Department of Chemistry, Yildiz Technical University, Istanbul, Turkey.
Prep Biochem Biotechnol. 2022;52(9):1019-1034. doi: 10.1080/10826068.2021.2023825. Epub 2022 Jan 11.
Polyphenol oxidase (PPO) was firstly purified from damson plum as a high antioxidant source. PPO was treated by 0-80% ammonium sulfate precipitation and dialysis. Characterization results were determined for catechol, 4-methyl catechol, pyrogallol and caffeic acid as 0.05 M/pH: 7.2/25 °C; 0.2 M/pH: 4.5/10 °C; 0.01 M/pH: 6.8/5 °C, and 0.2 M/pH: 8.5/10 °C, respectively. and values were calculated for same substrates as 17,219.97 U/(mLmin) and 11.67 mM; 7309.72 U/(mLmin) and 5 mM; 12,580.12 U/(mLmin) and 3.74 mM; 12,100.41 U/(mLmin) and 6.25 mM, respectively. Catechol gave the highest value among substrates. Affinity purification was performed by using Sepharose 4B-L-Tyrosine-p-aminobenzoic acid and Sepharose 6B-L-Tyrosine-p-aminobenzoic acid. Single bands were approximately observed at 50 kDa for each affinity sample in SDS-PAGE and Native-PAGE. 93.88 and 10.46 purification-folds were obtained for PPO by reference Sepharose-4B and original Sepharose-6B gels. Metal effects upon PPO activity were also investigated due to the importance of enzymatic browning in foods. Cu activation and Fe inhibition were observed with a final metal concentration of 1 mM at 219.66 and 43.18%, respectively. PPO purification from damson plum by affinity chromatography, its characterization, stability evaluation by statistically, and effects of metal ions on damson plum PPO have not been investigated in the literature.
多酚氧化酶（PPO）最初是从李子中作为高抗氧化剂来源进行纯化的。PPO 经过 0-80%硫酸铵沉淀和透析处理。用邻苯二酚、4-甲基邻苯二酚、焦儿茶酚和咖啡酸作为底物，测定其酶学特性，结果分别为：0.05M/pH：7.2/25°C；0.2M/pH：4.5/10°C；0.01M/pH：6.8/5°C 和 0.2M/pH：8.5/10°C。用同样的底物计算出 Km 和 Vmax 值分别为 17,219.97 U/(mLmin)和 11.67mM；7309.72 U/(mLmin)和 5mM；12,580.12 U/(mLmin)和 3.74mM；12,100.41 U/(mLmin)和 6.25mM。邻苯二酚是所有底物中 Km 值最小的，因此具有最高的亲和力。采用 Sepharose 4B-L-酪氨酸-p-氨基苯甲酸和 Sepharose 6B-L-酪氨酸-p-氨基苯甲酸进行亲和纯化。SDS-PAGE 和 Native-PAGE 中每个亲和样品的条带约为 50kDa。用参考 Sepharose-4B 和原始 Sepharose-6B 凝胶，PPO 的纯化倍数分别为 93.88 和 10.46。由于酶促褐变在食品中的重要性，还研究了金属离子对 PPO 活性的影响。Cu 激活和 Fe 抑制的最终金属浓度分别为 1mM 时，PPO 的活性分别为 219.66%和 43.18%。李子中 PPO 的亲和层析纯化、特性、统计稳定性评价以及金属离子对李子 PPO 的影响在文献中尚未报道过。
