酶催化分光光度法测定L-酪氨酸

利用L-酪氨酸对酶催化体系的抑制作用,建立了一种操作简便、灵敏的分析L-酪氨酸的新方法。优化了该抑制体系的实验条件,L-酪氨酸5.0×10-7mol/L~1.0×10-4mol/L浓度范围内与抑制率有较好的线性关系,检出限为2.6×10-7mol/L。将浓度为2.0×10-5mol/L的L-酪氨酸进行11次平行测定,其RSD为2.1%。该法已成功的应用于啤酒、葡萄酒中L-酪氨酸含量的测定。     

PHYSICO-CHEMICAL PROPERTIES of POLYPHENOL OXIDASE FROM d'ANJOU and BARTLETT PEARS (PYRUS COMMUNIS L.)

Polyphenol oxidase (PPO) and phenolics were studied in two pear cultivars, d'Anjou and Bartlett. PPO activity, total phenolics and chlorogenic acid concentration differed significantly with respect to cultivar. PPO activity, total phenolics and chlorogenic acid all decreased in overripe fruits. the pH optimum (d'Anjou, 4.7 and Bartlett, 5.5), and optimum temperature (d'Anjou 40C and Bartlett, 20C) for maximum PPO activity were determined. Among substrates, 4-methylcatechol followed by catechol and dopamine, was the most readily oxidized substrate of PPO from both pear cultivars. the enzyme from both cultivars did not show any activity with any of the monohydroxy substrates. K_m of 13.33 and 10.92 mM were determined with 4-methylcatechol for d'Anjou and Bartlett pear PPO, respectively. Benzoic and cinnamic acid series compounds were poor inhibitors of PPO. However, L-cysteine, sodium metabisulfite, ascorbic acid and thiourea proved to be effective inhibitors of this enzyme. Heating at 65C for 30 min resulted in a loss of 60–72% of PPO activity.     

基于ZIF-8@Ag/MWCNTs的绿原酸电化学传感器的构建及应用

构建了用于定量分析绿原酸的ZIF-8@Ag/MWCNTs电化学传感器。Ag纳米粒子具有良好的导电性和电催化能力,而ZIF-8优秀的比表面积能有效的分散Ag纳米粒子,因此在室温合成ZIF-8后,在ZIF-8表面原位还原Ag+制备ZIF-8@Ag复合材料。然后,以MWCNTs作为基底材料,复合ZIF-8@Ag获得修饰工作电极,获得高灵敏度的电化学传感器用于绿原酸的测定。通过循环伏安法（CV）、电化学阻抗谱（EIS）和差分脉冲伏安法（DPV）,探讨了ZIF-8@Ag/MWCNTs/GCE的电化学传感性能。在优化的实验条件下,绿原酸标准品浓度在5×10-8 mol/L~1×10-5 mol/L范围内与氧化峰电流具有良好的线性关系,检出限为1.36×10-8 mol/L（S/N=3）。探究了修饰电极的抗干扰性、重复性和再现性,结果表明电极抗干扰能力较强,重复性及再现性表现良好。用于实际样品绿咖啡豆稀释液的检测时,加标回收率在96.34%~103.34%。该方法简便、可靠,可用于绿原酸及绿原酸实际样品的快速定量分析。     

Purification and properties of polyphenol oxidase in head lettuce (Lactuca sativa)

Polyphenol oxidase (EC 1.10.3.1) in head lettuce (Lactuca sativa L) was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56 000 amu by Sephadex G-100 gel filtration. The purified enzyme quickly oxidised chlorogenic acid (5-caffeoyl quinic acid) and (—)-epicatechin. The K^m values for the enzyme, using chlorogenic acid (pH 4·5, 30°C) and (—)-epicatechin (pH 7·0, 30°C) as substrate, were 0·67 mM and 0·91 mM, respectively. The optimal pH of chlorogenic acid oxidase and (—)-epicatechin oxidase activities were 4·5 and 7·8, respectively, and both activities were stable in the pH range 6–8 at 5°C for 20 h. Potassium cyanide and sodium diethyldithiocarbamate markedly inhibited both activities of the purified enzyme. The inhibitory effect of metallic ions such as Ca²⁺, Mn²⁺, Co²⁺ and Ni²⁺ for chlorogenic acid oxidase activity was stronger than that for (—)-epicatechin oxidase activity.     

