*4.8. Construction of In Vitro Alginate-Metabolizing System from Recombinant Enzymes*

An in vitro alginate-metabolizing system was constructed using recombinant alginate lyases (recFlAlyA, recFlAlyB, and recFlAlex) [26,27], recombinant DEH reductase (recFlRed) [28], and recFlKin and recFlAld prepared in the present study. Alginate-metabolizing reaction was conducted at 25 ◦C in a mixture containing 0.2% (*w*/*v*) sodium alginate, 10 mM NADH, 10 mM ATP, 10 mM MgCl2, 20 mM sodium phosphate (pH 7.4), 1 mM DTT, and various combinations of recFlAlyA, recFlAlyB, recFlAlex, recFlRed, recFlKin, and recFlAld with each final concentration at 10 μg/mL, 10 μg/mL, 10 μg/mL, 2.5 μg/mL, 10 μg/mL, and 1 μg/mL, respectively. After 12-h reaction, unsaturated oligo-alginates, DEH, and KDG, were analyzed by TLC and TBA reaction [52]. KDPG and pyruvate concentrations were determined by the LDH–NADH reaction.

#### *4.9. Determination of Unsaturated Sugars*

Unsaturated sugars were determined by the TBA method [52]. The sample containing unsaturated sugars (150 μL) was mixed with 150 μL of 20 mM NaIO4–0.125 M H2SO4 and allowed to react for 1 h on ice. Then, 100 μL of NaAsO2–0.5 N HCl was added to the mixture and incubated for 10 min at room temperature. To the mixture, 600 μL of 0.6% (*w*/*v*) TBA was added and heated for 10 min at 100 ◦C. The unsaturated sugars were determined by measuring Abs 548 nm, adopting the absorption coefficient for DEH and KDG, <sup>ε</sup> = 41 × 103 <sup>M</sup>−1·cm−1, which we determined in the present study using KDG and DEH standards.

#### *4.10. Thin-Layer Chromatography*

TLC silica gel 60 plate was used for the analysis of the reaction products produced by recFlKin and recFlAld. The reaction product of recFlKin was prepared with a reaction mixture containing 2.5 mM KDG and 2.5 mM ATP and 200 μg/mL recFlKin. The reaction was carried out at 30 ◦C for 0–15 min and terminated by heating at 100 ◦C for 2 min. Four microliters of each reaction mixture was applied to a TLC plate. The reaction product was developed with 1-butanol:acetic acid:water = 2:1:1 (*v*:*v*:*v*) and detected by heating at 130 ◦C for 10 min after spraying 10% (*w*/*v*) sulfuric acid–90% (*w*/*v*) ethanol. The reaction product of recFlAld was prepared with a reaction mixture containing 5 mM KDPG and 1 μg/mL recFlAld. After the reaction at 30 ◦C for 0–15 min, six microliters of the reaction

mixture were applied to TLC plate and developed with the same solvent as described above. The reaction product on the plate was detected with 0.5% (*w*/*v*) 2,5-dinitrophenylhydrazine (DNP)–20% (*v*/*v*) sulfuric acid–60% (*v*/*v*) ethanol. In case of unsaturated sugars, they were visualized with 4.5% (*w*/*v*) TBA after the periodic acid treatment.

### *4.11. Mass Spectrometry*

Phosphorylation of KDG by recFlKin was detected by mass spectrometry. The KDG phosphorylated by recFlKin in the conditions described in Section 4.10 was mixed with 6.7 mg/mL 9-aminoacridine–methanol at 1:3 (*v*:*v*). One microliter of the mixture was applied to a sample plate and air-dried at room temperature. The sample was subjected to a matrix-assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOF-MS) (Proteomics Analyzer 4700, Applied Biosystems, Foster City, CA, USA) and analyzed in a negative-ion mode.
