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Behandling af biomasse med enzymer er en bæredygtig grøn metode hvorved mange produkter, såsom biobrændstof, foder og fødevare ingredienser kan fremstilles. Målet med dette PhD studie var at producere funktionelle fødevare ingredienser – oligosakkarider og poly-sakkarider ved hjælp af enzymatisk katalyse af forskellige biomasser; casein glycomacropeptide (cGMP) og kartoffel pulp. Disse sakkarider har et prebiotisk potentiale, dvs. de er svære at nedbryde af mave/tarm floraen, de kan fremme eller hæmme mikroorganismerne og give en helbredsmæssig effekt. De forskellige produkter blev derfor testede i vækstforsøg med enkelt celle kulturer fra mave/tarm systemet.I dette PhD studie blev der fremstillet en række af forskellige oligo-sakkarider. Den første gruppe var Humane Mælke Oligo-sakkarider (HMOs), der indeholder sialinsyre. Disse stoffer blev fremstillet ud fra cGMP (doner til sialinsyre) og forskellige acceptor molekyler (glycaner). Katalysen var Tr6, der er en muteret sialidase fra Trypanosomas rangeli, der har 6 mutanter og har en rimelig trans-sialidase aktivitet. Tr6 blev produceret i Pichia pastoris. Produktionen af sialyl-laktose blev udført i 5-L skala.Den anden gruppe af sialylerede oligo-sakkarider blev fremstillet med den samme donor, cGMP, men med Tr13, et mutant enzym med 13 mutationer, og en meget høj trans-sialidase aktivitet. Ved rationel mutagenese fik vi vendt hydrolyse (sialidase) til en Transferase (trans-sialidase). Nogle af de godkendte prebiotiske oligo-sakkarider, Galacto-oligosakkarid (GOS), og isomalto-oligosakkarid (IMO), samt andre glycaner såsom melibiose, maltose og fucose blev sialyleret ved hjælp af dette Tr13 enzym. Alle produkterne blev oprenset vha. membran-filtrering og anion-kromatografi og deres struktur blev bestemt ved LC-MS og NMR. De nye sialylerede oligo-sakkarider blev navngivet som HMO-lignende molekyler.Den tredje kategori af prebiotiske molekyler var galactooligo-sakkarider, der blev fremstillet ved enzymatisk katalyse fra kartoffel pulp galactaner også kaldet SPPP (”solubilized potato pulp polysaccharides”). Det anvendte enzym, en endo-1,4-ß- galactanase fra Emericella nidulans blev produceret i en rekombinant P. pastoris stamme, og blev oprenset ved søjle kromatografi, karakteriseret, hvorefter 3-D strukturen blev bestemt.Resultaterne in denne PhD Tese viser en ny vej til fremstilling af nye funktionelle fødevare ingredienser fra industrielle side-strømme og værdiskabende nye produkter med stor marked potentiale. Enzymatic treatment of biomass is an environmentally friendly method to obtain a range of products, such as biofuels, animal feed or food ingredients. The objective of this PhD study was to produce functional food ingredients – oligosaccharides and polysaccharides by means of enzymatic catalysis from biomass: casein glycomacropeptide (cGMP) and potato pulp. These saccharides should possess prebiotic properties, i.e., they should be non-digestable, selectively fermented and allow specific changes, both in the composition and/or activity of the gastrointestinal microbiota that confers benefits upon host well-being. Therefore the obtained compounds were incubated with single bacterial cultures to examine their prebiotic potential.Different types of oligosaccharides were produced in the present study. The first group of compounds was human milk oligosaccharides (HMOs) containing sialic acid in their structures. These were synthesized from cGMP (a donor of sialic acid) and appropriate glycan in trans-glycosylation reaction catalysed by mutant sialidase from Trypanosama rangeli expressed in Pichia pastoris, Tr6. Production of the model HMO, sialyllactose was enlarged to 5 L scale.The second type of sialylated oligosaccharides was obtained with the same donor of sialic acid – cGMP and different glycans with a new Trypanosoma rangeli transsialidase, Tr13. Well-documented prebiotics galactooligosaccharides (GOS), isomaltooligosaccharides (IMO) and lactulose, and three other compounds, i.e., melibiose, maltose, and fucose were sialylated with this enzyme resulting in creating novel human milk-like oligosaccharides. Both HMO and human milk- like oligosaccharides were purified by filtration and chromatography.The last compounds produced during this study were GOS and some galactopolysaccharides. They were generated from isolated galactan and galactan contained in solubilised potato pulp polysaccharides (SPPP). An endo-1,4-ß-galactanase from Emericella nidulans was produced in a recombinant P. pastoris strain to catalyse hydrolysis of galactan and SPPP. This enzyme was purified, characterized and its crystal structure was determined. The products of enzymatic hydrolysis were fractionated according to their molecular weight using membrane filtration.The results of this work pave the way for development of new functional food ingredients from industrial side-streams and generate value-added products with valuable biological properties and great market potential.

6 p.-5 fig.-2 tab., Glycan-lectin recognition is assumed to elicit its broad range of (patho)physiological functions via a combination of specific contact formation with generation of complexes of distinct signal-triggering topology on biomembranes. Faced with the challenge to understand why evolution has led to three particular modes of modular architecture for adhesion/growth-regulatory galectins in vertebrates, here we introduce protein engineering to enable design switches. The impact of changes is measured in assays on cell growth and on bridging fully synthetic nanovesicles (glycodendrimersomes) with a chemically programmable surface. Using the example of homodimeric galectin-1 and monomeric galectin-3, the mutual design conversion caused qualitative differences, i.e., from bridging effector to antagonist/from antagonist to growth inhibitor and vice versa. In addition to attaining proof-of-principle evidence for the hypothesis that chimera-type galectin-3 design makes functional antagonism possible, we underscore the value of versatile surface programming with a derivative of the pan-galectin ligand lactose. Aggregation assays with N,N′-diacetyllactosamine establishing a parasite-like surface signature revealed marked selectivity among the family of galectins and bridging potency of homodimers. These findings provide fundamental insights into design-functionality relationships of galectins. Moreover, our strategy generates the tools to identify biofunctional lattice formation on biomembranes and galectin-reagents with therapeutic potential. © 2019 National Academy of Sciences., Financial support from NSF Grants DMR-1066116, DMR-1720530, and DMR-1807127 (to V.P.), the P. Roy Vagelos Chair at the University of Pennsylvania (V.P.), the Alexander von Humboldt Foundation(V.P.), NSF Grant DMR-1120901 (toM.L.K. and V.P.), NIH National Cancer Institute Grant R21-CA178754 (to M.C.), a China Scholarship Council PhD scholarship (to H.M.), Science Foundation Ireland Grant 13/IA/1959 (S.O.), and Spanish Grant BFU2016-77835R (to A.R.) is gratefully acknowledged.