Escuela Nacional de Ciencias Biológicas, Departamento de Ingeniería Bioquímica Instituto Politécnico Nacional, México, DF, Mexico.
Phytochemistry. 2011 Jan;72(1):82-8. doi: 10.1016/j.phytochem.2010.10.011. Epub 2010 Nov 17.
While a long shelf life for fruit products is highly desired, enzymatic browning is the main cause of quality loss in fruits and is therefore a main problem for the food industry. In this study polyphenol oxidase (PPO), the main enzyme responsible for browning was isolated from mamey fruit (Pouteria sapota) and characterized biochemically. Two isoenzymes (PPO 1 and PPO 2) were obtained upon ammonium sulfate precipitation and hydrophobic and ion exchange chromatography; PPO 1 was purified up to 6.6-fold with 0.28% yield, while PPO 2 could not be characterized as enzyme activity was completely lost after 24 h of storage. PPO 1 molecular weight was estimated to be 16.1 and 18 kDa by gel filtration and SDS-PAGE, respectively, indicating that the native state of the PPO 1 is a monomer. The optimum pH for PPO 1 activity was 7. The PPO 1 was determined to be maximum thermally stable up to 35°C. Kinetic constants for PPO 1 were K(m)=44 mM and K(m)=1.3 mM using catechol and pyrogallol as substrate, respectively. The best substrates for PPO 1 were pyrogallol, 4-methylcatechol and catechol, while ascorbic acid and sodium metabisulfite were the most effective inhibitors.
虽然人们非常希望水果产品具有较长的保质期，但酶促褐变是导致水果质量损失的主要原因，因此也是食品工业的主要问题。在这项研究中，从人心果（Pouteria sapota）中分离出了多酚氧化酶（PPO），这是导致褐变的主要酶，并对其进行了生化特性分析。通过硫酸铵沉淀和疏水层析及离子交换层析得到了两种同工酶（PPO1 和 PPO2）；PPO1 经纯化后比活力提高了 6.6 倍，收率为 0.28%，而 PPO2 由于酶活性在储存 24 小时后完全丧失，因此无法进行特性分析。凝胶过滤和 SDS-PAGE 分别估计 PPO1 的分子量为 16.1 和 18 kDa，表明 PPO1 的天然状态为单体。PPO1 的最适 pH 为 7。PPO1 的热稳定性最高可达 35°C。用儿茶酚和焦儿茶酚作为底物时，PPO1 的动力学常数 K(m)分别为 44 mM 和 1.3 mM。PPO1 的最佳底物为焦儿茶酚、4-甲基儿茶酚和儿茶酚，而抗坏血酸和偏重亚硫酸钠是最有效的抑制剂。
