1283768-40-0Relevant articles and documents
β-D-Galactosidase from Paenibacillus thiaminolyticus catalyzing transfucosylation reactions
Benesova, Eva,Lipovova, Petra,Dvorakova, Hana,Kralova, Blanka
, p. 442 - 451 (2010)
A genomic library of bacterial strain Paenibacillus thiaminolyticus was constructed and the plasmid DNA of the clone, containing the gene encoding β-D-galactosidase with β-D-fucosidase activity, detected by 5-bromo-4-chloro-3-indoxyl β-D-galactopyranoside, was sequenced. Cells of Escherichia coli BL21 (DE3) were used for production of the enzyme in the form of a histidine-tagged protein. This recombinant fusion protein was purified using Ni-NTA agarose affinity chromatography and characterized by using p-nitrophenyl β-D-fucopyranoside (Km value of 1.18 ± 0.06 mmol/L), p-nitrophenyl β-D-galactopyranoside (Km value of 250 ± 40 mmol/L), p-nitrophenyl β-D-glucopyranoside (Km value of 77 ± 6 mmol/L), and lactose (Km value of 206 ± 5 mmol/L) as substrates. Optimal pH and temperature were estimated as 5.5 and 65°C, respectively. According to the amino acid sequence, the molecular weight of the fusion protein was calculated to be 68.6 kDa and gel filtration chromatography confirmed the presence of the enzyme in a monomeric form. In the following step, its ability to catalyze transfucosylation reactions was tested. The enzyme was able to catalyze the transfer of fucosyl moiety to different p-nitrophenyl glycopyranosides (producing p-nitrophenyl β-D-fucopyranosyl-(1,3)-β-D-fucopyranoside, p-nitrophenyl β-D-fucopyranosyl-(1,3)-α-D-glucopyranoside, p-nitrophenyl β-D-fucopyranosyl-(1,3)-α-D-mannopyranoside, and p-nitrophenyl β-D-fucopyranosyl-(1,6)-α-D-galactopyranoside) and alcohols (producing methyl β-D-fucopyranoside, ethyl β-D-fucopyranoside, 1-propyl β-D-fucopyranoside, 2-propyl β-D-fucopyranoside, 1-octyl β-D-fucopyranoside, and 2-octyl β-D-fucopyranoside). These results indicate the possibility of utilizing this enzyme as a promising tool for enzymatic synthesis of β-D-fucosylated molecules. The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.
