黑豆脂氧合酶的制备及其酶学活性的鉴定

Preparation and Identification of a Black Soybean Lipoxygenase

从黑豆种子中分离纯化1种黑豆脂氧合酶——bs LOX,对其酶学活性及降解花色苷等性质进行初步研究。采用缓冲液抽提、硫酸铵沉淀、透析、阳离子交换层析及凝胶层析等方法纯化出具有β-葡萄糖苷酶活性的bs LOX;分别以亚油酸钠和对硝基苯基-β-D-吡喃葡萄糖苷(p-nitrophenylβ-D-glucopyranoside,p NPG)为反应底物鉴定bs LOX的脂氧合酶活性和β-葡萄糖苷酶活性。从黑豆种子中纯化获得的bs LOX在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electropheresis,SDS-PAGE)图谱显示单一条带,分子质量约为95 k D。酶活性分析表明,纯化的bs LOX以亚油酸钠为底物时,酶活力为13 U/m L;当以p NPG为底物在405 nm波长处检测到了对硝基苯酚的生成,从而推测bs LOX具有β-葡萄糖苷酶活性。高效液相色谱实验结果表明bs LOX可以降解花色苷(矢车菊素-3-O-葡萄糖苷、矮牵牛素-3-O-葡萄糖苷)为花色素(矢车菊素、矮牵牛素)。由于降解作用不完全,继而发现花色素能抑制bs LOX的酶活性。bs LOX是黑豆中的一种兼具β-葡萄糖苷酶功能的蛋白质,而花色素可能是天然的bs LOX抑制剂。

Objective： To extract and purify black soybean lipoxygenase（bs LOX） containing β-glucosidase activity and investigate its anthocyanins-degrading properties and enzymatic activity. Methods： Purification of bs LOX was carried out by buffer extraction, ammonium sulfate precipitation, dialysis, cation exchange chromatography and gel filtration chromatography. The activities of lipoxygenase and β-glucosidase in bs LOX were analyzed by using sodium linoleate and p-nitrophenyl β-D-glucopyranoside（p NPG） as substrates, respectively. An LOX from black soybean seeds was obtained, which showed a single band in SDS-PAGE, with approximate molecular weight of 95 k D. The purified protein had a lipoxygenase activity of approximately 13 U/m L, and could produce nitro phenol using p NPG as substrate as detected at 405 nm, suggesting that bs LOX had β-glucosidase activity. HPLC results showed that bs LOX could only degrade part of anthocyanins（cyanidin-3-O-glucoside and petunidin-3-O-glucoside） into aglycone anthocyanidins（cyanidin and petunidin） and glucose; in turn, the residual anthocyanidins could inhibit the activity of bs LOX. Conclusion： LOX in black soybean may be a kind of protein with a variety of enzymatic functions, while anthocyanidins may be a natural bs LOX inhibitor.