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Sasaki et al. Microbiome Res Rep 2023;2:12 https://dx.doi.org/10.20517/mrr.2023.08 Page 3 of 15

METHODS
Materials
Gum arabic AGP was obtained from Sigma-Aldrich (St. Louis, MO, USA). Larch AGP was purchased from
Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Sugar beet arabinan was obtained from Megazyme
(Wicklow, Ireland). The polysaccharides were purified via ethanol precipitation of the aqueous solutions to
remove releasing sugars with low molecular weight from the original reagent. Wheat AGP was purified
[14]
according to the method described by Fincher et al. with some modifications . Briefly, wheat flour was
dried and heated to remove endogenous enzymes, and extraction was performed with 80% ethanol under
reflux for 30 min. Ethanol-insoluble residues were collected on a glass fiber filter and resolved in water at
37 °C for 60 min. After centrifugation, the water-soluble fraction was digested with α-amylase and
amyloglucosidase to remove starch. After dialysis, the solution was mixed gradually with ethanol until a
final concentration of 65% to separate arabinoxylan and AGP. The supernatant containing AGP was
collected, and ethanol was added to achieve a final concentration of 80%. The precipitate was collected via
glass fiber filtration, washed several times, and resolved in water. The oligosaccharides (S3-GA, S3-AA, and
S5-GA) were obtained from the supernatant following the ethanol precipitation of gum arabic reagent ,
[8]
based on the method described by Tischer et al. β-L-Arap-(1→3)-α-L-Araf-OMe and α-D-Galp-(1→3)-α-
[15]
L-Araf-OMe were prepared via the transglycosylation of GAfase with gum arabic and larch AGP,
[8]
respectively . pET23d_GAfase plasmid and recombinant GAfase were prepared as described previously .
[8]
Expression and purification of recombinant AAfase
The sequence of MCC10289_0425 was obtained from the genome of B. pseudocatenulatum MCC10289
(F03Father01; accession number: SAMN09671256) . The codon-optimized sequence of MCC10289_0425
[16]
without the N-terminal signal peptide (SP; aa 1-28) and with C-terminal His-tag and N-terminal SKIK-tag
was synthesized and cloned into a pET23a vector via GenScript (Nanjing, China). The synthesized pET23a-
MCC10289_0425 plasmid was transformed into Escherichia coli BL21 (DE3) pLysS cells (BioDynamics
Laboratory, Tokyo, Japan), which were cultured at 37 °C for 3 h. The cells were induced with 1 mM IPTG at
15 °C for 44 h. The cultured cells were centrifuged, and the resultant pellet was resuspended in xTractor™
buffer. The target protein was purified using Capturem™ His-Tagged Purification Maxiprep columns
(Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. Desalination and
concentration of the target protein were performed using ultrafiltration membranes [molecular-weight
cutoff (MWCO), 10 kDa; Millipore Co., Billerica, MA, USA].

Enzyme assay
The reactivity of the recombinant enzyme was analyzed using the following polysaccharide substrates: gum
arabic, larch, wheat AGP, and sugar beet arabinan. The hydrolytic activity of AAfase was evaluated using
polysaccharides (1.0%) in 40 µL of 50 mM sodium acetate buffer (pH 5.5). After incubating the mixture at
37 °C for 16 h, the reaction products were analyzed via thin layer chromatography (TLC) and high-
performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). For
TLC analysis, the reaction products were spotted on a silica gel 60 aluminum plate (Merck, Darmstadt,
Germany) with an n-propanol/ethanol/water mixture [7:1:2 (v/v/v)]. Sugars were visualized by spraying the
orcinol-sulfate reagent on the plates . For HPAEC-PAD, the reaction products were analyzed via
[17]
CarboPac PA-1 columns (φ, 4 mm × 250 mm; Dionex Corp., Sunnyvale, CA, USA) with a flow rate of 1.0
mL/min using the following gradient: 0-5 min, 100% eluent A (0.1 M NaOH); 5-30 min, 0%-100% eluent B
(0.5 M sodium acetate and 0.1 M NaOH); and 30-35 min, 100% eluent B.

The hydrolytic activity against oligosaccharides (S3-AA, S3-GA, and S5-GA) was analyzed as follows.
Briefly, oligosaccharides (final concentration: 1.25 µM) were incubated with 0.05 µg/mL AAfase in 50 mM
MES buffer (pH 6.5) or 0.05 µg/mL GAfase in 50 mM sodium acetate buffer (pH 4.5) at 37 °C for 24 h. The