PURIFICATION AND CHARACTERIZATION OF TRYPSIN INHIBITOR PROTEIN FROM SEEDS OF MOMORDICA DIOICA

摘要

Objective: To purify the trypsin inhibitor protein from the seeds of Momordica dioica and characterize the protein for its stability and effect on trypsin activity. Methods: The total protein was extracted from the seeds of M. dioica, and the purification of the protein was performed by ion exchange chromatography and ultrafiltration technique. The antitrypsin activity assay of the purified inhibitor protein was carried out using N-benzoylDL-arginine-p-nitroanilide (BAPNA) as the chromogenic substrate at various pH and temperature ranges to determine the stability of the protein. The inhibitory effect of purified protein on trypsin activity was characterized by enzyme kinetic study. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis was performed under the non-reducing condition to determine the presence of inhibitor protein in various fractions of elutes and its molecular mass. Results: The purified protein from M. dioica seeds showed almost 96.17±0.034% trypsin activity inhibition and 0.96±0.00 U trypsin inhibitory activity (TIA) at 30°C and pH 8. This trypsin inhibitory protein from M. dioica (MdTi) was found to be stable over a temperature range of 30-100°C and the pH range of 3-10 retaining the antitrypsin activity. The molecular mass of MdTi was found to be ?12 kDa. From the enzyme kinetics, it was found that Km value remains unaffected with decrease in the Vmax of trypsin in presence of inhibitor. Conclusion: M. dioica seeds were found to posses serine protease inhibitor protein. The protein was purified and characterized as thermostable as well as pH tolerant trypsin inhibitor with high TIA. It can be well explored for its use in the agriculture industry for pest management as well as the therapeutic applications.