Abd El-Latif Ashraf Oukasha
Department of Plant Protection, Faculty of Agriculture, Sohag University, Sohag, Egypt.
Pestic Biochem Physiol. 2015 Feb;118:26-32. doi: 10.1016/j.pestbp.2014.10.016. Epub 2014 Oct 30.
The cysteine inhibitors that are known as cystatin have been identified and characterized from several plant species. In the current study, 44 barley (Hordeum vulgare) genotypes including 3 varieties and 41 promising lines were screened for their potential as protease inhibitors. The barley genotypes showed low inhibitory activity against trypsin and chymotrypsin enzymes with a mean of 4.15 TIU/mg protein and 4.40 CIU/mg protein. The barley variety, Giza 123, showed strong papain inhibitory activity of 97.09 PIU/mg proteins and was subjected for further purification studies using ammonium sulfate fractionation and DEAE-Sephadex A-25 column. Barley purified proteins showed two bands on SDS-PAGE corresponding to a molecular mass of 12.4-54.8 kDa. The purified barley PI was found to be stable at a temperature below 80 °C and at a wide range of pH from 2 to 12. Barley PI was found to have higher potential inhibitory activity against papain enzyme compared to the standard papain inhibitor, E-64 with an IC50 value of 21.04 µg/ml and 25.62 µg/ml for barley PI and E-64, respectively. The kinetic analysis revealed a non-competitive type of inhibition with a Ki value of 1.95 × 10(-3 )µM. The antimetabolic effect of barley PI was evaluated against C. maculatus by incorporating the F30-60 protein of the purified inhibitor into the artificial diet using artificial seeds. Barley PI significantly prolonged the development of C. maculatus in proportion to PI concentration. Barley PI significantly increased the mortality of C. maculatus and caused a significant reduction in its fecundity. On the other hand, barley PI seemed to have non-significant effects on the adult longevity and the adult dry weight. The in vitro and in vivo results proved the efficiency of the papain inhibitory protein isolated from barley as a tool for managing the cowpea bruchid, C. maculatus.
被称为胱抑素的半胱氨酸抑制剂已从多种植物物种中被鉴定和表征。在本研究中，对包括3个品种和41个优良品系在内的44个大麦（Hordeum vulgare）基因型进行了蛋白酶抑制剂潜力的筛选。大麦基因型对胰蛋白酶和胰凝乳蛋白酶的抑制活性较低，平均分别为4.15 TIU/mg蛋白质和4.40 CIU/mg蛋白质。大麦品种吉萨123表现出97.09 PIU/mg蛋白质的强木瓜蛋白酶抑制活性，并使用硫酸铵分级分离和DEAE-葡聚糖A-25柱进行进一步纯化研究。大麦纯化蛋白在SDS-PAGE上显示出两条带，对应分子量为12.4 - 54.8 kDa。发现纯化的大麦蛋白酶抑制剂在低于80°C的温度和2至12的宽pH范围内稳定。与标准木瓜蛋白酶抑制剂E-64相比，发现大麦蛋白酶抑制剂对木瓜蛋白酶具有更高的潜在抑制活性，大麦蛋白酶抑制剂和E-64的IC50值分别为21.04 μg/ml和25.62 μg/ml。动力学分析显示为非竞争性抑制类型，Ki值为1.95×10(-3) μM。通过将纯化抑制剂的F30 - 60蛋白掺入使用人工种子的人工饲料中，评估了大麦蛋白酶抑制剂对黄斑豆象的抗代谢作用。大麦蛋白酶抑制剂与蛋白酶抑制剂浓度成比例地显著延长了黄斑豆象的发育时间。大麦蛋白酶抑制剂显著提高了黄斑豆象的死亡率，并使其繁殖力显著降低。另一方面，大麦蛋白酶抑制剂似乎对成虫寿命和成虫干重没有显著影响。体外和体内结果证明了从大麦中分离的木瓜蛋白酶抑制蛋白作为管理豇豆象甲黄斑豆象的工具的有效性。
