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Determination of phenolic compounds in wines with enzyme electrodes fabricated by immobilization of polyphenol oxidase in conducting copolymers
葡萄酒酚类化合物测定酶电极材料进行固定化多酚氧化共聚物里
Abstract—A copolymer of pyrrole with a new monomer (MBTA) containing an ester group derived from 3-thiophene acetic acid and (S)-(——)-2-methylbutanolwas used as the matrix for immobilization of polyphenol oxidase. Enzyme electrodes were constructed by entrapment of enzyme in the conducting copolymer during the electrochemical polymerization of pyrrole. The performance of enzyme electrodes was optimized by examining the effects of pH and temperature on enzyme activity。The changes in the maximum reaction rate (Vmax) and Michaelis–Menten constant (Km) upon immobilization were investigated in addition to shelf-life and operational stability. By using these enzyme electrodes the total amount of phenolic compounds in red wines of Turkey was also analyzed.
摘-吡咯共聚物用新单体含有酯(-MBA)集团从3-吩乙酸和(S)-(--)-2-methylbutanolwas 作为固定化多酚氧化基质. 酶电极构造坑害酶在电化学聚合导电聚合物吡咯. 酶电极的性能优化研究了pH、温度对酶的活性变化 最大反应速率(Vmax)和米氏-Michaelis-Menten常数(公里),经查,除了固定的保质期和运行稳定. 利用这些酶电极总额酚类化合物红酒土耳其也分析.
Keywords: Polyphenol oxidase; immobilization; electrochemistry;phenolic determination; wine.
关键词:多酚氧化; 固定; 电化学; 酚醛决心; 葡萄酒.
1. INTRODUCTION
1. 引言
The study of phenolic compounds is of great interest because of their contribution to the properties of fruits and beverages, such as color, astringency, bitterness, flavor and browning [1, 2]. Wine is one of the products that is at the center of interest in order to optimize wine quality [3, 4].
研究酚类化合物具有浓厚兴趣,因为它们对性能的水果和饮料, 例如色彩、收敛、苦味、香味和褐变〔1,2〕. 葡萄酒是一种产品为中心的兴趣就是为了优化葡萄酒品质[3、 4].
Polyphenol oxidase (PPO) is a bifunctional enzyme responsible for the formation of the natural macromolecule pigment melanin in different species [5]. In its first reaction, monooxygenase activity, PPO hydoxylates a phenolic substrate at the ortho-position to the hydroxyl group. In the second reaction, oxidase activity, the o-dihydroxy compound is oxidized to the pertinent o-quinone derivative.
多酚氧化酶(PPO)是一种双功能酶负责黑色素的形成天然色素大分子在不同物种 [5]. 它的第一个反应,单活动,在基板酚类多酚氧化hydoxylates一个邻位的羟基. 在第二次反应,氧化酶活性,邻羟基化合物是氧化有关邻醌衍生物.
Immobilization of PPO has been proven to be an alternative method to typical methods based on chromatographic techniques for determination of amount of phenolic compounds.
固定化多酚氧化酶已被证实是另一种典型的方式方法基于色谱技术测定 酚类化合物的数量.
In our previous study [6] immobilization of PPO was achieved in matrices of polypyrrole (PPy) and a copolymer of menthyl ester of 3-thiophene acetic acid (MM) with pyrrole. Results were compared with the MBTA matrice. Synthesis and characterization of both copolymers were studied earlier [7, 8].
在以前的研究[6]固定化多酚氧化取得方阵吡咯(吡咯)、薄荷共聚物 酯3-吩与吡咯乙酸(毫米). 结果与-MBA矩阵. 无论共聚物合成与表征研究较早〔7,8〕.
2. EXPERIMENTAL
2. 实验
2.1. Materials
2.1. 材料
Tyrosinase (polyphenol oxidase, PPO, EC 1.14.18.1) was purchased from Sigma. Pyrrole was purchased from Aldrich and sodium dodecyl sulfate (SDS) from Sigma.
Pyrrole was distilled before use. 3-methyl-2-benzothiozolinone (MBTH), acetone and sulfuric acid, used in spectrophotometric activity determination of PPO, were also obtained from Sigma. For preparation of citrate buffer, tri-sodium citrate-2 hydrate and citric acid were used as received.
酪氨酸(多酚氧化酶,多酚氧化酶、欧共体1.14.18.1)购买西格玛. 吡咯是爱和购买十二烷基硫酸钠(SDS)由西格玛. 吡咯是蒸馏后使用. 3-甲基-2-benzothiozolinone(试剂)、丙酮、硫酸、多酚氧化活性测定用光度,也从西格玛. 制备柠檬酸缓冲、柠檬酸三钠-水合物2、柠檬酸用刊出.
Catechol was purchased from Sigma. All catechol solutions were prepared in citrate buffer.
邻苯二酚是购自西格玛. 邻苯二酚解决一切准备柠檬酸缓冲.
