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.     

Interaction between chloroplast pigments and lipoxygenase enzymatic extract of olives.

Lipoxygenase activity in olive fruits increases considerably when the enzyme is extracted using extraction buffer with ethylenediaminetetraacetate, Triton X-100, dithiothreitol, sodium meta-bisulfite, and hydrated polyvinylpolypyrrolidone. In the absence of these compounds the activity measured of the crude extract is almost negligible, and further effects of the enzymatic reaction, such as bleaching activity on chloroplast pigments, do not appear. Moreover, when the oxidizing level of the medium is increased by the addition of linoleic acid or soybean lipoxygenase with linoleic acid, the crude enzymatic extracts of olives reduce the destructive capacity of soybean lipoxygenase on chlorophylls to one-sixth. These results suggest a double pigment protection against lipoxygenase in the olive crude extract, one inhibiting the enzyme and the other interrupting the chain of oxidative reactions beginning with hydroperoxide formation.     

Effect of titanium (IV) application on some enzymatic activities in several developing stages of red pepper plants

The effect of two titanium (IV) compounds, titanium ascorbate and titanium chloride, on some enzymatic activities, such as catalase, peroxidase, lipoxygenase and nitrate reductase in seeds, embryos, and seedlings and adult plants of red pepper (Capsicum annuum L.), was studied. A stimulatory effect of titanium was observed for every iron‐depending enzyme studied at all developing stages as well as for nitrate reductase but only for whole plants. This occurs because Ti⁺³/Ti⁺⁴ catalizes the activity of iron and enhances iron‐dependent enzymes.     

Determination of lipoxygenase activity in plant extracts using a modified ferrous oxidation-xylenol orange assay

The ferrous oxidation-xylenol orange (FOX) assay method for determination of lipid hydroperoxides is based on that under acidic conditions Fe2+ is oxidized to Fe3+, which then oxidizes xylenol orange to a product that absorb at 550 nm. The procedure has been adapted for determination of lipoxygenase activity in plant extracts. This enzyme is responsible for generation of off-flavors in vegetal foods, bleaching of pigments, and a lot of oxidative degradations. It is of interest to check the initial lipoxygenase activity in vegetal foods before the processing, using an assay that is rapid, reproducible, and easily adaptable to high throughput format. The enzymatic assay is based on a discontinuous determination of lipoxygenase activity using the FOX reagent for colorimetric determination of hydroperoxides accumulated in the medium by a period of incubation that is established by the addition of the extract (start of the reaction) and the addition of FOX reagent (finish of the reaction). The procedure is capable of detecting lipoxygenase activity in a number of vegetable homogenates, being especially useful for a rapid visual evaluation of this enzymatic activity.     

Purification and characterization of a lipoxygenase enzyme from durum wheat semolina.

Purification of a lipoxygenase enzyme from the cultivar Tresor of durum wheat semolina (Triticum turgidum var. durum Desf) was reinvestigated furnishing a new procedure. The 895-fold purified homogeneous enzyme showed a monomeric structure with a molecular mass of 95 +/- 5 kDa. Among the substrates tested, linoleic acid showed the highest k(cat)/K(m) value; a beta-carotene bleaching activity was also detected. The enzyme optimal activity was at pH 6. 8 on linoleic acid as substrate and at pH 5.2 for the bleaching activity on beta-carotene, both assayed at 25 degrees C. The dependence of lipoxygenase activity on temperature showed a maximum at 40 degrees C for linoleic acid and at 60 degrees C for bleaching activity on beta-carotene. The amino acid composition showed the presence of only one tryptophan residue per monomer. Far-UV circular dichroism studies carried out at 25 degrees C in acidic, neutral, and basic regions revealed that the protein possesses a secondary structure content with a high percentage of alpha- and beta-structures. Near-UV circular dichroism, at 25 degrees C and at the same pH values, pointed out a strong perturbation of the tertiary structure in the acidic and basic regions compared to the neutral pH condition. Moreover, far-UV CD spectra studying the effects of the temperature on alpha-helix content revealed that the melting point of the alpha-helix is at 60 degrees C at pH 5.0, whereas it was at 50 degrees C at pH 6.8 and 9.0. The NH(2)-terminal sequence allowed a homology comparison with other lipoxygenase sequences from mammalian and vegetable sources.     

