Part:BBa_K1339002

RBS-G3DH

G3DH

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.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2366
  Illegal NotI site found at 347
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2672
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 490
  Illegal NgoMIV site found at 587
  Illegal NgoMIV site found at 1803
  Illegal NgoMIV site found at 2099
  Illegal NgoMIV site found at 2652
  Illegal NgoMIV site found at 2717
  Illegal AgeI site found at 769
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1860
  Illegal SapI site found at 1964