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Microbiology 154 (2008), 2719-2729; DOI  10.1099/mic.0.2008/018978-0
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Microbiology 154 (2008), 2719-2729; DOI  10.1099/mic.0.2008/018978-0
© 2008 Society for General Microbiology

Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis

José Manuel Inácio1, Isabel Lopes Correia1 and Isabel de Sá-Nogueira1,2

1 Laboratory of Microbial Genetics, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, Apt 127, 2781-901 Oeiras, Portugal
2 Departamento de Ci�ncias da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal

Correspondence
Isabel de Sá-Nogueira
sanoguei{at}itqb.unl.pt

Bacillus subtilis produces {alpha}-L-arabinofuranosidases (EC 3.2.1.55; AFs) capable of releasing arabinosyl oligomers and L-arabinose from plant cell walls. Here, we show by insertion-deletion mutational analysis that genes abfA and xsa(asd), herein renamed abf2, encode AFs responsible for the majority of the intracellular AF activity in B. subtilis. Both enzyme activities were shown to be cytosolic and functional studies indicated that arabino-oligomers are natural substrates for the AFs. The products of the two genes were overproduced in Escherichia coli, purified and characterized. The molecular mass of the purified AbfA and Abf2 was about 58 kDa and 57 kDa, respectively. However, native PAGE gradient gel analysis and cross-linking assays detected higher-order structures (>250 kDa), suggesting a multimeric organization of both enzymes. Kinetic experiments at 37 °C, with p-nitrophenyl-{alpha}-L-arabinofuranoside as substrate, gave an apparent Km of 0.498 mM and 0.421 mM, and Vmax of 317 U mg–1 and 311 U mg–1 for AbfA and Abf2, respectively. The two enzymes displayed maximum activity at 50 °C and 60 °C, respectively, and both proteins were most active at pH 8.0. AbfA and Abf2 both belong to family 51 of the glycoside hydrolases but have different substrate specificity. AbfA acts preferentially on (1->5) linkages of linear {alpha}-1,5-L-arabinan and {alpha}-1,5-linked arabino-oligomers, and is much less effective on branched sugar beet arabinan and arabinoxylan and arabinogalactan. In contrast, Abf2 is most active on (1->2) and (1->3) linkages of branched arabinan and arabinoxylan, suggesting a concerted contribution of these enzymes to optimal utilization of arabinose-containing polysaccharides by B. subtilis.

Abbreviations: ABN, {alpha}-1,5-arabinanase; AF, {alpha}-L-arabinofuranosidase; GH 51, glycoside hydrolase family 51; pNPAf, p-nitrophenyl-{alpha}-L-arabinofuranoside; pNPAp, p-nitrophenyl-{alpha}-L-arabinopyranoside; pNPXp, p-nitrophenyl-β-D-xylopyranoside

The GenBank/EMBL/DDBJ accession number for the nucleotide sequence of the abf2 gene reported in this paper is EU073712.






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