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 degrees C and 7.38, respectively. After ste10 gene knock-out, the monosaccharide composition of EPS-m produced by the mutant Streptomyces sp. 139 (ste(10-)) was found changed in comparison with that of Ebosin while its antagonist activity for IL-IR 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. (c) 2008 Elsevier Inc. All rights reserved.