摘要
为建立水蜜桃多酚氧化酶(PPO)高效分离纯化方法,并明确该酶的酶学特性,本研究采用柱层析法对该酶进行分离纯化,对其反应特性及稳定性进行研究。结果表明,Phenyl-Sepharose CL-4B疏水柱层析法对水蜜桃PPO的纯化倍数与硫酸铵分级沉淀法相近,但其得率为89%远高于后者;采用PhenylSepharose CL-4B、DEAE Sepharose Fast Flow和Sephaoryl HR S-200三级柱层析联用的方法对PPO分离纯化,获得PPO总纯化倍数为12.40倍,总得率为20%;该PPO组分经SDS-聚丙烯酰胺凝胶电泳分析得到单一蛋白条带,其分子量为81 k Da。纯化后的水蜜桃PPO对(+)-儿茶素和(-)-表儿茶素的米氏常数分别为0.14 m M和0.12 m M,最适反应p H值为7.0,最适反应温度为40℃。纯化后的水蜜桃PPO对热以及低p H值都很不稳定。本研究结果为进一步研究PPO酶促褐变机制提供了理论指导,同时也为其他果蔬中PPO的分离纯化提供参考。
In an attempt to establish an effective method for the purification of polyphenol oxidase（ PPO） from peach pulp and clarify the characteristics of the PPO,a column chromatographic purification produce as well as the reaction properties and the stabilities of the PPO were investigated in this paper. Phenyl-Sepharose CL-4B column chromatography achieved a similar purification fold with ammonium sulfate fractional precipitation. However,the yield of PPO obtained by the former method was 89%,much higher than that of the latter. A three-step column chromatographic purification procedure involving Phenyl-Sepharose CL-4B,DEAE Sepharose Fast Flow and Sephaoryl HR S-200,had been established for the purification of the PPO. A 12. 40-fold purification of PPO with a total yield of 20% was achieved,and the SDS-PAGE electrophoresis analysis showed a single band with a molecular weight of 81 k Da. The Km values of the purified PPO were 0. 14 m M and 0. 12 m M for（ ＋）-catechin and（-）-epicatechin,respectively. The optimum p H and temperature were 7. 0 and 40 ℃,respectively. The thermostability results suggested that this PPO was unstable during the thermal treatment or in the low p H value solutions. The results could provide theoretical supports for the enzymatic browning mechanism of honey peach and references for the purification of PPO from other fruits and vegetables.
出处
《核农学报》
CAS
CSCD
北大核心
2015年第6期1121-1128,共8页
Journal of Nuclear Agricultural Sciences
基金
浙江省自然科学基金(LQ12C20002)
宁波市自然科学基金(2012A610133)
关键词
水蜜桃
多酚氧化酶
纯化
酶学特性
honeydew peach polyphenol oxidase purification properties
