龙井茶树多酚氧化酶蛋白提取方法的优化

目的优化缓冲液匀浆提取茶树多酚氧化酶的方法。方法以龙井43号茶树鲜叶为材料,采用缓冲液匀浆浸提法提取茶树多酚氧化酶,分别进行不同单因素浸提优化试验,测定比较提取的粗酶活性,确定适合的浸提条件。结果以鲜叶标准为一芽二叶,料液比1:2,加入内含5%PVP(w:v)、经预冷的p H5.6柠檬酸-磷酸盐缓冲液(0.15 mol/L),海砂适量,冰浴研磨,4℃条件下隔夜浸提12 h,于4℃、9000 r/min离心35 min,取上清液,为龙井43号鲜叶中多酚氧化酶缓冲液成浆浸提的最优条件。结论采用优化的浸提条件,可以制备得到高活性多酚氧化酶粗酶液,为茶树多酚氧化酶同工酶分离与酶性质研究提供了基础。     

多酚氧化酶的提取及分离纯化研究进展

多酚氧化酶(PPO)是自然界中分布极广的一种金属蛋白酶,能催化邻-苯二酚氧化成邻-苯二醌,也是许多果蔬等农产品酶促褐变的主要原因。本文综述了国内外多酚氧化酶的提取及分离纯化技术的研究进展,包括传统提取方法(如缓冲液匀浆法、丙酮提取法、丙酮粉提取法等)和新型提取方法(超声波辅助提取法和超高压提取法等),传统经典分离纯化方法(如溶剂法、沉淀法)和新型分离纯化方法(如凝胶色谱分离法和离子交换色谱分离法等),旨在为多酚氧化酶的研究和应用,特别是为抑制农产品酶促褐变提供参考。     

茶树单宁酶的原核表达及酶学性质表征

基于大肠杆菌的密码子偏好性,对茶树单宁酶基因CsTanA碱基进行优化,基因优化后GC含量为51.9%,密码子适应指数为0.8,优化前后基因序列的一致性为77.8%,以pET-30a为表达载体对优化基因进行重组表达及酶学性质分析。A600 nm约0.6的重组菌株以1 mmol/L的异丙基-β-D-硫代半乳糖苷在30 ℃诱导12 h,破碎上清液重组单宁酶rCsTanA比活力为0.35 U/mg,镍柱纯化后的rCsTanA比活力为1.53 U/mg,纯化倍数约为4.4。纯化重组单宁酶rCsTanA分子质量为39 kDa,其最适温度和最适pH值分别为40 ℃和7.0。不同金属离子浓度对酶活力的影响存在差异,在低浓度（1 mmol/L）条件下,K＋增强了rCsTanA 37.28%的酶活力,而Ag＋、Cu2＋和Fe3＋使该酶活力均丧失80%以上;而在高浓度（5 mmol/L）条件下,Cu2＋表现出完全抑制rCsTanA活性的特性。表面活性剂（十二烷基硫酸钠、吐温80和十六烷基三甲基溴化铵）和极性溶剂（甲醇、乙醇、丙三醇、异丙醇及丙酮）对rCsTanA活性具有较强抑制作用。本研究不仅实现了茶树单宁酶的表达和酶学性表征,同时也丰富了单宁酶资源,为植物单宁酶的进一步应用研究提供了必要的理论支撑。     

莲子多酚氧化酶的分离与纯化

由多酚氧化酶（polyphenol oxidase,PPO）导致的酶促褐变严重影响莲子的外观。本研究旨在探讨莲子PPO的提取纯化方法,为研究其蛋白结构、进一步探究莲子褐变机理提供理论依据。以酶活力和酶比活力为指标,对比了提取PPO的四种方法,发现匀浆吸附后丙酮沉淀法提取莲子PPO可获得较高的酶比活力,达289.04 U/mg;采用单因素实验对该提取方法进行优化,得最佳提取参数为:料液比1∶5、提取时间2 h、p H=7、含2%PVPP和0.8%Triton X-100提取溶液,优化后PPO粗酶液酶比活力为294.40 U/mg;采用DEAE Sepharose Fast FLow阴离子交换层析、Sephadex G-75凝胶柱层析对粗酶液进行纯化,得到分子量大约为58 ku莲子PPO,酶比活力为7926.68 U/mg,纯化倍数为26.92,回收率为38.48%。      

