Biochemical properties and potential endogenous substrates of polyphenoloxidase from chufa (Eleocharis tuberosa) corms

Polyphenoloxidase (PPO) was partially purified from chufa corms through ammonium sulphate precipitation and dialysis. Biochemical properties of chufa PPO were analysed using exogenous substrate catechol. Optimal pH and temperature for PPO activity were 5 and 45 °C. Ethylenediaminetetraacetic acid disodium salt and l-cysteine could not inhibit the PPO activity. However, sodium thiosulphate pentahydrate exhibited the strongest inhibiting effect, followed by ascorbic acid and anhydrous sodium sulphite. Except for K⁺, other metal ions such as Zn²⁺, Cu²⁺, Fe³⁺, Ca²⁺, Fe²⁺ and Na⁺ accelerated the enzymatic reaction between catechol and PPO. Kinetic analysis showed that the apparent K_m and V_max values were around 10.77 mM and 82 units/ml min. In addition, (−)-gallocatechin gallate, (−)-epicatechin gallate and (+)-catechin gallate isolated and identified from chufa corms were supposed to be the potential endogenous PPO substrates due to their ortho-diphenolic or pyrogallolic structures. These polyphenols might be catalysed by PPO, resulting in the browning of chufa corms after fresh-cut processing.     

Characterization of polyphenoloxidase (PPO) and total phenolic contents in medlar (Mespilus germanica L.) fruit during ripening and over ripening

Characterization of polyphenoloxidase (PPO) enzyme and determination of total phenolic concentrations during fruit ripening and over ripening in medlar (Mespilus germanica L.) were determined. During ripening, PPO substrate specificity, optimum pH and temperature, optimum enzyme and substrate concentrations were determined. Among the five mono- and di-phenolic substrates examined ((p-hydroxyphenyl) propionic acid, l-3,4-dihydroxyphenylalanine, catechol, 4-methylcatechol and tyrosine), 4-methylcatechol was selected as the best substrate for all ripening stages. A range of pH 3.0–9.0 was also tested and the highest enzyme activity was at pH 7.0 throughout ripening. The optimum temperature for each ripening stage was determined by measuring the enzyme activity at various temperatures over the range of 10–70 °C with 10 °C increments. The optimum temperatures were found to be 30, 20 and 30 °C, respectively, for each ripening stage. Optimum enzyme and substrate concentrations were found to be 0.1 mg/ml and 40 mM, respectively. The V_max and K_m value of the reaction were determined during ripening and found to be 476 U/mg protein and 26 mM at 193 DAFB (days after full bloom) – stage 1, 256 U/mg protein and 12 mM at 207 DAFB – stage 2, 222 U/mg protein and 8 mM at 214 DAFB – stage 3. For all ripening stages sodium metabisulfite markedly inhibited PPO activity. For stage 1 of ripening, Cu²⁺, Hg²⁺ and Al³⁺, for stage 2, Cu²⁺ and Hg²⁺, and for stage 3, Cu²⁺, Hg²⁺, Al³⁺ and Ca²⁺ strongly inhibited diphenolase activity. Accordingly, it can be concluded that as medlar fruit ripen there is no significant changes in the optimum values of polyphenoloxidases, although their kinetic parametres change. As the fruit ripening progressed through ripe to over-ripe, in contrary to polyphenoloxidase activity, there was an apparent gradual decrease in total fruit phenolic concentrations, as determined by using the aqueous solvents and water extractions.     

Role of endogenous and exogenous phenolics in litchi anthocyanin degradation caused by polyphenol oxidase

The degradation of anthocyanins and/or the oxidation of phenolics caused by polyphenol oxidase (PPO) results in an enzymatic browning reaction of fruits and vegetables. This work was conducted with a view to explaining the unexpected observation that litchi (Litchi chinensis Sonn.) PPO did not directly oxidise litchi anthocyanins. PPO and anthocyanin from litchi fruit pericarp were extracted and purified, respectively, and then the anthocyanin degradation by PPO in the presence of (−)-epicatechin (endogenous PPO substrate), and catechol and gallic acid (exogenous PPO substrates) were analysed comparatively. The results showed that catechol was the most effective in litchi anthocyanin degradation, followed by (−)-epicatechin and gallic acid, but no significant differences existed between catechol and (−)-epicatechin. The study suggested that litchi PPO directly oxidised (−)-epicatechin; then oxidative products of (−)-epicatechin in turn catalysed litchi anthocyanin degradation, and finally resulted in the browning reaction, which can account for pericarp browning of postharvest litchi fruit.     