Purification and some properties of polyphenoloxidase in eggplant (Solanum melongena)

Polyphenoloxidase (EC 1.10.3.1) in eggplant (Solatium melongena L) was purified by ammonium sulphate fractionation, DEAE-Cellulofine and DEAE-Toyopearl chromatography and Sephadex G-100 gel filtration. The enzyme was purified about 110-fold with a recovery of 5%. The purified enzyme more quickly oxidised chlorogenic acid (5-caffeoylquinic acid, IUPAC) than 10 other substrates used. The K_m value for the enzyme was found to be 0·50 mM with respect to chlorogenic acid; the optimum pH of the enzyme was about 4 with enzyme stability between pH 5 and 8. The enzyme was completely inactivated after heat treatment at 75°C for 30 min or 80°C for 5 min. Sodium metabisulphite, potassium cyanide and sodium fluoride markedly inhibited the enzyme activity.     

番石榴多酚氧化酶的部分纯化及其特性研究

番石榴PPO的粗提取液经过80%硫酸铵盐析和DEAE-Toyopearl 650M、CM-Sephadex C-50离子交换柱层析分离后,被纯化了约126倍,回收率为16.13%。该酶迅速地催化焦性没食子酸的酶促氧化反应,而对对苯二酚和绿原酸则完全无催化活性。该酶对焦性没食子酸的Km值为4.6 mmol/L,其最适pH为7.5,pH稳定性范围在pH4.0~11.0,最适温度为50℃,热稳定性相对较高,在≥90℃加热10 min后仍残留约15%的酶活性。该酶的最佳抑制剂是抗坏血酸和NaHSO3,其次是植酸、盐酸-L-半胱氨酸、柠檬酸,Ca2+、Mg2+等金属离子对该PPO也有较强的抑制作用。     

Heat inactivation and kinetics of polyphenoloxidase from palmito (Euterpe edulis)

Polyphenoloxidase (PPO, EC 1.14.18.1)was extracted from palmito (Euterpe edulis Mart) using 0.1 M phosphate buffer, pH 7.5. Partial purification of the enzyme was achieved by a combination of (NH₄)₂SO₄precipitation (35–90% saturation) and Sephadex G-25 and DEAE-cellulose chromatography. The purified preparation gave five protein bands on polyacrylamide gel electrophoresis, three of them with PPO activity. The K_mvalues for chlorogenic acid, caffeic acid, catechol, 4-methylcatechol and catechin were 0.57, 0.59, 1.1, 2.0 and 6.25 mM , respectively. PPO has a molecular weight of 51 000 Da, maximum activity at pH 5.6 with chlorogenic acid as substrate, and was stable between pH 5.0 and 8.0. The enzyme was heat stable at 50–60°C and inactivated at 75°C. The heat stability of palmito PPO was found to be pH dependent; at 50°C and pH 4.0 the enzyme was fully inactivated after 30 min. The pH/activity studies showed two groups with pK values c 4.6 and 6.7 involved in PPO catalysis.     

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.     

Biodegradation in vivo and in vitro of chlorogenic acid by a sunflower-seedling (Helianthus annuus) like-polyphenoloxidase enzyme

Sunflower seeds contain a high amount of chlorogenic acid (3–4% in weight) and a specific polyphenoloxidase that oxidize it. In this research, the activity of a sunflower seed enzymatic solution was investigated in vivo and in vitro on endogenous and exogenous chlorogenic acid. Phenol degradation was monitored by spectrophotometric analysis at 325 and 420 nm or by HPLC analysis. It was ascertained that during the germination, sunflower seeds synthesized an enzyme like-polyphenoloxidase after 24 h that got dark the seedlings ground and degraded endogenous chlorogenic acid. Sunflower-seedling like-polyphenoloxidase enzyme could be easily isolated in active form. Enzymatic extract catalysed in vitro the degradation of chlorogenic and also caffeic acid. In comparison to original value, after 60 min of incubation under environment temperature and pH 7, chlorogenic and caffeic acid have been reduced by 64 and the 90%, respectively. A high linear correlation was verified between enzymatic activity and original concentration of chlorogenic acid in a range from 0.01 to 0.1 mM. This linear correlation suggested a possible use of like-polyphenoloxidase enzyme for analytical application and other biotechnological uses in the treatment of medium containing natural phenols.     