不同反应条件对勐库大叶种多酚氧化酶合成茶黄素的影响

研究不同因素对勐库大叶种多酚氧化酶催化合成茶黄素的影响,结果表明在p H 4.0、37℃、茶多酚质量浓度为4.5 mg/mL的条件下,勐库大叶种多酚氧化酶催化1.0 h,最有利于茶黄素的合成,其总产量可达(461.12±15.01)μg/mL,转化率为(15.31±0.40)%。勐库大叶种多酚氧化酶对酯型儿茶素催化活性更强,催化合成酯型茶黄素为主,在催化过程中还伴随着表没食子儿茶素和没食子酸的生成,有利于茶黄素的合成积累,这可能与滇红优异品质形成有关。     

A comprehensive review of polyphenol oxidase in tea (Camellia sinensis): Physiological characteristics,oxidation manufacturing,and biosynthesis of functional constituents

Polyphenol oxidase (PPO) is a metalloenzyme with a type III copper core that is abundant in nature. As one of the most essential enzymes in the tea plant (Camellia sinensis), the further regulation of PPO is critical for enhancing defensive responses, cultivating high-quality germplasm resources of tea plants, and producing tea products that are both functional and sensory qualities. Due to their physiological and pharmacological values, the constituents from the oxidative polymerization of PPO in tea manufacturing may serve as functional foods to prevent and treat chronic non-communicable diseases. However, current knowledge of the utilization of PPO in the tea industry is only available from scattered sources, and a more comprehensive study is required to reveal the relationship between PPO and tea obviously. A more comprehensive review of the role of PPO in tea was reported for the first time, as its classification, catalytic mechanism, and utilization in modulating tea flavors, compositions, and nutrition, along with the relationships between PPO-mediated enzymatic reactions and the formation of functional constituents in tea, and the techniques for the modification and application of PPO based on modern enzymology and synthetic biology are summarized and suggested in this article.     

苦杏仁多酚氧化酶的分离纯化及部分特性分析

以苦杏仁为原料,采用(NH4)2SO4分级沉淀法对苦杏仁多酚氧化酶进行初步分离后经Sephadex G-75凝胶柱层析进一步纯化,冷冻干燥后即得苦杏仁多酚氧化酶纯品;通过凝胶电泳和热分析试验测定多酚氧化酶的相对分子质量和变性温度。结果表明:经分离纯化后苦杏仁多酚氧化酶的纯化倍数为49.03倍,该酶可能存在同工酶且相对分子质量分别约为21.9 k D和23.2 k D;苦杏仁多酚氧化酶变性温度为60.03℃。为苦杏仁加工过程中的酶促褐变控制提供部分理论参数。     

Comparison of two treatment methods on the purification of shrimp polyphenol oxidase

The effects of different stirring time and two treatment methods (salt and organic solvent) on the recovery of shrimp polyphenol oxidase (PPO) were investigated. Stirring for 30 min yielded maximal PPO recovery. With respect to PPO specific activity, yield and purification fold enhancement, the use of butanol treatment followed by Phenyl Sepharose CL-4B chromatography was shown to be better than ammonium sulphate fractionation and then Phenyl Sepharose chromatography. White shrimp PPO was more susceptible than pink shrimp PPO to inactivation during purification. © 1997 SCI.     

小麦主产区小麦粉多酚氧化酶特性分析

将收集我国10个小麦主产区省(区)200多份小麦样品磨成粉,对这些小麦粉多酚氧化酶活性进行测定,评估各主产区小麦粉多酚氧化酶活性含量分布情况。实验结果表明,在10个主产区省(区)中,以陕西省小麦粉多酚氧化酶活性最高,宁夏小麦粉多酚氧化酶活性最低;小麦粉多酚氧化酶活性在7~11min-1g-1范围内分布较集中,占样品总数70.3%。另外,小麦粉多酚氧化酶活性越小,鲜湿面片L*值变化越小。     

