α-glucans produced from sucrose by lactic acid bacteria

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Table of contents

INTRODUCTION
CHAPTER I: GH70 -TRANSGLUCOSYLASES: ATTRACTIVE TOOLS FOR THE PRODUCTION OF BIO-SOURCED TAILORED POLYSACCHARIDES, A LITERATURE REVIEW
I. α-glucans produced from sucrose by lactic acid bacteria
I.1. Structural diversity
I.2. Dextran applications
I.2.1. Pharmaceutical and medical sectors
I.2.2. Prebiotics sector
I.2.2.1. Definition and market
I.2.2.2. Dextrans and isomaltooligosaccharides as prebiotics
I.2.3. Agri-food sector
I.2.4. Analytical chemistry
I.2.5. Other applications
I.3. Dextran production processes from sucrose
II. -transglucosylases of the GH70 family: structure/function relationships and understanding of their catalytic mechanism
II.1. Generalities
II.2. Reactions catalyzed by GH70 glucansucrases
II.3. Particularity of the branching-sucrases and -4,6/-4,3 –glucanotransferases
II.3.1. Brief overview of the branching-sucrases
II.3.2. -4,6- and -4,3-glucanotransferases
II.4. Structures of GH70 family enzymes and understanding of their mechanism
II.4.1. Global structure and domain organization of GH70 enzymes
II.4.2. Catalytic core structure and reactional mechanism
II.4.2.1. Structure of the catalytic core
II.4.2.2. Catalytic mechanism
II.4.3. Focus on domain V
II.4.3.1. Structural organization of domain V
II.4.3.2. Functional role of domain V
II.4.4. Polymerization mode: processive / non-processive and product specificity determinants
II.4.4.1. Mode of polymer elongation by glucansucrases
II.4.4.2. Linkage specificity and polymer size
III. DSR-M and DSR-OK, the two templates of this study
III.1. DSR-M
III.2. DSR-OK
THESIS OBJECTIVES
CHAPTER II: INVESTIGATIONS ON THE DETERMINANTS RESPONSIBLE FOR LOW MOLAR MASS DEXTRAN FORMATION BY DSR-M DEXTRANSUCRASE
Abstract
Key-words
I. Introduction
II. Results
II.1. Design and characterization of truncated variants DSR-M1 and DSR-M2
II.2. Kinetics of polymer formation reveal that DSR-M 2 can accept different chain initiators
II.3. 3D structures of DSR-M2 and DSR-M2 E715Q in complex with sucrose or isomaltotetraose
II.4. Functional implications of domain V
III. Discussion
IV. Conclusion
V. Material and methods
V.1. Construction of DSR-M1, DSR-M2 and DSR-MV deletion mutants
V.2. Protein expression and purification
V.3. Activity assays
V.4. Enzymatic reaction and product characterization
V.5. Crystallization and Data collection
V.6. Structure determination
V.7. SAXS measurements and processing
V.8. Mutagenesis studies
VI. Acknowledgements
VII. Supplementary information
CHAPTER III : FUTILE CYCLE ENGINEERING OF THE DSR-M DEXTRANSUCRASE MODIFIES THE RESULTING POLYMER LENGTH
I. Introduction
II. Results and discussion
II.1. The crystal structure of DSR-MV in complex with an isomaltotetraose defines novel anchoring points
II.2. The W624A mutation changes the reaction rate and final length distribution.
II.3. Beyond a simple stacking platform: the W624A mutation equally influences the dynamics of the catalytic site.
II.4. Monte Carlo model of the chain elongation.
III. Conclusion
IV. Complementary work (not part of the publication)
IV.I. Further NMR analysis
IV.2. The DSR-M W624A mutant produces short oligosaccharides in good yield
IV.3. Discussion
IV. Complementary conclusion
V. Material and methods
V.1. Protein expression and purification
V.2. Activity assays
V.3. Enzymatic reaction
V.4. Kinetic analysis of acceptor reactions
V.5. Acceptor reactions on glucose with DSR-M W624A mutants
V.6. Product characterization
V.7 Crystallization and Data collection
V.8. Structure determination
V.9. Mutagenesis studies
V.10. 15N protein expression
V.11. 15NTrp protein expression
V.12. NMR analysis of DSR-M variants
V.13. Monte Carlo simulation.
VI. Supplementary information
VI.1. Protein purification procedures
VI.2. Recombinant DSR-MDV protein sequence
VI.3. Electron density map and structural data statistics
VI.4. Analysis of reaction medium after 15min reaction before and after invertase digestion.
VI.5. Monte Carlo model of the chain elongation.
VII. NMR analyses
CHAPTER IV: HIGH MOLAR MASS DEXTRAN SYNTHESIS BY DSR-OK DEXTRANSUCRASE FROM OENOCOCCUS KITAHARAE DSM 17330, ROLE OF THE DOMAIN V?
I. Introduction
II. Results & Discussion
II.1. Design and characterization of DSR-OK 1, a model of study
II.2. Monitoring of dextran synthesis by DSR-OK1
II.3. DSR-OK 1 structural insights
II.4. Functional implications of domain V
II.5. Effect of sugar binding pocket deletions
II.6. Effect of mutations targeting the aromatic residues of the sugar binding pockets
II.7. Construction of chimeric enzymes with domain V swapping
III. Conclusion
IV. Material and Methods
IV.1. Construction of DSR-OK1
IV.2. Protein expression and purification
IV.3. Activity assays
IV.4. Enzymatic reaction
IV.5. Product characterization
IV.6. SAXS measurements and processing
IV.7. Building the DSR-OK core models
IV.8. Circular dichroism analyses
IV.9. Chimera construction
IV.10. Mutagenesis study
IV.10.1. Construction of deletion mutants
IV.10.2. Site-directed mutagenesis
V. Supplementary information
CONCLUSION & PROSPECTS
DSR-M: short-chain polymerase from Leuconostoc citreum NRRL B-1299
DSR-OK: very long-chain polymerase from Oenococcus kitaharae DSM 17330
Discussion and perspectives
REFERENCES
ARTWORKS & TABLE CONTENTS
Figure content
NMR figures
Table content
Supplementary information content
Supplementary figures
Supplementary tables
ABBREVIATIONS