The effect of titanium and gallium on photosynthetic rate of algae

The influence of titanium (Ti) and gallium (Ga) on photosynthesis in synchronized algae (Anacystis nidulans) has been investigated. At moderately low concentrations, Ti and Ga enhanced photosynthetic oxygen evolution and a three‐fold increase in fructose‐1, 6‐bisphosphatase activity. In the presence of Ti, growth as determined by dry weight of Anacystis nidulans was increased, but not so influenced by Ga if the cultures were exposed to continuous illumination.     

Oxidation of resveratrol catalyzed by soybean lipoxygenase

In this work the oxidative degradation of resveratrol catalyzed by lipoxygenase-1 (LOX-1) has been studied. The process has been characterized by spectroscopic and polarographic measurements. The oxidation of resveratrol was dependent on the concentration of resveratrol and the enzyme. When resveratrol was incubated in the presence of lipoxygenase at pH 9.0, the reaction displayed a k(M) value of 18.6 x 10(-)(6) M and a catalytic efficiency (k(cat)/k(M)) of 4.3 x 10(4) s(-)(1) M(-)(1). These values are close to those shown by the enzyme when linoleic acid is used as the substrate. The effect of lipoxygenase inhibitors on the lipoxygenase-catalyzed resveratrol oxidation was also evaluated. The rate of resveratrol oxidation was markedly decreased by the presence of NDGA in the incubation mixture. From HPLC measurements, it can be deduced that resveratrol is oxidatively decomposed to a complex mixture of products similar to those obtained when the molecule is oxidized by hydrogen peroxide.     

Growth response of cabbage plants to transition elements under water culture conditions

Cabbage plants were water-cultured under 4 levels of titanium (0,0,4,4,20 ppm), vanadium (0, 0.4,4, 20 ppm), chromium (0, 0.2, 2, 10 ppm), manganese (0, 0.4, 4, 20 ppm), and iron (0, 1, 10, 50 ppm), and the effects of the elements supplied on the growth of the plants and on the distribution of the elements in the plants were studied.

The dry weight of a whole plant and of inner leaves (yield) without the addition of Ti, V, and Cr did not decrease, while that without the addition of Mn and Fe decreased slightly. The addition of Ti, V, Cr, Mn, and Fe (more than 0.4, 0.4, 2, 4, and 10 ppm, respectively) decreased the yield. Slight transition element-induced chlorosis was observed in the plots at high levels of Ti, Cr, and Mn.

The contents of the elements in the plants increased with an increase in the concentration of each element in the culture solution. The critical contents of Mn and Fe in plant-tops which resulted in a slight decrease in the yield due to the deficient injury were estimated to be about 10 and l00 ppm. The contents of Ti, V, Cr, Mn, and Fe when the yield decreased by 50% due to the excess injury were 4,6, 20, 3,000, and 300 ppm in outer leaves and 3,000, l,000, 2,000, 15,000, and 50,000 ppm in roots, respectively.     

Optimization of Temperature,Time, and Lactic Acid Concentration to Inactivate Lipoxygenase and Lipase and Preserve Phytase Activity in Barley (cv. Blenheim) During Soaking

A barley-soaking process was studied to find conditions that inactivate the prooxidative enzyme lipoxygenase and the lipolytic enzyme lipase but preserve phytase activity to develop possible procedures for production of barley products with potentially high mineral bioavailability and good oxidative stability. Lactic acid concentration, temperature, and soaking time were studied. The study was done using a multivariate experimental design. Lactic acid concentration varied between 0 and 1%, temperature varied between 45 and 70°C, and soaking time varied between 30 and 120 min. Although conditions under which lipoxygenase was inactivated were found, total inactivation of lipase was not obtained. Total lipoxygenase inactivation with <20% remaining lipase activity and >60% remaining phytase activity was reached after soaking in 1% lactic acid at suitable time-temperature combinations of 70–110 min and 53–58°C.     

