Pentane production by peanut lipoxygenase

Pentane and hexanal were the major volatile end-products of a peanut lipoxygenase and linoleic acid model system and were produced by both crude and purified enzyme preparations. The enzyme system did not require an anerobic condition for the production of pentane and hexanal, thus distinguishing it from other reported systems. A 122-fold purification of the enzyme was achieved.     

Anaerobic lipoxygenase activity from Chlorella pyrenoidosa responsible for the cleavage of the 13‐hydroperoxides of linoleic and linolenic acids

An enzyme from the alga Chlorella pyrenoidosa, previously identified as a hydroperoxide lyase (HPLS), cleaves the 13‐hydroperoxide derivatives of linoleic and linolenic acids into a volatile C5 fragment and a C13 oxo‐product, 13‐oxo‐9(Z),11(E)tridecadienoic acid (13‐OTA). Gas chromatography/mass spectrometry (GC/MS) headspace analysis of the volatile products indicated the formation of pentane when the substrate was the 13‐hydroperoxide derivative of linoleic acid, whereas a more complex mixture of hydrocarbons was formed when the 13‐hydroperoxide derivative of linolenic acid was the substrate. Analysis of the nonvolatile products by GC/MS and liquid chromatography/mass spectrometry (LC/MS) indicated the formation of 13‐OTA along with the 13‐ketone derivative. This enzymatic activity was inhibited by oxygen but was restored with nitrogen. The enzymatic cleavage activity was coincidental in purified fractions with lipoxygenase activity that produced the 13‐ and 9‐hydroperoxide derivatives of linolenic acid. The results suggest that the enzymatic cleavage activity in Chlorella pyrenoidosa was not a consequence of hydroperoxide lyase activity as previously thought, but was due to anaerobic lipoxygenase activity. This enzyme fraction was purified by (NH₄)₂ SO₄ precipitation, gel filtration, and hydrophobic interaction chromatography. The purified enzyme has an approximate MW of 120 KDa and maximum activity at pH 8.0.     

Aerobic pentane production by soybean lipoxygenase isozymes

The effects of oxygen on production of pentane and compounds absorbing at 234 nm and 285 nm by soybean lipoxygenase isozymes I and II were examined in a model system. Aerobic conditions increased pentane production. Differences in dienone formation (A₂₈₅) and diene conjugation (A₂₃₄) indicate the reaction sequences of the 2 isozymes are not the same. Paper No. 4551, Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh North Carolina.     

Determination of ethane and pentane in free oxygen radical-induced lipid peroxidation

It has been proposed that ethane and pentane reflect free oxygen radical-induced lipid peroxidation. However, methodological difficulties limit the use of these gases for assessment of free oxygen radical activity. In the present report we describe an improved method for the accurate analysis of picomole quantities (≥1 pmol) of ethane and pentane. They are first quantitatively trapped into an adsorbent and then heat-desorbed directly into a capillary column for gas chromatographic quantitation. During oxidation of linolenic (n−3) and linoleic (n−6) acid, ethane and pentane were formed, respectively. Nonstimulated granulocytes formed pentane. Upon addition of phorbol 13-myristate 12-acetate, the generation of pentane was increased by 540%. Addition of superoxide dismutase plus catalase inhibited lipid peroxidation in both a cell-free system and in isolated cells. The present method is useful in the evaluation of free oxygen radical induced damage.     

Formation of tetramethylethylene radical cations after pentane adsorption on sulfated zirconia

Tetramethylethylene radical cations have been registered by ESR after pentane adsorption on sulfated zirconia. The radical cations proved to be not stable in the presence of oxygen, only molecular oxygen radical anions being registered. Tetramethylethylene formation and pentane disproportionation are shown to occur under illumination within the same spectral region proving that the former is formed as a by-product of pentane transformations.     

Fatty acid hydroperoxide lyase: a plant cytochrome p450 enzyme involved in wound healing and pest resistance

