Purification, characterization, antibacterial activity and N-terminal sequencing of buffalo-milk lysozyme

Abstract

S. Lysozyme from buffalo milk was purified to homogeneity and its Nterminal amino acid sequence, biochemical properties and antibacterial spectrum were determined. The purification procedure, comprising ion-exchange chromatography using CM-cellulose and size-exclusion chromatography using Sephadex G-50, conferred 8622-fold purification and 39n3 % recovery of lysozyme. The purified enzyme migrated as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. Immunological purity of lysozyme preparation was confirmed by immuno-electrophoresis. Molecular weight of buffalomilk lysozyme as determined by SDS-PAGE was 16 kDa and its amino acid composition was determined by reverse phase high performance liquid chromatography (HPLC). The sequence of 23 amino acid residues at the N-terminal end showed 56n5 % homology with bovine milk lysozyme and 30n4 % with equine milk lysozyme. The specific activity of buffalo milk lysozyme was ten-times that of bovine milk lysozyme. Buffalo-milk lysozyme was active over a wide range of pH and its activity was strongly influenced by molarity of the medium. Antibacterial activity of buffalo-milk lysozyme was determined against 11 species of bacteria ; out of seven Gram-positive bacteria tested, four were inhibited, while Gram-negative bacteria were resistant.

Maternal Milk Contains Antimicrobial Factors That Protect Young Rabbits from Enteropathogenic Escherichia coli Infection

“…However, in this study, the transmission of specific antibodies by maternal milk was not involved, since all does were seronegative for the O103 LPS. More recently, other substances present in milk have also been shown to have antimicrobial properties: defensins (19); lactoferrin (42) and its derivative, the lactoferricin (15); various enzymatic complexes like lactoperoxidase, xanthine oxidoreductase, and lysozyme (28,36,40); fucosylated oligosaccharides (10); and various fatty acids (18,27,41). It has been suggested that these components could inactivate pathogens by diverse mechanisms (growth inhibition, perturbation of adhesion) and may thereby act additively and/or synergistically (18).…”

A comprehensive review on the composition and properties of buffalo milk

“…The specific activity of BM lysozyme was ten times that of CM and retained full activity after 1 min at 74°C or 30 min at 63°C (Priyadarshini and Kansal 2003). The sequence of 23 amino acid residues at the N-terminal end of buffalo lysozyme showed a 56.5% homology with CM lysozyme (Priyadarshini and Kansal 2002). Buffalo milk lysozyme inhibited four out of seven Gram-positive bacteria, but Gram-negative bacteria were resistant (Priyadarshini and Kansal 2002).…”

“…The sequence of 23 amino acid residues at the N-terminal end of buffalo lysozyme showed a 56.5% homology with CM lysozyme (Priyadarshini and Kansal 2002). Buffalo milk lysozyme inhibited four out of seven Gram-positive bacteria, but Gram-negative bacteria were resistant (Priyadarshini and Kansal 2002). Buffalo milk lysozyme was greatly influenced by the ionic strength and the concentration of metal ions in milk and in buffers (Priyadarshini and Kansal 2003).…”

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

“…Lysozyme has been well studied on its antibacterial activity against Gram-positive and Gram-negative bacteria, and it has been widely developed agricultural and industrial applications (During, Porsch, Mahn, Brinkmann, & Gieffers, 1999;Osman et al, 1995;Priyadarshini & Kansal, 2002). The catalytic mechanism of its bactericidal activity is hypothesized to reside in its muramidase activity, leading to degradation of the murein layer and reduction of the mechanical strength of the bacterial cell wall.…”