Isolation and Partial characterization of Cysteine Protease Inhibitor from Water Melon (Citrullus lanatus) Seeds

Cysteine protease inhibitors (CPIs) have a vital role in the strict regulation and control of cysteine protease activity. In this work, a cysteine protease inhibitor (CPI) from watermelon (Citrullus lanatus) seeds was isolated, purified, and partially characterised. Ammonium sulfate precipitation was used to extract CPI, then size exclusion chromatography and ion exchange were used to further purify it. Papain was used to assess the inhibitory action, and identifying the ideal temperature stability and method of inhibition were part of the biochemical characterisation. The protein content of the fractionated samples ranged from 3.6 to 6.5 mg/L, whereas the crude sample of C. lanatus seed had 9.2 mg/L. Cysteine protease inhibitor (CPI) was isolated from C. lanatus seeds, and the highest inhibitory activity occurred at 60–100% saturation of (NH4)2SO4. The CPI inhibitor exhibits a competitive mode of inhibition on papain, with the same Vmax = 1.06 μmol/min, Km = 2.5 μM, and Ki = 4.00 μM. Its activity is retained up to 65°C, but it drastically decreases beyond 70°C. The work advances knowledge of the biochemical characteristics of CPIs obtained from plants and highlights their applicability in the agricultural and pharmaceutical sectors. Keywords: Ammonium sulfate, Cysteine protease inhibitor (CPI), Isolation, Purification Watermelon (Citrullus lanatus)