Production of fucosyllactose with a fucosidase from Aspergillus niger

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.