东海中华管鞭虾多酚氧化酶（PPO）的纯化及特性研究

Purification and Characterization of the Polyphenoloxidase (PPO) from Solenocera crassicornis in the East Sea of China

摘要:

本文采用匀浆浸提法提取中华管鞭虾(Solenocera crassicornis，S. crassicornis)多酚氧化酶(polyphenoloxidase，PPO)，经硫酸铵质量分级、离子交换、凝胶过滤纯化后，以左旋多巴胺(L-DOPA)为底物研究其底物专一性和动力学特性，并选取不同抑制剂和金属离子探讨对该PPO的抑制影响。结果显示，匀浆浸提法提取PPO的最佳条件为料液比1:2(W/V)、pH 7.5、浸提3 h、温度50 ℃，从100 g中华管鞭虾中获取的PPO的产量和纯化倍数分别是1.2%和25.9，中华管鞭虾PPO对L-DOPA和儿茶酚具有很显著地催化氧化作用(p≤0.05)，Km值为分别是3.8 mM和8.12 mM，说明该酶为儿茶酚家族酶。PPO活性对苯硫脲、抗坏血酸、柠檬酸较为敏感，能显著被Cu2+、Zn2+、乙二胺四乙酸(EDTA)抑制。结果表明，中华管鞭虾PPO可能是活性部位含Cu2+的金属酶，为控制中华管鞭虾黑变提供基础的信息。

Abstract:

In this study, the polyphenoloxidase (PPO) was extracted using the homogenate extraction from Solenocera crassicornis (S. crassicornis) in the East China Sea, and purified through ammonium sulphate precipitation, ion-exchange and gel filtration. The substrate specificity (L-dihydroxyphenylalanine (L-DOPA) as the substrate) and kinetic properties of the purified PPO, along with the effects of different inhibitors and metal ions on the PPO, were determined. The obtained results showed the optimal conditions for the homogenate extraction were: the material to liquid ratio 1:2 (W/V), pH 7.5, time 3 h, and temperature 50 ℃. The yield and purification factor were 1.2% and 25.9, respectively. This PPO significantly catalyzed the oxidation of L-DOPA and catechol (p≤0.05), with the Km values being 3.8 mM and 8.12 mM, respectively, indicating that the PPO belongs to the catechol enzyme family. The PPO was sensitive to phenylthiourea, ascorbic acid, and citric acid, and its activity was strongly inhibited by Cu2+, Zn2+, and ethylenediaminetetraacetic acid (EDTA). Thus, the PPO from S. crassicornis could be a metalloenzyme containing Cu2+ in its active center. The results of this study provide the basis for controlling the melanotic blackening of S. crassicornis.