无核白葡萄多酚氧化酶的酶学特性

Characterization of polyphenol oxidase extracted from Thompson seedless grape

【目的】研究无核白葡萄多酚氧化酶(PPO)的酶学特性,为无核白葡萄干加工过程中褐变问题的解决和控制提供参考。【方法】利用McIlvaine缓冲液从无核白葡萄中提取PPO,研究了反应温度、pH、底物浓度、酶液用量对其催化活性的影响,并考察了该酶在不同温度条件下的稳定性,分析了其催化邻苯二酚的催化动力学方程及其参数。【结果】以10mmol/L的邻苯二酚McIlvaine溶液为底物时,无核白葡萄PPO的最适作用温度为25℃,最适反应pH为6.0。在最适作用条件下,米氏常数(Km)和最大反应速率(Vmax)分别为45mmol/L和500U/min。该酶在温度低于25℃时,能在60min内保持较稳定的活性,30～60℃的高温处理只能破坏其部分活性,在70℃条件下保温处理10min可使其活性全部丧失。【结论】高温短时处理能够破坏无核白葡萄的PPO活性,因此高温处理可在葡萄干加工的前处理中推广应用。

【Objective】 Polyphenol oxidase（PPO） was extracted from Thompson seedless,and its characteristics were studied to provide information for raisins processing.【Method】 Characteristics,including optimal temperature and pH for enzymatic reaction,and influences of enzyme dose and substrate concentration on the enzymatic activity were studied.Michaelis-Menten constant（Km） and maximum velocitiy of the reaction（Vmax）were also calculated on the basis of obtained kinetic functions for the enzymatic reaction when catechol was used as the substrate.【Result】 The results showed the optimal condition for the PPO was pH 6.0 and 25 ℃,with Km and Vmax values of 45 mmol/L and 500 U/min,respectively,when catechol was used as the substrate.The enzyme showed high stability for 60 min at temperatures lower than 25 ℃,its activity was partially daraged at 30-60 ℃ and could be inhibited completely at temperatures higher than 70 ℃ within 10 min.【Conclusion】 High temperature and short time treatment can reduce enzymatic activity and it can be extended,as a method of pre-treatment in raisins processing.