紫薯多酚氧化酶底物特异性分析

分别以愈创木酚、儿茶酚、间苯二酚、对苯二酚和绿原酸5种酚类物质为底物,加入紫薯多酚氧化酶,反应体系在300～400 nm范围内均有吸收峰,说明这5种物质都是紫薯多酚氧化酶的作用底物.紫薯多酚氧化酶对各酚类底物的亲合性依次为绿原酸>儿茶酚>间苯二酚>愈创木酚>对苯二酚.     

苹果梨中多酚氧化酶的性质及多酚成分定性

以苹果梨中的多酚氧化酶(PPO)为研究对象,对其酶的性质及多酚成分进行研究。采用分光光度法测定酶活,利用高效液相色谱法定性苹果梨果肉中的酚类物质。PPO在pH值为7.8时酶活波峰最大,在7.4时酶活下降趋势最不明显;当底物浓度大于3 mmol/L时,随着浓度的增加,PPO活性反而下降,底物为表儿茶酚时酶活最强;苹果梨果肉中可检测到的酚类物质包括儿茶酚、绿原酸、表儿茶素和槲皮素。结果表明:PPO的最适pH值为7.8,PPO在pH为7.4时最稳定;表儿茶酚和PPO的亲和力最高;苹果梨中的酚类物质绿原酸含量最多,其次含量较高的是儿茶素和槲皮素。     

鲜切慈菇酶促褐变底物的分析确定

鲜切慈菇在贮藏过程中极易发生酶促褐变。为进一步研究其褐变机理,分析测定了慈菇对不同酚类底物的亲和性,利用薄层层析、紫外吸收光谱及高效液相色谱法鉴定鲜切慈菇酶促褐变的主要底物。结果表明,慈菇中的酚类物质主要为愈创木酚和儿茶酚,慈菇中 POD 对不同底物的结合能力依次为愈创木酚＞焦性没食子酸＞绿原酸＞L-酪氨酸＞儿茶酚,引起酶促褐变的主要底物是愈创木酚。     

紫薯中绿原酸类化合物标准品的制备

从紫薯中提取制备高纯度、市场价值较高的绿原酸类化合物标准品。紫薯切片,采用沸水提取、大孔树脂吸附和凝胶柱色谱分段,然后以反相制备液相色谱获得高纯标准品,最后以核磁共振技术确定化学结构。从紫薯中制备得到异绿原酸A、异绿原酸B、异绿原酸C和3,4,5-O-三咖啡酰基奎宁酸,高效液相色谱检测表明这些化合物的纯度均在98%以上。本研究建立的制备工艺可高效地将紫薯中绿原酸类化合物富集,并快速获得高纯样品,有效地避免了紫薯花色苷类化合物对产品色泽和纯度的影响。     

紫薯愈伤组织诱导和培养条件优化及绿原酸类化合物的积累

以紫薯叶片为外植体,通过筛选培养基和培养条件建立了紫薯愈伤组织培养体系。研究6-苄基氨基嘌呤(6-BA)及萘乙酸(NAA)不同浓度组合对紫薯愈伤组织诱导的影响,之后分别考察基础培养基MS、1/2MS、B5、WPM、White及NAA、6-BA、2,4-二氯苯氧乙酸(2,4-D)三种激素对紫薯愈伤组织继代生长的影响。确定最适基础培养基后,对激素进行单因素实验后再做正交实验。结果表明1/2MS基本培养基中添加1.5 mg/L 6-BA和0.4 mg/L NAA适合紫薯愈伤组织诱导,诱导率为81.82%。MS为优选基础培养基,通过正交实验优化的激素配比组合为1.0 mg/L NAA,0.4 mg/L 6-BA,1.0 mg/L 2,4-D。最佳继代培养基为MS+1.0 mg/L NAA+0.4 mg/L 6-BA+1.0 mg/L 2,4-D+6 g/L琼脂+30 g/L蔗糖,此优化激素条件下愈伤组织的生物量增长倍数为23.46倍,3-咖啡酰奎宁酸、3,5-二咖啡酰宁酸、4,5-二咖啡酰宁酸三种绿原酸类化合物含量分别为0.30%、1.91%、1.10%,三者总含量3.31%,因此,通过紫薯愈伤组织培养生产绿原酸类化合物是可行的。     

