Part:BBa_K1339020

RBS-G3DH-T0 terminator

RBS-G3DH-dT

The enzyme, glucoside 3-dehydrogenase (E.C.1.1.99.13) or glucose-3-dehydrogenase (G3DH) catalyzes the oxidation of the C-3 hydroxyl group of the glucosides and converting them to corresponding 3-ketoglucosides (1). Because G3DH has wide substrate specificity, it can convert not only monosaccharides but disaccharides, including trehalose.


G3DH is composed of three subunits: catalytic subunit, cytochrome c subunit, and small subunit. The catalytic subunit has a flavin adenine dinucleotide (FAD) as cofactor, and the cytochrome c subunit is bound to the cytoplasmic membrane in the periplasm
We evaluated this gene which inserted into pTrc99a vector.

Fig.1 SDS-PAGE analysis of G3DH expression in each fraction.

We observed strong band around 68 kDa, which is the molecular mass weight of G3DH catalytic unit in soluble memblane fraction.(Fig.1)

So, we concluded that G3DH which induced trc promoter express at soluble membrane.

Fig.2 trehalase inhibition activity by PMS-DCIP assay
We evaluated 3,3'-dikeotrehalose production by G3DH via trehalase inhibition assay, which measure glucose amount converted from trehalose by trehalase by glucose dehydrogenase with PMS-DCIP. When we mixed trehalose with homogenate of E.coli houboring pTrc empty, we observed high glucose dehydrogenase activity dependent on trehalase reaction time. On the other hand, we observed lower glucose dehydrogenase activities when we mixed trehalose with that of pTrc-pTrc-RBS-G3DH-dT, indicating that the mixture inhibited trehalase activities. </partinfo>