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

Enzymatic synthesis is an elegant biocompatible approach to complex compounds such as human milk oligosaccharides (HMOs). These compounds are vital for healthy neonatal development with a positive impact on the immune system. Although HMOs may be prepared by glycosyltransferases, this pathway is often complicated by the high price of sugar nucleotides, stringent substrate specificity, and low enzyme stability. Engineered glycosidases (EC 3.2.1) represent a good synthetic alternative, especially if variations in the substrate structure are desired. Site-directed mutagenesis can improve the synthetic process with higher yields and/or increased reaction selectivity. So far, the synthesis of human milk oligosaccharides by glycosidases has mostly been limited to analytical reactions with mass spectrometry detection. The present work reveals the potential of a library of engineered glycosidases in the preparative synthesis of three tetrasaccharides derived from lacto-N-tetraose (Galβ4GlcNAcβ3Galβ4Glc), employing sequential cascade reactions catalyzed by β3-N-acetylhexosaminidase BbhI from Bifidobacterium bifidum, β4-galactosidase BgaD-B from Bacillus circulans, β4-N-acetylgalactosaminidase from Talaromyces flavus, and β3-galactosynthase BgaC from B. circulans. The reaction products were isolated and structurally characterized. This work expands the insight into the multi-step catalysis by glycosidases and shows the path to modified derivatives of complex carbohydrates that cannot be prepared by standard glycosyltransferase methods.

• Keywords
• enzymatic synthesis, glycosidase, human milk oligosaccharide, mutagenesis,
• MeSH
• Bifidobacterium bifidum * metabolism   MeSH
• Glycoside Hydrolases metabolism   MeSH
• Glycosyltransferases metabolism   MeSH
• Humans MeSH
• Milk, Human * metabolism   MeSH
• Infant, Newborn MeSH
• Oligosaccharides chemistry   MeSH
• Substrate Specificity MeSH
• Check Tag
• Humans MeSH
• Infant, Newborn MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Glycoside Hydrolases MeSH
• Glycosyltransferases MeSH
• Oligosaccharides MeSH

Biotransformation has accompanied mankind since the Neolithic community, when people settled down and began to engage in agriculture [...].

• MeSH
• Bacteria enzymology   MeSH
• Biocatalysis * MeSH
• Biosensing Techniques MeSH
• Biotransformation MeSH
• Glycomics MeSH
• Fungi enzymology   MeSH
• Humans MeSH
• Agriculture MeSH
• Check Tag
• Humans MeSH
• Publication type
• Introductory Journal Article MeSH
• Editorial MeSH