金冠豆角籽粒中α-淀粉酶抑制剂的纯化及抑制动力学的研究

Purification and inhibition kinetics of α-amylase inhibitor from Golden Crown snap bean seeds

中文关键词:  α-AI  金冠豆角籽粒  纯化  抑制类型

英文关键词:α-amylase inhibitor  Golden Crown snap bean seeds  purification  inhibition type

中文摘要:

      目的 提取金冠豆角籽粒中α-淀粉酶抑制剂(α-amylase inhibitor, α-AI)并将其进行纯化, 研究其抑制类型及抑制效果。方法 以金冠豆角籽粒为原料, 通过NaCl盐溶、(NH4)2SO4沉淀提取α-AI, 使用G50、G75葡聚糖凝胶柱进行层析, 用NaCl洗脱液进行梯度洗脱, 收集蛋白峰后, 检测α-AI对猪胰淀粉酶的抑制活性; 用Lineweaver-Burk双倒数绘图法和Michaelis-Menton方程分析酶反应动力学, 探究α-AI的抑制类型。结果 金冠豆角籽粒中提取的α-AI蛋白含量为1.72 mg/mL, 对猪胰淀粉酶抑制率为84.20%, 半抑制浓度(semi-inhibition concentration, IC50)为(6.77±0.63) mg/mL。不添加抑制剂组和添加抑制剂组的α-淀粉酶的酶解曲线几乎相交于原点, 并且酶最大反应速率(Vmax)随着抑制剂浓度的增大而减小, 米氏常数(Km)几乎不变。结论 α-AI与猪胰淀粉酶的结合呈现可逆型非竞争性抑制。

英文摘要:

      Objective To extract and purify α-amylase inhibitor (α-AI) from the Golden Crown snap bean seeds, and study its inhibition type and effect. Methods The Golden Crown snap bean seeds were used as raw material, and crude α-AI was extracted by salting in NaCl solution and precipitating with ammonium sulfate. The α-AI was then purified with G50 and G75 dextran gel columns under gradient NaCl eluent elution. The different protein peaks were collected for determining α-AI activity against porcine pancreatic amylase. Lineweaver Burk double reciprocal plot method and Michaelis-Menton equation were used to analyze the enzyme reaction kinetics and explore the inhibition types of α-AI. Results The α-AI content was 1.72 mg/mL, and inhibition rate against porcine pancreatic amylase was 84.20%, with semi-inhibition concentration (IC50) of (6.77±0.63) mg/mL. The enzyme reaction kinetics results showed that the enzymatic hydrolysis rate curves intersected almost at the origin for the groups with or without α-AI supplementation. The maximum enzyme reaction rate (Vmax) decreased with increasing inhibitor concentration, and the Michaelis constant (Km) was nearly constant. Conclusion The binding of α-AI to porcine pancreatic amylase shows reversible and non-competitive inhibition.