A lipoxygenase with dual positional specificity is expressed in olives (Olea europaea L.) during ripening.

International audiencePlant lipoxygenases (LOXs) are a class of widespread dioxygenases catalysing the hydroperoxidation of polyunsaturated fatty acids. Although multiple isoforms of LOX have been detected in a wide range of plants, their physiological roles remain to be clarified. With the aim to clarify the occurrence of LOXs in olives and their contribution to the elaboration of the olive oil aroma, we cloned and characterized the first cDNA of the LOX isoform which is expressed during olive development. The open reading frame encodes a polypeptide of 864 amino acids. This olive LOX is a type-1 LOX which shows a high degree of identity at the peptide level towards hazelnut (77.3%), tobacco (76.3%) and almond (75.5%) LOXs. The recombinant enzyme shows a dual positional specificity, as it forms both 9- and 13-hydroperoxide of linoleic acid in a 2:1 ratio, and would be defined as 9/13-LOX. Although a LOX activity was detected throughout the olive development, the 9/13-LOX is mainly expressed at late developmental stages. Our data suggest that there are at least two Lox genes expressed in black olives, and that the 9/13-LOX is associated with the ripening and senescence processes. However, due to its dual positional specificity and its expression pattern, its contribution to the elaboration of the olive oil aroma might be considered

Optimizing the Production of Recombinant Hydroperoxide Lyase in <i>Escherichia coli</i> Using Statistical Design

Hydroperoxide lyase (HPL) catalyzes the synthesis of volatiles C6 or C9 aldehydes from fatty acid hydroperoxides. These short carbon chain aldehydes, known as green leaf volatiles (GLV), are widely used in cosmetic industries and as food additives because of their “fresh green” aroma. To meet the growing demand for natural GLVs, the use of recombinant HPL as a biocatalyst in enzyme-catalyzed processes appears to be an interesting application. Previously, we cloned and expressed a 13-HPL from olive fruit in Escherichia coli and showed high conversion rates (up to 94%) during the synthesis of C6 aldehydes. To consider a scale-up of this process, optimization of the recombinant enzyme production is necessary. In this study, four host-vector combinations were tested. Experimental design and response surface methodology (RSM) were used to optimize the expression conditions. Three factors were considered, i.e., temperature, inducer concentration and induction duration. The Box–Behnken design consisted of 45 assays for each expression system performed in deep-well microplates. The regression models were built and fitted well to the experimental data (R2 coefficient > 97%). The best response (production level of the soluble enzyme) was obtained with E. coli BL21 DE3 cells. Using the optimal conditions, 2277 U L−1of culture of the soluble enzyme was produced in microliter plates and 21,920 U L−1of culture in an Erlenmeyer flask, which represents a 79-fold increase compared to the production levels previously reported

Optimizing the Production of Recombinant Hydroperoxide Lyase in Escherichia coli Using Statistical Design

Hydroperoxide lyase (HPL) catalyzes the synthesis of volatiles C6 or C9 aldehydes from fatty acid hydroperoxides. These short carbon chain aldehydes, known as green leaf volatiles (GLV), are widely used in cosmetic industries and as food additives because of their &ldquo;fresh green&rdquo; aroma. To meet the growing demand for natural GLVs, the use of recombinant HPL as a biocatalyst in enzyme-catalyzed processes appears to be an interesting application. Previously, we cloned and expressed a 13-HPL from olive fruit in Escherichia coli and showed high conversion rates (up to 94%) during the synthesis of C6 aldehydes. To consider a scale-up of this process, optimization of the recombinant enzyme production is necessary. In this study, four host-vector combinations were tested. Experimental design and response surface methodology (RSM) were used to optimize the expression conditions. Three factors were considered, i.e., temperature, inducer concentration and induction duration. The Box&ndash;Behnken design consisted of 45 assays for each expression system performed in deep-well microplates. The regression models were built and fitted well to the experimental data (R2 coefficient &gt; 97%). The best response (production level of the soluble enzyme) was obtained with E. coli BL21 DE3 cells. Using the optimal conditions, 2277 U L&minus;1of culture of the soluble enzyme was produced in microliter plates and 21,920 U L&minus;1of culture in an Erlenmeyer flask, which represents a 79-fold increase compared to the production levels previously reported

A novel lipoxygenase in olives (Olea europaea L.), with dual positional specificity, expressed late during fruit development

International audiencePlant lipoxygenases (LOXs) are a class of widespread dioxygenase catalyzing the hydroperoxidation of polyunsaturated fatty acids. Although multiple isoforms of LOX have been detected in a wide range of plants, their physiological role is still unclear. With the aim to clarify the occurrence of LOXs and their contribution to the elaboration of the olive oil aroma, we carried out the biochemical and molecular characterization of the LOX isoform expressed during olive development. A full-length cDNA was isolated by Reverse Transcription PCR and Rapid amplification of cDNA end carried out on total RNA from mature olives. The 2852 bp sequence displays an open reading frame of 2592 bp encoding a putative polypeptide of 864 amino acids with a calculated molecular mass of 98.4 kDa and a pI of 5.95. The olive LOX is a type-1 LOX and shows a high degree of identity towards hazelnut (77.3%), tobacco (76.3%) and almond (75.5%) LOXs. The recombinant enzyme produced in E. coli shows a dual positional specificity, as it releases both 9- and 13-hydroperoxide of linoleic acid in a 2:1 ratio. Although LOX activity was detected throughout the olive development, the 9/13-LOX is mainly expressed at late developmental stages. Our data suggest the presence of at least two LOX isoforms in black olives, and that the 9/13-LOX is associated with the ripening and senescence processes. However, due to its dual positional specificity ant its expression pattern, its contribution to the elaboration of the olive oil aroma might be considered

