个桃LOX基因家族成员与其他植物的LOX基因尽管在核酸水平上的差异比较大,但在蛋白水平上却有较高的同源性。通过对桃LOX基因蛋白序列和其他植物LOX进行系统进化树分析,发现PpLox1属于13-LOX类型,PpLox2-3属于9-LOX类型。     

Characterization and histochemical localization of nonspecific esterase from ascocarps of desert truffle (Terfezia claveryi Chatin)

An esterase activity from Terfezia claveryi Chatin ascocarps, a mycorrhizal hypogeous fungus, is described for the first time. The enzyme was partially purified using phase partitioning in Triton X-114 (TX-114), achieving a reduction of 87% in the triglyceride content and the removal of 63% of phenols. The enzyme showed maximum activity toward short-chain p-nitrophenyl esters, and no interfacial activation was observed, indicating that the enzyme responsible for this activity is an esterase and not a lipase. This esterase presented its maximum activity at pH 7.4 and 60 degrees C. The values obtained for Km at pH 7.4 were 0.3 mM for p-nitrophenyl butyrate and 0.6 mM for p-nitrophenyl acetate with catalytic efficiencies (Vmax/Km) of 0.23 and 0.32, respectively. T. claveryi esterase was inhibited by phenylboric acid, indicating that serine residues were involved in the enzyme activity. This activity was localized only in the hypothecium and was absent from the peridium and gleba.     

Eggplant lipoxygenase (Solanum melongena): product characterization and effect of physicochemical properties of linoleic acid on the enzymatic activity

Lipoxygenase (LOX) from eggplant (Solanum melongena L. cv. Belleza negra) was partially purified, and the products and kinetics of the enzyme were studied. Linoleic acid (LA) was the best substrate for this enzyme. Product analysis by HPLC and GC/MS revealed that, at its pH optimum (pH 7.0), the enzyme converted LA almost totally into the 9-hydroperoxy isomer, whereas the 13-hydroperoxy isomer was only a minor product. At this pH, the enzyme had K(m) and V(max) values for LA of 1.4 microM and 2.2 micromol min(-1) (mg of protein)(-1), respectively, when the monomeric form of LA was used as substrate. The dependence of eggplant LOX activity on the physicochemical properties of LA was also studied. Experiments revealed that LA aggregates were used more efficiently than monomeric LA as substrate. The apparent substrate cooperativity observed may be due to the different activities exhibited toward monomers and aggregates. This result can be interpreted as a substrate-aggregation dependent activity.     

A simple and efficient system for green note compound biogenesis by use of certain lipoxygenase and hydroperoxide lyase sources

Six-carbon (C(6)) aldehydes and alcohols are important components of the aroma and flavor of fruits and vegetables. Soybean lipoxygenase (LOX) isozyme LOX 3 was reported not only to produce less 13-hydroperoxides, precursors of C(6) aldehydes, but also to convert them to ketodiene products. Here, we examined the effects of LOX 3 on hexenal formation from linolenic acid homogenized with watermelon 13-hydroperoxide lyase (HL)-overexpressing Nicotiana tabacum leaves and soybean acetone powder. Compared to the wild type, which contains LOXs 1, 2, and 3, the elimination of LOX 3 in LOX 1 + 2 facilitates greater production of hexenals. The use of LOX 2 alone yielded the highest hexenal production, while a two-step conversion was required for LOX 1 to produce hexenals at high levels due to different pH optima of the enzymes involved. These results clearly demonstrate that the soybeans lacking LOX 3 in combination with watermelon HL-overexpressing leaf tissues greatly enhance hexenal formation.     

