Isolation of a novel leguminous lysozyme and study on the antifungal activity

A novel lysozyme was isolated from the Canadian cranberry beans (Phaseolus vulgaris) and its enzymatic properties, antifungal and antibacterial activities were investigated. The isolation procedure involved aqueous extraction, ammonium sulfate precipitation, and chromatography purification including affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sephadex, and high performance liquid chromatography (HPLC) on Mono S. The enzyme exhibited a molecular mass of 15.6 kDa in SDS-polyacrylamide gel electrophoresis both under reducing and non-reducing conditions, indicating that it was a monomeric protein. The 15 N-terminal amino acid sequences were determined to be KVDMPGRFALAMQSH, showing some homology to hen egg white lysozyme. The pI was determined to be 9.6 by isoelectric focusing electrophoresis. The specific activity of the lysozyme was 652 U/mg at pH 5.5 and 40 °C. The lysozyme exhibited a pH optimum at pH 5.5 and a temperature optimum at 50 °C. It exerted a potent inhibitory action toward fungal species including Alternaria alternata(Fr.) Keissl, Pythium aphanidermatum, Fusarium oxysporum and Botrytis cinerea, in addition to an antibacterial action against Staphylococcus aureus. However, it showed no effect on Gram-negative bacteria. The enzyme was thermostable below 58 °C in both enzymatic reaction and antifungal activity. The present findings further the research on leguminous lysozyme with potentially exploitable significance.