β-N-Acetylhexosaminidases (CAZy GH20, EC 3.2.1.52) are exo-glycosidases specific for cleaving N-acetylglucosamine and N-acetylgalactosamine moieties of various substrates. The β-N-acetylhexosaminidase from the filamentous fungus Talaromyces flavus (TfHex), a model enzyme in this study, has a broad substrate flexibility and outstanding synthetic ability. We have designed and characterized seven glycosynthase-type variants of TfHex mutated at the catalytic aspartate residue that stabilizes the oxazoline reaction intermediate. Most of the obtained enzyme variants lost the majority of their original hydrolytic activity towards the standard substrate p-nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside (pNP-β-GlcNAc); moreover, the mutants were not active with the proposed glycosynthase donor 2-acetamido-2-deoxy-d-glucopyranosyl-α-fluoride (GlcNAc-α-F) either as would be expected in a glycosynthase. Importantly, the mutant enzymes instead retained a strong transglycosylation activity towards the standard substrate pNP-β-GlcNAc. In summary, five out of seven prepared TfHex variants bearing mutation at the catalytic Asp370 residue acted as efficient transglycosidases, which makes them excellent tools for the synthesis of chitooligosaccharides, with the advantage of processing an inexpensive, stable and commercially available pNP-β-GlcNAc.

Center for Nanobiology and Structural Biology Institute of Microbiology of the Czech Academy of Sciences Zámek 136 Nové Hrady CZ 37333 Czech Republic

Institute of Biotechnology Slovak University of Technology Radlinského 9 Bratislava SK 81237 Slovakia

Laboratory of Biotransformation Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 Prague 4 CZ 14220 Czech Republic

Laboratory of Biotransformation Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 Prague 4 CZ 14220 Czech Republic ; Department of Biochemistry University of Chemistry and Technology Prague Technická 6 Prague 6 CZ 16000 Czech Republic

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Three-Step Enzymatic Remodeling of Chitin into Bioactive Chitooligomers

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