Title : Use of olive recombinant hydroperoxide lyases for synthesis of green leaf volatiles: Activation and stabilization of enzymes by selected additives
Green leaf volatiles (GLVs), especially C6-aldehydes and C9-aldehydes, produced by plants, are responsible for the characteristic odor of cut grass, called “green note”. GLVs are widely used in flavor and food industries. Their production represents an important industrial stake. In addition, consumers’ distrust of chemical flavors leads to a strong growth in the natural flavor market. GLVs are synthesized naturally in plants during a lipid metabolic pathway called lipoxygenase pathway. First, lipoxygenase (LOX) catalyzes regiospecific dioxygenation at the carbon 9 or 13 of C-18 polyunsatured fatty acids, mainly linoleic and α-linolenic acids, to form the corresponding fatty acid hydroperoxides. Subsequently, HPL can act on these 9-hydroperoxides or 13-hydroperoxides of to form C9-aldehydes or C6-aldehydes and C9-oxoacids or C12-oxoacids, respectively. Given the high demand for natural GLVs, biocatalytic processes using the enzymes of the LOX pathway have been developed for large-scale production. Plant extracts are used as a source of LOX and HPL to convert vegetable oils chosen for their fatty acid profile into natural GLVs. Soybean flour is successfully used as LOX source, while many plant extracts with HPL activities were tested (mint leaves, green bell pepper, or tomato leaves or guava fruit) and conversion yield remains very low. Large amounts of raw materials are required, which leads to dependence on environmental conditions, harvesting period, and plant heterogeneity. In addition, crude extracts contain other enzymes that use fatty hydroperoxides, leading to undesired products and loss of substrate. The use of recombinant HPLs in such processes constitute an interesting alternative to overcome drawbacks arising from the use of HPL from plant extracts. We isolated a cDNA encoding for olive HPL named HPL full-length from black olive fruit (Olea europaea L., Leccino variety), and then, we deleted the N-terminal transit peptide to produce the matured HPL. Both enzymes was expressed in Escherichia coli, purified and characterized biochemically. We showed that olive recombinant HPLs (rHPLs) are promising efficient biocatalysts for the production of natural C6-aldehydes. However, the use of HPLs for bioconversion reactions is limited by their poor stability. The stabilization and activation of olive rHPLs was investigated using selected chemical additives. Both olive rHPLs are relatively stable at 4°C, and enzyme activity can be preserved (about 100% of the rHPL activities are maintained) during 5 weeks of storage at −20 or at −80°C in the presence of 10% glycerol. Among the additives used in this study, 2.5% glycine, 0.5 M NaCl, and 0.25 M Na2SO4 provided the highest activation of HPL full-length activity, while the best matured HPL activity was obtained with 0.25 M Na2SO4 and 1 M NaCl. Results of C6-aldehyde production assays also showed that glycine, NaCl, and Na2SO4 are appropriate additives and that NaCl appears to be the best additive, at least for hexanal production.