Biotransformation of tea catechins into theaflavins with immobilized polyphenol oxidase

Theaflavins, an active antioxidant, a natural pigment and pharmacologically active molecule obtained from black tea were bioprocessed on an immobilized tea polyphenol oxidase (PPO) system by the conversion of tea catechins extracted from green tea leaves with an overall conversion efficiency of 85% about 14-fold increase over maximum achievable in normal black teas. The immobilized enzyme (IE) system consists of activated cellulose matrix on to which the freshly extracted tea leaf polyphenol oxidase was covalently linked. Cellulose as a matrix of choice was considered primarily for its non-toxic nature, natural origin, low cost and easy availability. The kinetic parameters of the IE system were; protein loading capacity 11.8 mg/g; pH optimum 5.9; temperature optimum 37.5 °C; K_m 4.76 ± 0.08 mM; V_max 20 ± 1.80 nmol/min; enzyme activity retention (83.58%) and number of batches per turnover without loss of activity was 14. The product from IE system was identified by HPLC and ESI-QTOF-MS spectrometry.     

Tea Leaf Polyphenol Oxidase

The effect of polyamide on the extraction of polyphenol oxidase in the leaves and the localization of the enzyme in tea leaf cell were investigated.

In the homogenate treated with polyamide, a major part of the enzyme activity appeared in the centrifugal precipitates in the range of l,400×g to 15,000×g, and polyphenol content decreased. Therefore, it was thought that the enzyme in tea leaves existed primarily on the particle as small as mitochondria, but that it coagulated easily in the homogenate containing a high concentration of polyphenol, and precipitated by centrifugation at l,400×g.

And also from the comparison of the ratio of the enzyme activity to chlorophyll concentration in each fraction separated by centrifugation, it was assumed that a major part of the enzyme did not exist in the chloroplast, but was bound on the precipitable particle in the range of l,400×g to 15,000×g.     

Reactive oxygen and ethylene are involved in the regulation of regurgitant-induced responses in bean plants

Application of regurgitant from Leptinotarsa decemlineata Say on wound surfaces of one wounded leaf of intact bean (Phaseolus vulgaris L.) plants resulted in activation of ethylene biosynthesis followed by an increase of both peroxidase and polyphenol oxidase activity. The aim of the present investigation was to study the source of increased oxidative enzyme activities in regurgitant-treated bean leaves and to determine if hydrogen peroxide and ethylene biosynthesis is responsible for regurgitant-induced amplification of wound responses in bean plants. As the regurgitant contained relative high activities of both peroxidase and polyphenol oxidase, there is a possibility that increased enzyme activities in bean leaves following regurgitant treatment is an artifact of insect-derived enzymes. Localisation experiments and electrophoretic analysis revealed that only part of the increased enzyme activities could be attributed to regurgitant-derived enzymes. Both increase of ethylene production and oxidative enzyme activities depended on protein synthesis. To demonstrate if the increase of oxidative metabolism was ethylene-dependent, seedlings were pretreated with aminooxyacetic acid, an inhibitor of ethylene biosynthesis, and 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene action. Increase of both peroxidase and polyphenol oxidase activity in wounded and subsequently regurgitant-treated leaf was abolished by both aminooxyacetic acid and 1-MCP. Inhibitor studies indicated that H2O2 generated through NADPH oxidase and superoxide dismutase is necessary for regurgitant-induced increase of ethylene production and oxidative enzyme activities.     

Changes in mulitiple forms of polyphenol oxidase during maturation of tea leaves

Changes in polyphenol oxidase activity during davelopment of tea leaves were investigate by CM-cellulose column chromatography and isoelectric focusing components I-III separate by CM-cellulose chromatography, II and II decreased markedly during maturation. polyphenol oxidase activity was separated int     

烟草愈伤组织多酚氧化酶研究

柳叶烟草愈伤组织中多酚氧化酶氧化邻苯二酚的活性明显高于氧化对苯二酚的活性。当以邻苯二酚为底物时,烟草愈伤组织多酚氧化酶分别在pH 5.6和pH 7.4有两个活性高峰。KCN、Dieca和m-CLAM对烟草愈伤组织多酚氧化酶活性都有明显抑制效应。根据凝胶电泳分析,继代培养愈伤组织多酚氧化酶同工酶有5条酶带,而巳分化出芽原基的愈伤组织和新分化长出的小叶都有7—8条酶带。继代培养的愈伤组织多酚氧化酶主要存在于除去线粒体的上清液中,线粒体部分也可测出酶活性。继代培养愈伤组织在接种后18天内,多酚氧化酶活性无重大改变。在此期间,Dieca对呼吸的抑制效应也变化不大。分化组织多酚氧化酶活性显著高于继代培养愈伤组织,在芽原基形成后,酶活性明显升高;此时Dieca对呼吸的抑制也由32%上升到47%。     

