title: Sequencing, cloning and high-yield expression of a fungal β-N-acetylhexosaminidase in Pichia pastoris.
abstract: The β-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 β-N-acetylhexosaminidase by a single-cell producer in liquid medium. It represents a promising solution for wider biotechnological applications of this outstanding enzyme.
authors.affiliation: Institute of Microbiology, Center for Biocatalysis and Biotransformation, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ 14220, Prague 4, Czech Republic.
authors.forename: Kristýna
authors.initials: K
authors.lastname: Slámová
authors.forename: Pavla
authors.initials: P
authors.lastname: Bojarová
authors.forename: Daniela
authors.initials: D
authors.lastname: Gerstorferová
authors.forename: Barbora
authors.initials: B
authors.lastname: Fliedrová
authors.forename: Jana
authors.initials: J
authors.lastname: Hofmeisterová
authors.forename: Martin
authors.initials: M
authors.lastname: Fiala
authors.forename: Petr
authors.initials: P
authors.lastname: Pompach
authors.forename: Vladimír
authors.initials: V
authors.lastname: Křen
mesh_terms: D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D032901:Talaromyces / Q000201:enzymology* / Q000235:genetics; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D001619:beta-N-Acetylhexosaminidases