Discovery and characterization of enzymes acting on chitin Oppdagelse og karakterisering av kitin-aktive enzymer Philosophiae Doctor (PhD) Thesis Tina Rise Tuveng Norwegian University of Life Sciences Faculty of Chemistry, Biotechnology and Food Science Ås 2017 Thesis number 2017:71 ISSN 1894-6402 ISBN 978-82-575-1467-9 TABLE OF CONTENTS TABLE OF CONTENTS ACKNOWLEDGEMENTS .................................................................................................................. i SUMMARY ........................................................................................................................................ iii SAMMENDRAG ............................................................................................................................... vii ABBREVIATIONS ............................................................................................................................. xi LIST OF PAPERS ............................................................................................................................. xiii 1 INTRODUCTION ................................................................................................................ 1 1.1 Chitin ..................................................................................................................................... 1 1.2 Chitosan and chitooligosaccharides ...................................................................................... 3 1.3 Carbohydrate-active enzymes ............................................................................................... 6 1.3.1 CAZymes in chitin degradation and modification ....................................................... 6 1.3.1.1 Chitinases ................................................................................................................. 6 1.3.1.2 β-N-acetylhexosaminidases .................................................................................... 11 1.3.1.3 Lytic polysaccharide monooxygensases................................................................. 11 1.3.1.4 Carbohydrate esterases ........................................................................................... 13 1.3.1.4.1 Carbohydrate esterase family 4 ........................................................................ 13 1.3.1.5 Chitosanases ........................................................................................................... 17 1.3.1.6 Carbohydrate-binding modules .............................................................................. 17 1.3.2 Biological roles of chitin-active enzymes .................................................................. 18 1.4 Microbial degradation and utilization of chitin ................................................................... 21 1.4.1 Chitin degradation by Serratia marcescens ............................................................... 21 1.4.2 Chitin degradation by bacteria in the Bacteriodetes phylum ...................................... 24 1.4.3 Chitin degradation by Thermococcus kodakaraensis ................................................. 25 1.4.4 Chitin degradation by fungi ........................................................................................ 26 1.5 Protein secretion in Gram-negative bacteria ....................................................................... 27 1.5.1 In silico prediction of secreted proteins...................................................................... 30 1.6 Proteomics as a tool for studying bacterial secretomes ....................................................... 32 2 OUTLINE AND PURPOSE OF THE RESEARCH PRESENTED IN THIS THESIS ..... 37 3 MAIN RESULTS AND DISCUSSION ............................................................................. 39 3.1 Paper I – Structure and function of a CE4 deacetylase isolated from a marine environment ......................................................................................................................... 39 3.2 Paper II – Proteomic investigation of the secretome of Cellvibrio japonicus during growth on chitin ............................................................................................................................... 46 3.3 Paper III – Chitin degradation by Cellvibrio japonicus is dependent on the non-redundant CjChi18D chitinase ............................................................................................................. 52 3.4 Paper IV – Genomic, proteomic and biochemical analysis of the chitinolytic machinery of Serratia marcescens BJL200............................................................................................... 60 4 CONCLUDING REMARKS AND PERSPECTIVES ...................................................... 65 5 REFERENCES ................................................................................................................... 68 PAPERS I-IV ...................................................................................................................... APPENDIX AKNOWLEDGEMENTS ACKNOWLEDGEMENTS The work presented in this thesis was carried out in the Protein Engineering and Proteomics (PEP) group, Faculty of Chemistry, Biotechnology and Food Science at the Norwegian University of Life Sciences in the period from 2013 to 2017. The project was financed by the Norwegian Research Council through the Marpol project (project code 221576) lead by Prof. Gudmund Skjåk-Bræk at the Norwegian University of Science and Technology. Firstly, I would like to express my gratitude to my main supervisor Prof. Vincent G. H. Eijsink for offering me this Ph.D. position. Your knowledge and enthusiasm is admirable and very inspiring. Thank you for being a great supervisor; a meeting with you is always good for the motivation. I would also like to express my gratefulness to my co-supervisor Assoc. Prof. Gustav Vaaje-Kolstad. Thank you for answering questions, sharing ideas, and being supportive. I am especially grateful for all the work both of you have done for helping me achieve today’s result. Furthermore, I would like to thank Dr. Magnus Ø. Arntzen for sharing your impressive knowledge of proteomics, patiently teaching me the laboratory techniques in this field, and for answering all my questions. A special thanks to Zarah Forsberg for all scientific and non-scientific discussions in the office. Thanks to Sophanit Mekasha for always being positive and for all the early morning drives to work, and late night drives home. I would also like to thank the rest of the PEP- group, you have made the last four years very enjoyable. To the “Biotech-girls”, thank you for the Tuesday evenings and annual weekend trips. If I’m worried about work, time with you always helps. To my family, especially Mum and Halvor, thank you for always being supportive and pretending to understand what I have done at work during the last four years. Last, but not least, Martin, thank you for just being you and reminding me of the important things in life. As my namesake Tina Turner sings; you’re simply the best! Tina R. Tuveng Ås, September 2017 i SUMMARY SUMMARY In the shift from a fossil-based to a bio-based economy, exploration of renewable recourses is needed. Chitin is considered as the second most abundant polysaccharide on Earth, after cellulose, and its water-soluble derivatives chitosan and chitooligosaccharides (CHOS) have several applications, for example in medicine, agriculture, and the food industry. Today, the extraction of chitin from chitin-rich biomasses and the subsequent production of chitosan and CHOS involve harsh chemicals. It is of interest to replace the current chemical processing technology with enzyme-driven processes, since this would be more environmentally friendly. In addition, enzymes can be used to produce well-defined chitosans and CHOS, which is of interest, since the bioactivity of these compounds depends on properties such as the fraction of acetylation (FA), the degree of polymerization (DP) and the pattern of acetylation (PA). Investigation of proteins utilized by microorganisms during growth on chitin might provide insight into natural chitin conversion and may yield enzymes that can aid in industrial valorization of chitin-rich biomasses. Paper I describes the characterization of a carbohydrate esterase family 4 (CE4) deacetylase, which was selected because of its potential application in the production of CHOS with defined FA and PA. To utilize these enzymes in an optimal way, good understanding of their substrate interactions and specificities is needed. Paper I includes the first enzyme-substrate complex of a CE4 deacetylase with an open active site, providing valuable insight into how the enzyme interacts with its substrate. The enzyme is able to deacetylate a variety of substrates at varying positions. This broad specificity and the presence of seemingly few subsites occupied by the substrate indicate that it may be difficult to use or develop this type of CE4 enzymes for enzymatic tailoring of the PA of CHOS. The genome of Cellvibrio japonicus encodes a large array of carbohydrate-active enzymes, including several putative chitinases and other enzymes possibly involved in chitin degradation. Whether these enzymes are actually involved in chitin utilization by this Gram- negative bacterium had not been investigated at the start of the work described in this thesis. Paper II describes a study of proteins that C. japonicus secretes during growth on chitin, using a novel, plate-based proteomics approach