Purification, characterization, and bioassay of putative protease inhibitors from Hevea brasiliensis latex

Abstract

Latex, a milky white liquid, is the main product from rubber tree (Hevea brasiliensis). Latex is the cytoplasm of complex cellular networks named laticifers in which it contains many different components, including important proteins. Various types of enzymes carrying functions associated with plant defense against pathogen and wounding have been detected in latex in which one of these enzymes is protease inhibitor (PI). Plant protease inhibitor has tremendous potential as an antifungal agent which can be developed as biofungicide. In this work, protease inhibitors from B-serum (lutoid) of rubber tree latex were isolated and purified using Ion Exchange Chromatography (IEC) technique. Of the total 70 fractions of proteins extracted from the columns, only 26 fractions showed measurable levels of protein. The concentration of obtained putative protease inhibitors (three fractions of IEC) ranged from 0.007 to 0.022 mL/g B-serum. Inhibitory activity against four protease enzymes (subtilisin A, trypsin, α-chymotrypsin, and papain) showed the characteristics of Hevea putative protease inhibitors from B-serum as serine and/or cysteine protease inhibitors with more than 15% inhibitory activity of target protease. Based on SDS-PAGE visualization, the molecular weight of dominant protein considered as Hevea putative protease inhibitors was 21.5 kDa. In vitro bioassay test of antifungal activity for Hevea putative protease inhibitors showed reduced mycelium growth of Ganoderma boninense, Sclerotium sp., and Rigidosporus lignosus.