*4.2. Phylogenetic Analysis for KDG Kinases and KDPG Aldolases*

Phylogenetic analysis was carried out using the amino acid sequences of KDG kinases or KDPG aldolases from *Proteobacteria, Bacteroidetes* and *Archaea* currently available. *Bacteroidetes* enzymes used are from *Gramella forsetii* KT0803, *Lacinutrix* sp. 5H-3-7-4, and *Dokdonia* sp. MED134, which were reported to be located in the alginolytic gene cluster of each species [51]. These amino acid sequences were first aligned with the sequences of FlKin or FlAld by the ClustalW program, then aligned sequences were trimmed with GBlocks. Phylogenetic trees were generated by the maximum likelihood algorithm on the basis of the LG model implemented in the Molecular Evolutionary Genetics Analysis version 6.0 (MEGA 6) software. The bootstrap values were calculated from 1000 replicates.

### *4.3. Cloning, Expression, and Purification of Recombinant FlKin and FlAld*

Genomic DNA of strain UMI-01 was prepared with ISOHAIR DNA extraction kit (Nippon Gene, Tokyo, Japan). Coding regions of *flkin* and *flald,* 1023 bp and 669 bp, respectively, were amplified by PCR using specific primers including restriction sites, *Nco*I and *Bam*HI, in the 5 -terminal regions (Table 1). Genomic PCR was performed in a medium containing 10 ng of genomic DNA, 0.2 μM each primer, and Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA). The reaction

medium was preincubated at 95 ◦C for 2 min, and a reaction cycle of 95 ◦C for 10 s, 55 ◦C for 20 s, and 72 ◦C for 60 s was repeated 30 times. The PCR product was ligated to pCold I vector pre-digested by *Nco*I and *Bam*HI using In-Fusion cloning system (Clontech Laboratories, Mountain View, CA, USA). Insertion of the genes in the vector was confirmed by nucleotide sequencing with DNA sequencer 3130xl (Applied Biosystems, Foster, CA, USA). Recombinant enzymes, recFlKin and recFlAld, were expressed with the pCold I–*E. coli* BL21 (DE3) system. The transformed BL21 (DE3) was inoculated to 500 mL of 2× YT medium and cultivated at 37 ◦C for 16 h. Then, the temperature was lowered to 15 ◦C and isopropyl β-D-1-thiogalactopyranoside was added to make the final concentration of 0.1 mM. After 24-h induction, bacterial cells were harvested by centrifugation at 5000× *g* for 5 min and suspended in a buffer containing 10 mM imidazole-HCl (pH 8.0), 0.5 M NaCl, 1% (*v*/*v*) TritonX-100, and 0.01 mg/mL lysozyme. The suspension was sonicated at 20 kHz (30W) for a total of 4 min (30 s × 8 times with each 1 min interval) and centrifuged at 10,000× *g* for 10 min. The supernatant containing recombinant proteins was mixed with 1 mL of Ni-NTA resin and incubated for 30 min on ice with occasional suspension. The resin was set on a disposal plastic column (1 × 5 cm) and washed three times with 20 mL of 30 mM imidazole-HCl (pH 8.0)–0.5 M NaCl. The recombinant proteins adsorbed to the resin were eluted with 250 mM imidazole-HCl (pH 8.0)–0.5 M NaCl and collected as 1 mL fractions. The fractions containing the recombinant proteins were pooled and dialyzed against 20 mM Tris-HCl (pH 7.4)–0.1 M NaCl.

#### *4.4. Preparation of KDG*

KDG was prepared from alginate using the crude enzyme of the strain UMI-01 as follows; 0.5% (*w*/*v*) sodium alginate (50 mL) was digested at 30 ◦C with 1 mg/mL of the crude enzyme, which contains alginate lyases and other metabolic enzymes. NADH was added to the mixture to make the final concentration 10 mM to reduce DEH with DEH reductase contained in the crude enzyme. After 12 h, four volumes of −20 ◦C 2-propanol were added to terminate the reaction and the proteins and NADH precipitated were removed by centrifugation at 10,000× *g* for 10 min. The supernatant containing KDG was dried up in a rotary evaporator at 35 ◦C. The dried powder was dissolved in 50 mL of distilled water and subjected to a TOYOPEARL SuperQ-650S column (2.4 × 22 cm) equilibrated with distilled water. The absorbed KDG and trace amount of unsaturated disaccharide were separately eluted by a linear gradient of 0–0.2 M NaCl in distilled water (total 400 mL). Elution of KDG and unsaturated disaccharide was detected by TBA reaction. In this chromatography, KDG was eluted at around 80 mM NaCl, while disaccharides were eluted at around 120 mM. Approximately 90 mg of KDG was obtained from 0.25 g of sodium alginate.