八角莲多酚氧化酶的酶学性质

以邻苯二酚为底物,研究八角莲多酚氧化酶（polyphenol oxidase,PPO）粗提液的酶学特性。结果表明,底物邻苯二酚最适浓度为1.0 mol/L,八角莲PPO的最适pH值为7.0,最适温度为30 ℃,90 ℃高温处理10 min,八角莲PPO酶活力仅剩11.92%。PPO催化的酶促褐变反应符合米氏动力学方程,相应动力学参数Km和vmax分别为0.230 7 mol/L和769.23 U/min。抗坏血酸、甘氨酸、乙二胺四乙酸、柠檬酸对酶活性具有抑制作用,十二烷基硫酸钠表现出激活作用,亚硫酸氢钠对酶活性的抑制作用强于硫代硫酸钠,Al3+和Cu2+对酶活性具有一定的激活作用,Ca2+对酶活性有一定的抑制作用,Mg2+、Fe2+和Fe3+对酶活性影响不显著。     

Role of anthocyanins,polyphenol oxidase and phenols in lychee pericarp browning

Postharvest browning of lychee fruits is investigated in relation to anthocyanins, polyphenol oxidase (PPO) and phenols, using a purified PPO, anthocyanin extract and phenolic extract. Lychee PPO cannot oxidise the degradation of anthocyanins, but the presence of phenolic extract stimulates pigment degradation by PPO, leading to the development of brown pigments. The absorbance at 410 nm of the reaction solution is correlated well with the peel browning index. Pyrogallol, catechol and 4‐methylcatechol are good stimulators, but no action with chlorogenic acid, p‐cresol, resorcinol or tyrosine is observed. A decrease in the ascorbic acid content of lychee pericarp is associated with an increase in the peel browning index, and the addition of ascorbic acid to the reaction mixture exerts a preventive action on the anthocyanin degradation. Data here suggest that anthocyanins, PPO and phenols contribute to lychee pericarp browning involved in anthocyanin–PPO–phenol reactions. © 2000 Society of Chemical Industry     

Use of response surface methodology to study the combined effects of UV-C and thermal processing on vegetable oxidative enzymes

The combined thermal (25–65 °C) and ultraviolet processing (UV-C) effects on lipoxygenase (LOX), peroxidase (POD) and polyphenoloxidase (PPO) at different pH values (4.0–7.0) were studied using a central composite design. An initial screening design revealed that all factors had a significant effect on enzymatic activity except wavelength which showed a negligible effect. A synergistic effect was found between temperature and UV exposure time for POD and PPO and between pH and exposure time for LOX. LOX enzyme was affected by acidic conditions. POD was UV-C labile whereas PPO was the most UV-C resistant enzyme but was thermolabile. Second-order polynomial equations indicated that enzyme activities were inactivated after exposure to 58.2 mJ/cm² UV at 60 °C or higher temperatures at any pH condition. Combination of UV and thermal processing allowed the use of low energy/doses to obtain complete enzymatic inactivation. This study may serve as a basis to design UV-C processes for the inactivation of enzymes in liquid matrices.     

马铃薯多酚氧化酶酶学特性及热稳定性模型的研究

目的：研究马铃薯中多酚氧化酶（polyphenol oxidase,PPO）的部分酶学性质及热稳定性并拟合数学模型预测酶活变化过程。方法：采用紫外可见分光光度计测定马铃薯多酚氧化酶活力,利用Matlab软件拟合二元多项式热稳定性数学模型,SPSS软件对多项式模型回归分析。结果：马铃薯多酚氧化酶的最适pH为5.5,最适反应温度为40℃,最适底物浓度为8.33mmol/L,加工温度、时间与酶活残存率的函数关系为：Z=24.5439＋6.1163X＋3.4064Y-0.0269X2-0.2361XY-0.0351Y2-0.0002836X3＋0.001326X2Y＋0.0012XY2,其中R2=0.981,F=100.695,p〈0.001。结论：二元三次多项式模型能够较准确的反映并预测马铃薯多酚氧化酶在热加工过程中酶活的变化趋势。     