Changes in lipoxygenase activity in relation to lipid degradation in plucked tea shoots

The activity of lipoxygenase in tea leaf increased after plucking of tea shoots. The changes of lipoxygenase activity were accelerated by the dehydration of tea leaves. The activity of lipoxygenase was higher in the good fermenting clones than in the poor fermenting clones of black tea. The content of peroxide in tea shoots increased with rising lipoxygenase activity.     

The pathway of betalain biosynthesis: Effect of cytokinin on enzymic oxidation and hydroxylation of tyrosine in Amaranthus tricolor seedlings

The effect of exogenously added tyrosine or l -3-(3,4-dihydroxyphenyl) alanine on the accumulation of betacyanin in response to cytokinin in Amaranthus tricolor (L.) var. tricolor half-seedlings depends on the age of the seedlings and the treatment of the seedlings prior to induction of pigment by benzyladenine. Neither extracted polyphenol oxidase, peroxidase or tyrosine hydroxylase activity, nor in vivo tyrosine hydroxylation is increased in response to exposure of seedlings to cytokinin. However a small percentage of the polyphenol oxidase activated or unmasked by Triton X-100 treatment of membrane fractions is increased after cytokinin treatment of half-seedlings for 22 h. It is concluded that cytokinin control may be on a multi-enzyme membrane-located complex involving part of the polyphenol oxidase activity of the tissue (possibly an isoenzyme), and that the majority of the polyphenol oxidase activity in Amaranthus is a constitutive membrane enzyme which is not involved in betacyanin synthesis. Although cytokinins do not affect in vivo tyrosine hydroxylation, this activity follows closely the accumulation of betacyanin which is first detectable about 6.5 h after the application of cytokinin. Only a very low level of in vivo hydroxylation can be demonstrated in half-seedlings treated for 6 h either with or without cytokinin but it begins to increase shortly after this time. A large increase in this activity by 16 h (independent of cytokinin) can be almost completely (79%) prevented by chloramphenicol (300 μM) although the drug increases accumulation of betacyanin. At this concentration about 30% of the protein synthesis in inhibited. In vitro tyrosine hydroxylation is, however, not reduced in half-seedlings treated with chloramphenicol during 16 h induction nor is extractable polyphenol oxidase reduced. It is concluded that chloramphenicol is inhibiting the synthesis of some protein essential for in vivo hydroxylation other than the activity measured during in vitro hydroxylation and that the inhibition of synthesis of 79% in vivo hydroxylation still leaves enough activity for maximum betacyanin synthesis.     

黑刺粉虱危害对黄金芽茶叶生化成分含量及抗氧化酶活力的影响

【目的】明确黑刺粉虱Aleurocanthus spiniferus为害对茶叶生化物质含量及抗氧化酶活性的影响,为精准、安全、高效、绿色防治黑刺粉虱,提高茶叶品质提供参考。【方法】测定黑刺粉虱为害不同级别下黄金芽芽下第3叶中生化成分(茶多酚、可溶性糖、游离氨基酸、咖啡碱、儿茶素)含量及3种抗氧化酶(SOD, POD和CAT)活性的变化。【结果】随着黑刺粉虱为害等级的提高,黄金芽芽下第3叶中茶多酚的含量显著性降低,且以为害级别为Ⅳ级时最低,为18.82%±0.21%,而游离氨基酸、咖啡碱、可溶性糖和儿茶素的含量在不同的为害级别间均无显著性变化。黄金芽芽下第3叶中POD和CAT活力随着黑刺粉虱为害等级的增加先显著升高后很快降低,均以为害等级Ⅰ时活性最高,分别为57.14±3.98和28.05±0.40 U/mg pro。SOD活力呈显著下降趋势,且以为害等级Ⅳ时SOD活力最低,为442.73±10.54 U/mg pro。【结论】黑刺粉虱为害对黄金芽茶叶中与抗性提高相关的生化物质咖啡碱、儿茶素含量均没有显著性影响,与抗逆补偿相关的游离氨基酸和可溶性糖含量也没有显著性变化,但茶多酚含量显著降低,3种抗氧化酶活性随为害等级升高呈现先升高后显著降低的趋势,说明黄金芽对黑刺粉虱的抵抗力较弱,需要加强黄金芽上黑刺粉虱发生的科学管理。     

