ABSTRACT
ß-N-acetylglucosaminidases from the family 84 of glycoside hydrolases form a small group of glycosidases in eukaryotes responsible for the modification of nuclear and cytosolic proteins with O-GlcNAc, thus they are involved in a number of important cell processes. Here, the first fungal ß-N-acetylglucosaminidase from Penicillium chrysogenum was expressed in Pichia pastoris and secreted into the media, purified and characterized. Moreover, homology modeling and substrate and inhibitor docking were performed to obtain structural information on this new member of the GH84 family. Surprisingly, we found that this fungal ß-N-acetylglucosaminidase with its sequence and structure perfectly fitting to the GH84 family displays biochemical properties rather resembling the ß-N-acetylhexosaminidases from the family 20 of glycoside hydrolases. This work helped to increase the knowledge on the scarcely studied glycosidase family and revealed a new type of eukaryotic ß-N-acetylglucosaminidase.
Subject(s)
Acetylglucosaminidase/isolation & purification , Penicillium chrysogenum/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Acetylglucosaminidase/chemistry , Acetylglucosaminidase/genetics , Acetylglucosaminidase/metabolism , Amino Acid Sequence , Molecular Docking Simulation , Molecular Sequence Data , Penicillium chrysogenum/enzymology , Pichia/genetics , Pichia/metabolism , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence AlignmentABSTRACT
The ß-N-acetylhexosaminidase from Talaromyces flavus has a remarkable synthetic ability, processing even carbohydrates with various functionalities. Its broader use is partially hampered by low-yield production in the native fungus. Here, we present an optimized 3-day production of this enzyme in the eukaryotic host of Pichia pastoris, in ca 10-fold higher volume activity (10 U/ml) and close-to-perfect purity (one chromatographic step needed). Importantly, the recombinant enzyme features the same biochemical and catalytic properties, including the syntheses with derivatized carbohydrate substrates. This is the first example of the overexpression of a fungal ß-N-acetylhexosaminidase by a single-cell producer in liquid medium. It represents a promising solution for wider biotechnological applications of this outstanding enzyme.
