黑豆脂氧合酶的制备及其酶学活性的鉴定

从黑豆种子中分离纯化1 种黑豆脂氧合酶--bsLOX，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bsLOX；分别以亚油酸钠和对硝基苯基-β-D-吡喃葡萄糖苷（p-nitrophenyl β-D-glucopyranoside，pNPG）为反应底物鉴定bsLOX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bsLOX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 kD。酶活性分析表明，纯化的bsLOX以亚油酸钠为底物时，酶活力为13 U/mL；当以pNPG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bsLOX具有β-葡萄糖苷酶活性。高效液相色谱实验结果表明bsLOX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bsLOX的酶活性。bsLOX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bsLOX抑制剂。

Preparation and Identification of a Black Soybean Lipoxygenase

Objective: To extract and purify black soybean lipoxygenase (bsLOX) containing β-glucosidase activity and
investigate its anthocyanins-degrading properties and enzymatic activity. Methods: Purification of bsLOX 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 bsLOX were analyzed by using sodium linoleate and
p-nitrophenyl β-D-glucopyranoside (pNPG) 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 kD. The purified protein had a
lipoxygenase activity of approximately 13 U/mL, and could produce nitro phenol using pNPG as substrate as detected at
405 nm, suggesting that bsLOX had β-glucosidase activity. HPLC results showed that bsLOX 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 bsLOX. Conclusion: LOX in black soybean
may be a kind of protein with a variety of enzymatic functions, while anthocyanidins may be a natural bsLOX inhibitor.