Resveratrol is a potent inhibitor of the dioxygenase activity of lipoxygenase

Resveratrol is a naturally occurring phytoalexin, present in grapes and other food products, with important antioxidant properties. Although still under debate, it is generally assumed that resveratrol has protective effects against heart diseases and probably tumor development. Lipoxygenase is a dioxygenase with peroxidase activity involved in the synthesis of mediators in inflammatory, atherosclerotic, and carcinogenic processes. Lipoxygenase activity is also involved in the generation of flavors and aromas in foods from animal or vegetal sources. The results presented here show that resveratrol was a potent inhibitor of the dioxygenase activity of lipoxygenase, with an IC(50) = 13 microM. Simultaneously, resveratrol was oxidized by the peroxidase activity of lipoxygenase with a V(max) = 0.28 microM min(-1) and a k(M) = 16.6 microM. Furthermore, oxidized resveratrol was as efficient a lipoxygenase inhibitor as in its reduced form. From the data obtained it can be concluded that both resveratrol and its oxidized form can act as inhibitors of the dioxygenase activity of lipoxygenase. In contrast, the hydroperoxidase activity of lipoxygenase was not inhibited by resveratrol. These results suggest that resveratrol may be used as an antioxidant food additive and as a pharmacological agent to prevent the generation of eicosanoids involved in pathological processes.     

Effect of high-pressure treatment on lipoxygenase activity

Solutions of commercial soybean lipoxygenase (100 microgram/ML in 0.2 M citrate-phosphate and 0.2 M Tris buffer were subjected to pressures of 0.1, 200, 400, and 600 MPa for 20 mm. The enzyme was stable at atmospheric pressure (0.1 MPa) over a wide pH range (5-9). In citrate phosphate buffer, the enzyme had maximum stability over the pH range 58 in untreated samples and after treatment at 200 MPa, but with increasing pressure, the pH stability range become narrower and centered around pH 78. The enzyme was more sensitive to acid than alkali, and at pH 9, it lost virtually all activity after pressurization at 600 MPa for 20 mm in both buffers. The activity of the crude enzyme extracted from tomatoes treated at 200 and 300 MPa for 10 mm was not significantly different from that of the untreated tomatoes, while a pressure of 400 MPa for 10 mm caused a significant decrease in activity and treatment at 600 MPa led to complete and irreversible activity loss. Compared to unpressurized tomatoes, treatment at 600 MPa gave significantly reduced levels of hexanal, cis-3-hexenal, and trans-2-hexenal, which are important contributors to "fresh" tomato flavor, and this was attributed to the inactivation of lipoxygenase.     

Modification of volatile compound profile of virgin olive oil due to hot-water treatment of olive fruit

The effect of hot-water treatments of olive fruits before processing on the biosynthesis of virgin olive oil aroma was investigated by quantifying the variation within the major classes of volatile compounds. Data showed that hot-water treatments gave rise to changes in the volatile aroma profile of virgin olive oil from the three olive cultivars under study, Manzanilla, Picual, and Verdial. Different effects by thermal treatments were observed according to cultivar. In general, these changes are mainly due to a decrease in the contents of C(6) aldehydes and C(5) compounds. Contents of C(6) alcohols and esters remained constant or decreased slightly when the temperature of the treatment was increased. Thus, heat treatments seemed to promote a partial deactivation of the lipoxygenase/hydroperoxide lyase enzyme system, whereas other enzymatic activities, within the lipoxygenase pathway, such as alcohol dehydrogenase and alcohol acyltransferase, remained apparently unaffected as a consequence of heat treatments.     

Response of ATP content, respiration rate and enzyme activities in an arable and a forest soil to nutrient additions

Glucose (C), glucose plus NO₃^- (C+N) or glucose plus NO₃^- plus PO₄^3- (C+N+P) were added to an arable and a forest soil at a single dose, or split into four equal doses over 4 consecutive days, and the response of several enzyme activities, ATP content and respiration rate were monitored for 11 days. ß-Glucosidase activity was reduced in the two soils during the first day by substrate addition. Thereafter, this enzyme activity varied only slightly in the arable soil with reference to the non-amended control, while it increased substantially in the beech forest when C+N and C+N+P were added. Casein-hydrolysing activity increased in the C treatment and decreased after C+N+P addition during the first 4 days in the two soils. After 11 days, protease activity was enhanced in the arable soil when C+N was applied in a split dose. Urease activity decreased during the first 4 h, particularly in the arable soil with the addition of C+N or C+N+P, applied in a single dose, and then continuously increased. Thus, urease responded to high nutrient availability, being firstly repressed or inhibited, and stimulated afterwards. Phosphatase activity was only slightly modified in the arable soil but substantially increased in the beech forest by C+N addition. The presence of P usually decreased phosphatase activity. Arylsulphatase activity was repressed after substrate addition, which was particularly evident in the arable soil. In the beech forest topsoil, C added alone increased this enzyme activity. Significant correlations between ATP content and enzyme activity were only observed for urease in the arable system treated with C+N and in the forest when C was applied in a split dose. The effect of C, C+N and C+N+P addition varied between the arable and the forest soil according to environmental conditions and microbial ecophysiology.     

