title: Cloning, expression, and characterization of an insoluble glucan-producing glucansucrase from Leuconostoc mesenteroides NRRL B-1118.
abstract: 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 α-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 45°C. The presence of dextran offered some protection from thermal inactivation between room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-D-glucan revealed that it had approximately equal amounts of α(1 → 3)-linked and α(1 → 6)-linked D-glucopyranosyl units and a low degree of branching.
authors.affiliation: Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, Peoria, IL 61604, USA. greg.cote@ars.usda.gov
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Côté
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Skory
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005936:Glucans / Q000378:metabolism*; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D007934:Leuconostoc / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D005936:Glucans; D016695:Glycosyltransferases; C033837:alternansucrase