从茶叶中克隆 β-primeverosidase，一种茶叶香气形成的关键酶。,Plant Physiology

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从茶叶中克隆 β-primeverosidase，一种茶叶香气形成的关键酶。
Plant Physiology ( IF 6.5 ) Pub Date : 2002-12-01 , DOI: 10.1104/pp.102.011023
Masaharu Mizutani ₁ , Hidemitsu Nakanishi , Jun-ichi Ema , Seung-Jin Ma , Etsuko Noguchi , Misa Inohara-Ochiai , Masako Fukuchi-Mizutani , Masahiro Nakao , Kanzo Sakata

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来自茶 (Camellia sinensis) 植物的 β-primeverosidase 是一种独特的二糖特异性糖苷酶，可水解 β-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) 的香气前体，释放出各种香气化合物，该酶与乌龙茶和红茶在制造过程中的花香形成密切相关。从绿茶品种（C. sinensis var sinensis cv Yabukita）的新鲜叶子中纯化β-primeverosidase，并确定其部分氨基酸序列。已使用简并寡核苷酸引物从 cv Yabukita 的 cDNA 文库中分离出 β-primeverosidase cDNA。cDNA 插入片段编码由 28 个氨基酸残基的 N 端信号肽和 479 个氨基酸的成熟蛋白组成的多肽。β-primeverosidase 蛋白质序列与来自各种植物的 β-葡萄糖苷酶具有 50% 至 60% 的相同性，并被归类为 1 型糖基水解酶。在大肠杆菌中表达的成熟形式的β-primeverosides 能够水解β-primeverosides 以释放primeverose 单元和苷元，但不作用于2-苯乙基β-D-吡喃葡萄糖苷。这些结果表明，β-primeverosides 酶选择性地将β-primeverosides 识别为底物并特异性水解二糖和苷元之间的β-糖苷键。由β-primeveroside 酶水解2-苯乙基β-primeveroside 的立体化学之后是(1) H-核磁共振波谱，揭示该酶通过保留机制水解β-primeveroside。

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Cloning of beta-primeverosidase from tea leaves, a key enzyme in tea aroma formation.

A beta-primeverosidase from tea (Camellia sinensis) plants is a unique disaccharide-specific glycosidase, which hydrolyzes aroma precursors of beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) to liberate various aroma compounds, and the enzyme is deeply concerned with the floral aroma formation in oolong tea and black tea during the manufacturing process. The beta-primeverosidase was purified from fresh leaves of a cultivar for green tea (C. sinensis var sinensis cv Yabukita), and its partial amino acid sequences were determined. The beta-primeverosidase cDNA has been isolated from a cDNA library of cv Yabukita using degenerate oligonucleotide primers. The cDNA insert encodes a polypeptide consisting of an N-terminal signal peptide of 28 amino acid residues and a 479-amino acid mature protein. The beta-primeverosidase protein sequence was 50% to 60% identical to beta-glucosidases from various plants and was classified in a family 1 glycosyl hydrolase. The mature form of the beta-primeverosidase expressed in Escherichia coli was able to hydrolyze beta-primeverosides to liberate a primeverose unit and aglycons, but did not act on 2-phenylethyl beta-D-glucopyranoside. These results indicate that the beta-primeverosidase selectively recognizes the beta-primeverosides as substrates and specifically hydrolyzes the beta-glycosidic bond between the disaccharide and the aglycons. The stereochemistry for enzymatic hydrolysis of 2-phenylethyl beta-primeveroside by the beta-primeverosidase was followed by (1)H-nuclear magnetic resonance spectroscopy, revealing that the enzyme hydrolyzes the beta-primeveroside by a retaining mechanism. The roles of the beta-primeverosidase in the defense mechanism in tea plants and the floral aroma formation during tea manufacturing process are also discussed.

更新日期：2019-11-01

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