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.     

Substrate specificity of beta-primeverosidase, a key enzyme in aroma formation during oolong tea and black tea manufacturing

We synthesized nine kinds of diglycosides and a monoglycoside of 2-phenylethanol to investigate the substrate specificity of the purified beta-primeverosidase from fresh leaves of a tea cultivar (Camellia sinensis var. sinensis cv. Yabukita) in comparison with the apparent substrate specificity of the crude enzyme extract from tea leaves. The crude enzyme extract mainly showed beta-primeverosidase activity, although monoglycosidases activity was present to some extent. The purified beta-primeverosidase showed very narrow substrate specificity with respect to the glycon moiety, and especially prominent specificity for the beta-primeverosyl (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosyl) moiety. The enzymes hydrolyzed naturally occurring diglycosides such as beta-primeveroside, beta-vicianoside, beta-acuminoside, beta-gentiobioside and 6-O-alpha-L-arabinofuranosyl-beta-D-glucopyranoside, but were unable to hydrolyze synthetic unnatural diglycosides. The purified enzyme was inactive toward 2-phenylethyl beta-D-glucopyranoside. The enzyme hydrolyzed each of the diglycosides into the corresponding disaccharide and 2-phenylethanol. These results indicate the beta-primeverosidase, a diglycosidase, to be a key enzyme involved in aroma formation during the tea manufacturing process.     

Long-term dietary supplementation with the green tea cultivar Sunrouge prevents age-related cognitive decline in the senescence-accelerated mouse Prone8

A majority of the potential health benefits of green tea, including the potential to prevent cognitive decline, have been attributed to epigallocatechin gallate (EGCG). Sunrouge is a green tea cultivar that contains EGCG and several other bioactive components such as quercetin, myricetin, cyanidin and delphinidin. We compared the effects of Sunrouge and Yabukita, the most popular Japanese green tea cultivar, on cognitive function in the senescence-accelerated mouse Prone8. These mice were fed an experimental diet containing Sunrouge extract (SRE) or Yabukita extract (YBE). SRE feeding significantly prevented cognitive decline, whereas YBE feeding had little effect. Moreover, SRE feeding prevented elevation of the amyloid-β42 level while improving the gene expression of neprilysin and decreasing beta-site APP-cleaving enzyme 1 in the brain. These preventive effects of SRE against cognitive decline were attributed to the characteristic composition of Sunrouge and strongly suggest that consumption of this cultivar could protect against age-related cognitive decline.     

Effects of anthocyanin-rich tea "Sunrouge" on dextran sodium sulfate-induced colitis in mice

Sunrouge, an anthocyanin-rich tea, has similar levels of catechins as "Yabukita," the most popular green tea cultivar consumed in Japan. Green tea polyphenols (GTPs) have attracted interest due to their potent antioxidative activities combined with a lack of side effects in humans at normal consumption levels. However, we previously reported that high doses (0.5 and 1%) of dietary GTPs can result in deterioration of colitis and failed to prevent colon carcinogenesis in inflamed colons. In the present study, we determined the inhibitory effects of Sunrouge on colitis in dextran sodium sulfate (DSS)-treated and untreated control mice. Five-week-old female ICR mice were administered a single dose of Yabukita or Sunrouge (extracts in 1 mL distilled water) via a stomach tube for 3 weeks. After 1 week of treatment, the mice were divided into four groups (two Yabukita and two Sunrouge groups) and given drinking water with or without 3% DSS for 2 weeks, then they were euthanized. Those treated with DSS developed watery diarrhea and bloody stools, and showed body weight loss, spleen hypertrophy, and shortening of the colon, as well as deteriorations in survival rate, liver function, colon mucosal interleukin-1β level and expression of phase II detoxification enzyme mRNA. Sunrouge improved these DSS-induced symptoms, at least in part, whereas Yabukita showed either no effect or adverse effects in regard to some those parameters. It is suggested that the differences between Yabukita and Sunrouge on DSS-induced colitis might be due to the high levels of anthocyanins found in Sunrouge tea.     

