Purification and characterization of a hygromycin B phosphotransferase from Streptomyces hygroscopicus

Abstract

A hygromycin B phosphotransferase activity from Streptomyces hygroscopicus has been highly purified by ammonium sulphate fractionation followed by affinity column chromatography through Sepharose-6B- hygromycin-B.The combined active fractions showed a singleprotein band (41 kDa) when subjected to polyacryl- amide gel electrophoresis in the presence of sodium dodecyl sulphate. When gel electrophoresis was performed under non-denaturing conditions, the single protein band promoted in situ phosphorylation of hygromycin B, indicating that this protein corresponded to the purified hygromycin B phosphotransferase. The enzyme has been purified 236-fold and approximate Km values of 0.56 pM and 36.4 pM for hygromycin B and ATP, respectively, were deduced.