Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand.
J Agric Food Chem. 2010 Feb 24;58(4):2131-7. doi: 10.1021/jf903411x.
The insect Callosobruchus maculatus causes considerable damage to harvested mungbean seeds every year, which leads to commercial losses. However, recent studies have revealed that mungbean seeds contain alpha-amylase inhibitors that can inhibit the protein C. maculatus, preventing growth and development of the insect larvae in the seed, thus preventing further damage. For this reason, the use of alpha-amylase inhibitors to interfere with the pest's digestion process has become an interesting alternative biocontrolling agent. In this study, we have isolated and purified the alpha-amylase inhibitor from mungbean seeds (KPS1) using ammonium sulfate precipitation, gel filtration chromatography and reversed phase HPLC. We found that the alpha-amylase inhibitor, isolated as a monomer, had a molecular weight of 27 kDa. The alpha-amylase inhibitor was purified 750-fold with a final yield of 0.4 mg of protein per 30 g of mungbean seeds. Its specific activity was determined at 14.5 U (mg of protein)(-1). Interestingly, we found that the isolated alpha-amylase inhibitor inhibits C. maculatus alpha-amylase but not human salivary alpha-amylase. After preincubation of the enzyme with the inhibitor, the mungbean alpha-amylase inhibitor inhibited C. maculatus alpha-amylase activity by decreasing V(max) while increasing the K(m) constant, indicating that the mungbean alpha-amylase is a mix noncompetitive inhibitor. The in vivo effect of alpha-amylase inhibitor on the mortality of C. maculatus shows that the alpha-amylase inhibitor acts on C. maculatus during the development stage, by reducing carbohydrate digestion necessary for growth and development, rather than during the end laying/hatching stage. Our results suggest that mungbean alpha-amylase inhibitor could be a useful future biocontrolling agent.
昆虫绿豆象每年都会对收获的绿豆种子造成严重损害，导致商业损失。然而，最近的研究表明，绿豆种子中含有α-淀粉酶抑制剂，能够抑制蛋白 C. maculatus，阻止昆虫幼虫在种子中的生长和发育，从而防止进一步的损害。因此，利用α-淀粉酶抑制剂来干扰害虫的消化过程，已成为一种有趣的生物防治替代方法。在这项研究中，我们使用硫酸铵沉淀、凝胶过滤层析和反相高效液相色谱法从绿豆种子中分离和纯化了α-淀粉酶抑制剂（KPS1）。我们发现，分离出的单体α-淀粉酶抑制剂的分子量为 27 kDa。α-淀粉酶抑制剂经过 750 倍的纯化，最终从 30 克绿豆种子中获得 0.4 毫克蛋白质，其比活为 14.5 U（mg 蛋白）（-1）。有趣的是，我们发现分离出的α-淀粉酶抑制剂抑制绿豆象α-淀粉酶，但不抑制人唾液α-淀粉酶。在酶与抑制剂预孵育后，绿豆α-淀粉酶抑制剂通过降低 Vmax 而增加 K(m)常数来抑制 C. maculatus α-淀粉酶活性，表明绿豆α-淀粉酶是一种混合非竞争性抑制剂。α-淀粉酶抑制剂对 C. maculatus 死亡率的体内效应表明，α-淀粉酶抑制剂在发育阶段作用于 C. maculatus，通过减少生长和发育所需的碳水化合物消化，而不是在产卵/孵化结束阶段。我们的结果表明，绿豆α-淀粉酶抑制剂可能是一种有用的未来生物防治剂。
