Location: Renewable Product Technology ResearchTitle: Cloning, expression, and characterization of an insoluble glucan-producing glucansucrase from Leuconostoc mesenteroides NRRL B-1118) Author
Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/17/2011
Publication Date: 9/14/2011
Publication URL: http://handle.nal.usda.gov/10113/55128
Citation: Cote, G.L., Skory, C.D. 2011. Cloning, expression, and characterization of an insoluble glucan-producing glucansucrase from Leuconostoc mesenteroides NRRL B-1118. Applied Microbiology and Biotechnology. 93:2387-2394. DOI: 10.1007/s00253-011-3562-2. Interpretive Summary: In order to produce renewable polymers for a variety of applications, we have cloned an enzyme from a food-grade microbe and characterized its properties. It produces a water-insoluble gel-like material from cane or beet sugar. The properties of the polymer suggest many uses, including several in foods, biomedical applications, and industrial films and fibers. By cloning the enzyme, we open the possibility of custom-tailoring these properties for specific applications.
Technical 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 a-D-glucan from sucrose (KM 12mM) 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 denatured at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal denaturation between room temperature and 40°C, but had little effect above 45°C. NMR and methylation analysis of the water-insoluble a-D-glucan revealed that it had approximately equal amounts of 1,3-linked and 1,6-linked D-glucopyranosyl units and a low degree of branching.