Abstract: Bacterial levansucrases polymerize fructose residues of sucrose to β-2,6 linked fructans—fructooligosaccharides (FOS) and levan. While β-2,1-linked FOS are widely recognized as prebiotics, the health-related effects of β-2,6 linked FOS are scarcely studied as they are not commercially available. Levansucrase Lsc3 (Lsc-3) of Pseudomonas syringae pv. tomato has very high catalytic activity and stability making it a promising biotechnological catalyst for FOS and levan synthesis. In this study we evaluate feasibility of several high-throughput methods for screening and preliminary characterization of levansucrases using 36 Lsc3 mutants as a test panel. Heterologously expressed and purified His-tagged levansucrase variants were studied for: (1) sucrose-splitting activity; (2) FOS production; (3) ability and kinetics of levan synthesis; (4) thermostability in a Thermofluor assay. Importantly, we show that sucrose-splitting activity as well as the ability to produce FOS can both be evaluated using permeabilized levansucrase-expressing E. coli transformants as catalysts. For the first time we demonstrate the key importance of Trp109, His113, Glu146 and Glu236 for the catalysis of Lsc3. Cost-effective and high-throughput methods presented here are applicable not only in the levansucrase assay, but have a potential to be adapted for high-throughput (automated) study of other enzymes.
Keywords: levansucrase; fructooligosaccharides; levan; Pseudomonas syringae; cell permeabilization; Thermofluor; prebiotics
Export to BibTeX
MDPI and ACS Style
Mardo, K.; Visnapuu, T.; Gromkova, M.; Aasamets, A.; Viigand, K.; Vija, H.; Alamäe, T. High-Throughput Assay of Levansucrase Variants in Search of Feasible Catalysts for the Synthesis of Fructooligosaccharides and Levan. Molecules 2014, 19, 8434-8455.
Mardo K, Visnapuu T, Gromkova M, Aasamets A, Viigand K, Vija H, Alamäe T. High-Throughput Assay of Levansucrase Variants in Search of Feasible Catalysts for the Synthesis of Fructooligosaccharides and Levan. Molecules. 2014; 19(6):8434-8455.
Mardo, Karin; Visnapuu, Triinu; Gromkova, Maria; Aasamets, Anneli; Viigand, Katrin; Vija, Heiki; Alamäe, Tiina. 2014. "High-Throughput Assay of Levansucrase Variants in Search of Feasible Catalysts for the Synthesis of Fructooligosaccharides and Levan." Molecules 19, no. 6: 8434-8455.