芋头多酚氧化酶的分离纯化与酶学特性

以鲜芋头为原料,通过提取、30%～80%饱和度硫酸铵沉淀、透析、超滤,以及柱层析等步骤对芋头多酚氧化酶（polyphenol oxidase,PPO）进行逐级分离纯化,并探讨其酶学特性及抑制剂对其作用规律。结果表明：经DEAE-Sepharose、Superdex G-75色谱柱纯化后,得到了电泳纯PPO,比活力为27 471.26 U/mg,纯化倍数达6.37 倍。纯化后的PPO经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析,其分子质量约为24 kDa。PPO与底物结合能力顺序为：邻苯二酚＞间苯二酚＞间苯三酚＞焦性没食子酸＞L-酪氨酸。PPO的最适反应温度、pH值和反应时间分别为30 ℃、6.8和60 s。抗坏血酸和L-半胱氨酸对提取的PPO有较强的抑制作用,其抑制类型分别属于反竞争性抑制和竞争性抑制方式。结果表明,在芋头加工过程中通过控制温度、pH值或加入适当的抑制剂处理,可以有效减少褐变的发生。     

Synergistic effect of high-intensity ultrasound and β-cyclodextrin treatments on browning control in apple juice

This work evaluated the synergistic effects of combined high-intensity ultrasound (HIU) with β-cyclodextrin (β-CD) treatments on inhibiting browning of apple juice and explored the mechanism through simulation system. The combined treatment of 300 W HIU with 0.006 g mL⁻¹ β-CD had a synergistic impact on maintaining juice colour, resulting in a 39.06% reduction in browning degree, only a 36.64% decrease in total phenolic content, and a 17.82% reduction in PPO activity. The inhibition of enzymatic browning in simulated system revealed that HIU suppressed the enzyme (Polyphenol oxidase, PPO) and β-CD inhibited enzyme (PPO) and embedded substrate (polyphenol). The results of spectroscopic analysis showed that the particle-size distribution of PPO narrowed, the content of α-helix in the secondary structure increased, the fluorescence intensity increased, and the maximum wavelength was red-shifted after HIU and β-CD treatment. Changes in structure could further result in PPO activity loss. Hence, the combined treatment could synthetically alleviate the browning of apple juice.     

雪梨多酚氧化酶酶学特性及其果汁褐变控制技术研究

雪梨在加工过程中极易发生酶褐变,为抑制雪梨汁的酶褐变,试验以雪梨为原料制备多酚氧化酶(Polyphenol oxidase,PPO)粗酶提取液,加入邻苯二酚为底物,采用分光光度法对其PPO的酶学特性及不同护色剂对PPO活性的影响进行了研究;并选择L-半胱氨酸、D-异抗坏血酸钠、抗坏血酸3种添加剂对雪梨汁进行护色,通过响应面优化试验确定雪梨汁最优护色组合。结果表明:雪梨PPO的最适pH为4.5,最适温度为30 ℃;雪梨PPO具有一定的热稳定性,随着温度的提高,抑制PPO活性所需要的时间逐渐减少;雪梨PPO催化底物邻苯二酚的酶促反应动力学与米氏方程高度符合,最大反应速率Vmax=217.39 U/min,米氏常数Km=0.152 mol/L。雪梨汁加工的最优护色剂组合为:6.56 mmol/L的L-半胱氨酸、4.58 mmol/L的D-异抗坏血酸钠和6.18 mmol/L的抗坏血酸,在此条件下对雪梨汁褐变的抑制率可达90.82%。     

芒果多酚氧化酶的特性及抑制研究

以邻苯二酚为底物,采用分光光度法在420nm处测定芒果多酚氧化酶(PPO)的活性,研究了温度、pH值、底物浓度对其活性的影响,并建立了酶促褐变反应动力学方程,探讨了L-半胱氨酸、抗坏血酸、亚硫酸氢钠、柠檬酸和醋酸五种抑制剂对芒果酶促褐变的抑制效果。结果表明,芒果多酚氧化酶的最适温度为50℃,最适pH值为6.86;酶促褐变反应动力学符合米氏方程所描述的单底物酶促反应动力学,以邻苯二酚为底物的最大反应速度Vmax=192(U/min·g),Km=0.0173mol/L,相应的动力学方程为v=192[S]/(0.0173+[S]);五种物质对该酶均表现出不同的抑制作用,抑制效果为:抗坏血酸>L-半胱氨酸>亚硫酸氢钠>柠檬酸>醋酸。     