2.2. Immobilization of PPO in PMBTA
220. 固定化多酚氧化pmbta
The homo-polymerization of MBTA (Scheme 2) was achieved by constant current
electrolysis in one compartment cell consisting of platinum working and counter electrodes. Experiments were carried out in dichloromethane (10ml)/tetrabutylammonium tetrafluoroborate (TBAFB) (0.2 M) solvent-electrolyte system with 50 mg monomer at 0°C using 30 mA for 10 min.
同源聚合-MBA(方案2)是通过一种恒流电解白金车厢组成工作电池 电极和柜台. 二氯甲烷进行实验(10ml组)/四丁基四氟硼酸(tbafb)(0.2米)溶剂电解质体系50毫克单体在0℃使用 3010马闵.
Immobilization of PPO was achieved by electropolymerization of pyrrole on a previously PMBTA-coated platinum electrode. The solution consists of 0.3 mg/ml PPO, 0.6 mg/ml supporting electrolyte (sodium dodecyl sulfate), 0.01 M pyrrole and 10 ml citrate buffer (pH 6.5). Immobilization was performed in a typical threeelectrode cell, consisting of the Pt working and counter electrodes and a Ag/Ag reference electrode. Immobilization was carried out at a constant potential of C1.0 V for 1 min at room temperature. Enzyme electrodes were kept at 4±C in citrate buffer solution when not in use.
固定化多酚氧化成一此前pmbta电吡咯包覆铂电极. 解分为0.3毫克/毫升的PPO,0.6毫克/毫升支持电解质(十二烷基硫酸钠) 吡咯0.01米和10毫升柠檬酸缓冲液(pH值6.5). 固定术的一个典型threeelectrode细胞 由铂电极、银柜、工作/银参考电极. 固定在一个常数进行潜力c1.01敏五室温. 酶电极保持在4+C在不使用时柠檬酸缓冲液.
2.3. Determination of PPO activity
230. PPO活性测定
The activities of free and immobilized PPO were determined by using Besthorn’s hydrazone method [9]. For determination of activity of immobilized PPO, different concentrations of catechol were prepared (3.0 ml) and put in a water bath at 25±C.1 ml MBTH solution was added. The enzyme electrode was immersed in the solution and shaken for 5 min. 1 ml sulfuric acid and 1 ml acetone were added for a total volume of 6 ml. After mixing, absorbances were measured at 495 nm.
固定化多酚氧化酶的活动自由和决心用besthorn的腙法[9]. 固定化多酚氧化酶活性测定, 备不同浓度的邻苯二酚(3.0毫升),花了25+水浴部分C.1毫升试剂溶液 . 酶电极在溶液中浸泡5分钟和动摇. 1毫升1毫升硫酸、丙酮等内容进行了总额6毫升. 混合后,在495nm处测定吸收.
2.4. Determination of optimum temperature and pH
240. 最适温度和pH的测定
Optimum temperature and pH determinations were carried out by changing incubation
temperature between 20 and 80±C and pH between 2 and 11, respectively. The rest of the procedure was the same as the determination of PPO activity.
最适温度和pH进行测定,通过改变孵化温度20至80月2日期间+三和pH 11美元. 其余的程序是一样的测定PPO活性.
2.5. Protein determination
250. 蛋白测定
Protein determination measurements were performed by Bradford’s method [10].
The protein determination procedure is explained in detail elsewhere [6].
蛋白质测定测量表演布拉德福方法[10]. 蛋白质测定过程详细讲解了别处[6].
3. RESULTS AND DISCUSSION
3.1. Protein determination of enzyme electrodes
As it was mentioned in the previous study [6], the reaction rate for free enzyme was 11.2 ¹mol/min per mg protein. Protein determination results for PMBTA/PPO electrode showed that 0.0043 mg protein was entrapped in the matrix.
3.2. Kinetic studies
Vmax and Km are parameters that give maximum reaction rate and Michaelis–Menten constant, respectively. These parameters were obtained from Lineweaver– Burk plots [11].
Vmax for PPy/PPO and MM/PPO was 0.11 and 0.10 umol/min per electrode, respectively [6], Vmax for the PMBTA/PPO enzyme electrode was found to be 0.048 ¹mol/min per electrode. When we compared this result with other two enzyme electrodes in the previous study we saw that Vmax of immobilized enzyme in PMBTA/PPO electrode was half that of the other two electrodes. These results were also confirmed by the protein amount entrapped in the electrodes.
3. 结果与讨论3.1. 酶电极测定蛋白质因为先前所提研究[6]、 酶的反应速度免费11.2¹摩尔/分钟每毫克蛋白. 蛋白测定结果pmbta/多酚氧化酶电极显示0.0043毫克蛋白质已然在阵. 320. 动力学研究公里的Vmax和参数,给予最高的反应速度和米氏-Michaelis-Menten常数. 这些参数是根据Lineweaver-Burk法阴谋[11]. 为吡咯的Vmax/PPO和毫米/为0.11和0.10μ的PPO/每分钟电极分别[6]、 为pmbta的Vmax/多酚氧化酶电极,发现是0.048¹摩尔/每分钟电极. 当我们相比这两种酶电极结果与先前其他研究我们看到固定的Vmax 酶pmbta/多酚氧化酶电极一半,其他两个电极. 这些结果也证实了蛋白质在电极数额已然.
