Downloaded from orbit.dtu.dk on: Oct 04, 2021 Production and characterisation of glycoside hydrolases from GH3, GH5, GH10, GH11 and GH61 for chemo-enzymatic synthesis of xylo- and mannooligosaccharides Dilokpimol, Adiphol Publication date: 2010 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Dilokpimol, A. (2010). Production and characterisation of glycoside hydrolases from GH3, GH5, GH10, GH11 and GH61 for chemo-enzymatic synthesis of xylo- and mannooligosaccharides. 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. chemo-enzymatic synthesis of xylo- and mannooligosaccharides of synthesis chemo-enzymatic GH3, GH5, GH10, GH11 and GH61for from hydrolases glycoside of and characterisation Production Production and Characterisation of Glycoside Hydrolases from GH3, GH5, GH10, GH11 and GH61 for Chemo-Enzy- matic Synthesis of Xylo- and Mannooligosaccharides Adiphol Dilokpimol Ph.D. Thesis June 2010 Adiphol Dilokpimol Adiphol Enzyme and Protein Chemistry Department of Systems Biology Production and characterisation of glycoside hydrolases from GH3, GH5, GH10, GH11 and GH61 for chemo-enzymatic synthesis of xylo- and mannooligosaccharides Adiphol Dilokpimol Ph.D. Thesis June 2010 Enzyme and Protein Chemistry Department of Systems Biology Technical University of Denmark Preface The present Ph.D. thesis entitled “Production and characterisation of glycoside hydrolases from GH3, GH5, GH10, GH11 and GH61 for chemo-enzymatic synthesis of xylo- and mannooligosaccharides” summarises my work carried out under the supervision of Prof. Birte Svensson and Assoc. Prof. Maher Abou Hachem at Enzyme and Protein Chemistry (EPC), Department of Systems Biology, Technical University of Denmark (DTU) for the period of June 2007 to June 2010. This study was supported by the Danish Strategic Research Council’s Committee on Food and Health (FøSu, to the project ‘Biological Production of Dietary Fibres and Prebiotics’ no. 2101-06-0067). This work has resulted in the following manuscripts: Dilokpimol, A., Nakai, H., Gotfredsen, C. H., Appeldoorn, M., Baumann, M. J., Nakai, N., Schols, H. A., Abou Hachem, M., Svensson, B. Enzymatic synthesis of β-xylosyl- oligosaccharides by transxylosylation using two β-xylosidases of glycoside hydrolase family 3 from Aspergillus nidulans FGSC A4, submitted to Carbohydrate Research (see 10.2.3.1) Dilokpimol, A., Nakai, H., Gotfredsen, C. H., Baumann, M. J., Nakai, N., Abou Hachem, M., Svensson, B. GH5 endo-β-1,4-mannanases from Aspergillus nidulans FGSC A4 producing hetero manno-oligosaccharides by transglycosylation, to be submitted to Biochimica et Biophysica Acta, Proteins and Proteomics (see 10.2.3.2) In addition, I am the coauthor of several publications and manuscripts (listed in 10.2) on results obtained in the same project. Kgs. Lyngby, 3rd June 2010 ____________________ Adiphol Dilokpimol i Acknowledgements This work could not have been accomplished without the help and support of many teachers, colleagues and friends. First, I would like to express my deep appreciation and gratefully thankful to Professor Birte Svensson and Associate Professor Maher Abou Hachem for giving me the opportunity to carry out my Ph.D. study. I deeply appreciate all of your invaluable guidance and mental supports throughout the whole period. I would like to extend my heartiest thanks to Dr. Hiroyuki Nakai for his excellent and inspirational scientific supports in this study. Professor Anne S. Meyer and Professor Jørn D. Mikkelsen, Center for Biological Production of Dietary Fibers and Prebiotics, DTU Chemical Engineering, and the Steering Committee for this project are sincerely thanked for their suggestions and discussions. Professor Stephen G. Withers, University of British Columbia, Canada, Professor Monica Palcic, Professor Ole Hindsgaul, and Dr. Karin Mannerstedt, Carbohydrate Chemistry group, Carlsberg Laboratory, are thanked for providing and deacetylation of α-xylosyl- and α-xylobiosyl fluorides. Professor Henk A. Schols, Dr. Maaike Appeldoorn, and Dr. Yvonne Westpahl, Laboratory of Food Chemistry, Wageningen University, The Netherlands, are thanked for their excellent electrospray ionization mass spectrometry analysis and scientific discussions. I would genuinely like to thank Associate Professor Charlotte H. Gotfredsen and Anne Hector, DTU Chemistry, not only for her superb nuclear magnetic resonance analysis of