茶多酚对金黄色葡萄球菌和铜绿假单胞菌的抑菌机理

以革兰氏阳性的金黄色葡萄球菌和革兰氏阴性的铜绿假单胞菌为试验菌,通过测定茶多酚与两种菌作用前后细菌培养液的电导率和可溶性总糖的变化,以及菌体在磷代谢和蛋白质表达方面的变化,初步阐明了茶多酚对这两种菌的抑菌机理。研究结果表明,茶多酚对金黄色葡萄球菌和铜绿假单胞菌均有抑菌活性,但对金黄色葡萄球菌的抑菌活性更强。经茶多酚处理后,细菌培养液的电导率和总糖浓度均增大,表明了茶多酚可破坏细胞膜的结构、导致细胞通透性增加,进而使细胞内容物外泄。另一方面,经茶多酚处理后的两种菌对磷的消耗量降低,以致严重影响了核酸、磷脂等细胞重要成分的合成以及能量代谢;通过SDS-PAGE分析,证实茶多酚可以阻碍细菌蛋白质的正常表达,以致影响其细胞的结构组成以及酶的催化活性,最终导致细菌正常生理功能的丧失。     

Changes in the location of polyphenol oxidase in potato (Solanum tuberosum L.) tuber during cell death in response to impact injury: comparison with wound tissue

In order to elucidate the nature of the response of potato to impact injury at the biochemical level, changes in the location of the enzyme responsible for the discoloration, polyphenol oxidase, were determined using immunogold location with an antibody specific for potato tuber polyphenol oxidase. Tissue printing revealed that the enzyme was distributed throughout the tuber. Following impact injury, both tissue printing and quantitative electron microscopy indicated that there was no increase in the level of the enzyme although there was subcellular redistribution of polyphenol oxidase. This redistribution was first apparent at 12 h after impact, as determined by the use of confocal immunolocation, and coincided with loss of membrane integrity. These changes were examined in parallel with a number of stress-related parameters in both impact and wound responses. Wounding was accompanied by active gene expression and protein synthesis, leading to metabolic activity and tissue repair. In contrast, the bruising response was characterised by a limited active response and vital-staining methods indicated that after 16 h the tissue undergoes cell death. Received: 4 June 1998 / Accepted: 18 September 1998     

Arthropod-induced neoplastic formations on trees change photosynthetic pigment levels and oxidative enzyme activities

Abstract

In order to test the hypothesis that arthropod-induced neoplastic formations on trees affect biochemical characteristics of both the newly formed galls and host plant tissues, biochemical characteristics with a possible adaptive role were determined in nine gall-former–host tree combinations. Photosynthetic pigments, extractable protein content, and oxidative enzyme activities were determined in gall tissues, leaf tissues of galled leaves, and leaves on ungalled tree branches. Neoplastic tissues were characterized by a low content of photosynthetic pigments, decreased chlorophyll a/b ratio, lower extractable protein content, and decreased activities of peroxidase and polyphenol oxidase as compared with ungalled host leaf tissues. In galled leaves or in leaves adjacent to galls, increased level of peroxidase activity was found. In several gall-inducer–host plant combinations, galled host plant tissues contained increased activity of polyphenol oxidase as well. The presented data reflect long-term systemic effects of neoplastic formation on host tree physiology suggesting that gall inducers affect potential adaptive responses of host plants.     

Biochemical processes at the stage of withering during black tea production

We determined the molecular weight and some properties of multiple forms of phenol oxidase from tea leaves and four other perennial plants. It was shown that multiple high- and low-molecular forms of phenol oxidase differed in substrate specificity. Low-molecular forms of the enzyme mostly demonstrated hydroxylase activity, while high-molecular forms showed catechol oxidase activity. It was revealed that the withering stage of black tea production is accompanied by the formation of only high-molecular forms of phenol oxidase, which possess catechol oxidase activity crucial for the procurement of oxidative reactions and the quality of the product.     