百合中多酚氧化酶的部分性质

通过丙酮酚法从百合中提取出多酚氧化酶(PPO)的粗酶液。以儿茶酚为底物时它有两个最适pH,分别为4.0、7.0,在pH5.0-6.5之间酶活力可以在4℃保持至少10h。PPO的最适温度为40℃,从40℃开始酶出现热失活,热失活速度符合一级反应动力学。PPO除对L-酪氨酸没有活力外,对儿茶酚、儿茶素、没食子酸均有活力,其中对儿茶素具有最好的底物特异性。亚硫酸钠对PPO的抑制作用最强,高浓度的抗坏血酸、半胱氨酸、硫脲也有很好的抑制作用,氯化钠、氯化钙、柠檬酸的抑制作用较差。     

Purification of polyphenol oxidase and the browning control of litchi fruit by glutathione and citric acid

Polyphenol oxidase (PPO, EC 1.10.3.2) was purified to homogeneity from litchi peel yielding a single protein with a molecular weight of about 75.6 kD by Sephadex G‐100 gel filtration, and a 108‐fold purification of PPO achieved. The enzyme was determined to be composed of two similar subunits. Glutathione, L ‐cysteine and citric acid suppressed PPO activity markedly, whereas ascorbic acid and n‐propyl gallate showed a little inhibition. Moreover, the effect was enhanced by the addition of citric acid. On the basis of the inhibition of PPO activity in vitro, the use of 10 mmol l ⁻¹ glutathione and 100 mmol⁻¹ l citric acid was found to give good control of the browning of litchi fruit, and an 80–85% inhibition of PPO activity was observed. It is suggested that application of glutathione in combination with citric acid may slow down the browning of litchi fruit. © 1999 Society of Chemical Industry     

蓝莓中多酚氧化酶的酶学特性

为分析蓝莓中多酚氧化酶的酶学特性,本文对影响蓝莓多酚氧化酶（PPO）的酶活力的主要因素:p H、温度、金属离子、抑制剂及热稳定性进行了研究。结果表明:蓝莓中PPO最适p H为6.5,最适温度为30℃,Ca²⁺和Mn²⁺对蓝莓PPO有抑制作用,Cu²⁺、Mg²⁺、Zn²⁺、Fe³⁺对蓝莓PPO有激活作用。柠檬酸、抗坏血酸、L-半胱氨酸及亚硫酸氢钠能抑制PPO酶活,但柠檬酸抑制效果较差。热稳定性实验结果表明:蓝莓果PPO耐热性较差,高温短时可显著抑制PPO酶活性。      

板栗仁中多酚氧化酶活性的研究

采用分光光度法对板栗仁中多酚氧化酶的活性进行了研究.结果表明,酶液加入量与吸光度值成正比关系,多酚氧化酶的最适反应温度为30～40 ℃,最适反应pH值为5.4,板栗仁表层多酚氧化酶的活性显著高于心部的活性.同时,对酶的热稳定性以及抑制剂亚硫酸钠、抗坏血酸对酶活力的影响进行了探讨.     

Biochemical studies of cocoa bean polyphenol oxidase

Polyphenol oxidase (EC 1.14.18.1) was isolated and partially purified from cocoa beans. The properties of the enzyme were studied. The Michaelis constant K_m for catechol was 1 × 10^?2 M . The pH optimum of polyphenol oxidase activity assayed with catechol as substrate occurred at pH 6.8 and was characterised by a relatively high thermal stability, 50% of its activity was lost after heating for 40, 25 and 5 min at 60, 69 and 80°C respectively. The optimum temperature for the enzyme activity with catechol as substrate was around 45°C. The enzyme was reactive towards 3-(3,4-dihydroxy phenyl)-DL -alanine, 3-hydroxytyramine hydrochloride and 4-methyl catechol but showed no activity towards tyrosine, p-cresol, and 4-hydroxy-phenol. A rapid deactivation of the enzyme was observed when catechol of concentration > 40 mM was used as substrate. The enzyme activity was inhibited by ascorbic acid, L -cysteine, sodium bisulphite and thiourea.     

Purification and properties of polyphenol oxidase from oil bean (Pentaclethra macrophylla Benth) seeds

Polyphenol oxidase extracted from oil bean (Pentaclethra macrophylla) seeds was purified 165-fold over the crude enzyme extract. The apparent molecular weight of the enzyme by gel filtration was 110.8 k ± 9.0 k while SDS-PAGE indicated a single species of molecular weight 28.0 k. A copper content of 1.9 mg g^?1 corresponds to one copper atom for each of the four subunits. The purified enzyme oxidised pyrogallol, catechol, 4-methylcatechol and L-dihydroxyphenylalanine but had low activity towards tyrosine. p-Cresol, caffeic acid and cholorogenic acid were not oxidised. Thio-compounds were strong inhibitors of the enzyme. The phenolic compounds tyrosine, resorcinol and orcinol inhibited catechol oxidation but became oxidised in the process.     