基于新型互作模型的多酚不同羟基的抗氧化性评价

目的 揭示咖啡酸和绿原酸不同酚羟基抗氧化活性的构效关系及内在规律。方法 建立咖啡酸和绿原酸与功能小分子的复合物作用模型，然后计算互作模型下的分子间氢键，比较分子中间位酚羟基与对位酚羟基的抗氧化性强弱。结果 咖啡酸和绿原酸的间位酚羟基氧原子电荷为-0.486和-0.483，对位酚羟基氧原子电荷为-0.441和-0.445，表明酚羟基氧原子电荷绝对值与酚羟基抗氧化性存在正相关性；酚羟基与功能小分子互作模型显示分子间氢键键长与抗氧化性存在负相关性。结论 酚羟基氧原子电荷和分子间氢键互作模型，可以快速、准确评价同一分子酚羟基中的关键活性位点，同时，理论模型评价与酶抑制实验结果一致。该新型互作模型的建立，有望为天然抗氧化剂开发、酚类化合物调控酶活性的研究提供新的理论参考。     

酶法辅助聚二乙醇-200提取杜仲叶中绿原酸的工艺

采用Box-Behnken中心组合实验和响应面分析法,在单因素试验基础上,以酶用量、料液比、pH和酶解时间为影响因素,绿原酸提取率为响应值,对酶法辅助聚乙二醇(PEG)-200提取杜仲叶绿原酸的工艺进行优化。结果表明,其最佳工艺为:酶用量0.8%、料液比1︰20(g/mL),pH 4.4,酶解时间1.6 h。在此工艺条件下杜仲叶绿原酸提取率可达3.15%。该工艺条件温和、易于控制、提取效率高,可为杜仲叶中绿原酸的进一步开发研究提供工艺参考。     

Production of L-lactic acid from corncob

The optimum temperature, initial pH, amount of added enzyme and substrate (corncob) for the hydrolysis of corncob by Acremonium cellulase were 35 degrees C, 4.5, 10 u/g-corncob and 100 g/l, respectively. Under the optimum conditions, more than 55 g/l of reducing sugars were hydrolyzed from 100 g/l of corncob to 34 g/l of glucose and 12 g/l of xylose based on dried corncob. More than 25 g/l of L-lactic acid was produced from this enzymatic hydrolyzate and less than 5 g/l of xylose remained in the 3-l airlift bioreactor. The production of L-lactic acid by simultaneous saccharification and fermentation (SSF) was also carried out in the 3-l airlift bioreactor using Acremonium thermophilus (cellulose-producer) and Rhizopus sp. MK-96-1196 (lactic acid-producer). More than 24 g/l of L-lactic acid was produced from 100 g/l of untreated raw corncob.     

二次正交旋转组合设计法优化咸蛋清酶解条件

在木瓜蛋白酶酶解咸蛋清的单因素实验基础上,采用二次正交旋转组合设计实验方法对酶解工艺进行了优化。建立了底物浓度(X1)、加酶量(X2)、温度(X3)、pH(X4)与水解度(Y)的经验数学模型:Y=25.77583-4.01800X1+1.42650X3-1.48800X4-1.52933X21-2.50133X22-3.44708X23-3.02933X24+1.05450X1X2-1.58850X2X3。该模型的R2=96.49%,R2adj=94.14%,说明回归方程对实际拟合情况较好,自变量和响应值之间线性关系显著。最终得到咸蛋清酶解最佳条件为:底物浓度3%、加酶量5000U/g、酶解温度50℃、pH5.5、酶解6h。最佳条件下酶解咸蛋清水解度高达26.18%;氮收率可达93.47%。     

