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Page 10 of 15 Sasaki et al. Microbiome Res Rep 2023;2:12 https://dx.doi.org/10.20517/mrr.2023.08

Table 2. Substrate specificities of AAfase, GAfase, and GAfase N119Y mutant
AAfase GAfase GAfase N119Y
Substrate
(U/mg) (U/mg) (U/mg)
α-D-Galp-(1→3)-α-L-Araf-OMe 0.0776 33.1 0.363
β-L-Arap-(1→3)-α-L-Araf-OMe 15.1 0.0595 7.87

Figure 4. Comparison of amino acid residues at the catalytic site among Bifidobacterium GH39s. A: Comparison of amino acid residues
at the catalytic sites, according to a previous report [24] . Catalytic residues (red) and residues with variation (yellow) across GH39
sequences are presented. The phylogenetic tree depicted on the left was constructed via the neighbor-joining method based on aligned
Bifidobacterium GH39 sequences. GH39 sequences were aligned using the Clustal W program via MEGA11 software; B: The predicted
structures of AAfase (left) and GAfase (right) are depicted as a surface representation. AlphaFold2 (ColabFold) was used to obtain the
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structural model with default settings. Predicted catalytic residues are highlighted in red, and different amino acid residues (Tyr in
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AAfase and Asn in GAfase) are highlighted in yellow.
[Figure 5]. Among AAfase-carrier strains, proteins encoded in the AAfase gene cluster share over 99% of
sequence identity with the corresponding proteins from B. pseudocatenulatum MCC10289 [Figure 5A, right
table]. The in vitro assimilation test of β-L-Arap-(1→3)-L-Ara and larch AGP was performed by measuring
the increase in absorbance and conducting residual sugar analysis in the culture supernatant via TLC and
HPAEC-PAD. As a result, only B. pseudocatenulatum MCC10289 showed growth [Figure 5B, left], and
HPAEC-PAD analysis indicated that β-L-Arap-(1→3)-L-Ara was completely utilized by B.
pseudocatenulatum MCC10289 [Figure 5C]. Unexpectedly, other AAfase-carrier strains, B.
pseudocatenulatum MCC10285 and B. kashiwanohense MCC10250 did not grow and utilize β-L-Arap-(1→
3)-L-Ara. The in vitro assimilation test of larch AGP showed that MCC10289 exhibited higher absorbance
than B. pseudocatenulatum MCC10285, MCC10311, and JCM1200. As β-L-Arap-(1→3)-L-Ara was present
only in ~1.2% (w/w) of the total sugar content in larch AGP , the difference was not so large because of the
[8]
insufficient amount of β-L-Arap-(1→3)-L-Ara. Interestingly, B. kashiwanohense MCC10250, which does not
grow on β-L-Arap-(1→3)-L-Ara, showed good growth on larch AGP [Figure 5B], and some released
oligosaccharides were detected in the culture medium after 48 h via TLC [Supplementary Figure 5].