돌멍게(Pyura vittata)로부터 항균성 펩타이드의 정제 및 특성

Abstract

This paper investigates and describes the antibacterial and antioxidant activities of Pyura vittata’s whole body extract and its biochemical properties. The antibacterial activity assay showed that the extract exhibited antimicrobial activity against a wide range of microbial strains including gram-positive bacteria, gram-negative bacteria, fish pathogens, and a fungus. The antibacterial activity of the extract was affected by proteolytic enzyme (trypsin) treatment. This result alludes that the extract’s antibacterial activity is due to proteinaceus materials contained in the extract. The antioxidant activity of the extract, which is calculated compared to the antioxidant activity of L-ascorbic acid, tend to increase in a concentration-dependent manner. Having confirmed the antimicrobial activity of the whole body extract, series of purification steps were taken using the extract of the sea squirt, during which the antibacterial activity of each fraction was investigated using Bacillus subtilis KCTC 1022. The purified antimicrobial peptides (AMPs) were named PvAMPs, which means AMPs isolated from Pyura vittata. A total of 8 antimicrobial peptides (PvAMP1-8) were purified. The molecular weights of PvAMP1-3 were analyzed, but the amino acid sequences could not be analyzed. Among them, PvAMP3 was treated with trypsin yielding multiple fragments on reversed phase HPLC, among which was used to obtain a partial amino acid sequence. Currently, research is underway to find out the complete amino acid sequence of these substances. The amino acid sequence of PvAMP4 was analyzed giving 23 amino acid residues after determining its molecular weight. The revealed amino acid sequence showed high identity with 40S ribosomal protein S30 (RPS30). cDNA cloning was conducted to reveal the entire sequence of PvAMP4, and found the complete sequence of PvAMP isotype, which shows that PvAMP4 precursor protein is consisted of ubiquitin-like protein and ribosomal protein S30. The molecular weight of PvAMP5 was obtained and the amino acid sequence was analyzed. The analyzed sequence comprising 12 amino acids was identical to that of the small regions from the retinol binding protein (RBP) of sea urchin. In addition, the molecular weights and amino acid sequences of PvAMP6-8 were analyzed, and the analyzed sequences had high homology with sea urchin sperm activating peptide (SAP). In this study, for the first time, 8 antimicrobial peptides were purified from sea squirts (Pyura vittata) and their properties were analyzed.|본 논문은 돌멍게(Pyura vittata)의 껍질을 제외한 전체 조직을 사용해 항균 및 항산화 활성을 확인하였고, 그 특성에 관한 분석 내용을 기술하고 있다. 항균활성 측정 결과, 추출물은 그람 양성균, 그람 음성균, 곰팡이 균 및 어병세균에 대해 광범위한 항균 활성을 나타냈다. 이후 돌멍게 추출물을 사용해 정제 단계를 진행했으며, 각 분획에 대한 항균활성은 B. subtilis KCTC 1022를 사용하여 확인하였다. 정제된 항균 활성 펩타이드 (AMP)는 돌멍게(Pyura vittata)에서 분리된 AMP라는 의미로 PvAMP라고 명명하였으며, 총 8개의 항균활성 펩타이드(PvAMP1~8)를 정제하였다. PvAMP1~3은 분자량을 확인하였으며, 아미노산 서열 분석이 추가적으로 필요하다. 다음으로 PvAMP4는 분자량 확인 후, N-말단부터 23번쨰 아미노산 서열까지 분석하였다. 분석한 아미노산 서열은 40S ribosomal protein S30 (RPS30)과 높은 identity를 나타내었다. 이후 cDNA cloning 결과, PvAMP4의 isomer로 예측되는 물질의 전체 서열을 확인하였으며, ubiquitin-like protein과 ribosomal protein S30이 연속적으로 나타나는 protein을 precursor로 가지고 있음을 확인하였다. PvAMP5 또한 분자량 확인 후, N-말단부터 12번까지 아미노산 서열을 분석하였다. 분석한 서열은 성게의 retinol binding protein (RBP)의 일부와 서열이 일치하는 것을 확인하였다. 이외에도 PvAMP6~8의 분자량 및 아미노산 서열을 분석하였으며, 분석한 서열은 성게의 sperm activating peptide (SAP)와 높은 identity를 가지는 것을 확인하였다. 이번 연구에서 최초로 돌멍게의 AMP를 8개 정제하였으며, 또한 그 특성을 분석하였다.