Downloaded from orbit.dtu.dk on: Sep 23, 2021 Keratin2Protein; Valorization of keratinous materials through microbial conversion Espersen, Roall Publication date: 2018 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Espersen, R. (2018). Keratin2Protein; Valorization of keratinous materials through microbial conversion. 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. Technical University of Denmark Department of Biotechnology and Biomedicine PhD Thesis Roall Espersen Keratin2Protein Valorization of keratinous materials through microbial conversion Main supervisor Professor Birte Svensson Technical University of Denmark Co-supervisor Associate Professor Per Hägglund Technical University of Denmark Preface This PhD thesis was prepared based research done in the Enzyme and Protein Chemistry group at the Department of Biotechnology and Biomedicine, Technical University of Denmark (DTU). Supervision was performed by Professor Birte Svensson and co-supervisor Per Hägglund in the period from 01-12- 2014 to 31-01-2018. This PhD was part of the project Keratin2Protein: Novel approach to protein recovery from unutilized slaughterhouse by-product through microbial conversion. The project as well as my PhD was partially funded by The Danish Council for Strategic Research | The Programme Commission on Health, Food and Welfare (grant number 1308-00015B) and a joint PhD stipend from DTU. The Keratin2Protein project involved several collaborations: - Professor Søren J. Sørensen (Department of Biology, Section for Microbiology, Molecular Microbial Ecology Group, University of Copenhagen, Denmark) - Associate Professor Waleed Abu Al-Soud (Department of Biology, Section for Microbiology, Molecular Microbial Ecology Group, University of Copenhagen, Denmark) - Associate Professor Anna E. Lantz (Department of Chemical and Biochemical Engineering, DTU, Denmark) - Professor Krist Gernaey (Department of Chemical and Biochemical Engineering, DTU, Denmark) - Professor Lene Lange (Department of Chemical and Biochemical Engineering, DTU, Denmark) - Senior Specialist Claus Mosby Jespersen (Danish Meat Research Institutet, Denmark) - Project head Jesper Frørup (Dansih Crown, Denmark) - Product developer Kim Schøn Ekmann (Biomar Group BM, Denmark) The Q-Exactive Orbitrap mass spectrometry instruments used in these studies were acquired based on grants from the Villum Foundation and The Danish Council for Independent Research | Natural Science (grant number 11-106246). The Orbitrap Fusion Tribrid instrument was granted by the Villum Foundation. Center for Advanced Food Studies (LMC) is thanked for supporting the acquisition of the Bruker Daltonics Ultraflex II MALDI-tof/tof. A number of presentations have been given during conferences and workshops, which are to be found in appendix A of the thesis. ii Acknowledgement Throughout the three years of my PhD studies, a number of people have contributed with valuable collaboration and support, which have aided me in my work and should be acknowledged. I am grateful to Professor Birte Svensson for giving me the opportunity to study under her supervision and allowing continued development of new knowledge and collaborations. I am also thankful to Per Hägglund for co-supervision and his sharing of knowledge within mass spectrometry. I furthermore wish to say a special thanks to all members (former and present) of both the EPC and PSSB group, for scientific discussions and for being good company during the long work hours in the lab. Thanks also goes to the technical staff, Karina Jansen, Lene Holberg Blicher, Marzanna Due and Anne Blicher for technical support and forcing me to keep a somewhat order workspace. As part of the Keratin2Protein project I have collaborated with people from many different branches of science, with whom great collaboration has allowed me to broaden my scientific knowledge. - Fellow PhD student Francesco Falco at DTU and supervisors Anna Lantz and Krist Gernaey are thanked for a great collaboration and taking the initiative to drive the project, when things were looking bleak. - The people from the University of Copenhagen, Milena Gonzalo, Sam Jacquiod, Waleed Abu Al-Soud should be thanked for their hard work on the assay and consortia development. Søren Sørensen is thanked for his role as leader on the Keratin2Protein project. - Huang Yuhong and Lene Lange are thanked for their expertise within the field of keratin degradation and for sharing their knowledge during fruitful project meetings. - Erasmus student Nazli Coşkun is thanked for her great eager to learn and help with cloning of proteases during her stay in the EPC group. I also wish to say my thanks to my parents for at least trying to understand what on earth I was doing with those pig shavings. Finally thanks to my girlfriend Sidsel for here love and understanding, keeping me tied to reality and helping me to remember to enjoy life outside the lab. iii Abstract The resource consumption by the human population is ever growing and with it comes an increasing amount of waste materials of which many are made from organic matter. The meat industry is generally considered as resource heavy, making it even more urgent that as much as possible of the by- product produced is valorized. The focus of the Keratin2Protein project is on pig bristles and nails produced by slaughterhouses during preparation of the pig carcasses. Bristles and nails are part of a large family of proteinaceous materials (e.g. hair, wool, horn and feathers), made mainly from keratin and keratin related proteins. Because the materials are mainly made from keratin protein, the overall protein content generally exceeds 90% of the total biomass of the keratin material. Both bristles and nails are, however, very recalcitrant of nature, because of a high amount of chemical bonds (disulfide bonds) that makes it resilient to degradation by the proteases present in the digestive systems of most animals. To make the proteins available for degradation, treatment of the keratin material is needed. An alternative method for valorization of keratinous materials is the use of microbial organisms that can degrade the keratin and liberate easily degradable protein. Keratinolytic microorganism produce specialized proteases called keratinases, which can degrade the keratin. Furthermore, can the microorganisms break the disulfide bonds and further the degradation to produce digestible protein. In this PhD work different aspect of the keratin degradation process is investigated An assay was developed based on the degradation of the slaughterhouse by-product (bristles and nails) and was used for the evaluation of bacterial consortia grown on the by-product, to enrich for keratin degraders. Though many interesting bacteria were discovered, they were unfortunately all found to be potential pathogenic, thus not usable in the application. We did however manage to gain knowledge about potentially novel keratin degrading bacteria and their protease profiles. A known keratinolytic bacterium called Amycolatopsis keratinophila was acquired and studied to gain knowledge about its degradation mechanisms. Using a combination of different approaches involving protease purification, characterization and mass spectrometry, knowledge was gained on the protease profile of the bacterium. Two proteases were shown to be abundant in the culture supernatant of the A. iv keratinophila, purified and characterized, which showed that they were capable of degrading the slaughterhouse by-product. Furthermore, one of the protease showed promise as a biotechnological application by working at elevated temperatures. The protease profile was further elucidated by mass spectrometry, showing a wealth of potential keratinases in the culture supernatant. Further investigation will shed light on the roles of each protease. The culture supernatant showed high potential for degradation of the slaughterhouse by-product and optimization of hydrolysis condition (elevated temperature and pH stabilization), resulted in the 75% of the protein material in the slaughterhouse by-product being solubilized. Testing the digestibility of the end product showed >95% digestibility, indicating a successful valorization of the slaughterhouse by- product. v Dansk resume Den menneskelige populations ressource forbrug er stigende og medfølgende er også en øget affalds produktion, hvor af meget er organisk materiale. Kød industrien bliver generelt anset som ressource tung, hvilket gør det desto mere kritisk at så meget som muligt af affaldet kan beriges. Fokus for Keratin2Protein projektet er grisebørster og negle produceret af slagterriger under klargøring af grisekroppene. Børster og negle er del af en stor familie af protein materialer