원문정보
Purification and Characterization of an Indican-hydrolyzing β-glucosidase from Agrobacterium tumefaciens
초록
영어
Indican (indoxyl-β-D-glucoside) is a colorless natural compound and can be used as a precursor for the production of indigo. This production step only require an enzyme, β-glucosidase, that readily screened from microbial resource by using selective media supplemented with indican as a sole carbon source. Agrobacterium tumefaciens was well grown in this media and thus presumed to produce a related enzyme. The corresponding gene, encoding a protein with a calculated molecular mass of 51 kDa, was cloned and overexpressed as MBP fusion proteins. The purified enzyme was determined to be a dimer and showed the maximum activity for indican at pH 7.0 and 40℃. The kinetic parameters for indican, Km and Vmax, were determined to be 1.4 mM and 373.8 μM/min/mg, respectively. The conversion yield of indican into indigo using this enzyme was about 1.7-1.8 folds higher than that of previously isolated enzyme from Sinorhizobium meliloti. Additionally, this enzyme was able to hydrolyze various β-1,4 glycoside substrates.
목차
1. 서론
2. 재료 및 방법
2.1. 사용 시약, 균주 및 배양 조건
2.2. 유전자의 증폭, cloning 및 아미노산 서열비교
2.3. 단백질 분리 및 활성 측정
2.4. Oligomeric structure와 zymogram 분석
2.5. 온도와 pH에 따른 효소활성 측정
2.6. Kinetic parameter와 기질 특이성 측정
2.7. 인디칸의 인디고 전환율 비교
3. 결과 및 고찰
3.1. 인디칸 분해 활성을 갖는 β-glucosidase의 발굴 및 유전자 분석
3.2. 대장균으로부터 재조합 β-glucosidase 의 생산
3.3. β-glucosidase의 생화학적 특성 분석
3.4. 인디칸을 이용한 인디고 전환률
4. 결론
감사
References