초록 ▼

o 연구결과
수집된 토양시료의 전처리 및 방선균 선태배지 내에 항생제 첨가로 채취된 13개의 토양시료로 부터 희귀방선균들을 분리하였다. 분리된 2500여 균주를 배양한 다음 elastase에 대한 저해활성 측정을 실시하여 SMFll을 채택하여 동정 결과 Streptomyces lavendulae SMFll으로 명명하였다. SMFll을 배양한 후 2개의 물질을 분리하였고 (SMFEI01, SMFEI02), 분리된 활성분획은 TLC 판에서 단일 spot로 발색되어 순수 분리되었음을 확인하였다.
분리된 SMFEI01과 SMFE

o 연구결과
수집된 토양시료의 전처리 및 방선균 선태배지 내에 항생제 첨가로 채취된 13개의 토양시료로 부터 희귀방선균들을 분리하였다. 분리된 2500여 균주를 배양한 다음 elastase에 대한 저해활성 측정을 실시하여 SMFll을 채택하여 동정 결과 Streptomyces lavendulae SMFll으로 명명하였다. SMFll을 배양한 후 2개의 물질을 분리하였고 (SMFEI01, SMFEI02), 분리된 활성분획은 TLC 판에서 단일 spot로 발색되어 순수 분리되었음을 확인하였다.
분리된 SMFEI01과 SMFEI02의 이화학적 특성을 살펴보았고，이들의 구조분석을 수행하였다. 적외선 흡수 분석법, 질량분석법，아미노산 분석법, NMR 분석을 종합하여 SMFEI01과 SMFEI02의 구조를 규명 하였다. 결과적으로 SMFEI01은 2-(methylamino)-isobutanoly-3-amino-4(prydin-3yl)-butanoyl- leucyl-leucine의 구조로 신물질의 구조로 밝혀졌고，SMFEI02는 cyclo(_L-Phe-_L -Pro)로 확정하였다. 대량화를 위해 elastase 저해물질 생성의 최적화를 실시하였다. 이 중 탄소원，질소원의 영향을 검토하여 대량생산을 위한 배지를 확정하였다.
또한 신약으로서의 가능성을 고찰하기 위해 cysteine과 serine protease에 속하는 수 종의 protease에 대한 저해활성을 조사하고 Ki 값 (10^-3 M to 10^-6 M) 을 결정하였고， 기존의 저해물질과 비교하였다.

Abstract ▼

Soil samples were collected from various sites in Korea and a number of typical and rare actinomycetes were isolated from the soil samples. Dry heat treatment at l00℃ for 1 hour was proved to be effective for the selective isolation of general actinomycetes. Wet heat at 70℃ for 15 minutes and phenol

Soil samples were collected from various sites in Korea and a number of typical and rare actinomycetes were isolated from the soil samples. Dry heat treatment at l00℃ for 1 hour was proved to be effective for the selective isolation of general actinomycetes. Wet heat at 70℃ for 15 minutes and phenol treatment was effective for the isolation of Micromonospora Actinoplanes, Microbispora and Nocardia were also isolated by various selective isolation methods.
Consequently a strain (SMFll) among over 3,800 isolants from the selection of strains producing low molecular weight elastase inhibitor was selected, which isolate was identified with numerical analysis of the morphological and chemotaxonomical characters to be a member of Streptomyces lavendulae.
The inhibitors in culture filtrate of Streptomyces lavendulae SMFll were purified by ultrafiltration, adsorption on XAD-7 resin, n-butanol extraction, Dowex-l (Cl^-1) ion exchange chromatography, sequential gel permeation chromatography using Biogel P-6 or Biogel P-2 and Sephadex G-15, and HPLC. Two kind of inhibitors, SMFEI01 and SMFEI02, were obtained from the purification steps and their chemical structures were determined by physicochemical properties.
The molecular weight of SMFEI01 was determined as 505 Da by F AB mass spectrometry. UV spectrum showed maximum absorbance value at 230 nm. The IR spectra showed typical peptide absorption bands around 1660 and 1550 cm^-1 and amide band at 3291 cm^-I. From the amino acid analysis, leucine and unknown compounds were combined as 2:1 or 2 molar stoichiometry. Therefore, SMFEI01 is thought to be composed of leucine and unknown amino acid(s). Molecular elemental formula of SMFEI01 was determined to C₂₆H₄₃O₅N₅ by high resolution mass analysis, and the molecular structure of SMFEI01 was supposed 2-(methylamino)-isobutanoyl-3-amino-4(prydin-3yl)-butanoyl-leucyl-Ieucine.
The UV spectrum of inhibitor SMFEI02 dissolved in methanol showed maximum absorption peaks at 221.4 nm and 258.0 nm, which indicated the presence of phenyl group. The result of amino acid analysis of inhibitor SMFEI02 indicated the presence of proline and phenylalanine in molar ratio 1:1. The spectrum of inhibitor SMFEI02 obtained from FAB-MS suggests that inhibitor SMFEI02 can be a cyclic peptide. The molecular formula of inhibitor SMFEI02 was established as C₁₄H₁₆N₂O₂ from the results of the NMR spectral analysis, and the structure of inhibitor SMFEI02 was established as cyclo(phenylalanylprolyI). By comparison of optical rotation and chemical shift in ¹H NMR spectrum with those of stereoi somers , inhibitor SMFEI02 was identified to be cyclo(_L-Phe-_L-Pro) form.
The production of EI was related to mycelial growth, of which production was stimulated by glucose(1-1.25%) in the medium containing casein-peptone. The inhibitor SMFEI02 showed inhibitory activities against cysteine protease (cathepsin B and papain) and serine protease (chymotrypsin, trypsin and elastase). The inhibition by the inhibitor SMFEI02 exhibited inhibition constants (Ki) from 10^-3 M to 10^-6 M range and showed competitive type of cathepsin B, chymotrypsin and elastase and noncompetitive type of papain and trypsin.

목차 Contents

• 표지 ... 1
• 연구개발 최종보고서 ... 3
• 제 출 문 ... 4
• 요 약 문 ... 5
• 영문요약문 ... 7
• 목 차 ... 9
• 제 1 장 서론 ... 10
• 제 1 절 Elastase와 관련된 질병과 그 치료제의 개발에 관한 고찰 ... 10
• 제 2 절 연구개발의 필요성과 중요성 ... 10
• 제 2 장 국내·외 기술개발 현황 ... 12
• 제 1 절 국내 ... 12
• 제 2 절 국외 ... 12
• 제 3 장 연구개발수행 내용 및 결과 ... 14
• 제 1 절 연구수행 방법 ... 14
• 제 2 절 연구 수행내용 및 결과 ... 15
• 제 4 장 연구개발목표 달성도 및 대외기여도 ... 89
• 제 1 절 1차년도 ... 89
• 제 5 장 연구개발결과의 활용성과 및 계획 ... 91
• 제 6 장 기타 중요변경사항 ... 92
• 제 7 장 참고문헌 ... 92
• 최종보고서 요약 (초록) ... 94
• 끝페이지 ... 94