(3S,4S,5R)-1,3,4,5,6-pentahydroxyhexan-2-one → D-Arabinose

The recombinant strains Corynebacterium glutamicum CJ-1-TNAI and Corynebacterium glutamicum CJ-7-TNAI (Accession Nos: KCCM10786P and KCCM10787P) prepared in Example 1 were inoculated in MB medium (Bacto-trypton 10 g/L, Bacto-yeast extract 5 g/L, NaCl 10 g/L, Soytone 5 g/L) containing 10 μg/Ml of kanamycin at the concentration of OD600=0.1, followed by culture at 30° C. for 24 hours to induce expression of the recombinant arabinose isomerase. To measure the enzyme activity of the expressed arabinose isomerase, the culture solution was centrifuged at 8,000× g for 10 minutes and cells were recovered. The cells were resuspended in 50 mM Tris-HCl (pH 7.0) buffer, followed by ultrasonification to lyse the cells. The supernatant was obtained as a crude enzyme solution, with which galactose isomerization was performed. Particularly, for the isomerization, 100 μl of an enzyme solution containing 40 mM of galactose as a substrate was mixed with 1 ml of a reaction buffer (50 mM Tris-HCl, pH 7.0). At that time, 5 mM of MnCl2 and 1mM of CoCl2 were added to the reaction mixture. The crude enzyme reaction mixture was reacted at 60° C. for 20 minutes. The activity of the crude enzyme was measured by the cystein-carbazol-sulfuric acid method (Dische, Z., and E. Borenfreund., A New Spectrophotometric Method for the Detection and Determination of Keto Sugars and Trioses, J. Biol. Chem., 192:583-587, 1951). The protein contained in the crude enzyme solution was quantified with a Bradford assay kit (Biorad, U.S.A.). As a result, the isomerase activity was 2.050 (mg-tagatose/mg-protein·h), indicating that the product of galactose isomerization, tagatose, was successfully generated.

1. custom(Accession Nos: KCCM10786P and KCCM10787P) prepared in Example 1
2. extractionwere inoculated in MB medium (Bacto-trypton 10 g/L, Bacto-yeast extract 5 g/L, NaCl 10 g/L, Soytone 5 g/L)
3. additioncontaining 10 μg/Ml of kanamycin at the concentration of OD600=0.1
4. waitg for 10 minutes
5. customcells were recovered
6. customThe supernatant was obtained as a crude enzyme solution
7. customThe crude enzyme reaction mixture
8. customwas reacted at 60° C. for 20 minutes

ORD record

ord-fd110bdf81f0444aa916d52407f01d92

Source dataset

uspto-grants-2014_08

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US08802393B2