미꾸라지(Misgurnus mizolepis) 가수분해물 유래의 β-secretase 저해 활성 펩타이드의 특성
Characterization of β-secretase inhibitory peptides purified from loach (Misgurnus mizolepis) enzymatic hydrolysate
- 주제(키워드) 도움말 β-secretase inhibitory activity , loach , Peptide , Purification , Alzheimer’s disease
- 발행기관 강릉원주대학교 일반대학원
- 지도교수 도움말 변희국
- 발행년도 2024
- 학위수여년월 2025. 2
- 학위명 석사
- 학과 및 전공 도움말 일반대학원 수산생명의학과
- 실제URI http://www.dcollection.net/handler/kangnung/000000012122
- UCI I804:42001-000000012122
- 본문언어 한국어
초록/요약 도움말
미꾸라지(Misgurnus mizolepis) 가수분해물 유래의 β-secretase 저해 활성 펩타이드의 특성. 한대성 강릉원주대학교 대학원 수산생명의학과 요약 우리나라는 초고령화 사회로 접어들면서 알츠하이머 치매로 인한 사회적인 문제가 심각하게 대두되고 있다. 본 연구에서는 미꾸라지 전어체의 효소적 가수분해물로부터 알츠하이머 유발 원인인 β-secretase를 저해하는 펩타이드를 개발하고자 하였다. 본 연구에서는 미꾸라지(Misgurnus mizolepis)전어체를 사용하여 7가지 상업적 효소(Alcalase, α-chymotrypsin, Flavourzyme, Neutrase, papain, pepsin, trypsin)를 사용해 가수분해물을 제조하고, 항알츠하이머 활성 펩타이드를 조사하였다. 미꾸라지 전어체의 일반성분 함량은 수분 77.63 ± 0.23%, 단백질 14.62 ± 0.32%, 지질 4.57 ± 0.10%, 회분 3.16 ± 0.11%로 확인되었다. 또한, 아미노산 함량 분석 결과, Glutamic acid가 15.3%로 가장 높았으며, Aspartic acid 9.7%, Lysine 8.9%, Glycine 8.6% 순으로 높은 함량을 나타내었다. 7가지 상업적 효소로 제조된 미꾸라지 가수분해물의 수율은 Alcalase 43.05 ± 0.45, α-chymotrypsin 43.17 ± 0.85, Flavourzyme 37.08 ± 0.15, Neutrase 39.41 ± 0.17%, papain 38.95 ± 0.62%, pepsin 13.03 ± 0.14%, trypsin 39.66 ± 0.61%로 측정되었다. Neutrase 가수분해물의 β-secretase 저해 활성은 IC50값으로 0.386 ± 0.065mg/ml로 나타났다. RP-HPLC울 사용해 Neutrase 가수분해물로부터 얻어진 펩타이드를 정제하여 MF5-2를 얻었으며, 이 펩타이드의 β-secretase 저해 활성은 IC50값으로 0.153 ± 0.018mg/ml로 나타났다. 정제된 펩타이드를 LC-MS/MS로 분석한 결과, 트리펩타이드와 테트라펩타이드인 Val-Gly-Leu(288.192 Da) 및 Val-Ser-Pro-Leu(415.256 Da)의 아미노산 서열과 분자량을 확인하였다. 정제된 펩타이드의 아미노산 서열을 기준으로 Val-Gly-Leu, Val-Ser-Pro-Leu, Gly-Leu, Pro-Leu 펩타이드를 합성하였다. 또한, 이들 펩타이드의 순도는 RP-HPLC를 통해 90% 이상임을 확인하였다. 합성된 펩타이드의 β-secretase 저해 활성은 IC50값으로 각 4.81 mM, 2.46 mM, 12.07 mM, 3.86 mM로 나타났다. 사람 신경모세포인 SH-SY5Y 세포에 정제된 펩타이드를 다양한 농도(10, 30, 50, 70, 100, 200ug/ml)로 처리한 결과, 세포독성이 나타나지 않음을 확인하였다. 따라서 본 연구에서 정제된 펩타이드는 알츠하이머 유발 원인인 β-secretase 효소를 효과적으로 저해하고 세포독성을 나타내지 않았기에 항알츠하이머성 소재로서 이용가능성을 확인하였다.
