G DH has gained interest because of its

G3DH has gained interest because of its broad substrate specificity and site-selective oxidation properties [9,10]. The conversion product, 3-ketoglycoside, possesses potential as a starting material for the chemical industry such as in polymer and surfactants because the keto group becomes a specific site for selective chemical synthesis. Moreover, it is very attractive to produce new 3-ketosugars or rare sugars with G3DH [5,11]. d-gulose is one of an aldohexose that rarely exists in nature thus its applications have not yet been studied thoroughly. Described by James et al., d-gulose in crystalline form could be used as an excipient or a chelating agent, an intermediate for pharmaceutical industries, as well as an additive in food [12]. Several glucose oxidoreductases are known and have been used as a component of enzyme biosensors for blood glucose level measurement. Nevertheless, G3DH has practical applications over other glucose oxidoreductases because of its cofactor binding structure that would not be affected by oxygen in the reaction mixture. Another advantage is the property of reducing glycosides that lack of a hydroxyl group at the C-1 position, for example, the 1,5-anhydro-d-glucitol measurement in clinical diagnostics [13,14]. After its discovery in A. tumefaciens, G3DH was found later in many strains; Flavobacterium saccharophilum [15], Cytophaga marinoflava [8], Halomonas (Deleya) sp. α-15 [16], Agaricus bisporus [17], Stenotrophomonas maltrophilia CCTCC M 204024 [18], and recently Sphingobacterium faecium ZJF-D6 [19]. Those G3DHs were purified and characterized, but only a few have been cloned and overexpressed in Escherichia coli. Unfortunately, recombinant G3DH from Halomonas (Deleya) sp. α-15 was less active than the wild type [20] and that from A. tumefaciens was inactive [21]. Another attempt to Dryocrassin ABBA a fragment containing the G3DH gene from A. tumefaciens and overexpressed into E. coli resulted in G3DH that mostly accumulated in the insoluble fraction [22]. Newly isolated S. faecium ZJF-D6 was cloned and functionally expressed in E. coli, but its activity was lower than that from the natural organism [23]. Recently, G3DH from R. radiobacter (A. tumefaciens) was expressed in E. coli by a co-expression vector as a heterodimeric complex that showed dye-mediated G3DH activity toward methyl-α-d-glucoside [24]. Sorbitol dehydrogenase (SDH) from Gluconobacter oxydans and 2-keto-d-gluconate dehydrogenases (2KGDH) from Eriwinia herbicola and Pantoea citrea belong to a group of membrane-bound dehydrogenases complexes which is consisting of three subunits; FAD-related catalytic subunit, cytochrome c subunit, and a small subunit [25]. Previous studies [20,24] revealed that the calculated primary and secondary structure of G3DH from Halomonas sp. α-15 resembled that of SDH from G. oxydans and 2KGDH from P. citrea. Consequently, G3DHs from Rhizobium sp. S10 may show the structural similarity and function as an oligomeric enzyme. Until now, only limited genetic information on G3DH has been analyzed and reported even though it is necessary to improve G3DH utilization by genetic engineering. In the present study, we described the cloning and sequencing of a G3DH gene from Rhizobium sp. S10 and its co-expression in E. coli. The recombinant enzyme, identified as rG3DH, was consequently purified, characterized and utilized in d-gulose production.
Materials and methods
Results
Discussion Over decades, many researchers have been exploring novel G3DH or more deeply analyzing those G3DHs discovered in different organisms [6] but only a limited number of studies about gene cloning and overexpression of this enzyme have been reported, for example, that of Halomonas [19] and Sphingobacterium faecium [22], but with quite low activity of 0.28 U/mg and 2.56 U/mg, respectively. Later, the expression of G3DH from R. radiobacter (A. tumefaciens) could be achieved by co-expressing G3DH with an activity of 13 U/mg toward methyl-α-d-glucoside [24]. In this paper, the DNA fragment containing the G3DH gene from a newly isolated Rhizobium sp. S10 was cloned in E. coli JM 109 cells. The recombinant protein was successfully expressed in the soluble fraction with an outstanding G3DH activity of 38.54 U/mg.