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Page 4 of 15 Sasaki et al. Microbiome Res Rep 2023;2:12 https://dx.doi.org/10.20517/mrr.2023.08

reaction products were analyzed via HPAEC-PAD with CarboPac PA-1 columns (φ, 2 mm × 250 mm;
Dionex Corp., Sunnyvale, CA, USA) with a flow rate of 0.25 mL/min using the following gradient: 0-5 min,
40% eluent A (0.25 M NaOH), 0.5% eluent B (1 M sodium acetate), and 59.5% eluent C (ultrapure water); 5-
30 min, 40% eluent A, 0.5%-50% eluent B, and 59.5%-10% eluent C; and 30-35 min, 40% eluent A, 50%
eluent B, and 10% eluent C.

Kinetic analysis
The AAfase kinetic parameter was determined using 0.313-20.0 mg/mL larch AGP as a substrate. Larch
AGP was incubated with 35.3 ng/mL AAfase in 50 mM sodium acetate buffer (pH 6.0) at 45 °C for 20 min.
The reactions were terminated by adding 5% trichloroacetic acid (TCA) to one-fifth of the reaction mixture.
The products were analyzed via HPAEC-PAD using CarboPac PA-1 columns (φ, 4 mm × 250 mm). The
concentrations of the reaction products were calculated based on the peak areas.

Optimal pH and temperature
The optimal pH for enzyme activity was determined by using larch AGP as a substrate in 50 mM sodium
acetate (pH 3.5-6.0), MES (pH 5.5-7.0), and HEPES (pH 7.0-8.0) buffers at 45 °C. The optimal temperature
for enzyme activity was determined using 50 mM sodium acetate buffer (pH 6.5) at 25 °C-60 °C. The
samples were preincubated at each temperature for 5 min and were then incubated with the enzyme for 20
min at each temperature. The reactions were stopped by boiling the samples for 3 min, and the BCA reagent
was used to measure the content of reducing sugar .
[18]
Site-directed mutagenesis
A KOD Plus mutagenesis kit (Toyobo Co., Ltd., Osaka, Japan) was used to introduce N119Y and N119D
amino acid substitutions into pET23d_GAfase using specific primers [Table 1]. pET23d_GAfase_N119Y
and pET23d_GAfase_N119D mutants were cloned into E. coli BL21 (λDE3) cells (Nippon Gene, Toyama,
Japan), which were cultured at 37 °C for 2 h followed by 25 °C for 17 h using the Overnight Express
autoinduction system (Novagen Inc., Wisconsin, USA). The cultured cells were centrifuged, and the
resultant pellet was resuspended in BugBuster protein-extraction reagent (Novagen). The C-terminal His-
tagged GAfase N119Y protein was purified using a column containing Talon metal-affinity resin (Clontech,
CA, USA). Then, 25 and 50 mM imidazole fractions containing GAfase N119Y were desalted and
concentrated using an ultrafiltration membrane (MWCO, 10 kDa; Millipore Co., Billerica, MA, USA). Since
the activity of GAfase N119D was lost after desalting, we used a predesalted fraction containing imidazole to
assess GAfase N119D activity after the resuspended pellet was purified via Capturem™ His-Tagged
Purification Miniprep columns (Takara) according to the manufacturer’s instructions.

Construction of structural models using AlphaFold2
The structural models of AAfase and GAfase were predicted based on the protein sequence using
AlphaFold2 (ColabFold) with default settings (https://alphafold.ebi.ac.uk/) [19,20] . Visualization was performed
using PyMOL 2.5.0 (Schrödinger LLC, NY, USA).

Substrate specificity of AAfase, GAfase, and GAfase N119Y mutants
α-D-Galp-(1→3)-α-L-Araf-OMe and β-L-Arap-(1→3)-α-L-Araf-OMe (final concentration, 0.5 mM) were
incubated with the enzymes at appropriate concentrations at 45 °C. A 50 mM MES buffer (pH 6.5) was used
for AAfase and 50 mM sodium acetate buffer (pH 4.5) was used for GAfase and GAfase_N119Y. α-D-Galp-
(1→3)-α-L-Araf-OMe was incubated with AAfase (final concentration, 12.5 µg/mL) for 90 min and GAfase
(final concentration, 0.125 µg/mL) or GAfase_N119Y (final concentration, 12.5 µg/mL) for 20 min. β-L-Ara
p-(1→3)-α-L-Araf-OMe was incubated with AAfase (final concentration, 0.125 µg/mL) or GAfase_N119Y
(final concentration, 0.625 µg/mL) for 20 min and GAfase (final concentration, 12.5 µg/mL) for 90 min. The