Plants continuously have to defend themselves against life-threatening events such as drought, mechanical damage, temperature stress, and potential pathogens. Nowadays, more and more similarities between the defense mechanism of plants and that of animals are being discovered. In both cases, the lipoxygenase pathway plays an important role. In plants, products of this pathway are involved in wound healing, pest resistance, and signaling, or they have antimicrobial and antifungal activity. The first step in the lipoxygenase pathway is the reaction of linoleic or linolenic acids with molecular oxygen, catalyzed by the enzyme lipoxygenase. The hydroperoxy fatty acids thus formed are highly reactive and dangerous for the plant and therefore further metabolized by other enzymes such as allene oxide synthase, hydroperoxide lyase, peroxygenase, or divinyl ether synthase. Recently, these enzymes have been characterized as a special class of cytochrome P450 enzymes. Hydroperoxide lyases cleave the lipoxygenase products, resulting in the formation of omega-oxo acids and volatile C6- and C9-aldehydes and -alcohols. These compounds are major contributors to the characteristic "fresh green" odor of fruit and vegetables. They are widely used as food flavors, for example, to restore the freshness of food after sterilization processes. The low abundance of these compounds in nature and the high demand make it necessary to synthesize them on a large scale. Lipoxygenase and hydroperoxide lyase are suitable biocatalysts for the production of "natural" food flavors. In contrast to lipoxygenase, which has been extensively studied, little is yet known about hydroperoxide lyase. Hydroperoxide lyases from different organisms have been isolated, and a few genes have been published lately. However, the structure and reaction mechanism of this enzyme are still unclear. The identification of this enzyme as a cytochrome P450 sheds new light on its structure and possible reaction mechanism, whereas recombinant expression brings a biocatalytic application into sight.     

Calcium activation of peanut lipoxygenase

Peanut lipoxygenase isozyme 1 (pH optimum, 8.3) was strongly activated by 0.5–1.0 mM Ca⁺⁺, and the rate of activation was maximum when the ratio of substrate to Ca⁺⁺ was ca. 2∶1. Peanut lipoxygenase isozymes 2 and 3 (pH optima, 6.2) were activated by calcium but did not have an optimum level of activity. Calcium differentially activated peanut lipoxygenase causing the rate of pentane production to increase much more rapidly than the rate of oxygen consumed by the enzyme reaction. At pH 6.2, in the absence of calcium, the percentages of the hydroperoxide isomers produced by peanut lipoxygenase were 74.9% 13-hydroperoxycis-9,trans-11-octadecadienoic acid (13 LOOHcis-trans), 2.6% 13-hydroperoxytrans-9,trans-11-octadecadienoic acid (13 LOOHtrans-trans) and 22.5% 9-hydroperoxy 10, 12-octadecadienoic acid (9 LOOH). The presence of 1 mM Ca⁺⁺ at pH 6.2 did not significantly affect the percentage distribution of the hydroperoxides produced. However, at pH 8.3, the percentage distribution of hydroperoxides produced was 45.2% 13 LOOHcis-trans, 10.9% 13 LOOHtrans-trans and 43.9% 9 LOOH in the absence of Ca⁺⁺ and 57.0% 13 LOOHcis-trans, 8.0% 13 LOOHtrans-trans and 35.0% 9 LOOH in the presence of 1 mM Ca⁺⁺. Paper No. 5110 of the Journal of the North Carolina Agricultural Experiment Station, Raleight, NC 27607.     

Staining for soybean lipoxygenase activity in electrophoretic gels

A staining method for lipoxygenase in electrophoretic gels is described which is based on the staining of hydroperoxides, as reaction products of added substrates, by N,N-dimethyl-p-phenylenediamine. Soybean lipoxygenase (EC 1.13.11.12) was detected after gel electrophoresis for anodic proteins under non-denaturating conditions and was compared to soybean lipoxygenase stained by Coomassie Brilliant Blue. Specifity of the method was checked by use of heat-denaturated lipoxygenase, tyrosinase and substrate absence. Staining intensity has been found to be proportional to the enzyme activity.     

Protection by different agents against inactivation of lipoxygenase by hydrogen peroxide

H₂O₂ is a potent inactivator of lipoxygenase. In his paper, the ability of different agents [mannitol, oleic, stearic and linoleic acid,n-butanol, and hydroperoxy octadecadienoic acid (HPOD)] to prevent the inactivation of tomato lipoxygenase by hydrogen peroxide has been studied. The involvement of OH^· in the inactivation process is suggested by the ability of mannitol to prevent the loss of activity. This radical would be produced by reaction of H₂O₂ with the Fe(II) lipoxygenase. The most effective protection was displayed by HPOD, the product of the reaction of lipoxygenase with linoleic acid. This result could be explained by the conversion of the native enzyme into the Fe(III) lipoxygenase in the presence of HPOD; the Fe(III) enzyme is not able to react with H₂O₂, and no OH^· will be produced. The protective effect obtained with oleic and stearic acid could be explained by an occupation of the active center by these inhibitors. The enzyme would not transform them, but their presence would hamper the conversion of H₂O₂ in OH^· and limit the damage in the active center.     

