β-N-Acetylhexosaminidase from Talaromyces flavus (TfHex; EC 3.2.1.52) is an exo-glycosidase with dual activity for cleaving N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared a library of ten mutant variants with their substrate specificity significantly shifted towards GlcNAcase activity. Suitable mutations were identified by in silico methods. We optimized a microtiter plate screening method in the yeast Pichia pastoris expression system, which is required for the correct folding of tetrameric fungal β-N-acetylhexosaminidases. While the wild-type TfHex is promiscuous with its GalNAcase/GlcNAcase activity ratio of 1.2, the best single mutant variant Glu332His featured an 8-fold increase in selectivity toward GlcNAc compared with the wild-type. Several prepared variants, in particular Glu332Thr TfHex, had significantly stronger transglycosylation capabilities than the wild-type, affording longer chitooligomers - they behaved like transglycosidases. This study demonstrates the potential of mutagenesis to alter the substrate specificity of glycosidases.

• Keywords
• Pichia pastoris, Talaromyces flavus, site-directed mutagenesis, site-saturation mutagenesis, substrate specificity, β-N-acetylhexosaminidase,
• MeSH
• Acetylgalactosamine metabolism   MeSH
• Acetylglucosamine * metabolism   MeSH
• Acetylglucosaminidase MeSH
• beta-N-Acetylhexosaminidases * metabolism   MeSH
• Kinetics MeSH
• Mutation MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetylgalactosamine MeSH
• Acetylglucosamine * MeSH
• Acetylglucosaminidase MeSH
• beta-N-Acetylhexosaminidases * MeSH