Purification and characterization of antimicrobial peptides from the sandworm Perinereis nuntia savigny

Abstract

Sandworm, Perinereis nuntia Savigny, (class Polychaeta) is widely used as live food in aquatic animal industry due to its nutritional values which are essential for maturation in many aquaculture species. Although farmed and wild sandworms are different in both diet and habitat, their nutritional values have never been comparatively reported. The proximate composition of farmed sandworm fed with a commercial shrimp diet and wild sandworm caught from shore line of Chonburi, Thailand, were investigated. Protein and fat contents in farmed and wild sandworms were not significantly different. Moisture content of wild animal was significantly higher than that of farmed animal while the ash content was in the contrary. The fatty acid profile of major saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) in both groups of sandworm were C16:0, C18:1 and C18:2 respectively. SFA and MUFA contents of wild sandworm were significantly higher, while its PUFA content was significantly lower than that of farmed sandworm. Amino acid profile of both groups of sandworm was not different and the major amino acids were glutamic acid and aspartic acid in both groups of worm. Mineral contents in farmed and wild sandworms were significantly different except potassium. The cholesterol and vitamin A contents in wild sandworm were higher than those of farmed sandworm while vitamin C, D3 and E contents were in contrary and vitamin B1was in the same order. Sandworms live in sandy beach which rich in microorganism and their abundance in this type of environment suggest that they have developed efficient immunodefense. They are able to protect themselves against invading pathogens due to efficient innate defense mechanism, one is antimicrobial peptides (AMPs). The AMPs were isolated and purified from sandworm P. nuntia tissue and coelomic fluid. The general protocol of peptides purification method including acid extraction, purification step by gel-filtration chromatography, solid phase extraction and reverse phase HPLC. Moreover, sandworms were challenged by Vibio harveyi before extraction and found the highest antimicrobial activity after 24 hr challenged. Each purified step was checked for antimicrobial activity by paper disc method against gram positive bacteria Bacillus subtilis. Three AMPs; AMP-F5(P), AMP-F15(P) and AMP-F40(P) were isolated from sandworm and they were mixed peptides. Molecular weight of major peptided of AMP-F5(P), AMP-F15(P) and AMP-F40(P) were 2461.797, 8564.595 and 2459.779 Daltons, respectively. Only AMP-F5(P) showed activity against B. subtilis after purification by the last step. Furthermore, 0.26 g of AMP-F5(P) was found from 1 g of wet weight of fresh sandworm.