Laboratório de Química e Função de Proteínas Bioativas, Departamento de Bioquímica, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil.
Plant Physiol Biochem. 2013 Sep;70:61-8. doi: 10.1016/j.plaphy.2013.04.023. Epub 2013 May 18.
The present study aims to provide new in vitro and in vivo biochemical information about a novel Kunitz trypsin inhibitor purified from Piptadenia moniliformis seeds. The purification process was performed using TCA precipitation, Trypsin-Sepharose and reversed-phase C18 HPLC chromatography. The inhibitor, named PmTKI, showed an apparent molecular mass of around 19 kDa, visualized by SDS-PAGE, which was confirmed by mass spectrometry MALDI-ToF demonstrating a monoisotopic mass of 19.296 Da. The inhibitor was in vitro active against trypsin, chymotrypsin and papain. Moreover, kinetic enzymatic studies were performed aiming to understand the inhibition mode of PmTKI, which competitively inhibits the target enzyme, presenting Ki values of 1.5 × 10(-8) and 3.0 × 10(-1) M against trypsin and chymotrypsin, respectively. Also, the inhibitory activity was assayed at different pH ranges, temperatures and reduction environments (DTT). The inhibitor was stable in all conditions maintaining an 80% residual activity. N-terminal sequence was obtained by Edman degradation and the primary sequence presented identity with members of Kunitz-type inhibitors from the same subfamily. Finally after biochemical characterization the inhibitory effect was evaluated in vitro on insect digestive enzymes from different orders, PmTKI demonstrated remarkable activity against enzymes from Anthonomus grandis (90%), Plodia interpuncptella (60%), and Ceratitis capitata (70%). Furthermore, in vivo bioinsecticidal assays of C. capitata larvae were also performed and the concentration of PmTKI (w/w) in an artificial diet required to LD50 and ED50 larvae were 0.37 and 0.3% respectively. In summary, data reported here shown the biotechnological potential of PmTKI for insect pest control.
本研究旨在为从 Piptadenia moniliformis 种子中纯化得到的新型 Kunitz 胰蛋白酶抑制剂提供新的体外和体内生化信息。该抑制剂命名为 PmTKI，通过 SDS-PAGE 显示其表观分子量约为 19 kDa，通过 MALDI-ToF 质谱证实其单同位素质量为 19.296 Da。该抑制剂对胰蛋白酶、糜蛋白酶和木瓜蛋白酶均具有体外活性。此外，还进行了酶动力学研究，旨在了解 PmTKI 的抑制模式，结果表明该抑制剂对靶酶具有竞争性抑制作用，对胰蛋白酶和糜蛋白酶的 Ki 值分别为 1.5×10(-8)和 3.0×10(-1)M。同时，还在不同的 pH 值范围、温度和还原环境（DTT）下测定了抑制活性。抑制剂在所有条件下均保持稳定，残留活性为 80%。通过 Edman 降解获得了 N 端序列，该序列与同一亚家族的 Kunitz 型抑制剂具有同源性。最后，在进行生化特性表征后，评估了 PmTKI 在不同目昆虫消化酶中的体外抑制效果，结果表明其对 Anthonomus grandis（90%）、Plodia interpuncptella（60%）和 Ceratitis capitata（70%）的酶具有显著活性。此外，还对 C. capitata 幼虫进行了体内生物杀虫试验，结果表明在人工饲料中 PmTKI 的浓度（w/w）达到 LD50 和 ED50 幼虫所需的浓度分别为 0.37%和 0.3%。综上所述，本文报道的数据表明 PmTKI 在昆虫防治方面具有生物技术潜力。
