Department of Biotechnology, Sri Jayachamarajendra College of Engineering, JSS Science and Technology University, JSS TI Campus, Mysuru, Karnataka, 570006, India.
Faculty of Natural Sciences, Adichunchanagiri University, Bellur Cross, B.G. Nagara, Mandya, Karnataka, India.
Mol Cell Biochem. 2020 Aug;471(1-2):71-80. doi: 10.1007/s11010-020-03766-y. Epub 2020 Jun 23.
Purification of DPP-IV enzyme from porcine serum, is presented in this study for the first time. The high molecular weight DPP-IV from porcine serum was fractioned using Sephadex G-75 gel filtration followed by DEAE Sephadex anion exchange and Sephadex G-100 gel filtration chromatography columns with a final yield of 11.25%. The SDS-PAGE of the purified sample showed a single band of molecular mass nearing 160 kDa. Distinct single band was observed after PAS staining confirmed it to be a glycoprotein. The purified enzyme showed an optimum pH and temperature of 8 and 37 °C, respectively. The enzyme effectively cleaved fluorogenic substrate Gly-Pro-AMC with Km and Vmax of 4.578 µM and 90.84 nmoles/min, respectively. Purified DPP-IV activity was inhibited by Diprotin A with an IC value of 8.473 µM. Among the three plant extracts used to study DPP-IV inhibition, the aqueous hot extract of Terminalia chebula showed the highest inhibition of 87.19%, followed by the aqueous cold extract of Momordica carantia, ( 31.6%) and Azadirachta indica (34.16%) at the concentration of 25 µg.
本研究首次从猪血清中纯化 DPP-IV 酶。采用 Sephadex G-75 凝胶过滤柱、DEAE Sephadex 阴离子交换柱和 Sephadex G-100 凝胶过滤柱对猪血清中的高分子量 DPP-IV 进行分级，最终收率为 11.25%。SDS-PAGE 纯化样品显示出一条接近 160 kDa 的单带分子量。过 PAS 染色后观察到明显的单带，证实其为糖蛋白。纯化后的酶在 pH8 和 37°C 时表现出最佳的 pH 和温度，该酶能有效切割荧光底物 Gly-Pro-AMC，Km 和 Vmax 分别为 4.578 μM 和 90.84 nmol/min。Diprotin A 抑制纯化的 DPP-IV 活性，IC 值为 8.473 μM。在用于研究 DPP-IV 抑制的三种植物提取物中，Terminalia chebula 的水热提取物显示出最高的抑制率 87.19%，其次是 Momordica carantia 的水冷提取物（31.6%）和 Azadirachta indica（34.16%），在 25μg 的浓度下。
