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United States Department of Agriculture

Agricultural Research Service

Research Project: NOVEL TECHNOLOGIES FOR PRODUCING RENEWABLE CHEMICALS AND POLYMERS FROM CARBOHYDRATES DERIVED FROM AGRICULTURAL FEEDSTOCKS

Location: Renewable Product Technology Research Unit

Title: Production of insoluble glucans using modified recombinant glycosyltransferase from Leuconostoc mesenteroides

Authors
item Skory, Christopher
item Cote, Gregory
item Rich, Joseph

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: July 24, 2014
Publication Date: N/A

Technical Abstract: Glucansucrases catalyze the transfer of D-glucopyranosyl units from sucrose to form a-glucan chains. Glucansucrases are capable of catalyzing the synthesis of several different a-glucosidic linkages that affect molecular mass, branching, and solubility of the polysaccharide. In general, a-glucans containing mostly a(1'6) linkages (e.g., dextran) are water-soluble, while those made primarily of a(1'3) linkages are water-insoluble. These water-insoluble polymers have potential for production of biodegradable gels, fibers and films that can be used in a broad number of applications. The amino acids immediately following the transition state stabilizer of the enzyme often determine the orientation of the acceptor molecules and therefore influence the type of glycosidic bond that is formed. The fifth amino acid after the transition stabilizer, associated with the subsite that binds the acceptor molecule, is almost universally an aspartate or threonine among Streptococcus and a threonine with Leuconostoc species. We substituted this position in a cloned glucansucrase from Leuconostoc mesenteroides NRRL B-1118 with twelve different amino acids. Several substitutions yielded enzymes that produced an increased percentage of 1,3-disubstituted a-D-glucopyranosyl units, with corresponding decreases in 1,6-disubstituted a-D-glucopyranosyl units. Only one substitution resulted in a significant increase in the percentage of 1,6-disubstituted a-D-glucopyranosyl units, with a concomitant increase in glucan yield. We also optimized recombinant expression of these enzymes in Lactococcus lactis using several different background vectors, signal peptides, strains, induction conditions, and bioreactor parameters to increase extracellular synthesis of the enzyme over 150-fold higher compared to the native L. mesenteroides strain.

Last Modified: 9/21/2014
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