Isolation, purification and characterization of antimicrobial protein from seedlings of Bauhinia purpurea L.

• A potent antimicrobial protein from seedlings of B. purpurea.
• Purification by CM-cellulose column chromatography and preparative native-PAGE.
• Characterized by CD spectrum, SDS-PAGE, 2-DE, HPLC and MALDI-TOF.
• Protein efficacy was tested against pathogenic bacteria.
• Protein degrading the bacterial cell wall was visualized from TEM images.

A novel antimicrobial protein was purified from the seedlings of Bauhinia purpurea by sequential procedures entailing ammonium sulfate precipitation, cation exchange chromatography, preparative native-PAGE and a yield of 2.7% was obtained from the crude extract. The purified antimicrobial protein appeared as a single protein band on SDS-PAGE with the molecular mass of 20.9 kDa. Purified antimicrobial protein exhibited a potent antimicrobial activity against both Gram-positive and Gram-negative bacteria. Analysis of the trypsin digested peptides of purified protein using the MALDI-TOF MS/MS resulted in the identification of 174 amino acids. The purified protein had an optimum of pH of 5.5 and was stable at 35 °C for exhibiting its maximal antibacterial activity. The addition of metal ions such as Mn2+ and Ca2+ to the purified protein enhanced the antimicrobial activity of purified protein. The MIC of purified protein against Bacillus cereus and Escherichia coli were 13 μg/ml and 15 μg/ml, respectively. The purified protein digested the peptidoglycan layer of bacteria which was visualized by TEM analysis.

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