title: High-throughput assay of levansucrase variants in search of feasible catalysts for the synthesis of fructooligosaccharides and levan.
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.
authors: Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia. kmardo@ut.ee.
authors: Karin
authors: K
authors: Mardo
authors: Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia. triinu.visnapuu@ut.ee.
authors: Triinu
authors: T
authors: Visnapuu
authors: Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia. manjunja@ut.ee.
authors: Maria
authors: M
authors: Gromkova
authors: Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia. anneli.aasamets@gmail.com.
authors: Anneli
authors: A
authors: Aasamets
authors: Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia. katrin66@ut.ee.
authors: Katrin
authors: K
authors: Viigand
authors: National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn, Estonia. heiki.vija@kbfi.ee.
authors: Heiki
authors: H
authors: Vija
authors: Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia. talamae@ebc.ee.
authors: Tiina
authors: T
authors: Alamäe
mesh_terms: D002384:Catalysis; D005630:Fructans / Q000378:metabolism*; D006602:Hexosyltransferases / Q000737:chemistry / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D056692:Prebiotics; D044224:Pseudomonas syringae / Q000201:enzymology
chemical_list: D005630:Fructans; D009844:Oligosaccharides; D056692:Prebiotics; C116580:fructooligosaccharide; C072599:levan; D006602:Hexosyltransferases; C021513:levansucrase