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 “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 <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

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

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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