双孢蘑菇酶促褐变特性及褐变的控制

为有效控制双孢蘑菇褐变,研究pH值、温度、底物浓度以及抑制剂对双孢蘑菇的多酚氧化酶(polyphenol oxidase,PPO)活性的影响。结果表明：以邻苯二酚为底物,双孢蘑菇PPO最适反应pH6.8,最适反应温度20℃,最适反应底物浓度为0.06mol/L,米氏常数Km为0.1072mol/L。采用计算机图像处理技术对双孢蘑菇褐变程度进行定量检测,得出对双孢蘑菇贮藏保鲜效果较好的褐变抑制剂及其最佳配比为0.30mmol/L半胱氨酸、0.06％抗坏血酸、0.05%乙酸。     

Characterisation and tissue distribution of polyphenol oxidase of deepwater pink shrimp (Parapenaeus longirostris)

Characterisation and tissue distribution of polyphenol oxidase (PPO) was studied in deepwater pink shrimp (Parapenaeus longirostris) post mortem. PPO activity was the highest in the carapace, followed by that in the abdomen exoskeleton, cephalotorax, pleopods and telson. No PPO activity was found in the abdomen muscle and in the pereopods and maxillipeds using the enzymatic assay. Storage of whole shrimps and of the different organs showed that melanosis (blackening) required the presence of the cephalotorax to be initiated, indicating that its development depends on other factors in addition to the PPO levels. Further characterisation was carried out in extracts partly purified using 40–70% ammonium sulfate fractionation. The enzyme had the highest activity at pH 4.5 and was most stable at pH 4.5 and 9.0. No clear maximum was observed in the 15–60 °C range but the higher stability was achieved at 30–35 °C. Apparent kinetic constants in the partly purified PPO from carapace were K_M = 1.85 mM and V_max = 38.5 U/mg of protein, pointing to a high affinity and reactivity of the enzyme when assayed with DOPA. Electrophoretic mobility was studied in native PAGE and non-reducing SDS–PAGE followed by staining with DOPA. Approximate MW of 500 kDa and 200 kDa were observed, respectively. These two forms could correspond to aggregates of minor PPO subunits that could not be resolved in these electrophoretic systems. The peptide mass fingerprinting obtained by MALDI-TOF analysis showed some peptides whose homology with hemocyanins and different PPO subunit precursors has already been demonstrated in the same species.     

苔干加工过程中控制酶促褐变的热处理法工艺条件

研究了热处理法钝化苔干多酚氧化酶 (PPO)活性 ,从而控制酶促褐变的工艺条件及效果。结果表明 ,采用邻苯二酚为底物 ,在 41 0nm处测定酶促反应 1min时OD值来表示酶活性高低是适宜的 ;以 85～ 1 0 0℃热水烫漂 1min处理或沸水蒸汽处理 1～ 2 5min后再于 70℃条件下干燥脱水至成品 ,能有效钝化苔干PPO活性 ,很好地控制了酶促褐变 ,产品质量好。也可先用 0 0 5 %NaHSO3浸泡处理苔干 5min后再于 3 5℃条件下干燥脱水至成品 ,得到的产品质量好 ,PPO活性被完全抑制 ;传统的晾、晒干制或烘干不是苔干干制加工的最佳方法。     

国产大麦多酚氧化酶酶学特性的初步研究

以邻苯二酚为底物通过分光光度计法,对国产大麦中多酚氧化酶（PPO）的酶学特性进行了研究。同时建立了 PPO 活性测定方法,确定了大麦 PPO 的米氏常数 Km 为1.96mol/L。结果表明,最适测定条件为酶添加量≤6mL、反应时间为4min,检测波长为415nm;国产大麦 PPO 的最适作用温度为80℃,最适作用 pH 为7.6。PPO 在沸水中处理6min 即可有效抑制其活性。实验中同时考察了柠檬酸、抗坏血酸对大麦 PPO 的抑制作用。     

Purification and enzymatic characteristics of a novel polyphenol oxidase from lotus seed (Nelumbo nucifera Gaertn.)