The (3R,9R)-3-hydroxy-7,8-dihydro-beta-ionol disaccharide glycoside is an aroma precursor in tea leaves

The disaccharide glycoside, (3R,9R)-3-hydroxy-7,8-dihydro-beta-ionyl 6-O-beta-D-apiofuranosyl-beta-D-glucopyranoside was isolated as an aroma precursor from the leaves of Camellia sinensis var. sinensis cv. Yabukita. Its stereochemistry was elucidated on the basis of spectral data and chemical synthesis.     

trans- and cis-Linalool 3,6-Oxide 6-O-β-d-Xylopyranosyl-β-d-glucopyranosides Isolated as Aroma Precursors from Leaves for Oolong Tea

New glycosidic aroma precursors (1 and 2) of the main volatile constituents, trans- and cis-linalool 3,6-oxides (linalool oxides I and II), were isolated from oolong tea leaves (Camellia sinensis var. sinensis cv. Maoxie). The isolation was guided by an enzymatic hydrolysis with acetone powder prepared from fresh tea leaves (cv. Yabukita) followed by GC or GC-MS analyses. Chromatographic purification of hot water extracts of the tea leaves on active charcoal, Amberlite XAD-2, and Sephadex LH-20 columns as well as HPLC gave two new glycosides, trans- and cis-linalool 3,6-oxide 6-O-β-d-xylopyranosyl-β-d-glucopyra-nosides (1 and 2).     

Expression and biochemical characterization of beta-primeverosidase and application of beta-primeverosylamidine to affinity purification

Beta-primeverosidase (PD) is a family 1 glycosidase catalyzing the hydrolysis of beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) to release a disaccharide primeverose. To investigate how PD recognizes the disaccharide moiety of beta-primeverosides, the recombinant PD was expressed by a baculovirus-insect cell system. The recombinant PD was secreted from High Five cells and was properly modified with N-glycosylation and correct cleavage at the N-terminal signal peptide. The recombinant PD exhibited high substrate specificity to beta-primeverosides in terms of the glycone moiety, consistently with the substrate specificity of native PD from Camellia sinensis. Next, beta-glycosylamidines were synthesized as substrate analog inhibitors. Beta-primeverosylamidine strongly inhibited PD activity, but beta-glucosylamidine did not. Hence beta-primeverosylamidine is an ideal chemical tool for probing disaccharide recognition in the active site of PD. An affinity adsorbent for PD was prepared using beta-primeverosylamidine as a ligand. Affinity chromatography gave large amounts of PD with high purity, permitting crystallographic study.     

Improvement of catechin productivity in suspension cultures of tea callus cells

In the suspension cultures of tea callus cells, C.sinensis cv. Yabukita, the effects of the culture conditions, such as culture period and light irradiation, on cell growth and catechin production were investigated. The production of flavonoids (catechins + proanthocyanidins) was promoted by inoculating the cells into the fresh medium at the culture period giving the maximum flavonoid content in the cells. The cultivation under light irradiation was repeated several times by inoculating the cells with the maximum flavonoid content. The flavonoid production was significantly increased without inhibiting the cell growth. We obtained the maximum flavonoid production, 1.5 g/dm(3) medium, and the maximum content, 150 mg/(g of dry cell weight (DCW)). The latter value was larger than that in the leaves of the tea plant.     

Analysis of tea plant resistance to tea green leafhopper,Empoasca onukii,by detecting stylet‐probing behavior with DC electropenetrography