Partial purification, characterization, and histochemical localization of fully latent desert truffle (Terfezia claveryi Chatin) polyphenol oxidase

In the present paper, a fully latent polyphenol oxidase (PPO) from desert truffle (Terfezia claveryi Chatin) ascocarps is described for the first time. The enzyme was partially purified by using phase partitioning in Triton X-114 (TX-114). The achieved purification was 2-fold from a crude extract, with a 66% recovery of activity. The interfering lipids were reduced to 13% of the original content. In addition, the purification gave rise to a reduction of phenolic compounds to only 37.5%, thus avoiding the postpurification tanning of the enzyme. Latent PPO was activated by the anionic surfactant sodium dodecyl sulfate (SDS) or by incubation with trypsin. The amount of SDS necessary to obtain a maximum activation was dependent on the nature of the substrate. The use of SDS also permitted the histochemical localization of the latent enzyme within the ascocarp. Terfezia polyphenol oxidase was kinetically characterized using two phenolic substrates (L-DOPA and tert-butylcatechol). The latter substrate presented inhibition at high substrate concentration with a K(si) of 6.3 mM. Different inhibiting agents (kojic and cinnamic acid, mimosine and tropolone) were also studied, tropolone being the most effective.     

Proteolytic activation of latent Paraguaya peach PPO. Characterization of monophenolase activity

The kinetics of the activation process of latent peach PPO by trypsin was studied. By coupling this activation process to the oxidation of 4-tert-butylcatechol (TBC) to its corresponding quinone, it was possible to evaluate the specific rate constant of active PPO formation, k(3), which showed a value of 0.04 s(-1). This proteolytic activation of latent peach PPO permitted us to characterize the monophenolase activity of peach PPO for the first time using p-cresol as substrate, and it showed the characteristic lag period of the kinetic mechanism of monophenols hydroxylation, which depended on the enzyme and substrate concentration, the pH and the presence of catalytic amounts of o-diphenol (4-methylcatechol). The enzyme activation constant, k(act), was 2 microM.     

Activation of a latent mushroom (Agaricus bisporus) tyrosinase isoform by sodium dodecyl sulfate (SDS). Kinetic properties of the SDS-activated isoform.

This study reports the activation of a latent mushroom tyrosinase isoform by sodium dodecyl sulfate (SDS). The activation process of latent mushroom tyrosinase by SDS is characterized by the presence of a lag period (tau) prior to the attainment of a steady-state rate (V(ss)). This could be related to a slow conformational change of the latent enzyme to render the active isoform. The molecular size of the latent isoform was 67 kDa as determined by SDS-PAGE and western-blotting assays. This size did not change after activation by SDS. The molecular size of the protease-activated isoform was 43 kDa. tau and V(ss) displayed a sigmoidal relationship to the concentration of SDS, but tau was not dependent on o-diphenol or enzyme concentration. Increasing SDS concentrations decreased tau, but then lower V(ss) values were detected because of a possible excess of unfolding and subsequent denaturation of the protein. The same reaction mechanism operated in both SDS-activated and protease-activated tyrosinase isoforms despite their different kinetic features. A possible mechanism for the activation of this latent tyrosinase by SDS is proposed.     

Dual positional and stereospecificity of lipoxygenase isoenzymes from germinating barley (green malt): biotransformation of free and esterified linoleic acid

The lipoxygenase isoenzymes LOX1 and LOX2 from green malt were separated by isoelectric focusing, and their catalytic properties regarding complex lipids as substrates were characterized. The regio- and stereoisomers of hydroperoxy octadecadienoates (HPODE) resulting from LOX1 and LOX2 enzymatic transformations of linoleic acid, methyl linoleate, linoleic acid glycerol esters monolinolein, dilinolein, and trilinolein, and 1-palmitoyl-2-linoleoyl-glycero-3-phosphocholine (PamLinGroPCho) were determined. In addition, biotransformations of polar and nonpolar lipids extracted from malt were performed with LOX1 and LOX2. The results show that LOX2 catalyzes the oxidation of esterified fatty acids at a higher rate and is more regioselective than LOX1. The dual position specificity of LOX2 (9-HPODE:13-HPODE) with trilinolein as the substrate (6:94) was higher than the resultant ratio (13:87) when free linoleic acid was transformed. A high (S)-enantiomeric excess of 13-HPODE was analyzed with all esterified substrates confirming the formation of 13-HPODE through the LOX2 enzyme; however, 9-HPODE detected after LOX2 biotransformations showed (R)-enantiomeric excesses. PamLinGroPCho was oxygenated by LOX1 with the highest regio- and stereoselectivities among the applied substrates.     

