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Abida Sultan 978 87 91494 66 6 Downloaded from orbit.dtu.dk on: Oct 07, 2021 A proteomics-based study of endogenous and microbial xylanases and xylanase inhibitors associated with barley grains used for liquid feed Sultan, Abida Publication date: 2013 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Sultan, A. (2013). A proteomics-based study of endogenous and microbial xylanases and xylanase inhibitors associated with barley grains used for liquid feed. Technical University of Denmark. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. A proteomics-based study of endogenous and microbial xylanases and xylanase inhibitors associated with barley grains used for liquid feed Abida Sultan Ph.D. Thesis Februray 2013 DTU Systems Biology A proteomics-based study of endogenous and microbial xylanases and xylanase inhibitors associated with barley grains used for liquid feed Ph.D. Dissertation 2013 Abida Sultan Enzyme and Protein Chemistry (EPC) Department of Systems Biology Technical University of Denmark (DTU) Supervisors: Associate Professor Christine Finnie Professor Birte Svensson 1 Copyright©: Abida Sultan 2013 Address: Enzyme and Protein Chemistry Department of Systems Biology Technical University of Denmark Søltofts Plads, Building 224 DK-2800 Kgs.Lyngby Denmark Phone: +4545252525 Fax: +4545886307 Web: www.bio.dtu.dk Print: J&R Frydenberg A/S København December 2013 ISBN: 978-87-91494-66-6 2 2 PREFACE The present thesis represents the results of my Ph.D. study carried out at Enzyme and Protein Chemistry (EPC), Department of Systems Biology, Technical University of Denmark (DTU) for the period of September 2009 to February 2013 under supervision of Associate Professor Christine Finnie and Professor Birte Svensson. The Ph.D. project was performed in collaboration with the University of Aarhus (Department of Molecular Biology and Genetics, Faculty of Agricultural Sciencies, University of Aarhus) under supervision by Associate Professor Henrik Brinch-Pedersen and Dr. Giuseppe Dionisio. The Ph.D. stipend was founded by DTU and the work was supported by the Directorate for Food, Fisheries and Agri Business (DFFE) for the project “Exploiting barley first wave enzyme activities for better feed”. Other partners in the project include Sejet Plantbreeding and Novozymes A/S. The work of this Ph.D. project has resulted in the following manuscripts: Sultan A, Andersen B, Christensen JB, Poulsen HD, Svensson B, Finnie C. Establishing the protein profile of liquid feed and analysis of application of protease inhibitors and NADPH-dependent thioredoxin reductase/thioredoxin (NTR/Trx) system. Manuscript to be submitted to Journal of Proteomics (Chapter 2). Sultan A, Andersen B, Frisvad JC, Svensson B, Finnie C. Exploring the plant-microbe interface – Profiling and characterizing the indigenous fungal community, proteins and enzymes associated to surface of barley grains and xylanolytic activities. Manuscript to be submitted to Journal of Proteome Research (Chapter 3 and 4). Sultan A, Dionisio G, Brinch-Pedersen H, Svensson B, Finnie C. Secretory expression of functional barley xylanase inhibitor protein (XIP-III) by yeast Pichia pastoris. In Prep. (Chapter 5). Finnie C, Sultan A, Grasser KD. A review: From protein catalogues towards targeted proteomics approaches in cereal grains. Phytochemistry 2011;72:1145-53. (Appendix). 3 3 ACKNOWLEDGEMENTS During my three-year-old journey of Ph.D. study, I have encountered a unique group of people, who went from being acquaintances to good friends. First and foremost, I express my profound gratitude to my supervisors Associate Professor Christine Finnie and Professor Birte Svensson, for the opportunity to carry out this study at Enzyme and Protein Chemistry and for their scientific guidance, inspiration and encouragement throughout the research work. In particular, I acknowledge their support, corrections, and patience during the last phase of writing my thesis. Birgit Andersen is genuienly thanked for brilliant technical assistance with mass spectrometry and other “small” tasks in the lab. Professor Jens C. Frisvad (Center for Microbial Biotechnology, Department of Systems Biology, DTU) is acknowledged for rewarding scientific discussions and technical expertise with isolation and identification of fungi. All the project collaborators are thanked for fruitful discussions during the half-annual meetings. Professor Henrik