more초록/요약 도움말
Characterization of β-secretase inhibitory peptides purified from loach (Misgurnus mizolepis) enzymatic hydrolysate Dea Sung Han Departmant of Aquatic Life Medicine Graduate School Gangneung-Wonju National University Abstract The elderly population in Korea is currently growing at nearly twice the annual rate of other countries, resulting in a significant increase in age-related issues, particularly Alzheimer’s dementia. To address this, the study aimed to develop a peptide that inhibits β-secretase, a key enzyme responsible for Alzheimer’s, using enzymatic hydrolysates derived from the whole body of loach (Misgurnus mizolepis). In this study, enzymatic hydrolysates were prepared from the whole body of Misgurnus mizolepis using seven commercial enzymes: Alcalase, α-chymotrypsin, Flavourzyme, Neutrase, papain, pepsin, and trypsin. The proximate composition of Misgurnus mizolepis was determined to be 77.63 ± 0.23% moisture, 14.62 ± 0.32% protein, 4.57 ± 0.10% lipid, and 3.16 ± 0.11% ash. The amino acid profile revealed Glutamic acid as the most abundant (15.3%), followed by Aspartic acid (9.7%), Lysine (8.9%), and Glycine (8.6%). The enzymatic hydrolysate yields for the seven enzymes were as follows: Alcalase 43.05 ± 0.45%, α-chymotrypsin 43.17 ± 0.85%, Flavourzyme 37.08 ± 0.15%, Neutrase 39.41 ± 0.17%, papain 38.95 ± 0.62%, pepsin 13.03 ± 0.14%, and trypsin 39.66 ± 0.61%. Among these, the Neutrase hydrolysate demonstrated β-secretase inhibitory activity with an IC50 value of 0.386 ± 0.065 mg/ml. The peptides obtained from Neutrase hydrolysate was further purified using RP-HPLC to isolate the MF5-2 peptide, which exhibited a β-secretase inhibitory activity with an IC50 value of 0.153 ± 0.018 mg/ml. The purified peptide was analyzed by LC-MS/MS, identifying its amino acid sequences and molecular weights as Val-Gly-Leu (288.192 Da) and Val-Ser-Pro-Leu (415.256 Da). Based on the purified peptide sequences, synthetic peptides were prepared: Val-Gly-Leu, Val-Ser-Pro-Leu, Gly-Leu, and Pro-Leu. The β-secretase inhibitory activities of these synthetic peptides were measured, with IC50 values of 4.81 mM, 2.46 mM, 12.07 mM, and 3.86 mM, respectively. Additionally, it was confirmed that the purified peptide exhibited no cytotoxicity at various concentrations (10, 30, 50, 70, 100, and 200 µg/ml) when tested on SH-SY5Y human neuroblastoma cells. Therefore, the peptides purified in this study are considered promising candidates for Alzheimer's treatment due to their effective inhibition of β-secretase, a key enzyme implicated in the disease.
more목차 도움말
1. 서론 ···········································································································1
2. 재료 및 방법 ···························································································6
2.1. 재료 ···································································································6
2.2. 원료 전처리 ·····················································································6
2.3. 일반성분 분석 ·················································································7
2.4. 아미노산 조성 분석 ·······································································7
2.5. 효소적 가수분해물 제조 ·······························································8
2.6. 효소적 가수분해물의 분자량 분포 ·············································9
2.7. β-secretase 저해 활성 측정 ·····················································10
2.8. HPLC를 사용한 펩타이드의 분리정제 ·····································11
2.9. 정제된 펩타이드의 아미노산 서열 분석 ·································13
2.10. 펩타이드 합성 ·············································································13
2.11. 세포 배양 ·····················································································14
2.12. MTT assay ··················································································14
3. 결과 및 고찰 ·························································································15
3.1. 일반성분 ·······················································································15
3.2. 아미노산 조성 ···············································································17
3.3. 효소적 가수분해물의 수율 ·························································20
3.4. 효소적 가수분해물의 분자량 분포 ···········································22
3.5. 효소적 가수분해물의 β-secretase 저해 활성 ·······················25
3.6. 정제된 펩타이드의 β-secretase 저해 활성 ···························28
3.7. 정제된 펩타이드의 아미노산 서열 ···········································32
3.8. 합성 펩타이드의 β-secretase 저해 활성 ·······························36
3.9. SH-SY5Y 세포의 생존율 ·····························································39
4. 결론 ·········································································································41
5. Abstract ···································································································42
6. 참고 문헌 ·······························································································44