Effect of titanium ascorbate on the lipoxygenase pathway of tomato and red pepper seedlings

The effect of titanium applied as titanium ascorbate (Titavit^®) on the lipoxygenase (LOX) pathway of tomato and red pepper seedlings was studied. Seeds were germinated in solutions containing titanium at 5 and 10 ppm. The uptake of titanium was significant and occurred at a similar amount for both tomato and red pepper. LOX and hydroperoxide decomposing (HPD) activities were determined in the crude extracts. The results showed that LOX activity in the extract of Titavit‐germinated seedlings was significantly higher than that of the control, whereas no effect was observed on the activity of HPD enzymes. It is suggested that activation of LOX by Titavit was due to a chemical interaction of titanium within the reaction mixture than to an induction of the biosyntheis of the enzyme.     

Soybean lipoxygenase inactivation by pressure at subzero and elevated temperatures

Soybean lipoxygenase (LOX) inactivation [0.4 mg/mL in Tris-HCl buffer (0.01 M, pH 9)] was studied quantitatively under constant pressure (up to 650 MPa) and temperature (-15 to 68 degrees C) conditions and kinetically characterized by rate constants, activation energies, and activation volumes. The irreversible LOX inactivation followed a first-order reaction at all pressure-temperature combinations tested. In the entire pressure-temperature area studied, LOX inactivation rate constants increased with increasing pressure at constant temperature. On the contrary, at constant pressure, the inactivation rate constants showed a minimum around 30 degrees C and could be increased by either a temperature increase or decrease. On the basis of the calculated rate constants at 102 pressure-temperature combinations, an iso-rate contour diagram was constructed as a function of pressure and temperature. The pressure-temperature dependence of the LOX inactivation rate constants was described successfully using a modified kinetic model of Hawley.     

Oxidation of salsolinol by banana pulp polyphenol oxidase and its kinetic synergism with dopamine

Salsolinol, a tethrahydroisoquinoline present in banana and biosynthesized from dopamine, was oxidized by banana pulp polyphenol oxidase to its corresponding salsolinol-o-quinone. This oxidation was pH-dependent and showed a maximum at acidic pH values. At physiological pH of 5.0, the values obtained for the kinetic parameter (V(m) and K(m)) were 62.5 microM/min and 1.7 mM, respectively. When dopamine was added to the reaction medium to imitate physiological conditions, salsolinol was co-oxidized by dopamine-quinone. When this phenomenon was studied oxygraphically, an unexpected activation of dopamine oxidation was found in the presence of salsolinol. This activation was related with the enzyme's kinetic mechanism and was named "kinetic synergism", because a bad substrate activated a good one. A possible physiological role is discussed.     

Diphenol activation of the monophenolase and diphenolase activities of field bean (Dolichos lablab) polyphenol oxidase

This paper reports a study on the hydroxylation of ferulic acid and tyrosine by field bean (Dolichos lablab) polyphenol oxidase, a reaction that does not take place without the addition of catechol. A lag period similar to the characteristic lag of tyrosinase activity was observed, the length of which decreased with increasing catechol concentration and increased with increasing ferulic acid concentration. The activation constant K(a) of catechol for ferulic acid hydroxylation reaction was 5 mM. The kinetic parameters of field bean polyphenol oxidase toward ferulic acid and tyrosine were evaluated in the presence of catechol. 4-Methyl catechol, L-dihydroxyphenylalanine, pyrogallol, and 2,3,4-trihydroxybenzoic acid, substrates with high binding affinity to field bean polyphenol oxidase, could stimulate this hydroxylation reaction. In contrast, diphenols such as protocatechuic acid, gallic acid, chlorogenic acid, and caffeic acid, which were not substrates for the oxidation reaction, were unable to bring about this activation. It is most likely that only o-diphenols that are substrates for the diphenolase serve as cosubstrates by donating electrons at the active site for the monophenolase activity. The reaction mechanism for this activation is consistent with that proposed for tyrosinase (Sanchez-Ferrer, A.; Rodriguez-Lopez, J. N.; Garcia-Canovas, F.; Garcia-Carmona, F. Biochim. Biophys. Acta 1995, 1247, 1-11). The presence of o-diphenols, viz. catechol, L-dihydroxyphenylalanine, and 4-methyl catechol, is also necessary for the oxidation of the diphenols, caffeic acid, and catechin to their quinones by the field bean polyphenol oxidase. This oxidation reaction occurs immediately with no lag period and does not occur without the addition of diphenol. The kinetic parameters for caffeic acid (K(m) = 0.08 mM, V(max) = 32440 u/mg) in the presence of catechol and the activation constant K(a) of catechol (4.6 mM) for this reaction were enumerated. The absence of a lag period for this reaction indicates that the diphenol mechanism of diphenolase activation differs from the way in which the same o-diphenols activate the monophenolase activity.     

