*3.2. Properties of recFlKin and recFlAld*

KDG kinase and KDPG aldolase are known to be the enzymes included in Entner–Doudoroff (ED) pathway. This pathway distributes over bacteria and archaea and play important roles in the metabolisms of glucuronate and glucose. In this pathway, KDG kinase phosphorylates KDG to KDPG, and KDPG aldolase split KDPG to pyruvate and GAP. Optimal temperature and pH of recFlKin were 50 ◦C and ~7.0, which were similar to those of KDG kinase from the bacteria *S. marcescens* [39]. While thermal stability of recFlKin was considerably low compared with the enzymes from archaea *S. tokodaii* [47] and *S. solfataricus* [40], e.g., these enzymes were stable up to 60–70 ◦C. recFlAld acts only on KDPG unlike archaea aldolases which split both KDG and KDPG [48,49]. Primary structures of bacterial aldolases showed low identity with those of archaea enzymes. The amino acid sequence of FlAld showed only 22%–25% identity with respect to *Proteobacteria* enzymes, while it showed 61%–65% identity with the *Bacteroidetes* enzymes. This suggests that somewhat deviated function between the *Proteobacteria* enzymes and *Bacteroidetes* enzymes. However, less different properties were found in recFlAld. Reverse reaction of bacterial aldolases was shown to be useful for the production of KDPG from pyruvate and GAP and also various compounds from pyruvate and aldehydes [44]. Our preliminary experiments also indicated that recFlAld could produce KDPG from pyruvate and GAP (data not shown, but see Section 2.3). Thus, recFlAld is also considered to be useful for producing novel compounds from pyruvate and various aldehydes.

#### *3.3. Construction of In Vitro Alginate-Metabolizing System*

An in vitro alginate-metabolizing system was successfully constructed from the recombinant enzymes, recFlAlyA, recFlAlyB, recFlAlex, recFlRed, recFlKin, and recFlAld. Accordingly, various kinds of intermediates could be produced by this system (Figure 8 and Table 2). Recently, alginate-assimilating enzymes of *S. degradans* 2-40<sup>T</sup> were used for the production of KDG, KDPG, GAP and pyruvate [24,25]. However, the reaction efficiency of KDG kinase of *S. degradans* 2-40<sup>T</sup> appeared to be lower than that of our system. Namely, the major part of KDG in the reaction mixture remained to be unphosphorylated in the *S. degradans* 2-40<sup>T</sup> system. On the other hand, recFlKin in our system could convert KDG to KDPG with ~80% efficiency. This difference in the reaction efficiency between *S. degradans* enzyme and recFlKin may be derived from the origin of this enzyme, namely, from *Proteobacteria* species or *Bacteroidetes* species. To confirm this, we have to directly compare the KDG kinase properties between the enzymes from *Proteobacteria* and *Bacteroidetes* in future.

#### *3.4. Production of a High-Value Product KDPG from Alginate*

KDPG is a valuable leading compound for novel drugs and medicines. Synthesis of KDPG has been attempted by several methods [44,48,50]. For example, KDPG was first produced from gluconate with archaea enzymes [48]. However, this method required high-temperature reaction since the archaea enzymes are thermophilic. Reverse reaction of KDPG aldolase was also used for the production of KDPG from pyruvate and GAP [44,50]. However, this method required GAP, a significantly expensive raw material. On the other hand, we could produce KDPG from a much cheaper material, alginate, using the enzymes from the strain UMI-01. High recovery of KDPG from alginate (~80%) also indicated the practical potentiality of this enzyme. Thus, *Flavobacterium* sp. strain UMI-01 was considered to be a useful enzyme source for the production of value-added materials from alginate.
