Glycosyl hydrolases β-d-galactosidase and α-l-fucosidase cloned from Paenibacillus thiaminolyticus, are interesting not only because of their hydrolase activity but mainly, for their transglycosylation activity, which is important for synthesis of interesting glycosylated molecules. At the beginning of this work, we had no detailed information about the structure of these enzymes, because none of these enzymes had been crystalized yet. In this project both enzymes were studied using the combination of theorethical and experimental methods. In the theorethical part, the structures of enzymes were predicted by homology modeling and further studies of these structures proposed the possible catalytic residues in both enzymes. The experimental part is focused to confirmation of the predictions of catalytic amino acid residues from the theoretical part. This poster presents the final results, where the mutated enzymes β-d-galactosidase_mut233, α-l-fucosidase_mut186 and α-l-fucosidase_mut239 were produced and purified. Furthermore β-d-galactosidase_mut157 was purified in earlier experiments. All mutants were tested on selected substrates. The results of these activity tests are helpfull for better understanding the catalytic machinery of studied enzymes and lead us to possibility of transforming them into glycosynthases i.e. modified glycosyl hydrolases that lack their natural hydrolytic activity because of mutation of nucleophilic amino acid residue, but maintain their transglycosylation activity.
The project was supported by COST actions MultiGlycoNano (CM1102, LD13024). Participation at the conference is supported by specific university research (MSMT No 20/2015, No MSMT 21/2015).