The tea green leafhopper, Empoasca onukiiMatsuda (Hemiptera: Cicadellidae: Typhlocybinae), is a serious pest of tea plants in East Asia. Previous work has shown that two tea germplasms, Cd19 and Cd289, sustain less hopperburn damage by E. onukii than does Camellia sinensis (L.) O. Kuntze cv. ‘Yabukita’ (Theaceae), and E. onukii excretes less honeydew on these germplasms than on the susceptible Yabukita. This study investigated the feeding behavior of E. onukii with a direct current electropenetrograph (DC EPG) to compare feeding behaviors, including ingestion, on resistant tea germplasms and Yabukita. Feeding behaviors on the resistant germplasms were significantly restricted, with few bouts of active ingestion of short duration and long periods of non‐probing, whereas E. onukii engaged in active ingestion of long duration many times on the susceptible cv. Yabukita. The tea germplasms, Cd19 and Cd289, therefore showed strong resistance to E. onukii. Furthermore, the shape of puncture holes left after probing was compared between the susceptible Yabukita and the resistant germplasms. The puncture holes on Cd19 and Cd289 were indistinct in shape and closed compared with those on Yabukita.     

A Primeverosidase as a Main Glycosidase Concerned with the Alcoholic Aroma Formation in Tea Leaves

We isolated and characterized a primeverosidase from fresh tea leaves (Camellia sinensis var. sinensis cv. Yabukita) as a main glycosidase involved in alcoholic aroma (geraniol, linalool, benzyl alcohol, 2-phenylethanol, linalool oxides etc.) formation from their aroma precursors (β-primeverosides: 6-O-β-D-xylopyranosyl-β-D-glucopyranosides) in tea leaves.     

Thermostabilities of plant phenol oxidase and peroxidase, determining the technology of their use in food industry

Stabilities of phenol oxidase and peroxidase from tea plant (Camellia sinensis L.) clone Kolkhida leaves, apple (Malus domestica L.) cultivar Kekhura fruits, walnut (Juglans regia L.) green pericarp, and horseradish (Armoracia lapathifolia Gilib) roots were studied using different storage temperature modes and storage duration. It was demonstrated that both enzymes retained residual activities (approximately 10%) upon 20-min incubation at 80 degrees C. Phenol oxidases from tea, walnut, and, especially, apple, as well as tea peroxidase were stable during storage. A technology for treatment of plant oxidases was proposed, based on the use of a natural inhibitor phenol oxidase and peroxidase, isolated from tea leaves, which solving the problem of residual activities of these enzymes, arising during pasteurization and storage of beverages and juices. It was demonstrated that browning of apple juice during pasteurization and beer turbidity during storage could be efficiently prevented using the natural inhibitor of these enzymes.     

Differentiation of Japanese green tea cultivars as revealed by RFLP analysis of phenylalanine ammonia-lyase DNA

Japanese green tea cultivars and 463 local tea plants including mountainous tea, yama-cha, were analyzed to determine the process of differentiation of Japanese tea plants using phenylalanine ammonia lyase (PAL) as a DNA marker. The main DNA fragments detected by RFLP analysis, which were named A, B and D, were inherited as multiple allelic genes at one locus. Japanese tea cultivars were divided into five groups according to RFLPs: AA, AB, AD, BD and DD. The AA group included many cultivars selected from local tea plants. The BD group consisted of cv Yabukita or descendants from Yabukita produced by artificial crossing. There was no BB group of cultivars. Allelic frequencies of A, B and D were 0.66, 0.08 and 0.22, respectively, and these values were same in tea plants collected from all regions of Japan. Since the frequencies in yama-cha and local tea plants were also the same, it is thought that these teas have the same origin. These results indicate a process of differentiation from the ancestral material presumably introduced from China to the local tea plants and, finally, cultivars which were produced by selecting from local tea plants and crossing.     

Metabolomics-driven nutraceutical evaluation of diverse green tea cultivars

Background

Green tea has various health promotion effects. Although there are numerous tea cultivars, little is known about the differences in their nutraceutical properties. Metabolic profiling techniques can provide information on the relationship between the metabolome and factors such as phenotype or quality. Here, we performed metabolomic analyses to explore the relationship between the metabolome and health-promoting attributes (bioactivity) of diverse Japanese green tea cultivars.