Some properties of polyphenol oxidase from lily

A study of crude polyphenol oxidase (PPO) from lily bulbs was carried out to provide information useful for guiding food processing operations. Optimum pH for the enzyme activity in the presence of catechol, were 4.0 and 7.0 at room temperature(approximately 20 °C) and the enzyme was stable in the pH range from 5.0 to 6.5 at 4 °C for 10 h. Its optimum temperature was 40 °C and the heat inactivation of the enzyme followed first‐order kinetics. Lily PPO possessed a diphenolase activity toward catechol, catechin and gallic acid; catechin was the best substrate for the enzyme considering the V_max/K_m ratio. The most effective enzyme inhibitor was sodium sulphite, although ascorbic acid, l ‐cysteine and thiourea were also effective inhibitors at high concentration. But NaCl and citric acid were poor inhibitors of the enzyme. Data generated by this study might help to better prevent lily bulbs browning.     

Properties of polyphenol oxidase in mango (Mangifera indica) kernel

Polyphenol oxidase (PPO) activity of filtered extract of ground mango kernel suspension (400 g litre⁻¹) was studied spectrophotometrically at 420 nm using catechol as substrate. The enzyme was most active at pH 6·0 and 25°C. Activity was reduced by 50% at pH values of 5·0 and 7·1, and also at temperatures of 14°C and 30°C. The calculated activation energy and the Michaelis constant (K_m) were 21·4 kcal mol⁻¹ °C⁻¹ and 24·6 mM , respectively. The V_max value was 2·14 units g⁻¹ mango kernel. The time to heat inactivate PPO decreased rapidly to < 10 min with increasing temperature of ⩾ 70°C at 50% activity. © 1998 SCI.     

油茶籽油及茶叶籽油特征组分分析与比较

研究比较了油茶籽油和茶叶籽油的脂肪酸组成、三酰甘油组成、甾醇、生育酚、角鲨烯等特征组分。结果显示:油茶籽油主要脂肪酸为油酸、棕榈酸和亚油酸;其单不饱和脂肪酸占77.50%~84.44%;茶叶籽油主要脂肪酸为油酸、亚油酸和棕榈酸;其饱和脂肪酸含量为18.86%~23.04%,单不饱和脂肪酸为56.53%~58.54%。2种油脂主要三酰甘油均为OOO、SLO、LOO,但相对比例各不相同;甾醇含量分别为1 099~2 298,499~927 mg/kg,主要组分为β-谷甾醇和Δ7-豆甾烯醇,占甾醇总量80%以上。油茶籽油和茶叶籽油中生育酚以α型为主,占总生育酚90%以上;前者角鲨烯含量为21~336 mg/kg,后者为108~198 mg/kg。研究可为油茶籽油和茶叶籽油的开发和应用提供基础数据支持。     

多种食用菌中多酚氧化酶活性的初步比较

以金针菇、双孢磨菇、凤尾菇、杏鲍菇、香菇、花菇和猪肚菇7种常见食用菌为研究对象,采用磷酸盐缓冲液提取法提取多酚氧化酶(PPO),比较食用菌中多酚氧化酶的活性及温度和pH对多酚氧化酶活性的影响.结果表明,在常温和中性pH条件下,双孢蘑菇PPO活性最高,达到942 U/mL,金针菇PPO和猪肚菇PPO次之,分别为62,58 U/mL.金针菇和双孢磨菇PPO活性的最适温度为20 ℃,凤尾菇、香菇和花菇PPO活性的最适温度为25 ℃,而猪肚菇PPO和杏鲍菇PPO的最适温度较高,分别达到了75,80 ℃.凤尾菇PPO、杏鲍菇PPO和猪肚菇PPO最适pH值均为3.5,金针菇PPO为4.0,香菇PPO为6.5,双孢磨菇PPO为7.0,而花菇PPO最适pH值为8.0.