Protein purification and gene isolation of chlamysin, a cold‐active lysozyme‐like enzyme with antibacterial activity

Abstract

An antibacterial V V11 kDa protein designated chlamysin was isolated from viscera of the marine bivalve Chlamys islandica. Chlamysin inhibited the growth of all Grampositive and Gram-negative bacteria tested. The isolated protein was highly efficient in hydrolyzing Micrococcus luteus cells only at low pH (4.5^6.2) and at low temperature (4^35³C). No significant loss of enzyme activity was observed after 30 days storage at room temperature or after heating to 70³C for 15 min, suggesting relatively high protein structure stability. Sequenceanalyzed fragments of the protein revealed data which guided the isolation of the cDNA gene, encoding a 137 amino acid chlamysin precursor in scallops. The deduced protein contains a high portion of cysteine, serine and histidine residues and has a predicted isoelectric point below 7. The chlamysin protein was found to have sequence homology to an isopeptidase and to a recently published bivalve lysozyme.z 1999 Federation of European Biochemical Societies.

Crystal Structure of Tapes japonica Lysozyme with Substrate Analogue

“…Aliquots (10 l) were withdrawn from the reaction mixture at the appropriate incubation intervals and analyzed by HPLC using a YMC-Pack A-014 column (6 ϫ 300 mm) with an acetonitrile/water (80:20) eluent at a flow rate of 1 ml/min. The elution of PNP-(NAG) 5 was detected by absorbance at 300 nm. The concentration of PNP-(NAG) 5 was determined by calculating the area of an eluted peak.…”

“…The elution of PNP-(NAG) 5 was detected by absorbance at 300 nm. The concentration of PNP-(NAG) 5 was determined by calculating the area of an eluted peak. …”

“…Among all lysozymes and members of the chitinase family, the glutamate residue is conserved as a catalytic residue (31). To confirm whether Glu-18 is a catalytic residue, we prepared mutant TJLs in which Glu-18 was mutated to Ala, and we examined the activities of the E18A mutants against PNP-(NAG) 5 at 40°C. We found that the E18A mutants exhibited no lysozyme activity (Table 4).…”

“…This was the first lysozyme to be identified as an i-type lysozyme based on determination of the complete amino acid sequence at the protein level. Furthermore, several lysozymes have been identified, including those from the following organisms: two coastal bivalves belonging to the genus Mytilus (3), four deep-sea bivalves belonging to the genera Bathymodiolus and Calyptogena (3), a bivalve belonging to Chamys islandica (4,5), and a starfish belonging to Asterias rubens (6). In 2002, alignment and phylogenic analyses using six bivalve lysozymes suggested that i-type lysozymes form a monophyletic family (7).…”

Molecular characterisation, evolution and expression analysis of g-type lysozymes in Ciona intestinalis

“…It catalyzes the hydrolysis of 1, 4-beta-linkages between N-acetyl-dglucosamine (NAG) and N-acetylmuramic acid (NAM) in peptidoglycan heteropolymers of prokaryotic cell walls, leading to the breakdown of bacterial cells (Smirnow and Wislowska, 2001;Nilsen et al, 1999). As a result, lysozyme acts directly on Grampositive bacteria, causing the lysis of their outermost peptidoglycan layer.…”