Cloning and characterization of alfalfa hydroperoxide lyase : a biocatalyst for the production of green note flavors

Plants continuously have to defend themselves against life threatening events such as drought, mechanical damage, temperature stress and potential pathogens. A main component of the plant defense mechanism is the lipoxygenase pathway. Products of this pathway are involved in wound healing, pest resistance, signaling, or 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 formed hydroperoxy fatty acids are highly reactive and dangerous for the plant, and are therefore further metabolized by other enzymes such as allene oxide synthase, hydroperoxide lyase, peroxygenase or divinyl ether synthase. Hydroperoxide lyases are heme-containing enzymes of the cytochrome P450 class (CYP74B). They cleave the C-C bond adjacent to the hydroperoxy group in the lipoxygenase products, resulting in the formation of w-oxo acids and volatile C6- and C9-aldehydes. The aldehydes and the corresponding alcohols cause the characteristic 'fresh green' odor of damaged 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. Biocatalytic production of 'natural' food flavors by lipoxygenase and hydroperoxide lyase however, is hampered by the low availability and stability of hydroperoxide lyase. In this study hydroperoxide lyase genes from alfalfa seedlings have been isolated and expressed in E. coli cells. The substrate and product specificities of the isoenzymes have been determined and the enzymes have been characterized. The high expression level, high stability and specificity make the cloned lyases interesting for application in a biocatalytic process

A simple active bandpass filter

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A simple active bandpass filter

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Alfalfa contains substantial 9-hydroperoxide lyase activity and a 3Z:2E-enal isomerase

Fatty acid hydroperoxides formed by lipoxygenase can be cleaved by hydroperoxide lyase resulting in the formation of short-chain aldehydes and omega-oxo acids. Plant hydroperoxide lyases use 13- or 9-hydroperoxy linoleic and linolenic acid as substrates. Alfalfa (Medicago sativa L.) has been reported to contain a hydroperoxide lyase specific for 13-hydroperoxy linoleic and linolenic acid only. However, in addition to 13-hydroperoxide lyase activity we found substantial 9-hydroperoxide lyase activity in alfalfa seedlings as well. The specific activity for 9-hydroperoxy fatty acids was about 50% of the activity for the 13-isomers. Furthermore, alfalfa seedlings contain a 3Z:2E-enal isomerase that converts the 3Z-enal products to their 2E-enal isoforms

A trapezium function generator

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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 -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 sigh

Oxygenation of (3Z)-alkenals to 4-hydroxy-(2E)-alkenals in plant extracts : A nonenzymatic process

There is large interest in 4-hydroxy-(2E)-alkenals because of their cytotoxicity in mammals. However, the biosynthetic pathway for these compounds has not been elucidated yet. In plants, 4-hydroxy-(2E)-alkenals were supposed to be derived by the subsequent actions of lipoxygenase and a peroxygenase on (3Z)-alkenals. The presence of 9-hydroxy-12-oxo-(10E)-dodecenoic acid (9-hydroxy-traumatin) in incubations of 12-oxo-(9Z)-dodecenoic acid (traumatin) in the absence of lipoxygenase or peroxygenase, has prompted us to reinvestigate its mode of formation. We show here that in vitro 9-hydroxy-traumatin, 4-hydroxy-(2E)-hexenal and 4-hydroxy-(2E)-nonenal, are formed in a nonenzymatic process. Furthermore, a novel product derived from traumatin was observed and identified as 11-hydroxy-12-oxo-(9Z)-dodecenoic acid. The results obtained here strongly suggest that the 4-hydroxy-(2E)-alkenals, observed in crude extracts of plants, are mainly due to autoxidation of (3Z)-hexenal, (3Z)-nonenal and traumatin. This may have implications for the in vivo existence and previously proposed physiological significance of these products in plants

Tribological behavior of short-cut aramid fiber reinforced SBR elastomers: The effect of fiber orientation

Elastomeric materials are widely used in daily applications, such as conveyor belts and wiper. Generally, elastomeric materials show poor tribological behavior. Adding fibers to an elastomer is a way to solve this problem. The orientation of the fibers influences the mechanical and tribological behavior of the elastomers. In the present study, the effect of short-cut aramid fiber orientation on the tribological behavior for a Styrene-Butadiene Rubber (SBR) was examined. Three types of compounds which have different fiber orientations were prepared, resulting in normal, transverse and longitudinal orientation to the sliding direction. A contact model of the viscoelastic-anisotropic behaving elastomer was used to calculate the contact area. The friction and wear of the compounds were evaluated using a pin-on-disc tribometer. The results show that the frictional shear stresses of all compounds are nearly the same due to the presence of fibers on the wear track. The normal orientation of fibers shows the most effective way to increase the wear resistance compared to the longitudinal and transverse orientation of the fibers

Characterization of three cloned and expressed 13-hydroperoxide lyase isoenzymes from alfalfa with unusual N-terminal sequences and different enzyme kinetics

Three full-length cDNAs from alfalfa seedlings coding for hydroperoxide lyases were cloned and expressed in Escherichia coli and characterized as cytochrome P450 enzymes. The isoenzymes were specific for 13-hydroperoxy linoleic and linolenic acids and did not use the 9-hydroperoxy isomers as substrates. Because alfalfa contains both specificities, this indicates the presence of two different types of hydroperoxide lyases, each specific for one kind of substrate. The enzymes contain 480 amino acids (54 kDa) and contain an unusual, nonplastidic N-terminal sequence of 22 amino acids, which strongly reduces the enzyme activity. The only known presequence of a hydroperoxide lyase (from Arabidopsis thaliana) was considered to be a transit sequence. The reduced enzyme activity, however, indicates that the hydroperoxide lyases with N-terminal extensions could be pro-enzymes. This hypothesis is supported by the fast release of hydroperoxide lyase products by plants upon wounding. One of the isoenzymes showed a strongly decreased Vmax and Km compared to the other two. Because this is probably due to the substitution of Ser377 by Phe; the residue at position 377 seems to be important. This is the first time that sufficient quantities of hydroperoxide lyase have been obtained for characterization studies, by circumventing difficult purification procedures and degradation of the enzyme. The high expression level, easy purification, good stability and high specificity make these cloned hydroperoxide lyases excellent tools to study the reaction mechanism and structure. We postulate an integrated reaction mechanism, based on the known chemistry of cytochrome P450 enzymes. This is the first mechanism that unifies all observed features of hydroperoxide lyases