Isolation, Characterization, Kinetics, and Enzymatic and Nonenzymatic Microbicidal Activities of a Novel c-Type Lysozyme from Plasma of Schistocerca gregaria (Orthoptera: Acrididae)

Abstract : A protein, designated as Sgl, showing a muramidase lytic activity to the cell wall of the Gram-positive bacterium Micrococcus lysodeikticus was isolated for the first time from plasma of Escherichia coli-immunized fifth instar Schistocerca gregaria. The isolated Sgl was detected as a single protein band, on both native-and SDS-PAGE, has a molecular weight of similar to 15.7 kDa and an isoelectric point (pI) of ca 9.3 and its antiserum has specifically recognized its isolated form. Fifty-nine percentage of Sgl lytic activity was recovered in the isolated fractions and yielded ca 126-fold increase in specific activity than that of the crude. The partial N-terminal amino acid sequence of the Sgl has 55 and 40% maximum identity with Bombyx mori and Gallus gallus c-type lysozymes, respectively. The antibacterial activity against the Gram-positive and the Gram-negative bacteria were comparatively stronger than that of the hen egg white lysozyme (HEWL). The detected Sgl poration to the inner membrane that reach a maximum ability after 3 h was suggested to operate as a nonenzymatic mechanism for Gram-negative bacterial cell lysis, as tested in a permease-deficient E. coli, ML 35 strain. Sgl showed a maximal muramidase activity at pH 6.2, 30-50 degrees C, and 0.05M Ca2+ or Mg2+; and has a K-m of 0.5 mu g/ml and a V-max of 0.518 with M. lysodeikticus as a substrate. The Sgl displayed a chitinase activity against chitin with a K-m of 0.93 mg/ml and a V-max of 1.63.