巨大口蘑多酚氧化酶的酶学特性  

Enzymatic Properties of Polyphenol Oxidase from Macrocybe gigantea

作　　者：刘欣芊 肖利丽 莫转林 周伟坚[1] 莫美华[1] LIU Xinqian;XIAO Lii;MO Zhuanin;ZHOU Weijian;MO Meihua(College of Food Science,South China Agricultural University,Guangzhou 510642,Guangdong,China;Center for Basic Experiment and Practical Training,South China Agricultural University,Guangzhou)

机构地区：[1]华南农业大学食品学院,广东广州510642 [2]华南农业大学基础实验和实践训练中心,广东广州510642

出　　处：《食品研究与开发》2024年第24期35-41,共7页Food Research and Development

基　　金：广东省自然科学基金面上项目(2022A1515010308);2022年省级乡村振兴战略专项资金种业振兴项目(2022‐WPY‐00‐003);河源龙川食用菌“永根科技站”建设项目(2023‐22)。

摘　　要：为考察巨大口蘑多酚氧化酶(polyphenol oxidase,PPO)的酶学特性,在不同种类缓冲液和不同pH值缓冲液的条件下以巨大口蘑为原料制备PPO粗酶液,以邻苯二酚为底物,测定不同的底物用量、温度、pH值、抑制剂和金属离子下PPO活性,并测定该酶的米氏常数。结果表明,以pH6.8的磷酸盐缓冲液进行粗提,提取液中PPO相对活性最高;PPO最适底物用量为1.5 mL,最适pH值为6.5,最适反应温度为30℃;该酶在30℃以上不稳定,在80℃时PPO相对活性仅为19.67%,在pH值为6~7时较稳定。抑制剂对PPO活性抑制效果由强到弱依次是抗坏血酸、柠檬酸和乙二胺四乙酸二钠,当抗坏血酸浓度为1000 mmol/L时PPO完全失活;金属离子中只有Fe^(2+)促进PPO活性,其余均抑制PPO活性。此外,以不同浓度邻苯二酚测定米氏方程,得到PPO米氏常数为0.114 mol/L。The polyphenol oxidase(PPO)from Macrocybe gigantea was investigated.The crude extracts of PPO from M.gigantea were prepared with different buffer solutions and at different pH values.Then,with cat‐echol as the substrate,the PPO activity was measured under different substrate dosages,temperatures,pH val‐ues,inhibitors,and metal ions.And,the Michaelis constant of the enzyme was determined.The results showed that the relative activity of PPO was the highest in the crude extract prepared with phosphate buffer at pH6.8.The optimum substrate dosage,pH,and reaction temperature of PPO were 1.5 mL,6.5,and 30℃,respectively.The enzyme was unstable at above 30℃,with the relative activity of only 19.67% at 80℃,and it was stable at pH6-7.The inhibitory effects of inhibitors on the relative activity of PPO were in a descending order of ascor‐bic acid,citric acid,and ethylenediaminetetraacetic acid disodium salt.When the concentration of ascorbic acid was 1000 mmol/L,PPO was completely inactivated.Among the metal ions,only Fe^(2+) increased the rela‐tive activity of PPO,and the others inhibited the relative activity of PPO.In addition,the Michaelis equation was determined with different concentrations of catechol,and the Michaelis constant of PPO was obtained to be 0.114 mol/L.

关 键 词：巨大口蘑 多酚氧化酶 酶活性 抑制剂 金属离子 

分 类 号：TS219[轻工技术与工程—粮食、油脂及植物蛋白工程]