Effect of regurgitant from Leptinotarsa decemlineata on wound responses in Solanum tuberosum and Phaseolus vulgaris

The effect of regurgitant from Leptinotarsa decemlineata Say larvae on wound-induced responses was studied using two plant species, Solanum tuberosum L. and Phaseolus vulgaris L. Wounding of one leaf of intact S. tuberosum plants differentially affected ethylene production and activities of peroxidase and polyphenol oxidase. Only polyphenol oxidase activity was stimulated by wounding in both wounded and systemic leaves. Peroxidase activity was not affected by wounding. Wounding caused only a transient increase of ethylene production from wounded leaves. The application of regurgitant to wound surfaces stimulated ethylene production as well as activities of peroxidase and polyphenol oxidase in both wounded and systemic leaves. Wounding significantly enhanced ethylene production and polyphenol oxidase activity in wounded and systemic leaves of P. vulgaris . The application of regurgitant caused an amplification of ethylene production, peroxidase activity, and polyphenol oxidase activity, in both wounded and systemic leaves of bean plants. Several substances were tested for their role as possible endogenous signals in P. vulgaris . Hydrogen peroxide and methyl jasmonate appeared as potential local and systemic signals of ethylene formation in wounded bean plants. Local ethylene production in leaf discs was differentially affected by the regurgitant application in potato versus bean plants. While all tested concentrations of regurgitant caused stimulation of ethylene formation from potato leaf discs, ethylene production was completely inhibited by increasing concentrations of the regurgitant in bean leaf discs. Our data present evidence that ethylene may play an important role in the interaction between plants and herbivores at the level of recognition of a particular herbivore leading to specific induction of signalling cascades.     

Tea Leaf Polyphenol Oxidase

The large part of the polyphenol oxidase was solubilized from tea leaf homogenate by addition of Tween-80. After filtration of the solubilized polyphenol oxidase fraction through a Sephadex G-25 column and fractionation of the filtrate with ammonium sulfate, the specific activity of the solubilized enzyme increased about 4 to 5 times as much as that of tea leaf homogenate. Optimum pH of the solubilized enzyme was 5.5, and was almost the same as that of water-insoluble enzyme in the acetone powder. The minimum concentrations required for the maximum activity were about 5×10^?3 m, 4.3×10^?3 m, and 3×10^?3 m for d-catechin, l-epigallocatechin, and l-epigallocatechin-gallate, respectively. d-Catechin showed the highest activity among them. The enzyme activity was inhibited by potassium cyanide and sodium diethyldithiocarbamate.     

Role of micronutrient elements in the metabolism of higher plants. I. Changes in oxidative enzyme constitution of tomato leaves deficient in micronutrient elements

Cell-free extracts of tomato leaves from plants grown on media individually deficient in each of the micronutrient elements showed marked changes in enzymatic constitution. Each deficiency caused an alteration in the over-all enzyme pattern which was characteristic of the micronutrient element in question. The metallo-enzymes such as polyphenol oxidase, ascorbic acid oxidase, and peroxidase lost approximately half their activity in plants deficient in the specific metal concerned. In the cases of all other metal deficiencies, however, polyphenol oxidase and peroxidase were elevated in concentration at least two to six times that of the control. Ascorbic acid oxidase was doubled in zinc- and manganese-deficient material. Glycolic acid dehydrogenase and lactic acid dehydrogenase showed smaller parallel rises averaging a one and a half fold increase in content in zinc- and manganese-deficient plants. Reduced diphosphopyridine nucleotide diaphorase was doubled by copper and manganese deficiencies and was increased to almost one and a half fold by zinc deficiency. Endogenous oxygen uptake in boron-deficient, copper-deficient, and manganese-deficient leaf homogenates was increased, and almost lacking in molybdenum-deficient material.     

Analysis of cellulase and polyphenol oxidase production by southern pine beetle associated fungi

In this study, the production of extracellular enzymes by fungi associated with southern pine beetle was investigated for the first time. Cellulase and polyphenol oxidase production were analyzed for three beetle associated fungi. Only the mutualistic symbiont Entomocorticium sp. A was found to produce cellulases and polyphenol oxidase. In time course analyses of cellulase production in batch cultures, Entomocorticium sp. A showed maximum activity of 0.109 U/ml and 0.141 U/ml for total cellulase and endoglucanase activity respectively. Polyphenol oxidase production was simultaneous with fungal growth. Characterization of polyphenol oxidase by activity staining suggests that the enzyme is a tyrosinase/catechol oxidase. Enzyme assays in the presence of polyphenol oxidase inhibitors support the results of the activity staining.     

An Enzyme Hydrolyzing l-Theanine in Tea Leaves

Theanine hydrolase activity in tea leaves was assayed by measuring enzymatically released ethylamine from l-theanine. The o-phthalaldehyde derivative of ethylamine was measured by reverse phase HPLC recorded with a spectrofluorometric detector.

