Development of New Technology for Plant Aroma Emission with New Type of Plant

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Floral tea aroma of oolong tea and black tea has been clarified to be generated form aroma precursors, most of which are present as β-primeverosides in tea leaves, by the action of β-primeverosidase during their manufacturing, so called fermentation processes. Recently these aroma formation mechanisms have been found to be concemed with the attractive aroma formation in flower, fruits, wine, etc.Based on these new findings, new type of aroma emission technology was attempted to be established by screening a new disaccharide specific glycosidase among microbial metabolites, its mass production and its application as follows :1) Clarification of substrate specificities of the tea β-primeverosidase, cloning of its cDNA and a trial for its large scale overexpression in E.coli.Several kinds of disaccharide glycosides with 2-phenylethanol as an aglycon were obtained by chemical synthesis or from nature. They were subjected to hydrolysis with the β-primeverosidase to know its substrate specif … More icities. The β-primeverosidase showed very high substrate specificity towards β-primeveroside. All the synthetic unntural disaccharide glycosides were not hydrolyzed at all. Only other natural disaccharide (β 1-6) glycosides were hydrolyzed some extent (0.07-3.0%).The amino acid alignment of the β-primeverosidase obtained by its gene cloning suggested some N-glycosyl modifications and fairly high homology (60%) with that of the β-glucosidase which hydrolyzes cyanogenic β-D-glucopyranosides. The recombinant enzyme easily agglutinated to loose its activity, suggested that the N-glycosyl modifications are necessary for the enzyme stability.2) Screening of microbials with the β-primeveroside hydrolyzing activity Soil-born microbial strains were screened using eugenyl β-primeveroside as a sole carbon sauce to give 4 active strains with the activity. The most active strain was identified as Aspergillus fumigatus Pr-20. The glycosidase purified from the culture medium of the strain showed a single band at 47 kDa in SDS-PAGE analysis and was found to hydrolyze other β (1→6)-disaccharide glycosides as well as 6-acylated β-D-glucopyranoslde.3) Gene cloning of the β-primeverosidase-like diglycosidase from A.fumigatusThe amine acid alignment of the diglycosidase revealed by the gene cloning showed only 14% identity with that of the tea leave β-primeverosidase. The overexpression of the gene was attempted in a yeast and a fungus to give a successful result with a fungus.4) A few trials to enhancing aroma from endogenous plant aroma precursors by treatment with the diglycosidaseThe diglycosidase was applied to crude tea leave extracts and grape juice and liberated aroma was analyzed by GC.Many kinds of aroma compounds were found to be much enhanced by this treatment, suggesting that this new enzyme will be applicable to enhance aroma in black tea and wine production and their related industries. Less