Listeria innocua 11262로부터 얻어지는 새로운 SGNH hydrolase의 특성규명과 amyloid의 형성 그리고 고정화
Characterization, amyloid formation, and immobilization of a novel SGNH hydrolase from Listeria innocua 11262
- 발행기관 아주대학교
- 지도교수 김두헌
- 발행년도 2012
- 학위수여년월 2012. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000012288
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
A novel oligomeric hydrolase (LI22) from Listeria innocua CLIP 11262 was identified, characterized, and immobilized for industrial application. Sequence analysis of LI22 revealed a putative catalytic triad (Ser10-Asp176-His179), and a conserved sequence motif Ser(S)10-Gly(G)77-Asn(N)79-His(H)179 with moderate identities (<30%) with other members of the SGNH-hydrolase superfamily. LI22 was able to hydrolyze p-nitrophenyl acetate, α- and β-naphthyl acetate, while the S10A mutant completely lost its activity. Structural properties of LI22 were investigated using gel filtration, circular dichroism (CD), fluorescence, molecular modeling, and gel filtration. We have shown that upon incubation in 30% TFE or 50% ethanol solution, LI22 was transformed into curly amyloid fibrils. Cross-linked enzyme aggregates of LI22 were prepared by precipitating the enzyme with ammonium sulfate and subsequent cross-linking with glu-taraldehyde. Higher thermal and chemical stability, as well as good durability after repeated use of the LI22-CLEA, highlight its potential applicability as a biocatalyst in the pharmaceutical and chemical industries.
more목차
Abstract ⅰ
List of Figures ⅱ
Ⅰ. Introduction 1
1.1. Backgrounds 1
1.2. SGNH superfamily esterase 1
1.3. Aims 2
Ⅱ. Materials and methods 3
2.1 Bacterial strains, plasmid, enzymes, and reagents 3
2.2 Cloning, expression, and purification of LI22 3
2.3 Primary sequence analysis and homology modeling 4
2.4. Chromatographic, circular dichroism and fluorescence analysis 5
2.5. Activity staining and enzyme assays 5
2.6. Thioflavin T fluorescence and electron microscopy 7
2.7. Cross-linked enzyme aggregates (CLEAs) 8
Ⅲ. Results and Discussion 10
3.1 Primary sequence analysis of LI22 10
3.2 Biochemical characterization of LI22 12
3.3 Molecular modeling and functional analysis of LI22 16
3.4 Circular dichroism (CD) analysis and fluorescence spectra 23
3.5 Amyloid formation of LI22 25
3.6 Cross-linked enzyme aggregates (CLEAs) 29
Ⅳ. Conclusions 34
Ⅴ. Appendix 35
5.1. Introduction 36
5.2. Methods 38
5.3. Results and Discussion 45
5.4. Conclusions 51
Reference 52
