Cloning, expression, and characterization of an insoluble glucan-producing glucansucrase from Leuconostoc mesenteroides NRRL B-1118

We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble alpha-D-glucan from sucrose (K (M) 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0-6.2. The enzyme was quickly inactivated at temperatures above approximately 45A degrees C. The presence of dextran offered some protection from thermal inactivation between room temperature and 40A degrees C but had little effect above 45A degrees C. NMR and methylation analysis of the water-insoluble alpha-d-glucan revealed that it had approximately equal amounts of alpha(1 -> aEuro parts per thousand 3)-linked and alpha(1 -> aEuro parts per thousand 6)-linked d-glucopyranosyl units and a low degree of branching.