Structural and Functianal Characterizations of SGNH-family Esterases from Sinorhizobium meliloti
- 주제(키워드) SGNH-superfamily , esterase , GDSL , p-nitrophenyl acetate
- 발행기관 아주대학교
- 지도교수 김두헌
- 발행년도 2010
- 학위수여년월 2010. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000010412
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Novel oligomeric SGNH-arylesterases from Sinorhizobium mellioti, SM23, SM24A and SM24B, were characterized using biochemical and biophysical methods. A sequence comparison of Sm23 with other SGNH members confirmed the presence of the catalytic triad (Ser10, Asp187, and His190) and oxyanion holes (Ser10-Gly50-Asn90). SM24A has a catalytic triad (Ser13, Asp187, and His190), but it does not have a consensus blockⅡ. SM24B as well as Sm23 have four conserved sequence blocks containing the catalytic triad (Ser15, Asp192, and His195), and oxyanion hole residues (Ser15-Gly57-Asn97). All these Enzymes were able to hydrolyze p-nitrophenyl acetate, α-, and β-naphthyl acetate, while the S10A of Sm23 mutant completely losts its activity. Structural properties of Sm23 were investigated using circular dichroism (CD), fluoroscence, dynamic light scattering (DLS), chemical cross-linking, electron microscopy (EM), and time of flight (TOF) mass spectrometry. Spherical or globular aggregates were observed with 1-butyl-3-methylimidazolium tetrafluoroborate, while amorphous aggregates were formed with 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide.
more목차
Abstract ⅰ
List of Tables ⅱ
List of Figures ⅲ
Ⅰ. Introduction 1
1.1. Backgrounds 1
1.2. SGNH superfamily esterase 1
1.3. Esterases from Sinorhizobium meliloti 2
1.4. Aims 2
Ⅱ. Materials and methods 5
2.1 Bacterial strains, plasmid, enzymes, and reagents 5
2.2 Cloning analysis of SM23 5
2.3 Expression and purification of SM23 5
2.4. Mass Spectrometry, chemical crosslinking, and chromatographic analysis 6
2.5. Circular dichroism (CD) and fluorescence analysis 6
2.6. Enzyme assays 7
2.7. Activity staining and enantioselectivity 8
2.8. TEM analysis 8
Ⅲ. Results and Discussion 10
3.1 Sequence analysis of esterases 10
3.1.1 Sequence analysis of SM23 10
3.1.2. Sequence analysis of SM24A and SM24B 10
3.2 Structural characterization of SM23 12
3.3 Circular dichroism (CD) analysis and fluorescence spectra 18
3.4 Enzyme assay 21
3.5 Activity staining and enantioselectivity 29
3.6 Self assembly of SM23 with solvent conditions 33
Ⅳ. Conclusions 35
Ⅴ. Appendix 36
5.1. Introduction 37
5.2. Materials and methods 38
5.3. Results and Discussion 41
5.4. Conclusions 45
Reference 46
