Engineering and Mechanistic Insights into Inactivation-Resistant Coenzyme B12-Dependent Glycerol Dehydratase Chae Yeong Na
- 주제(키워드) Protein Engineering , Enzyme Engineering , Glycerol dehydratase , Interface Engineering
- 주제(DDC) 547
- 발행기관 아주대학교 일반대학원
- 지도교수 Tae Hyeon Yoo
- 발행년도 2026
- 학위수여년월 2026. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000036677
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Coenzyme B12-dependent glycerol dehydratase （GDHt） is a key biocatalyst for the microbial conversion of glycerol into value-added chemicals such as 3-hydroxypropionic acid （3-HP）, but its industrial application is significantly limited by rapid enzyme inactivation caused by oxygen exposure and turnover-associated cofactor degradation, which reduce catalytic longevity and increase cofactor demand. This study aimed to engineer an inactivation-resistant GDHt through rational modification of the α–β subunit interface, where the coenzyme B12- binding site is located. A fused GDHt scaffold （fGDHt） was first constructed by covalently linking the α- and β-subunits, resulting in improved resistance to oxygen-mediated inactivation while preserving catalytic activity. Based on this scaffold, computational interface engineering identified beneficial mutations that further enhanced enzyme stability. Although several variants exhibited strong resistance to oxygen-mediated inactivation, some showed reduced catalytic turnover, revealing a trade-off between structural stabilization and enzymatic function. To overcome this limitation, combinatorial engineering was performed, leading to the identification of the α-A177M/β-M158W variant as the most functionally balanced mutant, showing improved resistance to both oxygen-mediated and turnover-associated inactivation while maintaining favorable catalytic performance. Whole-cell 3-HP production experiments further demonstrated that this engineered variant significantly improved productivity under cofactor-limited conditions, suggesting reduced dependence on exogenous adenosylcobalamin. Collaborative structural analysis revealed that subtle interface mutations improved enzyme robustness through complementary mechanisms, including enhanced interface stabilization and restricted oxygen accessibility. Collectively, these findings establish a rational framework for engineering robust cofactor-dependent enzymes for industrial biocatalytic applications.
more목차
Chapter 1. General Introduction 1
1.1 Industrial applications and limitations of GDHt 2
1.2 Classification of GDHts 7
1.3 Structural and catalytic features of GDHt 9
1.4 Inactivation and reactivation of GDHt 13
1.5 Previous engineering strategies and their limitations in GDHt 18
1.6 Aim of the study 22
Chapter 2. Interface engineering of fGDHt for improved resistance to inactivation 24
2.1 Introduction 25
2.2 Materials and methods 28
2.2.1 Plasmid construction 28
2.2.2 Expression and purification 30
2.2.3 Computational design of alpha-beta interface veriants 31
2.2.4 Determination of enzymatic activity and kinetic parameters 37
2.2.5 Characterization of oxygen-mediated inactivation 37
2.2.6 Production of 3-HP using recombinant Escherichia coli strains 39
2.3 Results and Discussion 41
2.3.1 Validation of fGDHt as an engineering scaffold 41
2.3.2 Computational Design of alpha-beta interface variants 47
2.3.3 Experimental screening of designed variants 51
2.3.4 Characterization of beneficial single interface variants 56
2.3.5 Whole-Cell evaluation of selected single variants 58
2.4 Conclusion 61
Chapter 3. Combinatorial engineering of the alpha-beta interface in fGDHt 63
3.1 Introduction 64
3.2 Materials and methods 66
3.2.1 Construction of combinatorial fGDHt variants 66
3.2.2 Expression and purification 67
3.2.3 Determinization of enzymatic kinetic parameters 68
3.2.4 Enzyme-coupled assay for monitoring catalytic stability during turnover 68
3.2.5 Whole-cell 3-HP production analysis under varying cofactor conditions 69
3.3 Results and discussion 71
3.3.1 Combinatorial engineering of beneficial interface mutations 71
3.3.2 Combiatorial variants show enhanced resistance to oxygen-mediated inactivation 72
3.3.3 Engineered variants exhibit enhanced catalytic persistence during continuous turnover 76
3.3.4 Engineered GDHt variants improve whole-cell 3-HP production under cofactor-limited condition 81
3.4 Conclusion 85
Chapter 4. Structural interpretation of engineered GDHt variants 87
4.1 Introduction 88
4.2 Materials and methods 90
4.2.1 Preparation of fGDHt and engineered variants for structural analysis 90
4.2.2 Crystallization and structure determination 90
4.2.3 Structural visualization and unnel analysis 91
4.3 Results and discussion 92
4.3.1 Structural conservation of the fused GDHt scaffold 92
4.3.2 Structural basis of alpha-beta interface engineering 96
4.4 Conclusion 100
Chapter 5. Summary 101
References 105
