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

研究表明，乌龙茶和红茶的花茶香气是由香气前体产生的，这些香气前体大多以β-春草苷的形式存在于茶叶中，是由β-春草苷酶在茶叶制造过程中的作用产生的，即所谓的发酵过程。在此基础上，通过在微生物代谢产物中筛选一种新的双糖特异性糖苷酶，并进行大规模生产和应用，尝试建立一种新型的香气释放技术：1)澄清茶β-素维苷酶的底物特异性；以2-苯乙醇为糖基化合物，通过化学合成或从自然界获得了多种双糖糖苷。它们被β-素维苷酶水解，以了解其底物特异性。β-primeverosidase对β- primeverosid苷表现出非常高的底物特异性。所有合成的非天然双糖甙均未被水解。只有其他天然双糖（β 1-6）糖苷有一定程度的水解（0.07-3.0%）。通过基因克隆获得的β-初维葡萄糖苷酶的氨基酸序列表明，该酶有n -糖基修饰，与水解氰源β- d -葡萄糖苷的β-葡萄糖苷酶具有较高的同源性（60%）。重组酶容易凝集而失去活性，表明n -糖基修饰对酶的稳定性是必要的。以丁香基β-primeveroside为唯一碳酱筛选土壤微生物菌株，得到4株具有水解活性的活性菌株。最活跃的菌株为烟曲霉Pr-20。从该菌株的培养基中纯化的糖苷酶在SDS-PAGE分析中显示为47 kDa的单链带，并且可以水解其他β(1→6)-双糖苷以及6-酰化的β- d -葡萄糖苷。3)烟曲霉β-primeverosidase样二糖苷酶基因克隆结果表明，该二糖苷酶的氨基酸序列与茶叶β-primeverosidase的同源性仅为14%。在酵母和真菌中尝试过表达该基因，并在真菌中获得了成功的结果。4)用二糖苷酶处理植物内源香气前体增强香气的试验。将二糖苷酶应用于粗茶叶提取物和葡萄汁中，用气相色谱法分析其释放香气。经此处理后，多种香气化合物均有明显的增强作用，表明该酶可用于红茶和葡萄酒生产及其相关行业的香气增强。少

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