Purificação parcial de inibidores proteoliticos em sementes de Hevea brasiliensis e Hevea spruceana

Abstract

Seeds of species of Hevea genus are a promising source of substances for biotechnological application, with emphasis on proteins, with contents in the order of 15 to 20% in mass basis. Therefore, studies on the characterization and applicability of some protein classes such as protease inhibitors from Hevea seeds reinforce the expectations of obtaining scientific knowledge for the application in bioindustry of products prospected in the Amazonian flora. Considering the heterogeneity of Hevea species and the biotechnological importance of inhibitors, the objective of this work was to partially purify protease inhibitors present in Hevea brasiliensis and Hevea spruceana seeds. The seeds were collected in two forest sites in Acre (Parque Zoobotânico and Embrapa, Rio Branco -AC), taken to the Laboratory of Physiology and Plant Biochemistry - INPA where they were processed, then, part of the seeds were freezedried and ground in analytical knife mill, until obtaining finely pulverized material. In these samples the lipids were removed so as not to interfere in the protein extractions. Another part of the seeds were placed for sowing in order to monitor the contents of inhibitors in the germinative stages. Once the protein extract was obtained, the inhibitor stability test was performed at temperature, and afterwards, the trypsin, chymotrypsin, papain and bromelain inhibition activity test was performed. The trypsin inhibitors are present in the protein extracts from seeds of wild genotypes of H. brasiliensis and H. spruceana, the differences of the inhibitory activity can be associated to the different germination stages, once the species showed high activity in the quiescent seeds. The tested inhibitors are not thermostable molecules, having stability near 30 to 40 °C. By affinity chromatography, both species showed significant results for the contents of this class of proteins. With important register for H. spruceana that stood out for the high inhibitory activity and higher protein yield, probably, for having large amount of proteolytic inhibitors, when compared to the species H. brasiliensis that showed low inhibitory activity. Key-words: Germination; affinity chromatography; Hevea trypsin inhibitors; thermostability.