Laboratory of Plant Toxins, Department of Biochemistry and Molecular Biology, Federal University of Ceara Fortaleza, Brazil.
School of Pharmacy, University of Fortaleza Fortaleza, Brazil.
Front Microbiol. 2014 Jan 30;5:5. doi: 10.3389/fmicb.2014.00005. eCollection 2014.
Jatropha curcas seed cake is a low-value by-product resulting from biodiesel production. The seed cake is highly toxic, but it has great potential for biotechnology applications as it is a repository of biomolecules that could be important in agriculture, medicine, and industry. To explore this potential, a novel trypsin inhibitor called JcTI-I was purified by fractionation of the crude extract with trichloroacetic acid (2.5%, v/v) followed by affinity chromatography (Trypsin-Sepharose 4B) and molecular exclusion (Sephacryl S-200). Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration showed that JcTI-I has approximately 20.0kDa. Mass spectrometry analysis revealed that the intact molecular mass of JcTI-I is 10.252kDa. Moreover, JcTI-I is a glycoprotein with 6.4% (m/m) carbohydrates, pI of 6.6, N-terminal sequence similarity around 60% to plant albumins and high stability to heat, pH, and salinity. JcTI-I presented antibacterial activity against the human pathogenic bacteria Salmonella enterica subspecies enterica serovar choleraesuis and Staphylococcus aureus, with minimum inhibitory concentration less than 5~μg/mL. Furthermore, JcTI-I did have inhibitory activity against the serine proteases from the tested bacteria. Otherwise, no hemolytic activity of human erythrocytes and signs of acute toxicity to mice were observed for JcTI-I. The results demonstrate the benefits of J. curcas seed cake as a source of trypsin inhibitor with potential for biotechnological application as a new antimicrobial agent against human pathogenic bacteria.
麻疯树种子饼是生物柴油生产的一种低附加值副产物。种子饼毒性很高，但它具有巨大的生物技术应用潜力，因为它是生物分子的储存库，这些生物分子在农业、医学和工业中可能很重要。为了探索这种潜力，一种新型的胰蛋白酶抑制剂 JcTI-I 通过用三氯乙酸（2.5%，v/v）对粗提取物进行分级分离，然后进行亲和层析（胰蛋白酶-Sepharose 4B）和分子排阻（Sephacryl S-200）来进行纯化。非还原十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和凝胶过滤显示，JcTI-I 的分子量约为 20.0kDa。质谱分析表明，JcTI-I 的完整分子量为 10.252kDa。此外，JcTI-I 是一种糖蛋白，含有 6.4%（m/m）的碳水化合物，等电点为 6.6，N-末端序列与植物白蛋白相似性约为 60%，并且对热、pH 值和盐度具有高稳定性。JcTI-I 对人类病原菌肠炎沙门氏菌亚种肠炎血清型和金黄色葡萄球菌具有抗菌活性，最小抑菌浓度小于 5μg/mL。此外，JcTI-I 对测试细菌的丝氨酸蛋白酶具有抑制活性。否则，JcTI-I 对人红细胞没有溶血活性，对小鼠也没有急性毒性迹象。这些结果表明，麻疯树种子饼作为胰蛋白酶抑制剂的来源具有优势，具有作为新型抗人类病原菌抗菌剂的生物技术应用潜力。
