Purification and Characterization of Wheat (Triticum aestivum) Polyphenol Oxidase
Purification and Characterization of Wheat (Triticum aestivum) Polyphenol Oxidase
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小麦多酚氧化酶的纯化和表征
DOI：
10.3136/fstr.11.87
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发表时间：
2005
影响因子：
0.6
通讯作者：
K. Komae
中科院分区：
文献类型：
--
作者：
T. Kihara;M. Murata;S. Homma;S. Kaneko;K. Komae
从日本小麦(Triticum aestivum cv.)东北198)麸皮，以4-甲基儿茶酚为底物。通过碱化、硫酸铵沉淀、阴离子交换层析、疏水层析和凝胶过滤层析等步骤，该酶纯化了1670倍，回收率为0.175%。经凝胶过滤层析和SDS-PAGE测定，其相对分子质量分别为35 kDa和40 kDa。N-端氨基酸序列与其他植物PPO的内部序列匹配良好。纯化酶和粗酶的最适pH分别为4.5和5.3。纯化后的酶在4℃、pH 6.5~7.5范围内稳定24小时。纯化后的酶在60℃热处理10min后稳定性较好。纯化的酶氧化L-酪氨酸、邻苯二酚、4-甲基儿茶酚、L-多巴、绿原酸、(+)-儿茶素和(-)-表儿茶素。纯化后的酶对4-甲基儿茶酚和L-多巴的Km值分别为3.3 mM和8.3 mM。二乙基二硫代氨基甲酸酯、硫脲、叠氮化钠、半胱氨酸、艾维酸、氟化钠、反式肉桂酸、对香豆酸、阿魏酸和香草酸对纯化的酶有较强的抑制作用。纯化后的阿魏酸酶的KI值为108μM。
Polyphenol oxidase was purified to homogeneity from Japanese wheat (Triticum aestivum cv. Tohoku 198) bran, using 4-methylcatechol as a substrate. The enzyme was purified 1670-fold with a recovery of 0.175% by alkalization, ammonium sulfate precipitation, anion exchange chromatography, hydrophobic chromatography and gel filtration chromatography. The molecular mass was estimated to be 35kDa and 40kDa by gel filtration chromatography and SDS-PAGE, respectively. The N-terminal amino acid sequence matched well with the internal sequences of other plant PPOs. The pH optima for the purified enzyme and the crude enzyme were 4.5 and 5.3, respectively. The purified enzyme was stable in the range of pH 6.5-7.5 at 4°C for 24hr. The purified enzyme was stable after heat treatment at 60°C for 10min. The purified enzyme oxidized L-tyrosine, pyrocatechol, 4-methylcatechol, L-DOPA, chlorogenic acid, (+)-catechin and (-)-epicatechin. The Km values of the purified enzyme for 4-methylcatechol and L-DOPA were 3.3mM and 8.3mM, respectively. The purified enzyme was strongly inhibited by dietyldithiocarbamate, thiourea, sodium azide, cystein, idoacetic acid, sodium fluoride, trans-cinnamic acid, p-coumaric acid, ferulic acid and vanillic acid. The KI value of the purified enzyme for ferulic acid was 108μM.