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.