Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

Abstract

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 (µ = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (µ = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (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.

Antibacterial activity of lysozyme in the desert locust, Schistocerca gregaria (Orthoptera: Acrididae)

“…Lysozymes are also recorded in the pericardial cells of M. sexta (Russell & Dunn, 1990), mid gut of several Diptera, such as, Anastrepha fraterculus (Lemos & Terra, 1991), Drosophila melanogaster (Kylsten et al, 1992;Daffre et al, 1994) and Musca domestica (Lemos & Terra, 1991;Cançado et al, 2008), salivary glands of Anopheles darlingi (Moreira-Ferro et al, 1998), D. melanogaster (Kylsten et al, 1992), Helicoverpa zea (Liu et al, 2004), Reticulitermes speratus (Fujita et al, 2002) and eggs of Anthonomus grandis (Ourth & Jones, 1980) and Ceratitis capitata (Fernandez-Sousa et al, 1977).…”

Molecular cloning, recombinant protein expression, tissue distribution and functional analysis of a new c-type lysozyme from Lezhi black goat rumen

“…Previous studies have revealed that the lysozyme has a new digestive function (Canc¸ado et al 2008;Xue et al 2010) and the presence of LZRLyz in the goat rumen further support that LZRLyz has potential digestive function. Interestingly, as mentioned above, the LZRLyz protein has a lower predicted pI than that of the cow non-stomach lysozyme and contains three arginine residues, which may help to maintain the proteins' stability in acidic and pepsin-rich stomach environment and further support the LZRLyz function in the digestive system (Irwin 2004;Jiang et al 2010;Zhang et al 2013).…”

Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels

“…Methylumbelliferyl-based substrates have been used for a wide range of enzyme activities, but the dependence of the fluorescence on ionization of the group at high pHs has led to obligatory discontinuous assays for the majority of cases (Yang and Hamaguchi, 1980; Hoppe, 1983). Particularly, most insect glycosidases have optimal activities in acidic pHs (Gontijo et al, 1998; Terra and Ferreira, 2005; Jacobson et al, 2007; Cançado et al, 2008; Moraes et al, 2012; Tamaki et al, 2014; Moreira et al, 2015). In this way, the development of substrates with groups that fluoresce in acidic pHs would be greatly beneficial for the study of those enzymes.…”