Sclerotiorin, a novel inhibitor of lipoxygenase from Penicillium frequentans

Foods rich with unsaturated fatty acids are prone to enzymatic and nonenzymatic lipid peroxidation; lipoxygenase, a metalloenzyme and a free radical former, oxidizes polyunsaturated fatty acids and is one of the key enzymes in lipid oxidation. Here, we report sclerotiorin, purified from the fermented broth of Penicillium frequentans, as a potent reversible, uncompetitive inhibitor against soybean lipoxygenase-1 (LOX-1) with a half-maximal value (IC50) of 4.2 microM. The inhibitor also showed an antioxidant property by scavenging free radical with an ED50 of 0.12 microM; in addition, nonenzymatic lipid peroxidation was inhibited with a PD50 value of 64 microM and did not show metal chelation. The observations made in this study suggest that sclerotiorin possibly inhibits LOX in two ways: one, by interacting with the enzyme-substrate complex, and two, as an antioxidant by quenching or trapping the free radical intermediates formed in the enzyme reaction. Sclerotiorin compares well with other known natural and synthetic lipoxygenase inhibitors.     

Responses of extracellular enzymes to simple and complex nutrient inputs

Soil microbes produce extracellular enzymes that mineralize organic matter and release carbon and nutrients in forms that can be assimilated. Economic theories of microbial metabolism predict that enzyme production should increase when simple nutrients are scarce and complex nutrients are abundant; however, resource limitation could also constrain enzyme production. We tested these hypotheses by monitoring enzyme activities and nutrient pools in soil incubations with added simple and complex nutrient compounds. Over 28 days of incubation, we found that an enzyme's activity increased when its target nutrient was present in complex but not simple form, and carbon and nitrogen were available. β-Glucosidase and acid phosphatase activities also increased in treatments where only carbon and nitrogen were added. Glycine aminopeptidase and acid phosphatase activities declined in response to ammonium and phosphate additions, respectively. In some cases, mineralization responses paralleled changes in enzyme activity—for example, β-glucosidase activity increased and respiration was 5-fold greater in soil incubations with added cellulose, ammonium, and phosphate. However, a doubling of acid phosphatase activity in response to collagen addition was not associated with any changes in phosphorus mineralization. Our results indicate that microbes produce enzymes according to ‘economic rules’, but a substantial pool of mineral stabilized or constitutive enzymes mediates this response. Enzyme allocation patterns reflect microbial nutrient demands and may allow microbes to acquire limiting nutrients from complex substrates available in the soil.     

Enzyme activity as an indicator of soil quality changes in degraded cultivated Acrisols in the Mexican Trans‐volcanic Belt

Soils located at the Mexican Trans‐Volcanic Belt (MTB) have a worrying degree of degradation due to inappropriate management practices. Early indicators of soil changes are very useful to alert about negative impacts of wrong managements on these volcanic soils. The aim of this work was to evaluate the short‐term effects (4 years) of different agricultural practices on soil organic matter (SOM) quality and to validate the potential of the selected biochemical properties as optimal early indicators of soil quality in Mexican cultivated Acrisols. During 2002–2005 four agronomic management systems: conventional (Tc); improved conventional (Ti); organic (To) and fallow (Tf) were assayed in plots located at the MTB. An uncultivated soil under grass cover (Sg) was used as reference. Soil samples were collected at 0–10 cm depth and were analysed chemically (soil organic C, total N, water‐soluble C and humic C), and biochemically (total and extra‐cellular enzyme activity). After 4 years, soil organic C, total N, water‐soluble C, and dehydrogenase activity had higher values in To, followed by Ti treatment. A similar response pattern was observed in the extra‐cellular enzyme activity. The highest total enzyme activity was found in Sg, followed by Ti and To treatments, and the lowest values appeared in Tc and Tf. To and Ti increased SOM contents of the degraded Acrisols studied, while Tc and Tf managements decreased the quality of these soils. The results showed that the assayed soil enzymes can be used as indicators of quality changes of these Mexican volcanic soils. Copyright © 2010 John Wiley & Sons, Ltd.     

