Mutant Glycosidases with Altered Substrate Specificity and Their Analysis

β-N-Acetylhexosaminidases (EC 3.2.1.52, GH 20) are enzymes are typical retaining exo-glycosidases that in vivo cleavage both β-N-acetylglucosamine (GlcNAc) or β-N-acetylgalactosamine (GalNAc) residues fom glycostructures [1]. Under suitable reaction conditions, these enzymes are able to synthesize the glycosidic bond in good yields. Substitution of selected amino acid(s) in the active site by site-direct mutagenesis may change the enzyme's substrate specificity or suppress the hydrolytic activity of the enzyme in favor of synthesis [2].

We present here three new mutant variants the of β-N-acetylhexosaminidase from Talaromyces flavus (TfHex) with altered substrate specificity. Genetic engineering afforded selective enzymes carrying either GlacNAcase or GalNAcase activity. The respective point mutations were identified by molecular modeling. Site-directed mutants were prepared, expressed in Pichia pastoris and purified to homogeneity. The pH and temperature optimum and the kinetic parameters of enzymes were determined. The synthetic capabilities of the prepared mutants were demonstrated in transglycosylation reactions.