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.
This is an open access article distributed under the
Creative Commons Attribution License which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited.
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.