鲜切山药酶促褐变机理的研究

本文研究了鲜切山药在贮藏期间BD及有关成分的变化，分析测定了鲜切山药多酚氧化酶(PP0)的酶学特性，利用薄层层析、HPLC及紫外吸收光谱鉴定引起酶促褐变的主要底物。结果表明：鲜切山药在贮藏过程中PP0活性和游离酚含量呈同步性变化且达显著相关(r＝0．9964)，表明褐变主要是酚类发生的酶促氧化。鲜切山药PP0的最适反应温度为45℃，最适反应pH为5．0和6．8；PP0与不同底物结合能力的强弱依次为绿原酸＞儿茶酚＞酪氨酸＞焦性没食子酸＞愈创木酚＞苯酚；化学抑制剂NaHS03、EDTA—2Na、Zn(Ac)2、CA、VC、L—Cys、NaCl均有不同程度的抑制作用，尤以NaHS03的抑制作用明显；引起酶促褐变的主要底物是绿原酸。     

溧阳白芹PPO特性的研究

以溧阳白芹为试材,冰水清洗、切分后用研钵碾成匀浆状,于低温4℃下10000r/min离心15min,获得的粗酶液用于多酚氧化酶(PPO)活力的测定和酶特性的研究.结果表明:溧阳白芹PPO的最适pH是6.0,最适温度是25℃,溧阳白芹PPO 对60℃以下的温度不敏感,而在80℃以上温度下很易失活.溧阳白芹PPO对酚底物的亲和力依次为:绿原酸＞咖啡酸＞没食子酸＞儿茶酚＞焦性没食子酸＞愈创木酚＞表儿茶酚,且与绿原酸结合时出现最大活力.应用抑制剂的实验表明,半胱氨酸对白芹PPO的抑制效果最好,抗坏血酸和亚硫酸氢钠的抑制效果也很明显.     

莲藕多酚氧化酶酶学性质的研究

系统地研究了底物浓度、pH、温度以及抑制剂对莲藕多酚氧化酶（PPO）的影响.结果表明：以儿茶酚为底物,米氏常数Km为0.01mol/L,莲藕PPO最适宜pH值为7.0,最适温度为35℃,高温可以钝化PPO活性,褐变抑制剂对莲藕PPO活性具有抑制作用.     

紫薯功能性与其食品开发研究进展

紫薯属于甘薯的新品种,具有极好的营养价值和极高的药用价值。这极大促进紫薯在国内乃至国际的种植产业以及产品加工技术的发展。除含有普通甘薯的营养成分外,紫薯还富含花青素、多糖、黄酮类、绿原酸、异绿原酸和硒等功能性成分。正因为紫薯含有这些丰富的功能性成分,所以迫切需要将其加工成营养保健食品,目前以紫薯为原料的饮料类、酒类、牛奶类、全粉类等产品已成为热点。多项研究表明,长期食用紫薯或紫薯产品,可延缓人衰老,抑制致瘤致癌物质的产生,降低血糖血脂水平,利于益生菌的生长,抑制有害菌体的增殖,改善肝功能。现国内外对紫薯功能性及其食品开发的研究颇多,本文将对此作一重点概述,旨在为紫薯的进一步研究开发提供参考。     

