山楂多酚氧化酶的酶学特性研究

Enzymological Characteristics of Polyphenol Oxidase from Hawthorn

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、p H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p H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。

The enzymatic characteirstics of Polyphenol oxidase(PPO) from hawthorn was studied The effects of substrate specificity, p H value, temperature, thermostability, substrate concentration, metal ions, and inhibitors on PPO activity were examined using spectrophotometry. The optimum substrate, p H 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, Na HSO3 > citric acid, where ascorbic acid showed the most significant inhibitory effect on PPO activity.