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Sasaki et al. Microbiome Res Rep 2023;2:12 https://dx.doi.org/10.20517/mrr.2023.08 Page 5 of 15

Table 1. Primers for site-directed mutagenesis
Sequence
GAfase_mut_for 5′-CTTCAGGATTGGTACCCGGA-3’
N119Ymut_rev 5′-ATAGACCACCAGCTCCTCG-3’
N119Dmut_rev 5′-ATCGACCACCAGCTCCTCG-3’

reaction was terminated by adding 5% TCA to one-fifth of the mixture. The diluted products were analyzed
via HPAEC-PAD using CarboPac PA-1 columns (φ, 4 mm × 250 mm), as described above. One unit of
enzyme activity was defined as the amount of enzyme required to produce 1 μmol α-D-Galp-(1→3)-L-Ara or
β-L-Arap-(1→3)-L-Ara per min.

In vitro assimilation test of β-L-Arap-(1→3)-L-Ara and larch AGP using Bifidobacterium
The three strains of B. pseudocatenulatum (MCC10289, MCC10285, and MCC10311) and one strain of B.
kashiwanohense (MCC10250) used in the assimilation tests of β-L-Arap-(1→3)-L-Ara and larch AGP were
obtained from the stock cultures maintained at the Morinaga Milk Industry Co., Ltd., Zama, Japan. The
genome sequences have been submitted to GenBank and the accession number are as follows: B.
[16]
pseudocatenulatum MCC10285 (F02Son03), SAMN09671251; B. pseudocatenulatum MCC10311
(F04Father01), SAMN09671277; and B. kashiwanohense MCC10250 (F01Mother01), SAMN09671224. B.
pseudocatenulatum JCM1200 was obtained from the Japan Collection of Microorganisms (RIKEN
T
Bioresource Center, Ibaraki, Japan). The strains were precultured at 37 °C under anaerobic conditions in
MRS broth containing 0.05% L-cysteine hydrochloride (MRS + Cys) using an AnaeroPack system
(Mitsubishi Gas Chemical, Tokyo, Japan). The precultured cells were inoculated into the MRS + Cys
medium containing 1% L-arabinose and 1% larch AGP and incubated at 37 °C under anaerobic conditions
until all strains showed adequate growth to induce gene expression prior to primary cultivation.
Subsequently, the precultured cells were inoculated into the MRS + Cys medium containing 0.2% β-L-Arap-
(1→3)-L-Ara as a sole carbon source and were cultured for 48 h at 37 °C under anaerobic conditions. The
absorbance of each culture medium was measured at time points of 17, 24, 41, and 48 h and a wavelength of
600 nm. The experiment was performed only once because of the insufficient sugar source. For the
assimilation test of larch AGP, bacterial cells were precultured (as described above), inoculated into MRS +
Cys medium containing 5% larch AGP, and cultured for 48 h at 37 °C under anaerobic conditions.
Absorbance was measured at time points of 18, 24, 42, and 48 h and a wavelength of 600 nm. The
experiment was performed in triplicates. To analyze residual sugar after cultivation, the supernatant of the
culture medium of β-L-Arap-(1→3)-L-Ara and larch AGP after 41 and 48 h, respectively, were analyzed
through TLC and HPAEC-PAD using CarboPac PA-1 columns (φ, 4 mm × 250 mm), as mentioned above.
To assess the enzymatic activity of bacterial cells and the supernatant of culture medium, pellets and
supernatant of all strains cultured in MRS + Cys medium containing larch AGP as a sole carbon source for
18 h were collected and incubated with larch AGP in 50 mM sodium acetate buffer (pH 6.0) for 22 h. The
reaction products were analyzed via TLC, as described above.

RESULTS
Gene cluster including GH39 and GH36 in B. pseudocatenulatum
We previously characterized GH39 GAfase and GH36 α-galactosidase in the gene cluster for utilizing gum
arabic AGP in B. longum JCM7052 [8,21] . A homology search of GAfase revealed that GH39 candidates were
conserved in other Bifidobacterium species. Based on sequence identity and peripheral genetic composition,
a GH39 candidate in B. pseudocatenulatum was expected to exhibit a different function from GAfase. In this
study, we focused on a gene cluster in B. pseudocatenulatum MCC10289 containing the GH39 GAfase
homolog MCC10289_0425 named “3-O-β-L-arabinopyranosyl-α-L-arabinofuranosidase (AAfase)”