鲜切芦蒿PPO特性的研究

芦蒿去除不可食部分、冰水清洗、切分后用研钵碾成匀浆状于低温(4℃)离心机中离心(10000r/min,30min),获得的粗酶液用于多酚氧化酶(PPO)活力的测定和酶特性的研究。结果表明:鲜切芦蒿PPO适宜的pH值为6.5,温度为40℃。鲜切芦蒿PPO对60℃以下的温度不敏感,而在80℃以上温度下很易失活。鲜切芦蒿PPO对酚底物的亲和力依次为:绿原酸、愈创木酚>焦性没食子酸苯酚>酪氨酸>儿茶酚>间苯二酚>苯酚,且与儿茶酚结合时出现最大活力。抑制鲜切芦蒿PPO活力以焦亚硫酸钠效果最好,当其浓度达到0.33mmol/L(即0.06%)时能使PPO完全失活;亚硫酸氢钠和L-Cys的抑制效果也十分强;而EDTA-2Na浓度达3.0mmol/L对鲜切芦蒿PPO的抑制也仅为29.55%,作用很微弱;植酸、AA、醋酸锌、CA和4-HR的抑制作用介于L-Cys和EDTA-2Na之间。     

Inhibition kinetic and mechanism of polyphenol oxidase from various sources by diethyldithiocarbamic acid

Inhibition kinetics and mechanism of polyphenol oxidases (PPO) partially purified from various sources such as Thymbra spicata L. var. spicata and Ocimum basilicum L., and of mushroom PPO bought from Sigma by diethyldithiocarbamic acid have been described using catechol, 4-methylcatechol and pyrogallol as substrates. The inhibition type was competitive for O. basilicum L. PPO using catechol and 4-methylcatechol as substrates, for mushroom PPO using catechol, 4-methylcatechol and pyrogallol as substrates, and for T. spicata L. var. spicata PPO using 4-methylcatechol as a substrate; uncompetitive inhibition for T. spicata L. var. spicata PPO using pyrogallol as a substrate; and non-competitive inhibition for O. basilicum L. and T. spicata L. var. spicata PPO using pyrogallol and catechol as substrates, respectively. The inhibition effect of diethyldithiocarbamic acid on enzymatic browning varied greatly from one phenol to another and from one enzyme to another. Hence, no general rule can easily be established with regard to the type of inhibition observed.     

Inhibition kinetics of polyphenol oxidase by glutamic acid

The inhibition of polyphenol oxidase (PPO) by glutamic acid was investigated. Application of different concentrations of glutamic acid to mushroom solution and Ocimum basilicum L. extract showed that glutamic acid appeared to be an effective browning inhibitor. Glutamic acid showed uncompetitive inhibition for mushroom and Ocimum basilicum L. polyphenol oxidases using 4-methylcatechol as a substrate, for mushroom PPO using catechol as a substrate and for Ocimum basilicum L. polyphenol oxidase using pyrogallol as a substrate; mixed-type inhibition for mushroom polyphenol oxidase using pyrogallol as a substrate; and non-competitive inhibition for Ocimum basilicum L. polyphenol oxidase using catechol as a substrate. Furthermore, sodium azide was used as an inhibitor for comparison with the inhibition potency of glutamic acid. It was found that glutamic acid was a more power inhibitor than sodium azide. The type of inhibition observed depended on the substrate, inhibitor and enzyme source used.     

Inhibition of polyphenol oxidase obtained from various sources by 2,3‐diaminopropionic acid

This paper reports for the first time the inhibition of the catecholase activities of mushroom, artichoke (Cynara scolymus L) and Ocimum basilicum L polyphenol oxidase by 2,3‐diaminopropionic acid. Polyphenol oxidases from artichoke and O basilicum L were purified by ammonium sulfate precipitation, dialysis and a Sepharose 4B‐L ‐tyrosine‐p‐aminobenzoic acid‐affinity column. In inhibition studies, 2,3‐diaminopropionic acid showed uncompetitive inhibition for mushroom PPO using catechol and pyrogallol as substrates, competitive inhibition for O basilicum L PPO using catechol as a substrate, and uncompetitive inhibition for artichoke PPO using catechol as a substrate. Furthermore, sodium azide, which is an inhibitor of PPO, was used as an inhibitor for comparison with the inhibition potency of 2,3‐diaminopropionic acid. The highest 2,3‐diaminopropionic acid inhibition observed with O basilicum L (K_i = 0.89 mM ), followed by artichoke (K_i = 1.42 mM ) and mushroom (K_i = 2.47 mM ), respectively. Copyright © 2005 Society of Chemical Industry     

