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Heterologous production and characterization of chitin deacetylases with chitin binding domains

Chitin Deacetylasen (CDAs) katalysieren die Acetamido-Hydrolyse von N-Acetylglucosamin zu Glucosamin in Chitin und Chitosan. Es ist nachgewiesen, dass chemisch produzierte Chitosane ein zufälliges Acetylierungsmuster (PA) aufweisen, welches wahrscheinlich die biologische Aktivität und damit die Anwendung des Polymers beeinflusst. Thema dieser Arbeit ist die Charakterisierung zweier pilzlicher CDAs (BC95 aus Botrytis cinerea und PaCDA aus Podospora anserina), welche zusätzlich zur katalytischen Domäne Chitin-Bindedomänen (CBDs) aufweisen. Die heterologe Produktion beider Enzyme wurde erfolgreich in der Hefe Hansenula polymorpha durchgeführt, jedoch zeigte nur PaCDA Enzymaktivität. Des Weiteren wurden Deletionsvarianten der PaCDA erstellt, um die Rolle der CBDs zu untersuchen. Die Wirkungsweise von PaCDA wurde anhand der Produkte analysiert und ist nicht prozessiv; dennoch produziert das Enzym ein spezifisches Acetylierungsmuster, welches durch alternierende Einheiten gekennzeichnet ist

Chitin deacetylases (CDAs) catalyze the acetamido hydrolysis of N-acetylglucosamine to glucosamine in chitin and chitosan. It has been demonstrated that chemically produced chitosans show a random pattern of acetylation (PA), which is supposed to influence the biological activity and thereby the application of the polymer. The subject of this study is the characterization of two fungal CDAs (BC95 from Botrytis cinerea and PaCDA from Podospora anserina), which possess additional chitin binding domains (CBDs) next to the catalytic domain. The heterologous production of both enzymes was successful in Hansenula polymorpha, but CDA activity was confirmed only for PaCDA. Additionally, deletion variants of PaCDA were constructed to analyze the role of the CBDs. The mode of action of PaCDA was determined by product analysis and is not processive; nevertheless the enzyme is producing a specific PA, which is characterized by alternating unit

