*2.3. Enzymatic Properties of recFlKin and recFlAld*

We first investigated the kinetic parameter for recFlAld, since recFlAld was necessary for the KDG kinase assay. In the present study, the kinase activity was assayed by quantifying the pyruvate produced from KDPG by the action of recFlAld. KDPG-derived pyruvate was determined by the lactate dehydrogenase (LDH)–NADH system as described in Section 4.6. In the equilibrium state of recFlAld reaction, pyruvate concentration reached 1.2 mM. Since the KDPG concentration was originally 5 mM, that in the equilibrate state was regarded as 3.8 mM. From these values the equilibrium constant (*K*eq) and <sup>Δ</sup>*G*◦ were calculated to be 3.8 × <sup>10</sup>−<sup>1</sup> M and +0.57 kcal/mol, respectively. This indicated that the equilibrium position of KDPG–aldolase reaction is slightly shifted toward the KDPG side. Next, we determined the reaction rate of recFlAld by the LDH–NADH method. By this method, the specific activity of recFlAld was estimated to be 57 U/mg at pH 7.4 and 30 ◦C. Coexistence of LDH–NADH in the reaction mixture could extend the aldolase reaction longer time by decreasing pyruvate concentration in the reaction equilibrium.

Next, KDG kinase activity of recFlKin was determined by using recFlAld and LDH–NADH. recFlKin was allowed to react with KDG in the presence of ATP at 30 ◦C and the reaction was terminated by heating at 100 ◦C for 3 min at the reaction times 1, 15, and 30 min. The KDPG produced in the reaction mixture was then split to pyruvate and GAP by recFlAld, and the pyruvate was quantified by the LDH–NADH system. At reaction time 90 min, recFlKin was found to produce 1.7 mM KDPG from 2.5 mM KDG at ~70% efficiency with the specific activity 0.72 U/mg. recFlKin showed an optimal temperature and pH at around 50 ◦C and 7.0, respectively, and was stable at 40 ◦C for 30 min.

#### *2.4. Construction of In Vitro Alginate-Metabolizing System Using Recombinant Enzymes*

In the present study, we identified *flkin* and *flald* in the genome of strain UMI-01 as KDG kinase and KDPG aldolase gene, respectively. Since alginate lyases and DEH reductase in this strain have already been characterized [26–28], here we examined if the sequential action of these alginate-degrading and -assimilating enzymes could convert alginate to pyruvate and GAP in vitro. Namely, recombinant alginate lyases (recFlAlyA, recFlAlyB, and recFlAlex) [26,27], DEH reductase (recFlRed) [28], KDG kinase (recFlKin), and KDPG aldolase (recFlAld) were allowed to react alginate in various combinations, and each reaction product was analyzed by TLC (Figure 8) and quantified by thiobarbituric acid (TBA) and LDH–NADH methods (Table 2). As shown in Figure 8, alginate was almost completely degraded to DEH by the simultaneous actions of recFlAlyA, recFlAlyB, and recFlAlex. The DEH was also almost completely reduced to KDG by recFlRed. Furthermore, a major part of the KDG was converted to KDPG by recFlKin, and the band of KDPG became faint by the reaction of recFlAld. This indicated the splitting of KDPG to pyruvate and GAP by the action of recFlAld. Accordingly, the sequential action of recombinant enzymes was considered to be capable of converting alginate to pyruvate and GAP in vitro. Then, the yields of intermediates in each reaction step were quantified by TBA and LDH–NADH methods (Table 2). Concentrations of the unsaturated oligo-alginates, DEH, KDG, KDPG, and pyruvate (and GAP), were determined to be 4.2 mM, 9.8 mM, 9.8 mM, 8.1 mM, and 3.8 mM, respectively. Since the initial concentration of alginate (0.2% (*w*/*v*)) corresponds to 10 mM monosaccharide, the yields of DEH and KDG were estimated to be ~100%, and the yields of KDPG and pyruvate were estimated to be ~80% and ~40%, respectively. These results indicated that high-value intermediates such as KDPG could be produced from alginate with fairly high efficiency by the recombinant enzymes of the strain UMI-01 in vitro.



<sup>a</sup> 0.2% (*w*/*v*) sodium alginate theoretically corresponds to 10 mM monosaccharide.

**Figure 8.** Construction of in vitro alginate-metabolizing system using recombinant enzymes. Alginate was allowed to react with recFlAlyA, recFlAlyB, recFlAlex, recFlRed, recFlKin, and recFlAld in various combinations at 25 ◦C for 12 h. The reaction products were subjected to TLC and detected by staining with 4.5% TBA. Presence and absence of each enzyme is indicated with '+' and '−', respectively. Detailed conditions are shown under Section 4.

#### **3. Discussion**

#### *3.1. Alginate-Metabolizing Enzymes of Flavobacterium* sp. *Strain UMI-01*

In the present study, *flkin* and *flald* in the genome of *Flavobacterium* sp. strain UMI-01 were confirmed to be the enzyme genes encoding KDG kinase and KDPG aldolase. The recombinant enzymes, recFlKin and recFlAld, showed KDG kinase and KDPG aldolase activity although low sequence identities were shown to the corresponding enzymes from other bacteria and archaea (Figures 2–4). Consequently, these genes, along with previously reported alginate lyase and DEH reductase genes were confirmed to be the genes responsible for alginate metabolism of this bacterium. The alginate-metabolizing pathway of this strain is summarized as in Figure 9. The alginate lyases degrade polymer alginate to unsaturated monomer (DEH) in the periplasmic space [24,25]. DEH reductase, KDG kinase and KDPG aldolase convert DEH to pyruvate and GAP in the cytosol. Therefore, DEH produced in the periplasmic space should be incorporated to the cytosol by certain transportation system(s). Such DEH transporters in this strain have not been identified yet; however, sugar permease-like gene *sugp* and membrane transporter-like genes *susc* and *susd* were found in the operons A and B, respectively (see Figure 1). Thus, the putative permease and transporters are also indicated in Figure 9. Another problem is how the expressions of alginate-metabolizing genes are regulated. We recently noticed that expression levels of alginate lyases were significantly low in the absence of alginate but strongly increased by the addition of alginate to the medium. This indicates that the expressions of alginate-metabolic enzymes are up-regulated by alginate. We are now searching regulatory genes for alginate-metabolizing enzyme genes in the UMI-01 strain genome.

**Figure 9.** Alginate-metabolic system of *Flavobacterium* sp. strain UMI-01.
