Team:Tokyo-NoKoGen/g3dh

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	Project	Team	Result	Parts	Protocols	Notebook	Safety	Human Practice	Achievement

About G3DH

The enzyme glucose-3-dehydrogenase (G3DH) converts trehalose to 3,3’-diketotrehalose (3,3’-dkT), a trehalase inhibitor. This enzyme can oxidize the 3rd hydroxyl group of pyranose (1). Because G3DH has wide substrate specificity, it can convert not only monosaccharides but disaccharides.

G3DH is composed of three subunits: a catalytic subunit, a cytochrome c subunit, and a small subunit. The catalytic subunit has a flavin adenine dinucleotide (FAD) cofactor, and the cytochrome c subunit is bound to the cytoplasmic membrane in the periplasm. In our project, we used G3DH derived from Rhizobium tumefaciens EHA101. This enzyme is a homolog of G3DH derived from Halomonas sp. α-15, which was reported to convert trehalose to 3,3’dkT.

About 3,3'-diketotrehalose (3,3'-dkT)

3,3’-dkT is a novel trehalose derivative in which the third hydroxyl group of both glucose moieties are oxidized. It was already reported that 3,3-dkT showed an inhibitory effect toward the trehalose from pig-kidney and Bombyx mori (silkworm).(2)

Reference

(1) K Kojima et al., (2001) Cloning and Expression of Glucose 3-Dehydrogenase from Halomonas sp. α-15 in Escherichia coli. 282, 21-27

(2) K Sode et al., (2001) Enzymatic synthesis of a novel trehalose derivative, 3,3’-diketotrehalose, and its potential application as the trehalase enzyme inhibitor. 489, 42-45.

1.Construction of Biobrick

　G3DH gene were cloned from pTrc99a-G3DH which originated from Agrobacterium tumefaciens. PCR products were inserted pSB1C3. Original G3DH gene has two illegal restriction sites. In order to remove these restriction sites, the G3DH gene were amplified by overlap extension PCR. We designed two primer sets for overlap extension PCR.

Fig.1. Remove illegal restriction sites

　G3DH gene fragments were amplified and three PCR products were connected.

　G3DH (removed illegal restriction sites) were ligated with four promoters and double terminator (BBa_B0010 and BBa_B0012).

　One of those promoters is arabinose inducible, and the others are constitutive promoter.

Table 1. Promoter

Fig 2. Constructed plasmid

2. Evaluation

This G3DH needs the maturation of cytochrome c subunit to have catalytic activity. Therefore, we transformed G3DH with the plasmid, pEC86; which expresses cytochrome c maturation (CCM) enzymes (1).

This is the method of culturing and extraction of production (Fig. 3).

Culture condition and evaluation method

We cultured E. coli TOP10 transformed with two plasmids; G3DH expression vector, and cytochrome c maturation enzymes expression vector (pEC86) in LB medium containing 20 mM Trehalose. For expression of G3DH, we used four different promoters.

When OD₆₆₀ achieves 0.6, 0.2 % arabinose was added to the medium for induction in the case we used P_BAD as a promoter.

After culturing for 20 hours at 37C, we extracted products by boiling and centrifugation.

0.007U trehalase and 20 mM trehalose mixed sample and incubated 30h.

First, we investigated expression of G3DH by SDS-PAGE analysis. Second, we measured the glucose dehydrogenase activity of G3DH. Finally we tried to detect 3,3’-dkT by thin-layer chromatography (TLC).

Fig. 3 evaluation of G3DH

SDS-PAGE

　By SDS-PAGE analysis, there was the band which showed about 68 kDa on G3DH under the constitutive promoter P_medium and P_high, P_BAD. Therefore, we confirmed the expression of G3DH (Fig. 2).

Fig. 4 SDS-PAGE analysis

Measurement of glucose dehydrogenase activity

We measured the oxidase activity of G3DH by PMS-DCIP assay (Fig. 5). On this assay, we measured the decrease in absorbance of DCIP at 600 nm. Substrates were glucose and trehalose. PMS stands for phenazine methosulfate, and DCIP stands for 2,6-dichlorophenol-indophenol.

Trehalase inhibition measurement (Fig. 3)

Fig. 3 Trehalose inhibition activity assay

This is the result of trehalase inhibition activity assay. Each value of activity was normalized at the value of activity of empty vector which were not induced by arabinose. Samples of G3DH which induced by P_high and P_medium activity was lower than that of empty vector.

We concluded that G3DH which induced by P_high and P_medium expressed G3DH. And G3DH converted trehalose to 3,3’-dkT.

Reference

(3) E Arslan et al., (1998) Overproduction of the Bradyrhizobium japonicum c-Type Cytochrome Subunits of the cbb3 Oxidase in Escherichia coli. 251, 744-747