Kinetics of activation of latent mushroom (Agaricus bisporus) tyrosinase by benzyl alcohol.

A latent isoform of Agaricus bisporus tyrosinase has been isolated and activated by benzyl alcohol, one of the major volatile compounds in mushrooms of this genus. The progress curve that describes the activation process reached the steady-state rate (V(ss)) after a lag period (tau). The rate of active tyrosinase formation was calculated by coupling the oxidation of o-diphenols to the activation process. V(ss) depended on benzyl alcohol, o-diphenol, and latent tyrosinase concentrations. The lag period depended on benzyl alcohol concentrations but not on o-diphenol and enzyme concentrations. The size of the latent mushroom tyrosinase was 67 kDa, determined by SDS-PAGE and Western blotting assays. This size was not modified after activation by benzyl alcohol. The presence of a lag period and the lack of change of the molecular mass of the protein after activation could indicate a slow conformational change of the protein to render the final active form. The values of the kinetic constants V(max) and K(m) on the o-diphenols 4-tert-butylcatechol, L-DOPA, and dopamine were different between the latent tyrosinase activated by benzyl alcohol and the commercial tyrosinase. They might indicate that a different final active tyrosinase, depending on the activator used, could arise.     

Influence of Barley and Malt Storage on Lipoxygenase Reaction

The dioxygenation of linoleic acid (LA) by aqueous flour suspensions of barley and malting samples was studied. The rate of this lipoxygenase (LOX) reaction varied as the malting process proceeded, giving a characteristic LOX reaction profile for a malting. The differences in the profiles from one malting to another were dramatic. It also appeared that during storage of dry, intact kernel samples from a single malting, a reduction in the rate of LOX reaction always occurred, and the rates of reduction with time were dependent on the stage of malting at the time of sampling. The kinetics of this aging could roughly be divided into four categories representing different stages of malting. Consequently, greatly varying LOX reaction profiles can be obtained from a single malting depending on the time of storage of kernels before assays. The results indicate that steeping, germination and the subsequent drying render the state of kernels unstable with respect to the LOX reaction for at least two to three weeks. Homogeneity of malt quality is important in the further applications of malt, especially in the brewing industry. Therefore, the rate of LOX reaction should be considered as a quality factor of malt.     

Kinetic study of the activation process of a latent mushroom (Agaricus bisporus) tyrosinase by serine proteases.

Latent mushroom tyrosinase can be considered as a zymogen when activated by proteases because the activation process fulfilled all of the kinetic dependencies predicted by a theoretical zymogen activation model previously reported. The activation was studied under two assay conditions: high and low ratio of latent tyrosinase/serine protease (trypsin and subtilisin Carlsberg) concentrations, in the presence and in the absence of a serine protease inhibitor (aprotinin). The size of the latent enzyme was 67 kDa, determined by denaturing SDS-PAGE electrophoresis and Western blot assays. After proteolytic activation, the size was 43 kDa, with an intermediate band of 58 kDa. The values of the catalytic () and Michaelis () constants for the active forms of tyrosinase resulting from the activation by subtilisin, trypsin, or sodium dodecyl sulfate on the substrate tert-butylcatechol were slightly different, which could support the idea of "one activator-one different active tyrosinase". Vacuum infiltration experiments tried to reproduce in vivo the role of mushroom serine proteases in the activation of latent tyrosinase. The use of serine protease inhibitors is proposed as a new alternative tool to prevent melanin formation.     

