title: α-L-fucosidase from Paenibacillus thiaminolyticus: its hydrolytic and transglycosylation abilities.
abstract: In this work, focused on possible application of α-L-fucosidases from bacterial sources in the synthesis of α-L-fucosylated glycoconjugates, several nonpathogenic aerobic bacterial strains were screened for α-L-fucosidase activity. Among them Paenibacillus thiaminolyticus was confirmed as a potent producer of enzyme with the ability to cleave the chromogenic substrate p-nitrophenyl α-L-fucopyranoside. The gene encoding α-L-fucosidase was found using the genomic library of P. thiaminolyticus constructed in the cells of Escherichia coli DH5α and sequenced (EMBL database: FN869117, carbohydrate-active enzymes database: Glycosidase family 29). The enzyme was expressed in the form of polyhistidine-tagged protein (51.2 kDa) in Escherichia coli BL21 (DE3) cells, purified using nickel-nitrilotriacetic acid agarose affinity chromatography and characterized using the chromogenic substrate p-nitrophenyl α-L-fucopyranoside (K(m) = (0.44 ± 0.02) mmol/L, K(S) = (83 ± 8) mmol/L (substrate inhibition), pH(optimum) = 8.2, t(optimum) = 48°C). By testing the ability of the enzyme to catalyze the transfer of α-L-fucosyl moiety to different types of acceptor molecules, it was confirmed that the enzyme is able to catalyze the formation of α-L-fucosylated p-nitrophenyl glycopyranosides containing α-D-galactopyranosidic, α-D-glucopyranosidic, α-D-mannopyranosidic or α-L-fucopyranosidic moiety. This enzyme is also able to catalyze α-L-fucosylation of aliphatic alcohols of different lenghs of alkyl chain and hydroxyl group positions (methanol, ethanol, 1-propanol, 2-propanol and 1-octanol) and hydroxyl group-containing amino acid derivatives (N-(tert-butoxycarbonyl)-L-serine methyl ester and N-(tert-butoxycarbonyl)-L-threonine methyl ester). These results indicate the possibility of exploiting this enzyme in the synthesis of different types of α-L-fucosylated molecules representing compounds with potential application in biotechnology and the pharmaceutical industry.
authors.affiliation: Department of Biochemistry and Microbiology, ICT Prague, Prague, Czech Republic. eva.benesova@vscht.cz
authors.forename: Eva
authors.initials: E
authors.lastname: Benesová
authors.forename: Petra
authors.initials: P
authors.lastname: Lipovová
authors.forename: Hana
authors.initials: H
authors.lastname: Dvoráková
authors.forename: Blanka
authors.initials: B
authors.lastname: Králová
mesh_terms: D005643:Fucose / Q000096:biosynthesis / Q000378:metabolism; D006031:Glycosylation; D006868:Hydrolysis; D056507:Paenibacillus / Q000201:enzymology*; D005644:alpha-L-Fucosidase / Q000378:metabolism*
chemical_list: D005643:Fucose; D005644:alpha-L-Fucosidase
keywords: Paenibacillus thiaminolyticus; l-fucose; transglycosylation; α-l-fucosidase