Abstract
Arabinoxylan (AX) and arabinoxylooligosaccharides (AXOs) are carbohydrate sources utilized by Bifidobacterium longum subsp. longum. However, their degradation pathways are poorly understood. In this study, we characterized two genes, BLLJ_1850 and BLLJ_1851, in the hemicellulose-degrading gene cluster (BLLJ_1836–BLLJ_1859) of B. longum subsp. longum JCM 1217. Both recombinant enzymes expressed in Escherichia coli exhibited exo-α-l-arabinofuranosidase activity toward p-nitrophenyl-α-l-arabinofuranoside. BlArafE (encoded by BLLJ_1850) contains the glycoside hydrolase family 43 (GH43), subfamily 22 (GH43_22), and GH43_34 domains. The BlArafE GH43_22 domain was demonstrated to release α1,3-linked Araf from AX, but the function of BlArafE GH43_34 could not be clearly identified in this study. BlArafD (encoded by BLLJ_1851) contains GH43 unclassified subfamily (GH43_UC) and GH43_26 domains. The BlArafD GH43_UC domain showed specificity for α1,2-linked Araf in α1,2- and α1,3-Araf double-substituted structures in AXOs, while BlArafD GH43_26 was shown to hydrolyze α1,5-linked Araf in the arabinan backbone. Co-incubation of BlArafD and BlArafE revealed that these two enzymes sequentially removed α1,2-Araf and α1,3-Araf from double-substituted AXOs in this order. B. longum strain lacking BLLJ_1850–BLLJ_1853 did not grow in the medium containing α1,2/3-Araf double-substituted AXOs, suggesting that BlArafE and BlArafD are important for the assimilation of AX.
Key points
• BlArafD GH43 unclassified subfamily domain is a novel α1,2- l -arabinofuranosidase.
• BlArafE GH43 subfamily 22 domain is an α1,3-l-arabinofuranosidase.
• BlArafD and BlArafE cooperatively degrade α1,2/3-Araf double-substituted arabinoxylan.
Graphical abstract
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Data availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
We thank Prof. Takane Katayama (Kyoto University, Japan) for providing E. coli BL21(λDE3)ΔlacZ.
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This work was supported by JSPS KAKENHI grant number 18K05494 (to HA).
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HA and KF conceived and designed the study. MK, YY, JK, WN, KM, and YS conducted the experiments. MK, YY, JK, KF, and HA analyzed the data. MK and HA wrote the manuscript. All authors read and approved the manuscript.
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Komeno, M., Yoshihara, Y., Kawasaki, J. et al. Two α-l-arabinofuranosidases from Bifidobacterium longum subsp. longum are involved in arabinoxylan utilization. Appl Microbiol Biotechnol 106, 1957–1965 (2022). https://doi.org/10.1007/s00253-022-11845-x
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DOI: https://doi.org/10.1007/s00253-022-11845-x