Purification and Characterization of a Small Chito-specific Lectin from Datura innoxia Seeds Possessing Anti-microbial Properties

Abstract

Aims: To purify and characterize the smallest lectin from Datura innoxia (DiL9) and to evaluate its antimicrobial application potential. Study Design: Protein purification and characterization using biochemical and biophysical techniques. MALDI-MS/MS for peptide mass finger printing. In vitro assay of antimicrobial activity. Place and Duration of Study: Biochemical Sciences Division, CSIR-National Chemical Laboratory, Pune, India, From Jan 2014 to July 2015. Methodology: Dried Datura seeds were taken as source of lectin and purified using ion-exchange and gel filtration chromatographic techniques. The rabbit erythrocytes were used for the study of hemagglutinating activity. Structural properties were studied using Fluorescence and Circular dichroism spectroscopic methods. The studied lectin was also examined for its antimicrobial activity using agar disc diffusion method and minimum inhibitory concentration (MIC) was calculated using microdilution method. Results: The lectin was purified from dried seeds of Datura innoxia. It was characterized as a chito-specific glycoprotein with 4% neutral carbohydrate content and molecular weight of 9 kDa. Circular dichroism and fluorescence studies showed it to be structurally different from other known lectins isolated from Datura. Protein was found to be highly thermostable with sugar binding capacity spread over a broad pH range (1-12), and showed resistance to effects of organic solvents, chemical denaturation and proteolytic digestion. DiL9 also demonstrated antibacterial activity against both Gram-positive and Gram-negative bacteria; however, more effective against Enterococcus faecalis and Bacillus cereus with MIC values at 0.325 and 0.15 mg ml-1, respectively. Conclusion: The present study revealed that the lectin purified from D. innoxia is a glycoprotein and agglutinates pronase treated rabbit erythrocytes. The lectin is highly thermostable with respect to its structure and activity at all denaturing conditions, also showed significant antibacterial activity. This data also supports the fact of its traditional use in folk or alternative medicines for treatment of bacterial infections.