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Jehanathan Nilojan*, S. D. N. K. Bathige, W. S. Thulasitha, Roopasingam Kugapreethan, Jehee Lee
Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Republic of Korea.

Lysozymes are bactericidal enzymes, ubiquitously distributed from bacteria to human. The major role of the lysozymes is to catalyze the hydrolysis of the β-1,4-glycosidic bond between 2 alternative sugar residues (N-acetylmuramic acid and N-acetylglucosamine) in peptidoglycan layer of bacterial cell wall, thereby causing bacterial cell lysis. In addition, lysozymes play an important digestive role in some animals. Lysozymes of the animal kingdom have been classified into 3 classes: chicken-type (LysC), goose-type (LysG) and invertebrate-type (LysI). LysG was first identified in egg whites of Embden goose and subsequently in other vertebrates. In this study, two isoforms of LysG were identified from black rockfish transcriptome database and designated as RfLysG1and RfLysG2. The cDNA sequences of RfLysG1 and RfLysG2, were of 1514 bp and 900 bp, respectively. Open reading frame (ORF) of RfGLys1 comprised of 567 bp encoding 188 amino acids with a molecular mass of 20.11 kDa, while ORF of RfGLys2 consisted of 600 bp encoding 199 amino acids of 22.19 kDa. Isoelectric points were 6.82 and 6.83 respectively. Both sequences possess a soluble lytic trans-glycosylase domain. No signal peptides were detected in both. Predicted three-dimensional structures share the similar structure with other LysGs. Homology studies indicated that the RfLysG1 shown highest identity (84.6%) with LysG-B of Oplegnathus fasciatus while RfLysG2 shown highest identity (74.4%) with LysG of Siniperca chuatsi. Multiple sequence alignment revealed that both LysGs are evolutionarily conserved. Transcriptional analysis of both genes shown constitutive expression with the highest level in blood under normal physiological conditions. Immune challenges: lipopolysaccharide (LPS), Streptococcus iniae and poly I:C injections significantly upregulated the expression of RfLysG1and RfLysG2 in blood and spleen tissues in a time-dependent manner. In comparison, the level of induction was high in both tissues upon LPS than other stimuli. Both Maltose-binding protein (MBP) tagged recombinant proteins exhibit potent bacteriolytic activity against several Gram-positive and -negative bacteria, where no activity was obsereved with MBP. Optimum temperatures for the recombinant RfLysG1 and RfLysG2 were 40 °C and 50 °C, respectively. Both were highly active at pH 3.0. Results from the present study suggest the vital role of RfLysG1and RfLysG2 against microbial invasion.

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