7699-38-9Relevant articles and documents
How Site-Directed Mutagenesis Boosted Selectivity of a Promiscuous Enzyme
Nekvasilová, Pavlína,Kulik, Natalia,Rychlá, Nikola,Pelantová, Helena,Petrásková, Lucie,Bosáková, Zuzana,Cva?ka, Josef,Slámová, Kristyna,K?en, Vladimír,Bojarová, Pavla
, p. 4138 - 4150 (2020/09/01)
β-N-Acetylhexosaminidases (GH20; EC 3.2.1.52) are exo-glycosidases with a dual activity for cleaving both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from glycostructures. This substrate promiscuity is a hurdle in the selective synthesis of N-acetylhexosamine oligosaccharides combining both GlcNAc and GalNAc units since there are hardly any GalNAc transferring enzymes available for synthetic applications. We present here site-directed mutagenesis of a synthetically potent promiscuous β-N-acetylhexosaminidase from Talaromyces flavus (TfHex), which, as a wild type, exhibits a GalNAcase/GlcNAcase ratio of 1.2. On the basis of molecular modeling, we identified crucial amino acid residues responsible for its GalNAcase/GlcNAcase selectivity. Six site-directed mutants were prepared, heterologously expressed in Pichia pastoris, purified, and kinetically characterized. As a result, novel engineered enzymes with an up to 7-times higher selectivity for either GalNAc or GlcNAc substrates were obtained, preserving the favorable properties of the wild type TfHex, mainly its transglycosylation potential and tolerance to functional groups in the substrate molecule. The substrate selectivity and transglycosylation yield were further corroborated by reaction engineering. The new selective and synthetically capable enzymes were applied in the preparation of tailored N-acetylhexosamines. (Figure presented.).
Synthesis of derivatized chitooligomers using transglycosidases engineered from the fungal GH20 β-N-acetylhexosaminidase
Slámová, Kristyna,Krejzová, Jana,Marhol, Petr,Kalachova, Lubica,Kulik, Natallia,Pelantová, Helena,Cva?ka, Josef,K?en, Vladimír
, p. 1941 - 1950 (2015/06/02)
Abstract The synthesis of oligosaccharides using mutant glycosidases has been dynamically developing due to the need for novel carbohydrate-based materials. Chitooligomers (β-1→4-linked oligomers of N-acetylglucosamine) are bioactive compounds applicable in many industrial and pharmacological areas; however, their accessibility is still rather low. In this work, GH20 β-N-acetylhexosaminidase from the fungus Talaromyces flavus was engineered by site-directed mutagenesis to obtain three efficiently transglycosylating variants with ca. 200-times suppressed hydrolytic activity. Thus, we have prepared the first GH20 transglycosidases. In the reactions catalyzed by these mutant β-N-acetylhexosaminidases we were able to easily prepare and isolate both natural and modified chitooligomers in sufficient amounts for their complete spectral characterization and possible further application. The presented method for the synthesis of chitooligomers with aglycones suitable for linking to other biological structures is simple and robust enough to be easily scaled up.
Properties and Transglycosylation Reaction of a Chitinase from Nocardia orientalis
Nanjo, Fumio,Sakai, Kazuo,Ishikawa, Masato,Isobe, Kiyoshi,Usui, Taichi
, p. 2189 - 2195 (2007/10/02)
The hydrolytic products of a chitinase purified from Nocardia orientalis were examined on reduced (GlcNAc)n (n=2-6).The rate of hydrolysis on reduced (GlcNAc)4-6 increased with increasing chain-length of N-acetylglucosamine residues,