Methodology/Principal Findings

We investigated the ability of leaf extracts from 43 Japanese green tea cultivars to inhibit thrombin-induced phosphorylation of myosin regulatory light chain (MRLC) in human umbilical vein endothelial cells (HUVECs). This thrombin-induced phosphorylation is a potential hallmark of vascular endothelial dysfunction. Among the tested cultivars, Cha Chuukanbohon Nou-6 (Nou-6) and Sunrouge (SR) strongly inhibited MRLC phosphorylation. To evaluate the bioactivity of green tea cultivars using a metabolomics approach, the metabolite profiles of all tea extracts were determined by high-performance liquid chromatography-mass spectrometry (LC-MS). Multivariate statistical analyses, principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA), revealed differences among green tea cultivars with respect to their ability to inhibit MRLC phosphorylation. In the SR cultivar, polyphenols were associated with its unique metabolic profile and its bioactivity. In addition, using partial least-squares (PLS) regression analysis, we succeeded in constructing a reliable bioactivity-prediction model to predict the inhibitory effect of tea cultivars based on their metabolome. This model was based on certain identified metabolites that were associated with bioactivity. When added to an extract from the non-bioactive cultivar Yabukita, several metabolites enriched in SR were able to transform the extract into a bioactive extract.

Conclusions/Significance

Our findings suggest that metabolic profiling is a useful approach for nutraceutical evaluation of the health promotion effects of diverse tea cultivars. This may propose a novel strategy for functional food design.     

Patterns of adenine metabolism and caffeine biosynthesis in different parts of tea seedlings

Contents of purine alkaloids in different parts of tea ( Camellia sinensis L. cv. Yabukita ) seedlings, seeds and tissue cultures were determined with high-performance liquid chromatography. More than 99% of the caffeine detected was in the leaves of the 4-month-old seedlings. The amount expressed per g fresh weight was higher in older leaves. Theobromine, a precursor of caffeine biosynthesis, was found only in younger leaves. Zero or only trace amounts of theophylline, a degradation product of caffeine, were found in the seedlings. Almost all the caffeine in tea seeds was found in the seed coats. Theobromine and theophilline could not be detected in any part of the seeds.
Tracer experiments using [8-¹⁴C]-adenine indicate that (i) caffeine biosynthesis from [8-¹⁴C]-adenine occurs only in younger leaves,(ii) "salvage" of [8-¹⁴C]-adenine for nucleic acid synthesis takes place in all parts of the seedlings, (iii) considerable degradation of [8-¹⁴C]-adenine by conventional purine degradation pathway via uric acid takes place in roots and lower parts of stem tissue.
The results strongly suggest that caffeine is synthesized in younger leaves and accumulated within the leaves. Both caffeine contents and its synthetic activity from adenine were extremely low in tissue culture of tea.     

Isotheasaponins B1-B3 from Camellia sinensis var. sinensis tea leaves

Three saponins, isotheasaponins B1-B3, were isolated from the leaves of the tea plant Camellia sinensis var. sinensis, and their structures were determined to be theasapogenol B [beta-D-galactopyranosyl(1-->2)][beta-D-xylopyranosyl(1-->2)-alpha-L-arabinopyranosyl(1-->3)]-beta-D-gulcopyranosiduronic acid with two acyl groups by spectroscopic analysis.     

茶小绿叶蝉在不同茶树品种上的生长发育,生殖及空间分布

研究表明茶小绿叶蝉(Empoasca flavescens Fabricius)在云南大叶茶无性系良种云抗十号、云抗十四号、长叶白毫、矮丰上的生长发育历期、成活率、田间自然种群虫口数量、卵数量、卵分布等方面,皆有明显差异;说明这些品种对茶小绿叶蝉的抗性有差异,上述品种中矮丰对茶小绿叶蝉的抗性最强,云抗十号的抗性较差。在生产实践中对不同品种间存在的抗性差异应加以充分合理的利用。茶小绿叶蝉的空间分布型为随机分布型;在空间分布型方面,各品种间无明显差异。     

