[关键词]
[摘要]
研究山楂多酚氧化酶(PPO)的酶学特性。采用分光光度法测定PPO的酶活性,考察底物特异性、pH值、温度、热稳定性、底物浓度、金属离子以及抑制剂对PPO酶活性的影响。实验结果表明,山楂PPO的最适底物为邻苯二酚;最适pH值为6.5;最适温度为40 ℃;90 ℃热处理1.0 min或85 ℃处理1.5 min PPO酶活基本完全丧失;山楂PPO最适底物浓度为0.10 mol/L,PPO酶促褐变反应动力学符合米氏方程,相应的动力学参数Km=55.83 mmol/L,Vmax= 98.04 U/min;Al3+对PPO酶活的抑制作用较强,Mn2+、Ca2+和Zn2+次之,Cu2+和Mg2+对PPO酶活抑制作用不明显,Fe3+对PPO酶活有一定的促进作用;四种抑制剂对山楂PPO酶活均有一定的抑制作用,且抑制效果与抑制剂浓度呈量效关系,相同浓度下抑制效果由强到弱顺序为:抗坏血酸>L-半胱氨酸>亚硫酸氢钠>柠檬酸,抗坏血酸的抑制效果最为显著。
[Key word]
[Abstract]
The enzymatic characteirstics of Polyphenol oxidase (PPO) from hawthorn was studied The effects of substrate specificity, pH value, temperature, thermostability, substrate concentration, metal ions, and inhibitors on PPO activity were examined using spectrophotometry . The optimum substrate, pH value, temperature, and substrate concentration for PPO were found to be catechol, 6.5, 40 ℃, and 0.10 mol/L, respectively. PPO was completely inactivated by heating at 90 ℃ for 1.0 min or 85 ℃ for 1.5 min. PPO activity followed Michaelis-Menten kinetics in the enzymatic browning reaction. The Km and Vmax values were 55.83 mmol/L and 98.04 U/min, respectively. Metal ion Al3+ showed relatively strong inhibitory effect on the enzyme activity, followed by Mn2+, Ca2+, and Zn2+, where all four substances showed dose-dependent effect. The inhibitory effects of Cu2+ and Mg2+ on enzyme activity were not obvious, while activity was promoted to some degree by Fe3+. At the same concentration, inhibitory effects were in the following order: ascorbic acid > L-cysteine, NaHSO3 > citric acid, where ascorbic acid showed the most significant inhibitory effect on PPO activity.
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[基金项目]
陕西省科技厅资助科研项目(2014JQ2055);陕西省教育厅资助科研项目(2014JK1145)