Mutation Hotspot for Changing the Substrate Specificity of β- N -Acetylhexosaminidase: A Library of GlcNAcases.

β- N -Acetylhexosaminidase from Talaromyces flavus ( Tf Hex; 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 Tf Hex 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 Tf Hex, 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.