Journal of Microbiology and Biotechnology
- Volume 9 Issue 2
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- Pages.206-212
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- 1999
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- 1017-7825(pISSN)
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- 1738-8872(eISSN)
Expression of orf7(oxi III) as dTDP-Glucose 4,6-Dehydratase Gene Cloned from Streptomyces antibioticus Tu99 and Biochemical Characteristics of Expressed Protein
- Yoo, Jin-Cheol (Department of Pharmacy, Chosun University) ;
- Han, Ji-Man (Department of Pharmacy, Chosun University) ;
- Sohng, Jae-Kyung (Department of Chemistry, SunMoon University)
- Published : 1999.04.01
Abstract
The gene orf7(oxi III) was expressed using an E. coli system in anticipation that it would encode dTDP-glucose 4,6-dehydratase which is involved in the biosynthesis of the olivose moiety of chlorothricin produced from Streptomyces antibioticus Tu99. The solubility of the expressed protein increased up to 20% under optimal induction conditions. The expressed protein was purified from the E. coli BL 21(DE3) cell lysate by a 28.5-fold purification in two chromatography steps with a 38% recovery to near homogeneity. The molecular weight and N-terminal amino acid sequence of the purified protein correlated with the predicted mass and sequence deduced from the orf7 gene. The purified protein was a homodimer with a subunit relative molecular weight of 38,000 Dalton. The expressed protein was found to exhibit dTDP-glucose 4,6-dehydratase activity and be highly specific for dTDP-glucose as a substrate. The values of K'm and V'max for dTDP-glucose were 28
Keywords
- Biosynthesis;
- chlorothricin;
- deoxysugar;
- olivose;
- orf7 dTDP-glucose 4,6-dehydratase;
- Streptomyces antibioticus Tu99