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 ofα-amylase inhibitor from Golden Crown snap bean seeds

目的提取金冠豆角籽粒中α-淀粉酶抑制剂(α-amylaseinhibitor,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Na Cl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Na Cl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1.72mg/mL,对猪胰淀粉酶抑制率为84.20%,半抑制浓度(semi-inhibition concentration,IC_(50))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(V_(max))随着抑制剂浓度的增大而减小,米氏常数(K_(m))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。

Objective To extract and purifyα-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 crudeα-AI was extracted by salting in NaCl solution and precipitating with ammonium sulfate.Theα-AI was then purified with G50 and G75 dextran gel columns under gradient NaCl eluent elution.The different protein peaks were collected for determiningα-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 ofα-AI.Results Theα-AI content was 1.72 mg/mL,and inhibition rate against porcine pancreatic amylase was 84.20%,with semi-inhibition concentration(IC_(50))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 withoutα-AI supplementation.The maximum enzyme reaction rate(V_(max))decreased with increasing inhibitor concentration,and the Michaelis constant(K_(m))was nearly constant.Conclusion The binding ofα-AI to porcine pancreatic amylase shows reversible and non-competitive inhibition.