Lipoxygenase from banana leaf: purification and characterization of an enzyme that catalyzes linoleic acid oxygenation at the 9-position

The objective of the present study was to purify and characterize the lipoxygenase (LOX) from banana leaf (Giant Cavendishii, AAA), an unutilized bioresource. LOX was extracted, isolated, and purified 327-fold using 25-50% saturation of ammonium sulfate fractionation, hydroxyapatite column separation, and gel filtration on Superdex 200. The molecular mass of the purified LOX was 85 kDa, K(m) was 0.15 mM, and V(max) was 2.4 microM/min.mg using linoleic acid as substrate. Triton X-100 was required in the extraction medium; otherwise, no LOX activity was detected. LOX activity increased with the concentration of Triton X-100 with an optimum at 0.1%. The optimal pH of the purified LOX from banana leaf was 6.2, and optimal temperature was 40 degrees C. The LOX showed the highest reactivity toward 18:2 followed by 18:3 and 20:4. A very low reaction rate was observed toward 20:5 and 22:6. On the basis of retention time in normal phase HPLC, the products of 18:2 or 18:3 catalyzed by purified LOX were hydroperoxyoctadecadienoic acid or hydroperoxyoctadecatrienoic acid. It seems that 9-LOX is the predominant enzyme in banana leaf. Banada leaf dried at 110 degrees C for 2 h developed algal aroma. Banana leaf extract stored at 10 degrees C for 12 h formed an oolong tea-like flavor. Banana leaf extract reacted with 18:2 or soybean oil pretreated with bacterial lipase produced green and melon-like aroma, whereas the same reaction with 18:3 produced a sweet, fruity, cucumber-like flavor note.     

Lipoxygenase distribution in coffee (Coffea arabica L.) berries

In this paper lipoxygenase (LOX) presence was investigated in coffee berries to determine its involvement in lipid degradative metabolism of plants grown in organic and conventional cultivations. An immunochemical analysis has evidenced a ca. 80 kDa protein, cross-reacting with an anti-LOX antibody, only in the pulp fraction of berries obtained from plants of both cultivations. LOX activity in this fraction could be monitored either as conjugated diene formation or reaction products (determined by HPLC) and was mainly associated with a heavy membrane fraction (HMF, enriched in tonoplast, endoplasmic reticulum, plasma membrane, and mitochondria) and a light membrane fraction (LMF, enriched in plasma membrane and endoplasmic reticulum, with low levels of tonoplast and mitochondria). The LOX activity of LMF from berries of both cultivations showed an optimum at pH 8.0. The HMF exhibited a different activity peak in samples from conventional (pH 8.0) and organic (pH 5.5) cultures, suggesting the presence of different isoenzymes. These findings were also confirmed by variation of the ratio of 9- and 13-hydroperoxides in organic (1:1) and conventional cultivations (1:10), indicating that the organic one was subjected to an oxidative stress in the coffee pulp fraction leading to the expression of an acidic LOX. Such de novo synthesized LOX activity could be responsible for the production of secondary metabolites, which may interfere with the organoleptic profile of coffee.     

Ascorbic acid-induced browning of (+)-catechin in a model wine system

The ability of ascorbic acid to induce browning of (+)-catechin in a model wine system has been studied. A significant increase in absorbance at 440 nm was observed over 14 days when ascorbic acid was incubated at 45 degrees C with (+)-catechin in a model wine base. The onset of browning was delayed for about 2 days, although the length of the lag period was dependent on the amount of molecular oxygen in the headspace of the reaction system. The lag period was not observed when a preoxidized solution of ascorbic acid was used, suggesting that a product of ascorbic acid oxidation is responsible for the onset of browning. Hydrogen peroxide, when added directly to (+)-catechin in the model system, was not capable of producing the same degree of browning as that generated by ascorbic acid. Liquid chromotography evidence is presented to show that different reaction products are produced by ascorbic acid and hydrogen peroxide.