A polyphenol oxidase (PPO) from lotus seed was purified by the procedures including ammonium sulphate precipitation and affinity chromatography. The apparent molecular mass was 38.6 kDa by SDS‐PAGE. Kinetic studies showed that the K_m and V_max values for catechol were 6.04 mm and 416.67 U, respectively. The PPO performed optimal activity in 20 °C and pH 7.0. The enzymatic activity could be mainly maintained up to 50 °C and pH 4.0–7.0. The activity could be inhibited by various inhibitors including thiourea, urea, sodium hydrogen sulphite, EDTA·2Na, SDS, citric acid, guanidine hydrochloride, ascorbic acid, sodium sulphite and sodium thiosulphate. The metal ions Ba²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺ and Zn²⁺ could inhibit the activity of PPO, while Cu²⁺ performed obvious enhancement. The enzymatic properties of PPO could probably provide practical application in inhibiting the PPO activity and preventing enzymatic browning in the process of picking, transportation, processing and storage of fresh lotus seeds.     

Purification and characterisation of polyphenol oxidase from red Swiss chard (Beta vulgaris subspecies cicla) leaves

We report purification and characterisation of a polyphenol oxidase from red Swiss chard (rcPPO). Our purification procedure resulted in a 39-fold enrichment in specific activity and 17% recovery of total enzyme activity. The purified rcPPO appeared as a monomeric protein of 41 kDa, with a specific conformation conserved in the Cu²⁺ combining region. It was optimally active at pH 7.5 and 45 °C. It had a diphenolase substrate preference towards l-DOPA, catechol and chlorogenic acid, but also exhibited weak monophenolase one toward 4-methoxyphenol and l-tyrosine. We also found that the enzyme was activated by K⁺, Na⁺, SDS and laurouyl sarcosine, but inhibited by divalent cations including Ca²⁺, Cu²⁺. Its activity was completely inhibited by ascorbic acid, cysteine, 1,4-dithiothreitol, β-mercaptoethanol, sodium diethyldithiocarbamate, sodium metabisulphite, sodium sulphite and thiourea. This first report on the purification and characterisation of red Swiss chard PPO provides a basis for understanding and use of this enzyme.     

Purification and characterisation of polyphenol oxidase (PPO) from eggplant (Solanum melongena)

Eggplant (Solanum melongena) is a very rich source of polyphenol oxidase (PPO), which negatively affects its quality upon cutting and postharvest processing due to enzymatic browning. PPO inhibitors, from natural or synthetic sources, are used to tackle this problem. One isoform of PPO was 259-fold purified using standard chromatographic procedures. The PPO was found to be a 112 kDa homodimer. The enzyme showed very low K_m (0.34 mM) and high catalytic efficiency (3.3 × 10⁶) with 4-methyl catechol. The substrate specificity was in the order: 4-methyl catechol > tert-butylcatechol > dihydrocaffeic acid > pyrocatechol. Cysteine hydrochloride, potassium metabilsulphite, ascorbic acid, erythorbic acid, resorcylic acid and kojic acid showed competitive inhibition, whereas, citric acid and sodium azide showed mixed inhibition of PPO activity. Cysteine hydrochloride was found to be an excellent inhibitor with the low inhibitor constant of 1.8 μM.     

Polyphenol oxidase from yali pear (Pyrus bretschneideri)

Polyphenol oxidase (PPO) was isolated from Yali pear (Pyrus bretschneideri R). At the end of purification by ion exchange chromatography on DEAE-cellulose, 10.8-fold purification was achieved. The enzyme showed activity to catechol, pyrogallol, chlorogenic acid and DL-DOPA; of these four, chlorogenic acid was the best substrate. The optimum pH for the PPO was 7.0. PPO activity was not destroyed by heating to 30° for 30 min. The effects of various compounds as inhibitors of the reaction catalysed by the enzyme were tested.     

Purification and Characterization of Polyphenol Oxidase from Akko XIII Loquat (Eriobotrya japonica cv Akko XIII)

Akko XIII is an important loquat variety grown in Turkey. As with many fruits and vegetables, enzymatic browning catalyzed by polyphenol oxidase (PPO) also occurs in loquats. PPO from Akko XIII loquat was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis and ion exchange chromatography. The enzyme showed several peaks with PPO activity on DEAE-Toyopearl 650 M column, of which only two (isoenzyme A and isoenzyme B) were characterized. Assay of activity of the isoenzymes between pH 3.04 and 7.80 using catechol as substrate showed two activity peaks, one at acidic pH and the other at neutral pH. pH optima of isoenzyme A and B were found to be at 7.4 and 4.98, respectively. The Km values of isoenzyme A and B using catechol as substrate were found to be 152.3 mM and 5.4 mM, respectively. They both displayed maximal activity at 30^oC. The two isoenzymes displayed different heat resistance and sensitivity towards various inhibitors.