The endopolysaccharide metabolism of the hyperthermophilic archeon Thermococcus hydrothermalis: polymer structure and biosynthesis.

The endopolysaccharide accumulated by Thermococcus hydrothermalis was extracted and purified from a 4 h culture. It presented an "amylopectin-like" structure with an average chain length of 14 and a ramification degree of 7.5%. The glucosyltransferase was isolated, partially purified and characterized. The molecular mass was 42 kDa by SDS PAGE and 85 +/- 5 kDa by gel filtration. This enzyme was able to use both Uridine-5'-DiPhosphoGlucose (UDPG) and Adenosine-5'-DiPhosphoGlucose (ADPG) as substrates. Optimal pH and temperature for the enzyme were 5.5 and 80 degrees C, respectively. In the presence of 3.2 mM ADPG, the half life of the protein was 6 min at 110 degrees C. The apparent Km value with the two substrates was 0.9 mM, but the Vmax was 9.7 fold higher for ADPG. A branching activity was also detected at high temperature, up to 80 degrees C by different methods: phosphorylase stimulation, iodine, and branching linkage assays.