Purificação e caracterização de um inibidor de tripsina com atividade antimicrobiana da torta de pinhão-manso (Jatropha curcas L.)

Abstract

The residue (cake) obtained after the extraction of oil from jatropha (Jatropha curcas) seeds for biodiesel production is toxic. Despite of the obstacles related to its use in the animal feed, there are evidences that this residue has great potential for biotechnology applications due to its arsenal of molecules. Thus, the present study aimed to purify and characterize a trypsin inhibitor from jatropha cake, in order to make a better use of this residue. The centesimal composition analysis showed to be the jatropha cake mainly composed of proteins (37.21%), fiber (34.26%) and lipids (19.16%). The crude extract obtained from the jatropha cake under alkaline condition (100 mM sodium borate buffer, pH 10.0) showed the presence of lectin (319.76 HU/gF), papain inhibitor (1,287.38 IU/gF), protease (2.69 AU/gF) and, especially, trypsin inhibitor (1,649.7 IU/gF). The trypsin inhibitor, named JcTI, was purified by fractionation of the crude extract with trichloroacetic acid (2.5%) followed by affinity chromatography (trypsin-sepharose-4B) and molecular exclusion (sephacryl S-200). JcTI is a glycoprotein (6.4% carbohydrates) with molecular mass in the range of 20-21 kDa, pI of 6.6, NH2-terminal sequence (VRDICKKEAERQDLSSCENYITQRRGY) showing identity around 60% with plant albumins and highly stable to heat, pH and salinity. JcTI (500 µg/mL) slowed the growth of important phytopthogenic fungi, including Colletotrichum gloeosporioides, Colletotrichum lindemuthianum, Fusarium oxysporum and Fusarium solani. This inhibitor also presented antibacterial activity against human pathogenic bacteria such as Bacillus subtilis, Salmonella choleraesuis and Staphylococcus aureus, with minimum inhibitory concentration less than 5 µg/mL. The results demonstrate the potential of JcTI for biotechnological application as a new defense protein against phytopthogenic fungi and human pathogenic bacteria.