Isolation, purification, and characterization of a stable defensin-like antifungal peptide from Trigonella foenum-graecum (fenugreek) seeds

A novel defensin-like antifungal peptide (Tf-AFP) with molecular mass of 10.3 kDa was isolated from seeds of Trigonella foenum-graecum (fenugreek) by ammonium sulfate precipitation, cation-exchange, gel-filtration, hydrophobic chromatography, and RP-HPLC. Mass spectroscopic analysis revealed the intact mass of the purified antifungal peptide as 10321.5 Da and high similarity to plant defensins and other antifungal proteins in database search. 2D-PAGE showed pI value to be 8.8 and absence of isoforms. Isolated Tf-AFP inhibited growth of fungal species such as Fusarium oxysporum, Fusarium solani, and Rhizoctonia solani. The antifungal activity was inhibited in the presence of 50 mM NaCl. Circular dichroism analysis demonstrated that the protein is rich in β-sheet structure and highly stable over a wide range of temperatures. Surprisingly, reduction of disulfide bridges and chemical denaturation did not produce large changes in secondary structure as judged by circular dichroism as well as by fluorescence spectroscopy.