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Molecules 2014, 19(6), 8434-8455; doi:10.3390/molecules19068434
Article

High-Throughput Assay of Levansucrase Variants in Search of Feasible Catalysts for the Synthesis of Fructooligosaccharides and Levan

1
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2
 and
1,*
1 Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia 2 National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn, Estonia
* Author to whom correspondence should be addressed.
Received: 28 March 2014 / Revised: 10 June 2014 / Accepted: 10 June 2014 / Published: 20 June 2014
(This article belongs to the Special Issue Oligosaccharides and Glyco-Conjugates)
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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 levansucrase; fructooligosaccharides; levan; Pseudomonas syringae; cell permeabilization; Thermofluor; prebiotics
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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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.

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