Theanine hydrolase activity was purified about 4.6-fold by DEAE-cellulose column chromatography. Although this active fraction also had glutaminase activity, the yield of the glutaminase activity was about 50% of that of theanine hydrolytic activity. The theanine hydrolytic activity was inhibited by acidic amino acid and l-alanine, and stimulated by l-malic acid. The purified enzyme solution hydrolyzed not only theanine but also γ-glutamylmethylamide, γ-glutamyl-n-propylamide, γ-glutamyl-n-butylamide, γ-glutamyl-iso-butylamide, and γ-glutamyl-n-amylamide, which were synthesized from l-pyroglutamic acid and corresponding alkylamines. However, N-methylpropionamide and N-ethylpropionamide were not hydrolyzed. The theanine hydrolase activity and glutaminase in tea leaves showed the same pH optimum (8.5).

The activity of theanine hydrolase in tea leaves increased during the first lOhr after plucking but then decreased gradually, while that of glutaminase decreased constantly and was almost lost     

Ethylene production in rice bronzing leaves induced by ferrous iron

Bronzing, a nutritional disorder of rice plants which is widely distributed in tropical lowlands, was induced by dipping the cut end of rice leaves into FeSO₄ solution (pH 3.5). Ethylene production; the activities of peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase; and the effects of Co²⁺, aminoethoxyvinylglycine, Ag⁺, cycloheximide, and 1-aminocyclopropane-1-carboxylate, were investigated in the course of bronzing development. It was found that ethylene production could be stimulated up to about 20 times that of the control by Fe²⁺, and a peak could be reached at about 24 h after incubation. The Fe²⁺-treated leaves also had 10-fold higher peroxidase activity than the control, whereas in vitro enzyme activity was inhibited by Fe²⁺. Cycloheximide retarded in vivo stimulation of peroxidase, indicating that in vivo stimulation resulted from inducing de novo synthesis of the enzyme. No changes in the activities of phenylalanine ammonia-lyase and polyphenol oxidase were observed. The results, obtained from the incubation of leaves with Co²⁺, aminoethoxyvinylglycine, Ag⁺, cycloheximide, or 1-aminocyclopropane-1-carboxylate, showed that ethylene production was the effect of Fe²⁺ stress and that it was not involved in the process of bronzing development, which is probably an acclimation process to enable plants to cope with stress. The accelerated peroxidase activity may be associated with bronzing development.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - EFE ethylene forming enzyme - PAL phenylalanine ammonia-lyase - POD peroxidase - PPO polyphenol oxidase - SE standard error     

Activity of tocopherol oxidase in Phaseolus coccineus seedlings

In the present study, we have demonstrated that membrane-free extracts of etiolated shoots of Phaseolus coccineus seedlings show tocopherol oxidase activity. For this reaction, presence of membrane lipids, such as lecithin and mixture of plant lipids was required. The rate of the reaction was the highest for α-tocopherol and decreased in the order α ? β > γ > δ tocopherols. In the case of α-tocopherol, the main oxidation product was α-tocopherolquinone, while for the other tocopherol homologues the dominant products were other derivatives. When the enzyme activity was measured in leaves, hypocotyls and roots of etiolated seedlings of P. coccineus, the oxidase activity was the highest in extracts of leaves and decreased towards the roots where no activity was detected. The effect of hydrogen peroxide and of different inhibitors on the reaction suggest that tocopherol oxidase does not belong to peroxidases or flavin oxidases but rather to multi-copper oxidases, such as polyphenol oxidases or laccases. On the other hand, catechol, the well-known substrate of polyphenol oxidases and laccases, was not oxidized by the enzyme, indicating a high substrate specificity of the tocopherol oxidase.     

Polyphenol oxidase produced during encystation of Acanthamoeba castellanii

Acanthamoeba castellanii has a phenol oxidase activity that is believed to be a laccase. Enzyme activity was found in the outer cyst wall, in the cytoplasm of encysting amoebae and in the encystment medium. Encystment procedures were modified to promote an increase in the amount of soluble enzyme secreted during encystation. Acanthamoeba polyphenol oxidase has a pH optimum of 6.0 and a Km value of 0.21 mM with dihydroxyphenylalanine. The enzyme does not oxidize tyrosine, and it is inhibited by chloride but not by inhibitors of peroxidase. Its synthesis coincides with encystation, and known inhibitors of polyphenol oxidase prevent encystation. Polyphenol oxidase may have a role in making the cyst resistant to mechanical and chemical breakdown.