Cucumber cotyledon lipoxygenase oxygenizes trilinolein at the lipid/water interface

The reactivity of cucumber cotyledon lipoxygenase with trilinolein was examined. The activity of the enzyme against linoleic acid rapidly decreased with increasing pH of the assay solution, and essentially no activity could be detected above pH 8.5. The rapid decrease in activity was not the result of an inactiveness of the enzyme at alkaline pH, because with trilinolein, the enzyme showed a broad pH-activity profile, and substantial activity could be detected even at pH 9.0. Rather, the decrease in activity was due to the dissociation of the linoleic acid emulsion into acid-soap aggregates and/or the monomeric form, depending on the ionization of the terminal carboxylic group. This suggests that cucumber cotyledon lipoxygenase acts only on an insoluble substrate at the lipid/water interface but not on a soluble one. High-performance liquid chromatography analyses of the products formed from trilinolein revealed that the enzyme inserted oxygen into the acyl moiety of trilinolein without hydrolysis of the ester bonds. Preincubation of the enzyme with triolein emulsions effectively abolished its activity against trilinolein added afterward. Furthermore, the enzyme was adsorbed on the trilinolein or triolein emulsion droplets in an essentially irreversible manner. A reaction velocity curve of the enzyme with trilinolein showed saturation kinetics. This is thought to be due to a regional substrate deficiency as the reaction proceeds. These lines of evidence indicate that the enzyme, once bound to the lipid/water interface, is unable to break free and bind to other emulsions.     

Photoactivation of an inhibitor of the 12/15-lipoxygenase pathway

Lipoxygenases are lipid-peroxidizing enzymes that have been implicated in the pathogenesis of inflammatory diseases and lipoxygenase inhibitors may be developed as anti-inflammatory drugs. Structure comparison with known lipoxygenase inhibitors has suggested that (2Z)-2-(3-benzylidene)-3-oxo-2,3-dihydrobenzo[b]thiophene-7-carboxylic acid methyl ester might inhibit the lipoxygenase pathway but we found that it exhibited only a low inhibitory potency for the pure 12/15-lipoxygenase (IC(50) = 0.7 mM). However, photoactivation, which induces a Z-to-E isomerization of the double bond, strongly augmented the inhibitory potency and an IC(50) value of 0.021 mM was determined for the pure E isomer. Similar isomer-specific differences were observed with the recombinant enzyme and its 12-lipoxygenating Ile418Ala mutant, as well as in intracellular lipoxygenase activity. Structure modeling of the enzyme/inhibitor complex suggested the molecular reasons for this isomer specificity. Since light-induced isomerization may proceed in the skin, such photoreactive compounds might be developed as potential drugs for inflammatory skin diseases.     

Lipoxygenase activity in olive (Olea europaea) fruit

The present work was designed to characterize lipoxygenase activity in olive fruit pulp, in order to determine its significance in the biosynthesis of virgin olive oil aroma. Lipoxygenase activity has been detected in particulate fractions of enzyme extracts from olive pulp subjected to differential centrifugation. The activity in different membrane fractions showed similar properties, with optimal pH in the range of 5.0–5.5 and a clear specificity for linolenic acid, which was oxidized at a rate double that of linoleic acid under the same reaction conditions. The enzyme preparations displayed very low activity with dilinoleoyl phosphatidylcholine, suggesting that olive lipoxygenase acts on nonesterified fatty acids. The enzyme showed regiospecificity for the Δ-13-position of both linoleic and linolenic acid, yielding 75–90% of Δ-13-fatty acid hydroperoxides. Olives showed the highest lipoxygenase activity about 15 wk after anthesis, with a steady decrease during the developmental and ripening periods. Olive lipoxygenase displayed properties that support its involvement in the biogenesis of six-carbon volatile aldehydes, which are major constituents of the aroma of virgin olive oil, during the process of oil extraction.     

Extraction of soybeans with four hydrocarbon solvents

Soybean flakes were extracted in glass extraction apparatus with four hydrocarbon solvents: hexane, isohexane, pentane, and isopentane. The amount of extracted oil was determined at 10 min intervals for 60 min. The extraction rates of the solvents increased in the following order: isopentane, pentane, isohexane, and hexane. Quality and fatty acid composition of the oils extracted by the four solvents showed no significant differences.     

Edible oil quality as measured by thermal release of pentane

As part of their thermal decomposition products, fatty hydroperoxides produce normal hydrocarbons. The extent of hydrocarbon formation can be measured and associated with the quality and potential stability of an oil. Edible oils containing linoleic acid develop 13-hydroperoxy-9,11-octadecadienoic acid as one product of autoxidation. On thermal decomposition this hydroperoxide yields pentane; the amount released has been correlated with the flavor scores of fresh and aged soybean and cottonseed oils and with the peroxide values of these oils. The quantity of pentane released has an inverse linear relationship to flavor score and a direct linear relationship to peroxide values. Edible oils exposed to light exhibit a different relationship between flavor score and thermally derived pentane than do the same oils when autoxidized in the dark. Presented at AOCS Meeting, New Orleans, May 1967.     