Studies on the enzymic browning of apples. Inhibition of apple 0-diphenol oxidase by phenolic acids

The nature of the inhibition of a particulate preparation of apple o-diphenol oxidase by substituted cinnamic acids has been investigated for three substrates: 4-methylcatechol, chlorogenic acid and catechin. Solubilisation of the enzyme preparation altered the pattern of inhibitor/substrate relationships, and these results are discussed on the basis of the occurrence of separate catalytic and inhibitor sites on the enzyme.     

Mechanism of Browning in Fresh Highbush Blueberry Fruit (Vaccinium corymbosum L). Partial Purification and Characterisation of Blueberry Polyphenol Oxidase

The most efficient method for polyphenol oxidase (PPO) extraction from Highbush blueberry fruits was the preparation of an acetone powder. No activity was detected after direct extraction with phosphate buffer (pH 6·5) and detergents such as Triton X-100. PPO has been purified 19-fold, by ultrafiltration, ammonium sulphate precipitation and hydrophobic chromatography. Native-PAGE of the purified fraction revealed the presence of two isoforms. PPO has an observed optimum pH at 4·0, followed by a shoulder at pH 5·0. Caffeic acid is the best substrate (100%), followed by chlorogenic acid (60%) and pyrocatechol (32·5%). No activity was detected towards catechin, protocatechuic acid, resorcinol and monophenols. © 1997 SCI.     

绿原酸对苹果采后灰霉病抗性的影响

绿原酸是苹果中含量最高的酚类物质,为探究外源绿原酸对苹果灰霉病的影响,本研究首先通过体外抑菌实验证实绿原酸可抑制灰葡萄孢（Botrytis cinerea）生长,并确定了最佳抑菌质量浓度（300 μg/mL）。以此质量浓度的绿原酸浸泡富士苹果30 min,24 h后损伤接种灰葡萄孢,以清水浸泡30 min的富士苹果作为对照,同时测定相关代谢酶活力及总酚、类黄酮和木质素含量。结果表明：与对照组相比,300 μg/mL绿原酸处理能显著降低苹果灰霉病发病率且减缓病斑扩展（P＜0.05）;提高几丁质酶和β-1,3-葡聚糖酶活力,诱导酚类代谢途径中多种酶类的响应,具体表现为显著提高苯丙氨酸解氨酶、肉桂酸-4-羟化酶以及4-香豆酰辅酶A连接酶活力（P＜0.05）,促进苹果果实总酚、类黄酮和木质素等抗性物质的积累,从而减缓灰霉病的发展。研究可为绿原酸在苹果采后灰霉病防治中的应用提供理论依据。     

ISOLATION AND SOME PROPERTIES OF AN ACIDIC FRACTION OF POLYPHENOL OXIDASE FROM JERUSALEM ARTICHOKE(HELIANTHUS TUBEROSUS L.)

An acidic fraction of polyphenol oxidase (EC 1.14.18.1), representing the bulk activity, has been isolated from Jerusalem artichoke tubers by copper affinity chromatography, followed by DEAE-Sepharose ion-exchange chromatography. Isoelectric focusing analysis showed a cluster of activity bands (microheterogeneity) in the pH region of 4.5. The amino acid composition of the enzyme was also determined. The pH optimum for oxidation of chlorogenic acid, 4-methylcatechol and catechol was 6.0. Substrate inhibition was observed by high concentrations of chlorogenic acid. Thermal inactivation data indicated an apparent activation energy of 26.2 kcal/mol. Kinetics of inactivation, upon copper removal by KCN treatment, and reconstitution reactions revealed that the former is a much slower process than reactivation.     