几种抑制剂对小麦多酚氧化酶活性抑制效果研究

使用3-吗啉丙磺酸(MOPS)缓冲液从全麦粉中提取多酚氧化酶(PPO),以邻苯二酚为底物,研究不同抑制剂在不同浓度下对PPO的抑制作用。结果表明抑制剂浓度为1.0 mmol/L时,各种抑制剂的作用效果分别为:L-抗环血酸90.6%,L-半胱氨酸86.5%,NaHSO3 84%,曲酸75.5%。L-抗环血酸、L-半胱氨酸、NaHSO3、曲酸几种抑制剂浓度减小抑制作用降低。响应面分析得到的L-抗坏血酸、L-半胱氨酸和NaH-SO3最佳抑制浓度组合为:L-抗坏血酸0.4 mmol/L,L-半胱氨酸0.11 mmol/L,NaHSO3 0.25 mmol/L,抑制作用为82.03%。     

APPLICATION OF A NEW CONTINUOUS FLOW SPECTROPHOTOMETRIC METHOD FOR THE CHARACTERIZATION OF POLYPHENOL OXIDASE NATURALLY IMMOBILIZED ON COCONUT FIBER

The association of continuous flow injection and spectrophotometry affords a simple, novel and rapid way of monitoring continuously the activity of naturally immobilized enzymes in their natural environment, thus eliminating cumbersome purification. The method was applied to determine the activity of polyphenol oxidase (PPO) enzymes naturally immobilized on coconut (Cocus nucifera, L.) fiber tissues. Maximum enzyme activity occurred at a temperature of 25C and at pH 6.0 using catechol as substrate. Thermal stability was assayed in a temperature range of 20 to 75C. The PPO exhibited excellent thermal stability, with only 50% loss in its activity at 75C after 4.3 min exposure. For catechol apparent Michaelis‐Menten constant (apparent Km), apparent Vmax and the apparent activation energy were 9.1 × 10^?3 mol L^?1, 0.20 abs min^?1 and 10.5 kcal mol^?1, respectively. The immobilized PPO showed high activity for o‐diphenols. The reactivity order was caffeic acid > pyrogallol > catechol. Complete inhibition of the enzyme was observed with 1 × 10^?3 mol L^?1 concentration of cyanide, thiourea, L‐cysteine, ascorbic acid, sodium sulfite, nitrates of cadmium, zinc and mercury, individually. Benzoic acid, 3‐hydroxy‐benzoic acid, 4‐acetamidephenol, sodium azide, resorcinol, L‐cystine and EDTA at equal concentrations inhibited PPO partially.     

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.     

Characterization and Inhibitors of Polyphenol Oxidase from Chinese Toon

Chinese toon is the unique and traditional woody vegetable in China. Enzymatic browning catalyzed by polyphenol oxidase (PPO) is prone to happen during the harvest, storage, and processing of Chinese toon so that the sensory quality and the nutritional content of Chinese toon products are seriously influenced. In order to prolong shelf life and storage period, the characterization of Chinese toon PPO (CtPPO) has been analyzed in this study. The PPO was extracted and fractionated by 25–70% (NH₄)₂SO₄, and the biochemical characteristics were analyzed. Based on the V_max /K_m ratio, pyrogallol was the most suitable substrate, followed by catechol and gallic acid. CtPPO exhibited no affinity with methyl gallate. The molecular mass of CtPPO was approximately 84.55 kDa estimated by SDS-PAGE. The native PAGE showed four prominent bands. Optimal pH and temperature were 6.2, 40°C and 8.5, 80°C for catechol and pyrogallol, respectively. CtPPO showed high and stable activity at pH ranging from 5.0 to 7.2 for catechol and pH 7.4 to 9.5 for pyrogallol. Activation energy (Ea) values were 263.79 KJ/mol for catechol and 103.91 KJ/mol for pyrogallol. In addition, CtPPO showed stronger heat resistance above 70°C for pyrogallol than catechol in the half-life values, D-values and other thermodynamic parameters. Ascorbic acid was the most effective inhibitor, followed by L-cysteine and citric acid. Purple onion peel extract and pomegranate rind extract were effective natural inhibitors, but the former was more valid.     

