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Synthesis and Structural Characterization of Glucooligosaccharides and Dextran from Weissella Confusa Dextransucrases

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Synthesis and Structural Characterization of Glucooligosaccharides and Dextran from Weissella Confusa Dextransucrases YEB Recent Publications in this Series Dextran from and and Structural Characterization of Glucooligosaccharides QIAO SHI Synthesis 4/2016 Hany S.M. EL Sayed Bashandy Flavonoid Metabolomics in Gerbera hybrida and Elucidation of Complexity in the Flavonoid Biosynthetic Pathway 5/2016 Erja Koivunen Home-Grown Grain Legumes in Poultry Diets 6/2016 Paul Mathijssen DISSERTATIONES SCHOLA DOCTORALIS SCIENTIAE CIRCUMIECTALIS, Holocene Carbon Dynamics and Atmospheric Radiative Forcing of Different Types of Peatlands ALIMENTARIAE, BIOLOGICAE. UNIVERSITATIS HELSINKIENSIS 21/2016 in Finland 7/2016 Seyed Abdollah Mousavi Revised Taxonomy of the Family Rhizobiaceae, and Phylogeny of Mesorhizobia Nodulating Glycyrrhiza spp. 8/2016 Sedeer El-Showk Auxin and Cytokinin Interactions Regulate Primary Vascular Patterning During Root QIAO SHI Development in Arabidopsis thaliana 9/2016 Satu Olkkola Antimicrobial Resistance and Its Mechanisms among Campylobacter coli and Campylobacter Synthesis and Structural Characterization of upsaliensis with a Special Focus on Streptomycin 10/2016 Windi Indra Muziasari Glucooligosaccharides and Dextran from Impact of Fish Farming on Antibiotic Resistome and Mobile Elements in Baltic Sea Sediment Weissella confusa Dextransucrases 11/2016 Kari Kylä-Nikkilä Genetic Engineering of Lactic Acid Bacteria to Produce Optically Pure Lactic Acid and to Develop a Novel Cell Immobilization Method Suitable for Industrial Fermentations 12/2016 Jane Etegeneng Besong epse Ndika Molecular Insights into a Putative Potyvirus RNA Encapsidation Pathway and Potyvirus Particles as Enzyme Nano-Carriers 13/2016 Lijuan Yan Bacterial Community Dynamics and Perennial Crop Growth in Motor Oil-Contaminated Soil in a Boreal Climate 14/2016 Pia Rasinkangas New Insights into the Biogenesis of Lactobacillus rhamnosus GG Pili and the in vivo Effects of Pili 15/2016 Johanna Rytioja Enzymatic Plant Cell Wall Degradation by the White Rot Fungus Dichomitus squalens 16/2016 Elli Koskela Genetic and Environmental Control of Flowering in Wild and Cultivated Strawberries 17/2016 Riikka Kylväjä Staphylococcus aureus and Lactobacillus crispatus: Adhesive Characteristics of Two Gram- Weissella confusa Positive Bacterial Species 18/2016 Hanna Aarnos Photochemical Transformation of Dissolved Organic Matter in Aquatic Environment – From a Boreal Lake and Baltic Sea to Global Coastal Ocean 19/2016 Riikka Puntila Trophic Interactions and Impacts of Non-Indigenous Species in the Baltic DEPARTMENT OF FOOD AND ENVIRONMENTAL SCIENCES Sea Coastal Ecosystems Dextransucrases 20/2016 Jaana Kuuskeri FACULTY OF AGRICULTURE AND FORESTRY Genomics and Systematics of the White-Rot Fungus Phlebia radiata: DOCTORAL PROGRAMME IN FOOD CHAIN AND HEALTH Special Emphasis on Wood-Promoted Transcriptome and Proteome UNIVERSITY OF HELSINKI 21/2016 Helsinki 2016 ISSN 2342-5423 ISBN 978-951-51-2451-7 Department of Food and Environmental Sciences Faculty of Agriculture and Forestry University of Helsinki Doctoral Programme in Food Chain and Health Doctoral School in Environmental, Food and Biological Sciences Synthesis and structural characterization of glucooligosaccharides and dextran from Weissella confusa dextransucrases Qiao Shi Doctoral Thesis To be presented, with the permission of the Faculty of Agriculture and Forestry, University of Helsinki, for public examination in Auditorium 2, Infocenter Korona (Viikinkaari 11), on September 29th, 2016, at 12 o’clock noon. Helsinki 2016 Custos Professor Maija Tenkanen Department of Food and Environmental Sciences University of Helsinki, Finland Supervisor Professor Maija Tenkanen Department of Food and Environmental Sciences University of Helsinki, Finland Reviewers Dr. Gregory L. Côté Agricultural Research Service United States Department of Agriculture, USA Professor Markus Linder School of Chemical Technology Aalto University, Finland Opponent Professor Henk Schols Laboratory of Food Chemistry Wageningen University, The Netherlands Cover picture: Representation of a dextransucrase (based on Protein Data Bank no. 3HZ3) and its oligosaccharide products, made by Qiao Shi. ISBN 978-951-51-2451-7 (Paperback) ISBN 978-951-51-2452-4 (PDF) ISSN 2342-5423 (Print) ISSN 2342-5431 (Online) Unigrafia Helsinki 2016 ABSTRACT Many lactic acid bacteria are able to synthesize dextrans from sucrose by dextransucrases. Weissella confusa strains have attracted increasing attention due to their production of texture-modifying dextrans during food fermentation. Potential prebiotic oligosaccharides have also been produced by dextransucrases in the presence of sucrose and acceptor sugars. However, only few