Novel type hydrolase인 Bacillus licheniformis로 부터 유래한 BL28의 Characterization, immobilizaion, Crystallization
Characterization, Immobilization and Crystallization of a Novel Type of Hydrolase (BL28) from Bacillus licheniformis
- 주제(키워드) "Hydrolase" , "BL28" , "Stereoselectivity" , "CLEA"
- 발행기관 아주대학교
- 지도교수 김두헌
- 발행년도 2014
- 학위수여년월 2014. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000016467
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
A novel type of hydrolase (BL28) from Bacillus licheniformis was identified, expressed in Escherichia coli, characterized, and immobilized for industrial applications. Biochemical characteristics of BL28 were investigated by performing SDS-PAGE, mass spectrometry, enzyme assays, CD spectroscopy, fluorescence, and in silico analysis. Using fluorescence microscopy, we investigated the effects of several chemical compounds on aggregate formation of BL28. Furthermore, cross-linked enzyme aggregates (CLEAs) of BL28 were prepared and scanning electron microscopy images were obtained. These CLEA-BL28 aggregates exhibited improved catalytic efficiencies and stabilities compared to free BL28 against harsh conditions of thermal or chemical stress. The characteristics of the CLEA-BL28 aggregates highlight their great potential in pharmaceutical and chemical industries.
more목차
Abstract ⅰ
List of Figure ⅱ
Ⅰ. Introduction 1
Ⅱ. Materials and methods 4
2.1 Bacterial strains, enzymes, and reagents 4
2.2 Cloning, expression, and purification of BL28 4
2.3 Primary sequence analysis and homology modeling 6
2.4. Enzyme assays 7
2.5. Circular dichroism analysis and fluorescence spectra 8
2.6. Substrate specificities of BL28 8
2.7. Aggregates formation of BL28 9
2.8. Cross-linked enzyme aggregates (CLEAs) 10
2.9 Crystallization of BL28 11
Ⅲ. Results and Discussion 12
3.1 Bioinformatic analysis of BL28 12
3.2 Purification and mass analysis of BL28 16
3.3 Urea-induced unfolding analysis of BL28 18
3.4 Biochemical characterization of BL28 20
3.5 Regio- and stereo- specificity of BL28 20
3.6 Aggregates formation of BL28 26
3.7 Cross-linked enzyme aggregates (CLEAs) 28
3.8 Crystallization of BL28 31
Ⅳ. Conclusion 33
Reference 67
Appendix - Characterization, Immobilization, and Gelation of a Novel Hydrolase from Listeria monocytogenes
Abstract-34
Ⅰ. Introduction 36
Ⅱ. Materials and methods 38
2.1 Bacterial strains, enzymes, and reagents 38
2.2 Cloning, expression, and purification of EstLM 38
2.3 Primary sequence analysis and homology modeling 39
2.4. Enzyme assays 40
2.5. Circular dichroism analysis and fluorescence analysis 41
2.6. Substrate specificities of EstLM 41
2.7. Cross-linked enzyme aggregates (CLEAs) 42
2.8. Hydrogel formation of EstLM 43
Ⅲ. Results and Discussion 45
3.1 Bioinformatic analysis of EstLM 45
3.2 Biochemical analysis of EstLM 48
3.3 Chemical stabilities of EstLM 53
3.4 Regio- and stereo- specificity of EstLM 56
3.5 Cross-linked enzyme aggregates (CLEAs) 58
3.6 Hydrogel formation of EstLM 62
3.7 Structural analysis of EstLM 65
Ⅳ. Conclusions 66
Reference 67
