Glycoside hydrolases and their sequence based classification

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Table of contents

I. Introduction
I.1 The marine ecosystem
I.1.1 Heterotrophic bacteria as key player in marine carbon cycle
I.1.2 Polysaccharides are an important part of the marine DOM and POM pools The « Sweet Ocean »
I.1.3 Bacterial enzymatic decomposition of marine organic matter
I.1.4 Why enzymatic degradation of gel forming marine galactans impacts the carbon cycle 17 The ocean is a « Sweet Jelly »
I.2 Marine polysaccharides
I.2.1 The cell walls of marine macrophytes
I.2.2 The skeleton component of algal cell walls
I.2.3 The matrix component of algal cell walls
I.2.4 The green algae
I.2.5 The brown algae
I.2.6 The cell wall matrix of marine red algae: agars and carrageenans
I.2.7 Agarose 3D structure
I.3 The marine heterotrophic bacteria
I.3.1 Marine bacteroidetes
I.3.2 Zobellia galactanivorans
I.4 The glycoside hydrolases
I.4.1 Enzymatic transformation of HMW compounds
I.4.2 Glycoside hydrolases and their sequence based classification
I.4.3 The catalytic mechanism of glycoside hydrolases
I.4.4 Mode of action in glycoside hydrolases
I.4.5 Subsites in glycoside hydrolases
I.4.6 Agarolytic bacteria and glycoside hydrolases
I.4.7 Family classification of agarases
I.4.8 The glycoside hydrolase family GH
I.4.9 The β-agarases from Z. galactanivorans
I.5 The “knowledge gap” of marine polysaccharides degrading enzymes
I.6 The aim of the thesis: finding new glycoside hydrolases by analysing the agarolytic system of Zobellia galactanivorans Structural and Functional Organisation of the Agarolytic Enzyme System of the Marine Flavobacterium Zobellia galactanivorans
II. Results
II.1 Medium throughput cloning and expression strategy
II.2 The targets for further characterization: AgaD, PorA and PorB
III. The Structural and Biochemical Characterization of the new -agarase AgaD
III.1 Introduction for manuscript1: Protein crystallization of AgaD
III.2 Manuscript 
III.2.1 Abstract
III.2.2 Introduction
III.2.3 Material and Methods
III.2.4 Results and discussion
III.2.5 Conclusion
III.2.6 Acknowledgments
III.2.7 References
III.3 The crystal structure of AgaD
III.4 Biochemical characterization of AgaD
III.4.1 Catalytic behaviour of AgaD
III.4.2 AgaD is an endo β-agarase cleaving the β-1,4 linkages in agarose
III.4.3 Agarase specificities are different on natural substrates extracted from the agarophytes Gelidium, and Porphyra
III.4.4 PACE and HPLC analysis of porphyran degradation by AgaA, AgaB and AgaD
III.5 Conclusion: The new agarase AgaD together with AgaA,B as part of the agarolytic
IV. The first -porphyranases PorA and PorB
IV.1 Crystallisation of PorA
IV.1.1 3D structure solution of PorA using a gold derivative
IV.1.2 Crystallization of PorB
IV.1.3 The crystal structures of PorA and PorB
IV.2 The discovery of the β-porphyranase activity
IV.3 Introduction for manuscript2: Porphyranases and agarases constitute the first example of a nutrition derived CAZyme update into human gut bacteria
IV.4 Manuscript 
IV.4.1 Abstract
IV.4.2 Discovering a new enzyme activity
IV.4.3 Structural determinants of porphyran active enzymes
IV.4.4 β-Porphyranases are abundant in marine bacteria
IV.4.5 Horizontal gene transfer from marine to human gut bacteria
IV.4.6 Discussion
IV.4.7 Methods summary
IV.4.8 Methods
IV.4.9 References
IV.4.10 Supplementary material
IV.5 Introduction for manuscript3: Production of porphyran oligosaccharides with porphyranase
IV.6 Manuscript 
IV.6.1 Abstract
IV.6.2 Introduction
IV.6.3 Material and Methods
IV.6.4 Results and discussion
IV.6.5 Conclusion
IV.6.6 References
V. Material and Methods
V.1 Expression and purification of PorA and PorB
V.2 DNA techniques and plasmid construction
V.3 The medium throughput cloning strategy
V.4 Screening for crystallization conditions
V.5 Kinetic studies
V.6 Sequences and phylogeny
V.7 Fluorophore-assisted carbohydrate electrophoresis analysis (PAGE)
V.8 Enzyme activity essays
VI. Final discussion and outlook
VI.1 The agarolytic system of Z. galactanivorans
VI.2 Screening for new marine glycoside hydrolases
VI.3 β-porphyranases discovery in marine bacteria
VI.4 Seaweed polysaccharide degrading CAZymes in human gut bacteria
VI.5 Marine glycoside hydrolases as tools to analyse marine POM and DOM
VII. References