Production of fucosyllactose with a fucosidase from Aspergillus niger

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dc.contributor Aalto-yliopisto fi
dc.contributor Aalto University en
dc.contributor.advisor Usvalampi, Anne Ruvalcaba Medrano, Marcela 2017-09-04T12:59:57Z 2017-09-04T12:59:57Z 2017-08-21
dc.description.abstract The interest on Human Milk Oligosaccharides (HMOs) has been increasing throughout the last years due to their reported beneficial effects on the infant’s nutrition. Therefore, researchers have tried to develop effective strategies to produce HMOs in large scale. Fucosyllactose for instance, one of the most abundant HMOs, has been proven to display prebiotic effects, enhancing the babies’ immune system and preventing infections. Other beneficial effects are the antiadhesive properties against microorganisms and toxins, regulation of gut motility and a postulated ability to improve brain development. The main approaches to produce the fucosyllactose include the chemical synthesis or in vitro and in vivo biosynthesis. During this thesis, an α-fucosidase from Aspergillus niger was used to synthesize fucosyllactose through a transglycosylation reaction. Due to inconsistencies in gene annotations, two different versions of the gene encoding the desired fucosidase were cloned into Pichia pastoris strains SMD1168H and KM71H. Expression of both gene versions was attempted in order to find out which one produced the enzyme with the highest enzymatic activity. The results showed that the shorter version of the gene is not active, therefore, the longer gene version was selected for further studies. The produced α-fucosidase had an apparent molecular mass of 95 kDa and was highly N-glycosylated with the N-glycans contributing about 15 kDa to the molecular mass. The enzyme was most active at pH 3.8 with an optimal temperature of 45 °C for the hydrolysis reaction. The K_m and V_max values were 0.385 mM and 0.0296 mM/min, respectively. The transglycosylation activity was studied using either free fucose or pNP-fucose as donor and lactose as acceptor. Reaction products were analysed with LC-MS and HPLC. LC-MS results showed that fucosyllactose was detected in very low concentrations. This was probably due to the possible hydrolysis of the molecule of interest, formation of different products, or due to the very strict substrate specificity from the enzyme. Nevertheless, the HPLC analysis showed that about 20% of the initial substrate was consumed, suggesting that a reaction is taking place. Hence, further research needs to be conducted in order to determine if the studied α-fucosidase can produce HMOs or different kind of molecules through transglycosylation reaction. en
dc.format.extent 88+3
dc.language.iso en en
dc.title Production of fucosyllactose with a fucosidase from Aspergillus niger en
dc.type G2 Pro gradu, diplomityö fi Kemian tekniikan korkeakoulu fi
dc.subject.keyword fucosyllactose en
dc.subject.keyword human milk oligosaccharides en
dc.subject.keyword alpha-fucosidase en
dc.subject.keyword transglycosylation en
dc.identifier.urn URN:NBN:fi:aalto-201709046889
dc.programme.major Biosystems and Biomaterials Engineering fi
dc.programme.mcode CHEM3028 fi
dc.type.ontasot Master's thesis en
dc.type.ontasot Diplomityö fi
dc.contributor.supervisor Frey, Alexander
dc.programme Master’s Programme in Life Science Technologies fi
dc.ethesisid Aalto 9544
dc.location P1

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