金银花过氧化物酶的三相分离纯化及酶学性质

采用三相分离法提取纯化金银花中过氧化物酶,结果表明最优纯化条件为pH?5.60、硫酸铵质量浓度39.49?g/100?mL、提取液与叔丁醇体积比1∶1.38,在该条件下纯化倍数为5.849,回收率为87.64%。金银花过氧化物酶比活力为1 021.6 U/mg,色素清除率为92%;酶学性质研究表明:金银花过氧化物酶最适温度为30℃,热稳定范围为10~40℃;最适pH值为5,pH值稳定范围为4~7。在金银花过氧化物酶催化的双底物酶促反应中,当H_2O_2浓度一定时,酶对愈创木酚的Km值为8.12?mmol/L,vmax值为1.71?mmol/(min·L)。当愈创木酚浓度一定时,H_2O_2的Km值为0.822?mmol/L,vmax值为1.38?mmol/(min·L)。金银花过氧化物酶与底物的亲和力由强到弱依次是邻苯三酚、邻苯二酚、联甲氧基苯胺、愈创木酚。Ca~(2+)、Cu~(2+)、Zn~(2+)对金银花过氧化物酶有激活作用,Mg~(2+)、Mn~(2+)、柠檬酸、抗坏血酸、L-半胱氨酸、亚硫酸钠、偏重亚硫酸钠、十二烷基磺酸钠对金银花过氧化物酶有不同程度的抑制作用。     

EFFECT OF HYDROXYLAMINE, p-AMINOBENZOIC ACID AND p-AMINOSALICYLIC ACID ON THE OXIDATION OF O-DIHYDROXY- AND TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE

The effect of hydroxylamine (NH₂OH), p-aminobenzoic acid (PABA) and p-aminosalicylic acid (PASA) on the spectrum of the final product (s) formed when o-dihydroxy- and trihydroxyphenols were oxidized by tyrosinase was examined. New pigmented product(s), probably oximes, were formed by the interaction of NH₂OH with the o-quinones of 4-methyl catechol, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylpropionic acid (3,4-DPPA) but not with the o-quinones of catechol or protocatechuic acid. Interaction of PABA or PASA with the o-quinones of catechol, 4-methyl catechol, protocatechuic acid, DOPAC and 3,4-DPPA also yielded pigmented oximes. The interaction of the o-quinones of trihydroxyphenols with NH₂OH, PABA or PASA had little effect on the spectrum of the final product (s), suggesting that oximes are not formed in these reactions.     

In vitro anti-platelet effects of simple plant-derived phenolic compounds are only found at high, non-physiological concentrations

Scope : Bioactive polyphenols from fruits, vegetables, and beverages have anti‐platelet effects and may thus affect the development of cardiovascular disease. We screened the effects of 26 low molecular weight phenolic compounds on two in vitro measures of human platelet function. Methods and results : After platelets had been incubated with one of 26 low molecular weight phenolic compounds in vitro, collagen‐induced human platelet aggregation and in vitro TRAP‐induced P‐selectin expression (as marker of platelet activation) were assessed. Incubation of platelet‐rich plasma from healthy volunteers with 100 μmol/L hippuric acid, pyrogallol, catechol, or resorcinol significantly inhibited collagen‐induced platelet aggregation (all p<0.05; n≥15). Incubation of whole blood with concentrations of 100 μmol/L salicylic acid, p‐coumaric acid, caffeic acid, ferulic acid, 4‐hydroxyphenylpropionyl glycine, 5‐methoxysalicylic acid, and catechol significantly inhibited TRAP‐induced surface P‐selectin expression (all p<0.05; n=10). Incubation with lower concentrations of phenolics affected neither platelet aggregation nor activation. Conclusion : As concentrations of 100 μmol/L are unlikely to be reached in the circulation, it is doubtful whether consumption of dietary phenolics in nutritionally attainable amounts plays a major role in inhibition of platelet activation and aggregation in humans.