Determination of phenolic compounds in wines with enzyme electrodes fabricated by immobilization of polyphenol oxidase in conducting copolymers

葡萄酒酚類化合物測(cè)定酶電極材料進(jìn)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-吡咯共聚物用新單體含有酯(-MBA)集團(tuán)從3-吩乙酸和(S)-(--)-2-methylbutanolwas 作為固定化多酚氧化基質(zhì). 酶電極構(gòu)造坑害酶在電化學(xué)聚合導(dǎo)電聚合物吡咯. 酶電極的性能優(yōu)化研究了pH、溫度對(duì)酶的活性變化 最大反應(yīng)速率(Vmax)和米氏-Michaelis-Menten常數(shù)(公里),經(jīng)查,除了固定的保質(zhì)期和運(yùn)行穩(wěn)定. 利用這些酶電極總額酚類化合物紅酒土耳其也分析.

Keywords: Polyphenol oxidase; immobilization; electrochemistry;phenolic determination; wine.

關(guān)鍵詞:多酚氧化; 固定; 電化學(xué); 酚醛決心; 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].

研究酚類化合物具有濃厚興趣,因?yàn)樗鼈儗?duì)性能的水果和飲料, 例如色彩、收斂、苦味、香味和褐變〔1,2〕. 葡萄酒是一種產(chǎn)品為中心的興趣就是為了優(yōu)化葡萄酒品質(zhì)[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)是一種雙功能酶負(fù)責(zé)黑色素的形成天然色素大分子在不同物種 [5]. 它的第一個(gè)反應(yīng),單活動(dòng),在基板酚類多酚氧化hydoxylates一個(gè)鄰位的羥基. 在第二次反應(yīng),氧化酶活性,鄰羥基化合物是氧化有關(guān)鄰醌衍生物.

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證實(shí)是另一種典型的方式方法基于色譜技術(shù)測(cè)定 酚類化合物的數(shù)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陣吡咯(吡咯)、薄荷共聚物 酯3-吩與吡咯乙酸(毫米). 結(jié)果與-MBA矩陣. 無論共聚物合成與表征研究較早〔7,8〕.

2. EXPERIMENTAL

2. 實(shí)驗(yàn)

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、歐共體1.14.18.1)購買西格瑪. 吡咯是愛和購買十二烷基硫酸鈉(SDS)由西格瑪. 吡咯是蒸餾后使用. 3-甲基-2-benzothiozolinone(試劑)、丙酮、硫酸、多酚氧化活性測(cè)定用光度,也從西格瑪. 制備檸檬酸緩沖、檸檬酸三鈉-水合物2、檸檬酸用刊出.

Catechol was purchased from Sigma. All catechol solutions were prepared in citrate buffer.

鄰苯二酚是購自西格瑪. 鄰苯二酚解決一切準(zhǔn)備檸檬酸緩沖.

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)是通過一種恒流電解白金車廂組成工作電池 電極和柜臺(tái). 二氯甲烷進(jìn)行實(shí)驗(yàn)(10ml組)/四丁基四氟硼酸(tbafb)(0.2米)溶劑電解質(zhì)體系50毫克單體在0℃使用 3010馬閔.

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電吡咯包覆鉑電極. 解分為0.3毫克/毫升的PPO,0.6毫克/毫升支持電解質(zhì)(十二烷基硫酸鈉) 吡咯0.01米和10毫升檸檬酸緩沖液(pH值6.5). 固定術(shù)的一個(gè)典型threeelectrode細(xì)胞 由鉑電極、銀柜、工作/銀參考電極. 固定在一個(gè)常數(shù)進(jìn)行潛力c1.01敏五室溫. 酶電極保持在4+C在不使用時(shí)檸檬酸緩沖液.

2.3. Determination of PPO activity

230. PPO活性測(cè)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動(dòng)自由和決心用besthorn的腙法[9]. 固定化多酚氧化酶活性測(cè)定, 備不同濃度的鄰苯二酚(3.0毫升),花了25+水浴部分C.1毫升試劑溶液 . 酶電極在溶液中浸泡5分鐘和動(dòng)搖. 1毫升1毫升硫酸、丙酮等內(nèi)容進(jìn)行了總額6毫升. 混合后,在495nm處測(cè)定吸收.

2.4. Determination of optimum temperature and pH

240. 最適溫度和pH的測(cè)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.

最適溫度和pH進(jìn)行測(cè)定,通過改變孵化溫度20至80月2日期間+三和pH 11美元. 其余的程序是一樣的測(cè)定PPO活性.

