Mutant Glycosidases with Altered Substrate Specificity and Their Analysis

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Mutant Glycosidases with Altered Substrate Specificity and Their Analysis

Pavlína Nekvasilová 1,2 , Natalia Kulik 3 , Vladimír Křen 1 , Pavla Bojarová 1

1 Institute Of Microbiology, Czech Academy Of Sciences, Prague 4, Czech Republic, 2 Department of Analytical Chemistry, Charles University, Faculty of Science, Hlavova 2030/8, CZ-12843, Praha 2, Czech Republic, 3 Center for Nanobiology and Structural Biology, Institute of Microbiology of the Czech Academy of Sciences, Zámek 136, CZ-37333 Nové Hrady, Czech Republic

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.

Figure 1. A TfHex active site with docked thiazoline, an β-N-acetylhexosaminidase inhibitor [3]; B synthetic reaction yielding β-N-acetylhexosamines.

K. Slámová, P. Bojarová, L. Petrásková a V. Křen, Biotechnol. Adv. 2010, 28, 682-693.

P. Bojarová, N. Kulik, M. Hovorková, K. Slámová, H. Pelantová a V. Křen, Molecules 2019, 24, 599-613.

K. Slámová, J. Krejzová, P. Marhol, L. Kalachová, N. Kulik, H. Pelantová, J. Cvačka a V. Křen, Adv. Syn Cat. 2015, 357, 1941-1950

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