W. confusa dextransucrases have been previously studied, and the reactions of Weissella dextransucrases to synthesize oligosaccharides have not been investigated. In this thesis, two W. confusa dextransucrases from efficient dextran-producers were studied, and their products were structurally characterized because this information is essential for a better usability of these enzymes and corresponding bacteria in food and health applications. The biochemical and kinetic properties of one of the W. confusa dextransucrases were studied. Two activity assays were compared to determine the kinetic parameters. A sucrose radioisotope assay gave a KM of 14.7 mM and a Vmax of 8.2 µmol/(mg∙min), whereas a Nelson-Somogyi assay gave values of 13.0 mM and 19.9 µmol/(mg∙min), respectively. The dextrans from the two W. confusa dextransucrases were found by, e.g., NMR analysis to consist of mainly α-(1→6) linkages and 3% α-(1→3) branches, of which some were elongated. A high-performance size-exclusion chromatography analysis of the dextrans revealed high molar masses of 107‒108 g/mol. Weissella dextransucrases were also studied with acceptor sugars for the synthesis of glucooligosaccharides. The most efficient acceptor was maltose, followed by isomaltose, maltotriose, and nigerose, which formed series of glucooligosaccharides by the further elongation of intermediate acceptor products. The products derived from maltose formed two homologous series, with one series being predominant and the other being minor. The major maltose product series were linear isomaltooligosaccharides (IMOs) with reducing-end maltose units, as identified by multistage mass spectrometry (MSn) analysis. The minor maltose series were revealed by an NMR analysis of the isolated minor trisaccharide product to bear a novel branched structure. These products contained an α-(1→2)-linked glucosyl residue on the reducing residue of the linear IMOs. These structures have not been previously obtained by a dextransucrase. They probably formed by the attachment of a single-unit branch to linear IMOs. For the acceptor analogs lactose and cellobiose, their main acceptor products were identified by NMR analysis to be branched trisaccharides, with a glucosyl residue α- (1→2) linked to the acceptor’s reducing end. Surprisingly, a side product, isomelezitose 1 (6Fru-α-Glcp-sucrose), was produced when using lactose as an acceptor. The synthesis of this nonreducing trisaccharide by a dextransucrase was reported here for the first time. Linear IMOs with a degree of polymerization ≥3.3 serve as prebiotics for their resistance to digestion. However, industrial IMO production often leads to a high portion of unwanted digestible sugars. The dextransucrase reaction in the presence of the efficient acceptor maltose was demonstrated here as a promising alternative synthesis process to control IMO size distribution by varying the sucrose/maltose ratio. The effects of substrate concentrations (0.15–1 M) and dextransucrase dosage (1–10 U/g sucrose) on the IMO yield and profile were modeled. High sucrose (1 M) and medium maltose (0.5 M) concentrations were found to be optimal for the synthesis of long-chain IMOs, with 366 g/L of total IMOs attained. 2 ACKNOWLEDGEMENTS This thesis was carried out at the Department of Food and Environmental Sciences, Faculty of Agriculture and Forestry, University of Helsinki. The work was financially supported by the Finnish Graduate School on Applied Bioscience: Bioengineering, Food & Nutrition, Environment (ABS Graduate School), the Academy of Finland via project “Molecular and functional characterization of dextran production in Weissella spp. ‒ Superior dextran producers for cereal applications (WISEDextran)” and the Finnish Food and Drink Industries' Federation (ETL). I am deeply indebted to my supervisor Professor Maija Tenkanen. She provided me with the great opportunity to pursue a doctoral degree in her research group and has offered me insightful scientific guidance, advice, and encouragement throughout the years. Her open-mindedness, curiosity, and enthusiasm towards science have inspired me. It was also a privilege for me to work with Docent Liisa Virkki, who introduced me to NMR spectral interpretation. I feel grateful to Professor Vieno Piironen and Research Professor Kristiina Kruus for participating in my follow-up group during my thesis work. I also want to express my sincere gratitude to Dr. Gregory L. Côté and Professor Markus Linder for their pre-examination of this thesis. I owe my gratitude to Docent Hannu Maaheimo, Assistant Professor Kati Katina, Docent Päivi Tuomainen, Minna Juvonen, Dr. Ndegwa Henry Maina, Kaj-Roger Hurme, Dr. Sanna Koutaniemi, and Dr. Leena Pitkänen, who gave me invaluable help in their fields of expertise, as well as Yaxi Hou, who assisted me with laboratory work. I also thank the project collaborators Dr. Riikka Juvonen, Ilkka Kajala, and Dr. Antti
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