Plants Cp are widely distributed in the plant kingdom and
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 . 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 serine protease inhibitors 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 . 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.
Materials and methods
Results and discussion In plants, Cp are involved in protein maturation, degradation and protein rebuilding in response to various foreign stimuli and also play a housekeeping function to remove misfolding protein. Cp is believed to have an important role in protecting plants from various pathogens. Since hydrolytic enzymes are involving in the plant defense system . As only less information are available in the literature about optimization of cysteine protease extraction and purification from stem of C. quadrangularis, therefore, an attempt was made to study the plant Cp. A detailed knowledge on the extraction and purification is a prerequisite for efficient utilization of the enzyme for any application. Herein, we have purified the Cp from C. quadrangularis, characterized and evaluated its antibacterial potential against pathogenic bacteria. This study mainly emphasizes the possibility of a relationship between the extraction, purification conditions and enzymatic properties of Cp from C. quadrangularis and discusses the development of an extraction process technique by response surface methodology and subsequently evaluating its antibacterial property . In our study, we have determined the ideal conditions for purified Cp which has a maximum specific activity, storage and thermal stability. The purified Cp from C. quadrangularis was analyzed for its antibacterial activity against pathogenic bacteria B. cereus, B. subtilis, B. megaterium and B. licheniformis.
Conclusion We found that the 39kDa cysteine protease purified from C. quadrangularis possessed antibacterial activity. The physiochemical parameters such as, different buffer system, temperature, inhibitors, metallic ions and organic solvents were analyzed for purified Cp. The Cp was characterized by SDS-PAGE, 2D gel and HPLC analyses. Based on the characteristic determined during the course of retina study, the ability of this enzyme to have antibacterial activity produced by C. quadrangularis holds an immense scope for utilization as an antibacterial drug. Further, there is an ample scope for its future research on application study, structure elucidation and enzyme engineering of this cysteine protease towards the improvement of the enzyme for a wide range of application.
Acknowledgement We gratefully acknowledge University Grants Commission, New Delhi, India for the financial support.