alpha-l-Fucosidase from Paenibacillus thiaminolyticus: Its hydrolytic and transglycosylation abilities

作者：Benesova Eva^*; Lipovova Petra; Dvorakova Hana; Kralova Blanka

来源：Glycobiology, 2013, 23(9): 1052-1065.

DOI：10.1093/glycob/cwt041

• alpha-l-fucosidase
• l-fucose
• Paenibacillus thiaminolyticus
• transglycosylation

In this work, focused on possible application of alpha-l-fucosidases from bacterial sources in the synthesis of alpha-l-fucosylated glycoconjugates, several nonpathogenic aerobic bacterial strains were screened for alpha-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 alpha-l-fucopyranoside. The gene encoding alpha-l-fucosidase was found using the genomic library of P. thiaminolyticus constructed in the cells of Escherichia coli DH5 alpha 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 alpha-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 degrees C). By testing the ability of the enzyme to catalyze the transfer of alpha-l-fucosyl moiety to different types of acceptor molecules, it was confirmed that the enzyme is able to catalyze the formation of alpha-l-fucosylated p-nitrophenyl glycopyranosides containing alpha-d-galactopyranosidic, alpha-d-glucopyranosidic, alpha-d-mannopyranosidic or alpha-l-fucopyranosidic moiety. This enzyme is also able to catalyze alpha-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 alpha-l-fucosylated molecules representing compounds with potential application in biotechnology and the pharmaceutical industry.

• 出版日期2013-9