[Cloning and expression of UDP-glucose-4-epimerase gene from Streptomyces and characteristics of the enzyme].
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100050, China.ACTA MICROBIOLOGICA SINICA 01/2006; 45(6):881-4.
The ste 19 gene is identified to encode a UDP-Glucose-4-epimerase by data base comparison and experimental validation. The enzyme catalyzes the interconversion of UDP-Glucose and UDP-Galactose. The gene amplified by PCR with the total DNA of Streptomyces sp. 139 as template was cloned into plasmid pET30a at Xho I - Nde I sites. After the recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG, the protein was expressed as high as 26% of the total cell proteins and dominantly as soluble protein form. The high GC content (73.8%) and the preferential usage of G or C as third base of codons (96.2%) seems not to affect its good expression in E. coli BL21 (DE3). The purity of the recombinant protein purified by Ni2+ affinity chromatography is 92.9% by HPLC analysis. Enzyme activity assay shows the recombinant protein is a UDP-Glucose-4-epimerase and its activity is not dependent on exogenous NAD+. The above research lays a very good foundation for study on functions of the ste 19 gene in Ebosin biosynthesis.
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ABSTRACT: Ebosin produced by Streptomyces sp. 139 is a novel exopolysaccharide (EPS) with medicinal activity. This paper describes the functional study of ste10, a putative Ebosin biosynthesis gene. ste10 was cloned and expressed in Escherichia coli BL21 and the purified recombinant protein characterized. Ste10 was shown to be able of catalyzing the transfer of amide nitrogen of glutamine to the side chain of aspartate to produce asparagine. Its Km, optimum temperature and pH were determined to be 0.9 mM, 37 °C and 7.38, respectively. After ste10 gene knock-out, the monosaccharide composition of EPS-m produced by the mutant Streptomyces sp. 139 (ste10−) was found changed in comparison with that of Ebosin while its antagonist activity for IL-1R decreased significantly. Based on these results, it is concluded that ste10 codes for an asparagine synthetase which may function as a modificator gene of Ebosin during its biosynthesis.
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