Purification and functional characterization of a novel α-L-arabinofuranosidase from Bifidobacterium longum B667

The gene encoding a novel alpha-L-arabinofuranosidase from Bifidobacterium longum B667, abfB, was cloned and sequenced. The deduced protein had a molecular mass of about 61 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with alpha-L-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. Regions flanking the gene comprised two divergently transcribed open reading frames coding for hypothetical proteins involved in sugar metabolism. A histidine tag was introduced at the C terminus of AM, and the recombinant protein was overexpressed in Lactococcus lactis under control of the tightly regulated, nisin-inducible nisA promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 260 kDa, suggesting a homotetrameric structure. AbfB was active at a broad pH range (pH 4.5 to 7.5) and at a broad temperature range (20 to 70degreesC), and it had an optimum pH of 6.0 and an optimum temperature of 45degreesC. The enzyme seemed to be less thermostable than most previously described arabinofuranosidases and had a half-life of about 3 h at 55degreesC. Chelating and reducing agents did not have any effect on its activity, but the presence Of Cu2+, Hg2+, and Zn2+ markedly reduced enzymatic activity. The protein exhibited a high level of activity with p-nitrophenyl alpha-L-arabinofuranoside, with apparent K-m and V-max values of 0.295 mM and 417 U/mg, respectively. AbfB released L-arabinose from arabinan, arabinoxylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. No endoarabinanase activity was detected. These findings suggest that AbfB is an exo-acting enzyme and may play a role, together with other glycosidases, in the degradation of L-arabinose-containing polysaccharides.