8-氧化咖啡因和嘧啶类生物碱在普洱熟茶中的存在

应用柱层析分离技术,从普洱熟茶中首次分离到8-氧化咖啡因,嘧啶类生物碱(胸腺嘧啶脱氧核苷、胸腺嘧啶和尿嘧啶) ,黄酮类配糖体(黄杞甙) ,以及简单酚类化合物(1 ,2 ,4-苯三酚、1 ,3-苯二酚和4-甲基-1 ,2-二苯酚)。由于普洱熟茶是由大叶茶经微生物后发酵生产的, 8-氧化咖啡因显然是茶叶中的咖啡因在微生物作用下形成的转化产物。胸腺嘧啶脱氧核苷亦可能是茶叶中的嘧啶类生物碱与微生物中的核苷类化合物在后发酵过程中缩合形成的。二者均为新发现的普洱熟茶的特征性成分。     

Furcatin hydrolase from Viburnum furcatum Blume is a novel disaccharide-specific acuminosidase in glycosyl hydrolase family 1

Furcatin hydrolase (FH) is a unique disaccharide-specific acuminosidase, which hydrolyzes furcatin (p-allylphenyl 6-O-beta-D-apiofuranosyl-beta-D-glucopyranoside (acuminoside)) into p-allylphenol and the disaccharide acuminose. We have isolated a cDNA coding for FH from Viburnum furcatum leaves. The open reading frame in the cDNA encoded a 538-amino acid polypeptide including a putative chloroplast transit peptide. The deduced protein showed 64% identity with tea leaf beta-primeverosidase, which is another disaccharide glycosidase specific to beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides). The deduced FH also shared greater than 50% identity with various plant beta-glucosidases in glycosyl hydrolase family 1. The recombinant FH expressed in Escherichia coli exhibited the highest level of activity toward furcatin with a Km value of 2.2 mm and specifically hydrolyzed the beta-glycosidic bond between p-allylphenol and acuminose, confirming FH as a disaccharide glycosidase. The FH also hydrolyzed beta-primeverosides and beta-vicianoside (6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside) but poorly hydrolyzed beta-gentiobiosides (6-O-beta-D-glucopyranosyl-beta-d-glucopyranosides), indicating high substrate specificity for the disaccharide glycone moiety. The FH exhibited activity toward p-allylphenyl beta-D-glucopyranoside containing the same aglycone as furcatin but little activity toward the other beta-D-glucopyranosides. Stereochemical analysis using 1H NMR spectroscopy revealed that FH is a retaining glycosidase. The subcellular localization of FH was analyzed using green fluorescent protein fused with the putative N-terminal signal peptide, indicating that FH is localized to the chloroplast. Phylogenetic analysis of plant beta-glucosidases revealed that FH clusters with beta-primeverosidase, and this suggests that the disaccharide glycosidases will form a new subfamily in glycosyl hydrolase family 1.     

湖南茶叶植物资源的评价

对城步峒茶、江华苦茶、汝城白毛茶和云台山种等湖南茶叶植物资源的主要化学成分、光合特性和生产性状进行了研究。结果表明,茶多酚与儿茶素含量从高到低依次为汝城白毛茶、江华苦茶、城步峒茶、云台山种;各资源的游离氨基酸、茶氨酸和咖啡碱含量均为正常水平;汝城白毛茶属高茶多酚资源,它所含生物碱的组成方式与毛叶茶龙门模式种不同,虽可可碱和茶叶碱含量比茶种高,但仍以咖啡碱为主。在阴天阴凉条件下的净光合速率为汝城白毛茶＞城步峒茶＞江华苦茶＞云台山种,净光合速率与光量子通量、叶面温度呈极显著正相关,与气孔阻力呈极显著负相关,相关系数分别为０９２７８、０９１１５和－０８９３７。在晴天高温度强光条件下,净光合速率为云台山种＞城步峒茶＞江华苦茶＞汝城白毛花,净光合速率与光量子通量、叶面温度和气孔阻力都呈极显著负相关,相关系数分别为－０９１６５、－０９３２８、－０９０３１。在主要生产性状中,汝城白毛茶能安全通过－６℃的低温,在高茶多酚资源中,抗寒性突出,是选育红茶良种的一个非常有益的关键性状。