2.5. Protein determination

250. 蛋白測(cè)定

Protein determination measurements were performed by Bradford’s method [10].

The protein determination procedure is explained in detail elsewhere [6].

蛋白質(zhì)測(cè)定測(cè)量表演布拉德福方法[10]. 蛋白質(zhì)測(cè)定過程詳細(xì)講解了別處[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. 結(jié)果與討論3.1. 酶電極測(cè)定蛋白質(zhì)因?yàn)橄惹八嵫芯縖6]、 酶的反應(yīng)速度免費(fèi)11.2¹摩爾/分鐘每毫克蛋白. 蛋白測(cè)定結(jié)果pmbta/多酚氧化酶電極顯示0.0043毫克蛋白質(zhì)已然在陣. 320. 動(dòng)力學(xué)研究公里的Vmax和參數(shù),給予最高的反應(yīng)速度和米氏-Michaelis-Menten常數(shù). 這些參數(shù)是根據(jù)Lineweaver-Burk法陰謀[11]. 為吡咯的Vmax/PPO和毫米/為0.11和0.10μ的PPO/每分鐘電極分別[6]、 為pmbta的Vmax/多酚氧化酶電極,發(fā)現(xiàn)是0.048¹摩爾/每分鐘電極. 當(dāng)我們相比這兩種酶電極結(jié)果與先前其他研究我們看到固定的Vmax 酶pmbta/多酚氧化酶電極一半,其他兩個(gè)電極. 這些結(jié)果也證實(shí)了蛋白質(zhì)在電極數(shù)額已然.

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.

酶電極研究公里價(jià)值觀上有非常高的工作相比,游離酶. 參數(shù)公里,是與此有直接關(guān)系的形態(tài)矩陣. 回望掃描電子顯微圖的三個(gè)電極. 1,可以看出兩種吡咯/PPO和毫米/多酚氧化酶電極都非常緊湊,使家庭綜合形態(tài). 邪教的基板漫入陣來. 不過,pmbta/多酚氧化酶電極易產(chǎn)生擴(kuò)散,使酶更可為基板, 造成一小公里價(jià)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. 效果圖2溫度對(duì)酶電極溫度顯示固定化酶活性. 在溫度50+70+C和C組的酶電極高電阻溫度變化證物. 幾乎沒有損失,觀察這些活動(dòng)拉開帷幕. 340. pH對(duì)酶電極作用于以往工作[6],吡咯/PPO和毫米/多酚氧化酶電極進(jìn)行了研究, 此消彼長(zhǎng)的最適pH值為堿性方解釋劃分為質(zhì)子. 同一行為觀察pH值的依賴性pmbta/多酚氧化酶電極 但這個(gè)展品更加穩(wěn)定邁向高pH電極(圖3). pH值7至11月,沒有任何改變酶活性. 這說明這個(gè)酶電極可以保護(hù)他們免受高濃度哦. 350. 運(yùn)行穩(wěn)定和貨架期酶電極酶容易變性而失去活性, 所以小心存放和處理是必要的. 確定對(duì)重復(fù)使用的穩(wěn)定性和貨架期pmbta/多酚氧化酶活電極電極遏制. 40在同一天進(jìn)行測(cè)量,測(cè)試運(yùn)行穩(wěn)定. 逐步減少直至十五法觀察,然后下榻在60%活動(dòng)不斷(圖4A)條. 經(jīng)審查活性隨時(shí)間(圖4B)款 我們看到有一個(gè)快速下降的活動(dòng)放慢后20天. 360. 總金額測(cè)定酚醛紅葡萄酒的pmbta/多酚氧化酶電極用于分析酚醛金額在兩 土耳其紅葡萄酒、K及品牌四品牌如果比較兩個(gè)牌子酚醛金額、 結(jié)果顯示,含有鉀一倍的品牌,品牌丁酚(表1). 這一結(jié)果同意其他兩位酶電極研究結(jié)果以前[6]. 結(jié)果報(bào)子酸當(dāng)量(游戲)[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. 這項(xiàng)研究的結(jié)論顯示pmbta電極可用于固定化多酚氧化成功. 可以斷定,從所得結(jié)果的分析基于動(dòng)力學(xué)研究 溫度和pH優(yōu)化研究與穩(wěn)定都是非常好的學(xué)習(xí). 像這種電極研究工作電極上也可作為替代的方法 共測(cè)定酚類化合物.

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其中有一小部分是我用翻譯軟件翻譯了一點(diǎn)，抱歉！