Mohamed Saleh A, El-Badry Mohamed O, Drees Ehab A, Fahmy Afaf S
Molecular Biology Department, National Research Centre, Cairo, Egypt.
Appl Biochem Biotechnol. 2008 Aug;150(2):127-37. doi: 10.1007/s12010-008-8142-2. Epub 2008 Feb 15.
The major pool of peroxidase activity is present in the peel of some Egyptian citrus species and cultivars compared to the juice and pulp. Citrus jambhiri cv. Adalia had the highest peroxidase activity among the examined species. Four anionic and one cationic peroxidase isoenzymes from C. jambhiri were detected using the purification procedure including ammonium sulfate precipitation, chromatography on diethylaminoethanol-cellulose, carboxymethyl-cellulose, and Sephacryl S-200 columns. Cationic peroxidase POII is proved to be pure, and its molecular weight was 56 kDa. A study of substrate specificity identified the physiological role of POII, which catalyzed the oxidation of some phenolic substrates in the order of o-phenylenediamine > guaiacol > o-dianisidine > pyrogallol > catechol. The kinetic parameters (K (m), V (max), and V (max)/K (m)) of POII for hydrolysis toward H2O2 and electron donor substrates were studied. The enzyme had pH and temperature optima at 5.5 and 40 degrees C, respectively. POII was stable at 10-40 degrees C and unstable above 50 degrees C. The thermal inactivation profile of POII is biphasic and characterized by a rapid decline in activity on exposure to heat. The most of POII activity (70-80%) was lost at 50, 60, and 70 degrees C after 15, 10, and 5 min of incubation, respectively. Most of the examined metal ions had a very slight effect on POII except of Li+, Zn2+, and Hg2+, which had partial inhibitory effects. In the present study, the instability of peroxidase above 50 degrees C makes the high temperature short time treatment very efficient for the inactivation of peel peroxidase contaminated in orange juice to avoid the formation of off-flavors.
与果汁和果肉相比，一些埃及柑橘品种和栽培变种的果皮中存在主要的过氧化物酶活性池。在被检测的品种中，酸橙（Citrus jambhiri cv. Adalia）的过氧化物酶活性最高。通过包括硫酸铵沉淀、在二乙氨基乙醇纤维素、羧甲基纤维素和Sephacryl S - 200柱上进行层析的纯化程序，从酸橙中检测到四种阴离子和一种阳离子过氧化物酶同工酶。阳离子过氧化物酶POII被证明是纯的，其分子量为56 kDa。底物特异性研究确定了POII的生理作用，它催化某些酚类底物的氧化，顺序为邻苯二胺＞愈创木酚＞邻联茴香胺＞连苯三酚＞儿茶酚。研究了POII对过氧化氢和电子供体底物水解的动力学参数（Km、Vmax和Vmax/Km）。该酶的最适pH和温度分别为5.5和40℃。POII在10 - 40℃稳定，在50℃以上不稳定。POII的热失活曲线是双相的，其特征是受热后活性迅速下降。分别在50℃、60℃和70℃孵育15分钟、10分钟和5分钟后，POII的大部分活性（70 - 80%）丧失。除Li +、Zn2 +和Hg2 +有部分抑制作用外，大多数被检测的金属离子对POII的影响非常小。在本研究中，过氧化物酶在50℃以上的不稳定性使得高温短时处理对于灭活橙汁中污染的果皮过氧化物酶以避免异味形成非常有效。