Pigments present in virgin olive oil

The qualitative and quantitative control of pigments in ripe olives and in extracted virgin olive oil has increased our knowledge of the influence on these compounds in the areas of ripening of the fruit, storage time in the factory and the oil extraction process. As the harvesting time of the fruits increases, pigment content decreases. During storage, the presence of lipoxygenase has been detected, as well as a considerable decrease in chlorophylls and a small decrease in carotenoids. During the extraction process, the chlorophyllic fraction is destroyed in the greater part, and although the carotenoid fraction is also affected, its concentration increases in the oil with respect to that in the fresh fruit. In the pigment degradation, in addition to the acid-catalyzed reaction, the presence of lipoxygenase suggests a role for this enzyme.     

超临界沥青喷雾造粒中的戊烷闪蒸CFD模拟

重油梯级分离技术作为重油轻质化的新工艺,它通过耦合渣油萃取和沥青喷雾造粒过程,有效的简化重油处理的工艺流程,减少设备成本。在此工艺流程中,处于超临界状态的戊烷的闪蒸速率对沥青造粒质量起到了关键作用。根据其减压过程中的质量和热量传递机理,改进了戊烷闪蒸的经验模型,并将其植入到CFD软件FLUENT中,使其能较为准确的预测闪蒸雾化的非平衡热力学过程。同时对气液戊烷和沥青的三相闪蒸流动过程进行了数值模拟,结果显示喷嘴结构是控制闪蒸速率的关键,直接影响了沥青造粒的质量;此外沥青相的加入为戊烷的相变过程提供了更多能量,提高了戊烷在喷嘴内的汽化率。     

Evidence of an Enzymatic Source of Off Flavors in “Lipoxygenase-Null” Soybeans

The utilization of soybean products as food ingredients and foods is often limited by their beany-grassy flavor. Eliminating seed lipoxygenase (LOX) isozymes 1, 2 and 3 reduces the amounts of volatile off-flavor compounds in stored soybeans and soy products significantly, but they are not completely eliminated. The present work presents evidence that lipoxygenase-null (LOX-null) soybeans contain a LOX-like enzyme that is responsible for the off-flavors in LOX-null soybeans. Volatiles production in triple LOX-null soybeans was terminated by heat treatment, which suggests an enzymatic cause to the off-flavors. The source is LOX-like in that the volatile compounds produced are similar to LOX-generated products of polyunsaturated fatty acids. Oxygen was consumed when a LOX-null protein solution was incubated with crude soybean oil suggesting that the enzyme catalyzed oxygen consuming reactions. The generation of flavor compounds was inhibited by the typical LOX inhibitors propyl gallate and nordihydroguaiaretic acid (NDGA). The enzyme appears to be more active with phosphatidylcholine than with other lipid substrates. The cause of the off-flavors in LOX-null beans appears to have enzyme-like characteristics. This is the first report of the initial characterization of this LOX-like enzyme.     

A device for production of microbial extracellular enzymes in concentrated form

A novel device has been constructed that permits the concentrated production of microbial extracellular enzymes. The apparatus is an all glass structure containing a number of ports, two of which extend to form cylindrical projections, three-quarters the length of the vessel. These projections are highly perforated and do not impede nutrient movement to the inner growth chambers which are constructed of visking tubing. The concentrated enzyme is recovered from the growth chamber after removal of microorganisms by centrifugation. The applicability of this apparatus to studies with different physiological types has been established using Bacillus polymyxa, Serratia marcescens and Streptococcus thermophilus for exo-protease production and Clostridium acetobutylicum for elaboration of exo-amylase. In each instance, the level of enzyme produced was concentrated in a small volume, although the total yield was lower.     

采用填料强化的鼓泡塔直接接触换热性能研究

以正戊烷-水为物系,进行了采用填料强化的鼓泡塔直接接触蒸发换热实验。实验采用顺流操作,考察了分散相流量、温差以及分布器孔径对体积换热系数和汽化高度的影响。实验得出：在戊烷流量为23.868 L/h,分布器孔径为2.5 mm时加填料的鼓泡塔的体积换热系数约为未加填料的2倍;在一定的操作条件下,加填料的鼓泡塔中汽化高度随分散相流量和分布器孔径的增大而增加,随温差的增大而减小;加填料的鼓泡塔中体积换热系数随分散相流量的增加而增加,随分布器孔径的增大而减小,与温差成负幂指数关系。     

Lipoxygenase inChlorella pyrenoidosa

The presence of lipoxygenase enzyme is observed when cells ofChlorella pyrenoidosa are homogenized under anaerobic conditions. This is the first report of this enzyme in a lower form of plant life. The major product ofChlorella lipoxygenase with linoleic acid as substrate is 13-hydroperoxyoctadecadienoic acid. Research Chemist, ARS, USDA, in cooperation with the North Dakota Agricultural Experiment Station.