Effect of allyl isothiocyanate on antioxidant enzyme activities,flavonoids and post-harvest fruit quality of blueberries (Vaccinium corymbosum L., cv. Duke)

The effect of allyl isothiocyanate (AITC) on antioxidant enzyme activities, flavonoid content, and fruit quality of blueberries var. Duke (Vaccinium corymbosum L.) was evaluated. Results from this study showed that AITC was effective in maintaining higher amounts of sugars and lower organic acids compared to untreated fruit during storage at 10 °C. However, AITC reduced antioxidant enzyme activities [superoxide dismutase (SOD), guaiacol peroxidase (G-POD), glutathione-peroxidase (GSH-POD), ascorbate peroxidase (AsA-POD), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDAR) and glutathione reductase (GR)] and non-enzyme components, ascorbate (AsA) and glutathione (GSH). AITC treatments also reduced the amount of phenolic acids (chlorogenic acid, myricetin 3-arabinoside, quercetin 3-galactoside, quercetin 3-arabinoside, and kaempferol 3-glucoside) and anthocyanins (delphinidin 3-galactoside, delphinidon 3-glucoside, delphinidin 3-arabinoside, petunidin 3-galactoside, petunidin 3-glucoside, petunidin 3-arabinoside, malvidin 3-galactoside, and malvidin 3-arabinoside) during storage at 10 °C. The results from this study indicate that AITC does not promote antioxidant property or scavenge constitutive reactive oxygen species (ROS), but maintain blueberry fruit quality through other mechanisms.     

枸杞中绿原酸对转化生长因子-β1诱导心肌成纤维细胞纤维化的影响

目的：考察枸杞中分离得到的绿原酸对转化生长因子-β1（transforming growth factor-β1，TGF-β1）诱导心肌成纤维细胞（cardiac fibroblasts，CFs）纤维化的干预效果。方法：质量浓度10 ng/mL TGF-β1诱导CFs建立心肌纤维化细胞模型，用不同质量浓度绿原酸处理纤维化细胞，细胞毒性实验（cell counting kit-8，CCK-8）检测绿原酸对CFs增殖的影响；酶联免疫吸附试验（enzyme linked immunosorbent assay，ELISA）检测纤维化标志蛋白胶原蛋白（collagen，Col）I、Col III和免疫荧光检测α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）的表达；实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction，qPCR）检测Twist1、Acta2、Snail2及纤维化蛋白S100A4等mRNA表达水平；Western blot检测TGF-β1/Smads信号通路相关蛋白及其磷酸化表达水平。结果：CCK-8检测表明枸杞中分离得到的绿原酸能抑制细胞异常增殖；ELISA和免疫荧光检测表明，枸杞中分离得到的绿原酸能下调纤维化标志蛋白Col I、Col III和α-SMA的表达；qPCR检测表明枸杞中分离得到的绿原酸能高度显著下调纤维化转录调控因子Acta2、Twist1、Snail1、Snail2、Smad4及纤维化蛋白S100A4的mRNA表达（P＜0.001）；Western blot检测表明枸杞中分离得到的绿原酸能高度显著下调TGF-β1/Smads信号通路相关蛋白的表达（P＜0.001）。结论：在本研究质量浓度范围内，枸杞中分离得到的绿原酸抑制TGF-β1诱导的CFs纤维化过程，其机制可能与TGF-βl/Smads信号通路有关。     

On lipoxygenase and enzymes which decompose linoleic acid hydroperoxides in rye (author's transl)]

An enzyme fraction from rye containing lipoxygenase activity was investigated. The molecular weight of lipoxygenase was found to be about 102000. Two bands groups with isoelectric points between 5.1-5.5 and 5.8-6.4 were obtained by isoelectric focusing. Three isoenzymes could be separated by ion exchange chromatography. Lipoxygenase has optimum activity at pH 7.3-7.5 and predominantly forms 13-hydroperoxy-9-cis, 11-trans-octadecadienoic acid (13-LHPO). In rye the 13-LHPO is converted to alpha-ketols by a high molecular protein fraction. This isomerase converts the LHPO formed by rye lipoxygenase predominantly to 12,13-ketohydroxy acids. The Michaelis Constant of isomerase is 3-5 X 10(-5), using LHPO as substrate. At low protein concentrations the reaction velocity of LHPO-conversion increases linearly with protein concentration.