Isolation and Characterization of a Cysteine Protease from the Latex of <i>Araujia hortorum</i> Fruits

Abstract

A new protease (<i>araujiain h l</i>) was purified to mass spectroscopy homogeneity from the latex of Araujia hortorum Fourn. (Asclepiadaceae) fruits by ultracentrifugation and ion exchange chromatography. The enzyme has a molecular mass of 24,031 (mass spectrometry) and an isoelectric point higher than 9.3. The optimum pH range for casein hydrolysis was 8.0–9.5. The enzyme showed remarkable caseinolytic activity at high temperatures, although its thermal stability decayed rapidly. The proteinase was activated by thiol compounds and inhibited by common thiol-blocking reagents, particularly E-64 and HgCl₂, suggesting the enzyme belongs to the cysteine protease family. The concentration of active sites as determined by titration with E-64 was 3.3 μM. When assayed on N-α-CBZ-amino acid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative, followed by those of alanine, asparagine, glycine, and leucine, in decreasing order. Partial homology (36–48%) with other plant cysteine proteinases was observed in an internal fragment obtained by Protease V8 treatment.