Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae.
cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (mu = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (mu = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotriozide (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.
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Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brasil.
Chimoy Effio
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Terra
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Marana
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D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D019976:Cloning, Organism; D018076:DNA, Complementary / Q000235:genetics; D004064:Digestive System / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006793:Houseflies / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007814:Larva / Q000201:enzymology; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics / Q000302:isolation & purification; D010843:Pichia; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity
D018076:DNA, Complementary; D009113:Muramidase