A functional role identified for conserved charged residues at theactive site entrance of lipoxygenase with double specificity

International audiencePlant lipoxygenases (LOXs) are a class of widespread dioxygenases catalyzing the hydroperoxidation offree polyunsaturated fatty acids, producing 9-hydroperoxides or 13-hydroperoxides from linoleic and-linolenic acids, and are called 9-LOX or 13-LOX, respectively. Some LOXs produce both 9- and 13-hydroperoxides. The models proposed to explain the reaction mechanism specificity fail to explain the“double specificity” character of these LOXs. In this study, we used the olive LOX1 with double specificityto investigate the implication of the charged residues R265, R268, and K283 in the orientation of thesubstrate into the active site. These residues are present in a conserved pattern around the entrance ofthe active site. Our results show that these residues are involved in the penetration of the substrate intothe active site: this positive patch could capture the carboxylate end of the substrate, and then guide itinto the active site. Due to its position on 2 helix, the residue K283 could have a more important role, itsinteraction with the substrate facilitating the motions of residues constituting the “cork of lipoxygenases”or the 2 helix, by disrupting putative hydrogen and ionic bonds

A functional role identified for conserved charged residues at theactive site entrance of lipoxygenase with double specificity

International audiencePlant lipoxygenases (LOXs) are a class of widespread dioxygenases catalyzing the hydroperoxidation offree polyunsaturated fatty acids, producing 9-hydroperoxides or 13-hydroperoxides from linoleic and-linolenic acids, and are called 9-LOX or 13-LOX, respectively. Some LOXs produce both 9- and 13-hydroperoxides. The models proposed to explain the reaction mechanism specificity fail to explain the“double specificity” character of these LOXs. In this study, we used the olive LOX1 with double specificityto investigate the implication of the charged residues R265, R268, and K283 in the orientation of thesubstrate into the active site. These residues are present in a conserved pattern around the entrance ofthe active site. Our results show that these residues are involved in the penetration of the substrate intothe active site: this positive patch could capture the carboxylate end of the substrate, and then guide itinto the active site. Due to its position on 2 helix, the residue K283 could have a more important role, itsinteraction with the substrate facilitating the motions of residues constituting the “cork of lipoxygenases”or the 2 helix, by disrupting putative hydrogen and ionic bonds

Identification of putative residues involved in the accessibility of the substrate-binding site of lipoxygenase by site-directed mutagenesis studies.

International audienceLipoxygenases (LOXs) are a class of widespread dioxygenases catalyzing the hydroperoxidation of polyunsaturated fatty acids (PUFA). Recently, we isolated a cDNA encoding a LOX, named olive LOX1, from olive fruit of which the deduced amino acid sequence shows more than 50% identity with plant LOXs. In the present study, a model of olive LOX1 based on the crystal structure of soybean LOX-1 as template has been generated and two bulky amino acid residues highly conserved in LOXs (Phe277) and in plant LOXs (Tyr280), located at the putative entrance of catalytic site were identified. These residues may perturb accessibility of the substrate-binding site and therefore were substituted by less space-filling residues. Kinetic studies using linoleic and linolenic acids as substrates were carried out on wild type and mutants. The results show that the removal of steric bulk at the entrance of the catalytic site induces an increase of substrate affinity and of catalytic efficiency, and demonstrate that penetration of substrates into active site of olive LOX1 requires the movement of the side chains of the Phe277 and Tyr280 residues. This study suggests the involvement of these residues in the accessibility of the substrate-binding site in the lipoxygenase family

Olive Recombinant Hydroperoxide Lyase, an Efficient Biocatalyst for Synthesis of Green Leaf Volatiles

International audienceVolatile C6-aldehydes are the main contributors to the characteristic odor of plantsknown as Bgreen note^ and are widely used by the flavor industry. Biotechnological processeswere developed to fulfill the high demand in C6-aldehydes in natural flavorants and odorants.Recombinant hydroperoxide lyases (HPLs) constitute an interesting alternative to overcomedrawbacks arising from the use of HPL from plant extracts. Thus, olive recombinant 13-HPLwas assayed as biocatalysts to produce C6-aldehydes. Firstly, a cDNA encoding for olive HPLof Leccino variety was isolated and cloned in pQE-30 expression vector. In order to improvethe enzyme solubility, its chloroplast transit peptide was deleted. Both enzymes (HPL wild typeand HPL deleted) were expressed into Escherichia coli strain M15, purified, characterized, andthen used for bioconversion of 13-hydroperoxides of linoleic and linolenic acids. Aldehydesproduced were extracted, then identified and quantified using gas chromatography and massspectrometry. Recombinant HPL wild type (HPLwt) allowed producing 5.61 mM of hexanaland 4.39 mM of 3Z-hexenal, corresponding to high conversion yields of 93.5 and 73 %,respectively. Using HPL deleted (HPLdel) instead of HPLwt failed to obtain greater quantitiesof hexanal or 3Z-hexenal. No undesirable products were formed, and no isomerization of 3Zhexenalin 2E-hexenal occurred. The olive recombinant HPLwt appears to be a promisingefficient biocatalyst for the production of C6-aldehyde

Activation and Stabilization of Olive Recombinant 13-Hydroperoxide Lyase Using Selected Additives

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