Purification, characterization, antibacterial activity and N-terminal sequencing of buffalo-milk lysozyme.
Lysozyme from buffalo milk was purified to homogeneity and its N-terminal 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 39.3% 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 buffalo-milk 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 56.5% homology with bovine milk lysozyme and 30.4% 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.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects; D002020:Buffaloes; D002850:Chromatography, Gel / Q000662:veterinary; D002851:Chromatography, High Pressure Liquid / Q000662:veterinary; D002852:Chromatography, Ion Exchange / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D005260:Female; D006863:Hydrogen-Ion Concentration; D007122:Immunoelectrophoresis / Q000662:veterinary; D008826:Microbial Sensitivity Tests; D008892:Milk / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009113:Muramidase / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D000596:Amino Acids; D000900:Anti-Bacterial Agents; D009113:Muramidase