Structure and Properties of Thermus aquaticus α-Glucan Phosphorylase Expressed in Escherichia coli

Abstract

The α-glucan phosphorylase gene from Thermus aquaticus was isolated using partial amino acid sequences of purified enzyme. The identity of the gene was confirmed by expression in Es cherichia coli resulting in thermostable glucan phosphorylase activity. The open reading frame of this gene consisted of 2460 by and encoded a polypeptide of 819 amino acids. The deduced amino acid sequence exhibits high identity (32-43%) to 7 putative and 2 characterized glucan phosphorylases, but showed weak similarity to other well characterized glucan phosphorylases from various sources. Due to its high expression level and thermal stability, the recombinant enzyme was easily purified from E. coli cell extracts, and employed to characterize its activity. The smallest primer molecule for a synthetic reaction was maltotriose and the smallest effective substrate for a degrada tion reaction was maltotetraose. These results suggest that T. aquaticus glucan phosphorylase, and at least 9 other enzymes, form a new group of glucan phosphorylases whose structure and substrate specificity differ from the traditional glucan phosphorylases. The purified enzyme was also em ployed to investigate the effect of temperature and pH on the activities in both directions. The ac tivity exhibited a pH optimum of 8.0 for phosphorolytic reaction but 7.0 for synthesis reaction. The optimum temperature for phosphorolytic reaction was 80-85°C, while the one for synthesis reaction was 50°C.