원문정보
Gene Cloning and Enzymatic Properties of Thermostable Laccase from Thermus thermophilus HJ6
초록
영어
The gene encoding Thermus thermophilus HJ6 laccase (Tt-laccase) was cloned, sequenced, and comprised of 1,389 nucleotides encoding a protein (462 amino acids) with a predicted molecular mass of 51,049 Da. The deduced amino acid sequence of Tt-laccase showed 99.7% and 44.3% identities to the Thermus thermophilus HB27 laccase and Synechococcus sp. RS9917 laccase, respectively. Tt-laccase gene was expressed as a fusion protein with six histidine residues in E. coli Rosetta-gami (DE3) cells, and the recombinant protein was purified to homogeneity. UV-Vis spectrum analysis revealed that the enzyme has copper atoms, a type I Cu(II) and a type III binuclear Cu(II). The optimum pH for the oxidation of guaiacol was 5.0 and the optimum temperature was 90℃ The half-life of heat inactivation was about 120 min at 90℃ The enzyme reaction was inhibited by sodium azide, L-cystein, EDTA, dithiothreitol, tropolone, and kojic acid. The enzyme oxidized various known laccase substrates, its lowest Km value being for 4-hydroxyindole, highest kcat value for syringaldazine, and highest kcat/Km for guaiacol.
목차
1. 서론
2. 재료 및 방법
2.1. 유전자 클로닝 및 발현 vector 구축
2.2. 효소 생산 및 정제
2.3. UV-visible 흡수 스펙트럼 측정
2.4. 효소활성측정
3. 결과 및 고찰
3.1. Tt-laccase 유전자의 클로닝 및 염기서열분석
3.2. E. coli로부터 재조합 Tt-laccase의 생산
3.3. Tt-laccase의 UV-visible 흡수 스펙트럼
3.4. Tt-laccase의 생화학적 특징
3.5. Tt-laccase의 활성에 대한 금속이온 및 저해제의 효과
4. 결론
감사
References