all transglycosylation products but also for generous and fruitful discussions. Professor Jens Ø. Duus and Bent O. Petersen, Carlsberg Laboratory, are thanked for assisting with the NMR assignment. Professor William G. T. Willats and Dr. Henriette Lodberg Pedersen, Faculty of Life Sciences, University of Copenhagen, are thanked for performing analysis of the GH61 putative endo-β-glucanase on carbohydrate microarray analysis. Professor Atsuo Kimura and Associate Professor Haruhide Mori, Faculty of Agriculture, Hokkaido University, Japan, as well as Dr. Nathalie Juge and Dr. Jean- Guy Berrin, Institute of Food Research, Norwich Research Park, United Kingdom, are thanked for their kind support. Professor Peter Roepstorff and Dr. Martin Zehl, Department of Biochemistry and Molecular Biology, University of Southern Denmark, are thanked for electrospray ionization mass spectrometry analysis of Xyl-Cys. Associate Professor Leila Lo Leggio, Department of Chemistry, University of Copenhagen is thanked for stimulating discussions on β-xylosidase structure. Associate Professor Mads H. Clausen, DTU Chemistry, is thanked for the preparation of β-xylosyl fluoride. I apologise for my careless handling of the compound. I wish to thank Anne Blicher for ii her excellent amino acid analysis. PhD student Louise E. Rasmussen, DTU Chemical Engineering, is thanked for kind assistance in the beginning of this project. My thankfulness also goes to all former and present members of the Enzyme and Protein Chemistry, DTU Systems Biology, for their help in one way or another to make this study possible especially Associate Professor Susanne Jacobsen, Dr. Martin J. Baumann, Dr. Per Hägglund, Dr. Eun Seong Seo, Dr. Malene B. Vester-Christensen, Susanne Blume, Karina Jansen, Birgit Andersen, and Bjarne G. Schmidt. Special thanks to Karen Marie Jakobsen for her excellent administrative skills as well as PhD student Marie S. Møller, PhD student Anders D. Jørgensen, and Brian S. Lassen for kindly helping to translate ‘Danske resumé’. The Danish Strategic Research Council’s Committee on Food and Health (FøSu) and Danisco, Denmark, are thanked for co-funding the PhD project in the Center for Biological Production of Dietary Fibers and Prebiotics with DTU. Last but not least, I am thankful to my beloved parents, my family and my friends both in Denmark and Thailand, for their endless support and understanding throughout the difficult times. Without your love and support, I am sure that I would not be able to continue up till now. iii Summary Plant cell wall hydrolysate by-products are resources for oligosaccharides which potentially can act as prebiotics stimulating the growth of probiotic bacteria and thus provide a number of health benefits to the host. The objective of the present research project is to produce novel prebiotics or biologically active oligosaccharides by enzyme catalysed transglycosylation of starting materials that are related to or derived from industrial plant by-products. Five recombinant glycoside hydrolases, i.e. one GH3 β- xylosidase, two GH5 mannanases, one GH10 xylanase, and one GH11 xylanase from Aspergillus nidulans FGSC A4 were investigated for their transglycosylation activity aiming at production of β-xylo- and mannooligosaccharides. The project includes characterisation of the produced enzymes. The GH3 β-xylosidase (BxlA) was produced as a His-tag fusion protein secreted by Pichia pastoris X-33 in a yield of 16 mg/L culture. BxlA displayed a Km and kcat towards para-nitrophenyl β-D-xylopyranoside of 1.3 mM and 112 s-1, respectively, and also hydrolysed para-nitrophenyl α-L-arabinofuranoside albiet with two orders of magnitude lower catalytic efficiency (kcat/Km). Among xylooligosaccharides (degree of polymerisation 2−6), BxlA preferably hydrolysed xylobiose, while the catalytic efficiency decreased slightly with increasing chain length. Transglycosylation reactions using 10 mono- and six disaccharides, two sugar alcohols, and two amino acids revealed that BxlA possesses broad acceptor specificity, while mannose, lyxose, and talose are preferred acceptors for BxlA with transglycosylation yields of 25%, 23%, and 22%, respectively. Moreover, four di- and two trisaccharides/glycosides produced with lyxose, L-fucose, talose, cysteine, sucrose, and turanose as acceptors are novel compounds and the structures of β-D-Xylp-(1→4)-D-Lyxp,