Brinch-Pedersen and Dr. Giuseppe Dionisio (Department of Molecular Biology and Genetics, Faculty of Science, University of Aarhus) are thanked for a great collaboration, support and technical assitance in the lab. Post doc Anne Rosenkilde is thanked for a colorful collaboration, technical help and good friendship. Ph.D. student Jesper B. Christensen is thanked for a good collaboration and for supplying us with liquid feed samples. Students Mik Marstand (M.Sc.), Maria Bach og Julie Sørensen (B.Sc.), whom I co-supervised, are acknowledged for their work. All former and present members of the EPC group are thanked for providing a good and cheerful working environment. Finally, no words would suffice to express my feelings for my family, especially my mom and dad, siblings Alia, Zaki, Shahina and Jasmin, whose endless love, encouragement and support have continued to enrich my life and made me into the person I am today. Dedicated to the Sultans 4 4 ABSTRACT The mature barley grain contains a complement of enzymes that are synthesized during seed development for degradation of seed storage reserves during germination. These enzyme activities (first wave enzymes) are considered important for maximizing nutrient digestibility in food and feed. Several strategies, such as liquid feed and supplementation of amino acids and microbial exogenous enzymes, are applied to improve protein absorption. A diverse commensal microbial community populates the cereal grains. The colonizing microflora constitute an integrated part of the seeds and interact/influence the plant and/or competitors via secretion of an array of enzymes and compounds/metabolites. The occurrence of these enzyme activities both of plant and fungal origin present a great potential for improvement of grain nutritional components for feed applications. Knowledge is lacking in the variation of these enzyme activities between barley cultivars, as well as the distribution and composition of the residing commensal fungal community and the grain surface- associated proteins. To obtain new insight into the interplay between barley grains and the colonizing fungi, we set out to study the activities of barley grains as well as the populating fungal community using different approaches. The present thesis aimed at profiling the changes in proteome of liquid feed using gel- based proteomics. Comparative proteomics analysis was performed on liquid feed supplemented with protease inhibitors and/or the thioredoxin system (NTR/Trx). Addition of protease inhibitors to liquid feed seemed to some extent hamper protein degradation. With the NTR/Trx, the 2D-gels clearly showed an altered protein pattern and increased solubility of storage proteins, CM-proteins and protease inhibitors. The variability of microbial and endogenous xylanase, as well as xylanase inhibition activities across a set of barley cultivars were determined using activity assays and gel-based proteomic analysis. Considerable inter-cultivar variability was found in the level of both microbial and endogenous xylanase, as well as xylanase inhibition activity levels. Harvest year and cultivar/genotype had a significant impact on the variability of all three parameters. Harvest year/weather conditions seem to largely affect xylanases and inhibition activity levels, thus indicating the instability of these parameters. 5 5 A Reference map over the surface-associated proteins (surfome) constitutively present on barley grains of two barley cultivars were established using 2-DE and mass spectrometry. The majority of the identified proteins was of plant origin and ascribed to play a role in defense and/or oxidative stress mechanisms. A metaproteomics approach was applied to profile and characterize the composition of the fungal community populating the surface of barley grains across different cultivars and their secretomes. It was revealed, the barley cultivars with high microbial xylanase activity levels were found to contain high numbers of storage fungi e.g. Aspergilli and Penicillia. A detailed analysis of the secretomes of two of the isolated fungi, i.e Aspergillus niger and Fusarium poae, grown on barley flour and wheat arabinoxylan enabled identification of a collection of amylopectic and xylanolytic enzymes involved in cell wall degradation and carbohydrate catabolism. To gain a better understanding of the role of these xylanase inhibitors, the barley
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