Molecular characterization and antibacterial activity of a phage-type lysozyme from the Manila clam, Ruditapes philippinarum

A phage-type lysozyme, designed as RpPLYZ, was cloned and characterized from the clam Ruditapes philippinarum. The full-length cDNA of RpPLYZ was of 699 bp with an open reading frame (ORF) of 534 bp, encoding a polypeptide of 177-amino acid with a calculated molecular mass of 19.6 kDa and an isoelectric point of 9.05. Multiple alignments and phylogenetic analysis strongly suggested that RpPLYZ was a new member of the phage-type lysozyme family. The mRNA transcript of RpPLYZ was found to be constitutively expressed in a wide range of tissues and mainly in hemocytes and mantle. The relative expression of RpPLYZ mRNA in hemocytes was significantly up-regulated at 6, 24, 48 and 72 h after Vibrio anguillarum challenge. The recombinant RpPLYZ (rRpPLYZ) showed high activity against Entherobacter cloacae and Staphyloccocus aureus, and less effective towards Entherobacter aerogenes and V. anguillarum. Moreover, the optimal pH, temperature and ionic strength for rRpPLYZ activity was determined to be 5.5, 50 °C and 5 mM, respectively. These results suggested that RpPLYZ was a member of the phage-type lysozyme family and perhaps played an important role in the immune responses against bacterial invasion.