Km values of enzyme electrodes studied in the previous work were very high when compared to that of free enzyme. Km is a parameter that is directly related with morphology of the matrice. When we examine the scanning electron micrographs of the three electrodes in Fig. 1, one can see that both PPy/PPO and MM/PPO electrodes have very compact morphology that make it dif. cult for the substrate to diffuse into the matrix. However,PMBTA/PPO electrode gives rise to easy diffusion for substrate by making enzymes more available, resulting in a small Km value, 18 mM.
酶电极研究公里价值观上有非常高的工作相比,游离酶. 参数公里,是与此有直接关系的形态矩阵. 回望扫描电子显微图的三个电极. 1,可以看出两种吡咯/PPO和毫米/多酚氧化酶电极都非常紧凑,使家庭综合形态. 邪教的基板漫入阵来. 不过,pmbta/多酚氧化酶电极易产生扩散,使酶更可为基板, 造成一小公里价值18毫米.
3.3. Effect of temperature on enzyme electrode
Figure 2 shows the temperature dependence of the activity of immobilized enzyme. At temperatures between 50±C and 70±C, the enzyme electrode exhibits high resistivity to temperature change. Almost no activity loss was observed between these temperatures.
3.4. Effect of pH on enzyme electrodes
In the previous work [6], where PPy/PPO and MM/PPO electrodes were studied, the shift in the optimum pH values towards the alkaline side was explained as the partitioning of protons. The same behavior in pH dependence was observed for the PMBTA/PPO electrode, but this electrode exhibits greater stability towards high pH (Fig. 3). From pH 7 to 11 there is no change in the enzyme activity. This shows that this electrode can protect enzymes against high OH concentration.
3.5. Operational stability and shelf-life of enzyme electrodes
Enzymes can easily lose their catalytic activity and denatured, so careful storage and handling are essential. To determine the stability against repetitive use and shelf-life of PMBTA/PPO electrode the activity of electrode was checked. 40 measurements were done on the same day to test the operational stability. Gradual decrease was observed up to the 15th assay and then stayed constant at 60% activity (Fig. 4a). Upon examining the activity change with time (Fig. 4b), we see that there is a rapid decrease in the activity which slows down after the 20th day.

3.6. Determination of total phenolic amount in red wines
The PMBTA/PPO electrode was used for analysis of phenolic amount in two Turkish red wines, Brand K and Brand D.
When we compare the phenolic amount of two brands, results reveal that Brand K contains twice the amount of phenols of that of Brand D (Table 1). This result agrees with the result of other two enzyme electrodes studied previously [6]. Results are reported in Gallic Acid Equivalent (GAE) [12].
330. 效果图2温度对酶电极温度显示固定化酶活性. 在温度50+70+C和C组的酶电极高电阻温度变化证物. 几乎没有损失,观察这些活动拉开帷幕. 340. pH对酶电极作用于以往工作[6],吡咯/PPO和毫米/多酚氧化酶电极进行了研究, 此消彼长的最适pH值为碱性方解释划分为质子. 同一行为观察pH值的依赖性pmbta/多酚氧化酶电极 但这个展品更加稳定迈向高pH电极(图3). pH值7至11月,没有任何改变酶活性. 这说明这个酶电极可以保护他们免受高浓度哦. 350. 运行稳定和货架期酶电极酶容易变性而失去活性, 所以小心存放和处理是必要的. 确定对重复使用的稳定性和货架期pmbta/多酚氧化酶活电极电极遏制. 40在同一天进行测量,测试运行稳定. 逐步减少直至十五法观察,然后下榻在60%活动不断(图4A)条. 经审查活性随时间(图4B)款 我们看到有一个快速下降的活动放慢后20天. 360. 总金额测定酚醛红葡萄酒的pmbta/多酚氧化酶电极用于分析酚醛金额在两 土耳其红葡萄酒、K及品牌四品牌如果比较两个牌子酚醛金额、 结果显示,含有钾一倍的品牌,品牌丁酚(表1). 这一结果同意其他两位酶电极研究结果以前[6]. 结果报子酸当量(游戏)[12].
4. CONCLUSIONS
This study shows that PMBTA electrode can be successfully used for the immobilization of PPO. We can conclude that obtained results from the analysis based on kinetic studies, temperature and pH optimization studies and stability studies are very good. Like the electrodes studied in the previous work this electrode also can be used as an alternative method for the determination of total phenolic compounds.
4. 这项研究的结论显示pmbta电极可用于固定化多酚氧化成功. 可以断定,从所得结果的分析基于动力学研究 温度和pH优化研究与稳定都是非常好的学习. 像这种电极研究工作电极上也可作为替代的方法 共测定酚类化合物.
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