Cysteine proteases Cp are widely distributed

Cysteine proteases (Cp) are widely distributed among living organisms, found in both prokaryotes and eukaryotes (e.g. bacteria, parasites, plants, invertebrates and vertebrates) [13], [14]. The catalytic mechanism of these ONO-8711 involves a cysteine group in the active site. Cp comprise a family of enzymes, consisting of papain and related plant proteases such as chymopapain, caricain, bromelain, actinidin, ficin, aleurain and the mammalian lysosomal cathepsins [15]. Most plant Cp belongs to the papain family, including those of Asclepiadaceae, the milkweed family [19]. High proteolytic activity has been reported in crude enzyme preparations of latex of different species of this family. Inhibition analysis, where specific protease inhibitors are employed to identify catalytic groups within the active centre of the protease, suggesting that these proteases belonged to the cysteine type [8]. Proteases like asclepain from Asclepias curassavica[8], A. syriaca [18], A.glaucescens[19], A.fruticosa [20], calotropin from Calotropis gigantea[21], procerain from Calotropis procera[22], araujain from Araujia hortorum[23], actinidin from Actinidia chinesis[24], funastrain from Funastrum clausum[25] and philibertain from Philibertia gilliessi[26] have been isolated and characterized. Based on the review, the intra molecular disulphide bridges are presumably responsible for the functional stability of Kunitz type protease inhibitors in the presence of physical and chemical denaturants such as temperature, pH and reducing agents [27]. Extreme pH conditions will alter the structure of the inhibitor such that they no longer bind with the enzymes or with their substrates. Under strong acidic or alkaline conditions, the proteinaceous inhibitors get denatured and as a consequence they lose their activity partially or completely [28]. Plants Cp are widely distributed in the plant kingdom and are believed to act as virulence/defense factors for both hosts and pathogens. Cp are also found in plants, animals and bacteria and are known to be virulence factors involved in bacterial pathogenicity [29]. Further, Cp are involved in peptidoglycan turnover in both Gram-negative and Gram-positive bacteria, with disruption of amide-hydrolyzing autolysins in Bacillus subtilis, leading to defective cell wall division and thereby affecting bacterial viability. More recently, the growing resistance of microorganisms to conventionally used antibiotics is restricted due to it side effect and leading resistant bacteria meant so that the cysteine proteases have attracted attention as possible targets for antimicrobial therapy as organic molecule [30]. In this view, it is important to pursue a study on these enzymes i.e plant proteases. Such studies on comparative proteomics may provide a better insight into the antibacterial property of plant proteases. In this article, we have isolated, purified and characterized the Cp from the extract of C. quadrangularis. Cp was purified using ammonium sulfate fractionation, Sephadex G-100 and CM-Cellulose cation exchange column chromatography techniques to its homogeneity. The purity of the purified Cp was verified by SDS-PAGE, 2D gel and HPLC analyses. The antibacterial activity of the purified Cp against pathogenic gram positive bacteria has been evaluated. The effects of Cp on the structural destabilization of bacterial membrane were analyzed by transmission electron microscopy.