Functional properties, lipoxygenase activity, and health aspects of Lupinus albus protein isolates

To utilize lupin seeds for food and pharmaceutical applications, lupin seeds were pretreated to remove oil using hexane or carbon dioxide. Two types of lupin protein isolate were prepared. Both types of protein isolate showed good foaming activity, comparable to egg white. Protein isolate extracted under acid conditions showed higher foaming activity than protein isolate extracted at neutral pH. The lipoxygenase activity was much reduced in both of the protein isolates. The protein isolate extracted at neutral pH showed a stronger angiotensin converting enzyme inhibition than the protein isolate extracted under acidic pH. In contrast, the protein isolate extracted under acid conditions had a greater sodium cholate binding capacity, comparable to that of cholestyramine. Lupin samples showed less DPPH radical scavenging activity than deoiled soybean. The deoiling method did not affect the functional properties, lipoxygenase activity, angiotensin converting enzyme inhibition, sodium cholate binding, and radical scavenging activity.     

Effect of pepper lipoxygenase activity and its linked reactions on pigments of the pepper fruit.

The products formed during the enzymatic reaction catalyzed by the lipoxygenase of pepper (variety Agridulce) have in vitro a strong destructive action on the carotenoid pigments of the fruit. When conditions and proportions of enzyme and pigments are similar to those found in the fruit, and at a reaction temperature of 20 degrees C, almost 30% of the pigments are destroyed after 24 h of reaction. Of this amount, 2.5% is due to autoxidation of pigments, 4. 5% to oxidation induced by the presence of linoleic under saturating conditions, and the remaining 22% to the presence in the medium of reaction products of the lipoxygenase-catalyzed reaction. When the enzyme acts under substrate-saturating conditions, the rate of pigment destruction by lipoxygenase can be considered maximal at the experimental temperature. The fact that in vitro pepper lipoxygenase induces a heavy destruction of pigments and that, in vivo, its activity remains almost constant during over-ripening could explain why up to 40% of the pigment content in some varieties is lost during the postharvest period.     

Activity and stability of urease-hydroxyapatite and urease-hydroxyapatite-humic acid complexes

In agricultural calcareous soils, hydroxyapatite (APA) may well represent an important support for urease immobilisation and could be present in both mineral and organo-mineral complexes. In this paper we studied the formation of APA-urease-humic acid (HA) complexes after the addition of urease either before or after HAs. We then proceeded to evaluate the role of HAs on the activity and stability of the complexes as compared to the APA-urease complexes and free urease. When increasing amounts of HAs were added after urease, they did not affect the activity of the enzymes that had already adsorbed onto the complexes. On the contrary, adding the same amount of HA before the enzyme caused a significant reduction in the amount of enzyme adsorbed. However, when urease adsorption onto the APA-HA complexes was carried out in the presence of NaCl, the enzyme activity of the complexes increased sharply to 86% of the initial activity. The immobilisation of the enzyme on the support increased urease stability against pronase treatment as well as directly in soil over time. The inhibition of urease activity by Cu ²⁺was reduced by urease immobilisation. However, the presence of HA did not alter the stabilisation capability of APA when alone.     

响应面法优化重组枯草芽孢杆菌产脂肪氧合酶条件

采用Plackett-Burman设计法,对影响重组枯草芽孢杆菌发酵产脂肪氧合酶的7个相关因子进行筛选,并建立枯草芽孢杆菌产脂肪氧合酶的最佳工艺。结果表明对重组菌发酵产酶具有显著影响的因子是乳糖、发酵时间和发酵温度。依据Plackett-Burman设计法的筛选结果,进一步采用响应面中心组合设计(Central Composite Design)对显著影响因子的最佳水平和交互作用进行研究,通过模型方程的3-D图和等高线图发现,当乳糖浓度为16.9g/L、发酵时间为88.49 h、发酵温度为29.71℃和装液量为61.31ml时,出现酶活最大预测值164.90 U/ml,验证试验证实了在上述条件下得出的酶活为167.32U/ml,与预测值高度吻合。优化后的培养基和培养条件使得重组脂肪氧合酶产量较优化前提高了2.37倍。