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KMID : 0613820180280030300
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Journal of Life Science
2018 Volume.28 No. 3 p.300 ~ p.306
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Isolation of an Invertebrate-type Lysozyme from the Body Wall of Spoon Worm, Urechis unicinctus
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Oh Hye-Young
Park Nam-Gyu
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Abstract
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Lysozymes are innate immune factors that play a critical role in the defense against pathogens in various invertebrate animals including spoon worms. In this study, an invertebrate-type lysozyme was isolated from the body wall of spoon worm, Urechis unicinctus. The acidified body wall extract was partially separated using a Sep-Pak C18 cartridge. Among the fractions, the materials that were eluted with 60% methanol/0.1% trifluoroacetic acid showed the most potent antimicrobial activity against Bacillus subtilis KCTC 1021. A series of high performance liquid chromatography (HPLC) steps were then utilized to isolate a single antimicrobial absorbance peak. The molecular weight of the antimicrobial peak was approximated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which was approximately 13 to 14 kDa. The partial primary structure of this antimicrobial protein that was analyzed, using LC-MS/MS, was CTGGRPPTCEDYAK (1611.69 Da). Homology search of these fourteen residues, using the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST), revealed that the isolated protein was similar to the invertebrate-type lysozymes described in other animals. Then, the antimicrobial and lysozyme enzymatic (muramidase) activities of this protein were assessed. The isolated protein possessed antimicrobial activity and potent muramidase activity, which were comparable to those of hen egg white lysozyme. Therefore, the isolated protein was designated as Urechis unicinctus invertebrate-type lysozyme from the body wall, Uu-iLysb.
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KEYWORD
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Antimicrobial protein, lysozyme, muramidase activity, purification, Urechis unicinctus
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