Titel: Heterologous production and characterization of chitin deacetylases with chitin binding domains
Verfasser: Hoßbach, Janina GND
Gutachter: Moerschbacher, Bruno M.
Organisation: FB 13: Biologie
Dokumenttyp: Dissertation/Habilitation
Medientyp: Text
Erscheinungsdatum: 2015
Publikation in MIAMI: 09.11.2015
Datum der letzten Änderung: 09.11.2015
Schlagwörter: Chitin; Chitosan; Acetylierungsmuster; Chitin Deacetylase; Enzym; Pilz; Podospora anserina
chitin; chitosan; pattern of acetylation; chitin deacetylase; enzyme; fungal; Podospora anserina
Fachgebiete: Biowissenschaften; Biologie
Sprache: Englisch
Format: PDF-Dokument
URN: urn:nbn:de:hbz:6-57289607713
Permalink: https://nbn-resolving.org/urn:nbn:de:hbz:6-57289607713
Onlinezugriff:
Inhalt:
Table of Contents
Summary 1
1. Introduction 4
1.1. Chitin and Chitosan 4
1.1.1. Structure and properties 4
1.1.2. Biological activities 5
1.1.3. Pattern of acetylation 6
1.2. Chitin deacetylases 7
1.2.1. Occurrence of CDAs and role in nature 8
1.2.2. Characterization of CDAs 9
1.3. Carbohydrate-binding domains 11
1.3.1. Chitin binding domains 11
1.4. Aim of the thesis 13
2. Expression during conidia germination and heterologous production 14
of Botrytis cinerea putative chitin deacetylases
2.1. Introduction 15
2.2. Material and Methods 17
2.2.1. Bioinformatics 17
2.2.2. Cultivation of Botrytis cinerea 17
2.2.3. DNA/RNA isolation and RT-PCR 18
2.2.4. CDA activity tests 18
2.2.5. Heterologous production of BC95 19
2.2.5.1. Nucleic acid manipulations: Cloning of BC95 19
2.2.5.2. Biochemical analytics 19
2.2.5.3. S. pombe – cloning 20
2.2.5.4. S. pombe – screening 21
2.2.5.5. E. coli – cloning 21
2.2.5.6. E. coli – screening 22
2.2.5.7. H. polymorpha – cloning 22
2.2.5.8. H. polymorpha – screening 23
2.3. Results 24
2.3.1. Bioinformatical analysis 24
2.3.2. Gene expression during conidia germination 25
2.3.3. CDA activity during conidia germination 25
2.3.4. Heterologous production of BC95 26
2.3.4.1. Schizzosaccharomyces pombe 26
2.3.4.2. Escherichia coli 28
2.3.4.3. Hansenula polymorpha 29
2.4. Discussion 31
2.4.1. Botrytis cinerea putative chitin deacetylases 32
2.4.2. Heterologous production of BC95 34
3. A chitin deacetylase from Podospora anserina with two carbohydrate- 37
binding domains produces chitosans with non-random patterns of
acetylation
3.1. Introduction 38
3.2. Materials and Methods 40
3.2.1. Bioinformatics 40
3.2.2. Production and purification of PaCDA 40
3.2.3. Substrates 42
3.2.4. Characterization of PaCDA 42
3.2.5. Mode of action 44
3.2.6. Mass spectrometry (MS) 44
3.2.7. Nuclear magnetic resonance spectroscopy (NMR) 45
3.2.8. Biological activities 46
3.3. Results 47
3.3.1. Bioinformatics 47
3.3.2. Production and purification of PaCDA 48
3.3.3. Substrates 49
3.3.4. Characterization of PaCDA 50
3.3.5. Mode of action and pattern of acetylation 51
3.3.6. Biological activities – Oxidative Burst assay 54
3.4. Discussion 55
3.4.1. Bioinformatics 55
3.4.2. Production and purification of PaCDA 55
3.4.3. Substrates 55
3.4.4. Characterization of PaCDA 56
3.4.5. Mode of action and pattern of acetylation 57
3.4.6. Biological activities 58
3.5. Conclusions 59
4. Analysis of chitin binding domain deletion variants of Podospora 60
anserina chitin deacetylase
4.1. Introduction 61
4.2. Materials and Methods 62
4.2.1. Generation of PaCDA deletion constructs 62
4.2.2. Screening, production and purification of PaCDA deletion variants 62
4.2.3. Comparative characterization of PaCDA and PaCDA-ΔN 63
4.2.4. Enzymatic/mass-spectrometric fingerprinting of CDA products 63
4.2.5. PaCDA production in E. coli 64
4.3. Results 65
4.3.1. Bioinformatics and cloning 65
4.3.2. Screening of transformants 66
4.3.3. Production and purification 68
4.3.4. Comparison of PaCDA and PaCDA-ΔN 70
4.3.5. Enzymatic/mass-spectrometric fingerprinting 71
4.3.6. PaCDA production in E. coli 72
4.4. Discussion 73
4.4.1. PaCDA-ΔN 73
4.4.2. PaCDA-ΔC and ΔNC 75
4.4.3. PaCDA production in E. coli 76
4.4.4. Conclusion and Outlook 77
5. Discussion 79
5.1. Heterologous production of chitin deacetylases with chitin binding domains 80
5.1.1. Escherichia coli 80
5.1.2. Schizosaccharomyces pombe 81
5.1.3. Hansenula polymorpha 82
5.2. Characteristics of chitin deacetylases with chitin binding domains 84
5.2.1. Botrytis cinerea putative CDAs 84
5.2.2. Podospora anserina CDA 86
5.2.2.1. PaCDA characteristics 87
5.2.2.2. PaCDA mode of action 88
5.2.2.3. Role of CBDs for PaCDA 89
5.3. Chitosans with non-random pattern of acetylation 91
5.3.1. Biological activities of chitosans with non-random PA 92
5.4. Conclusions 93
5.4.1. Perspectives 94
6. References 95
Acknowledgements / Danksagung 109
Lebenslauf 110