High-throughput assay of levansucrase variants in search of feasible catalysts for the synthesis of fructooligosaccharides and levan.

Karin Mardo,Heiki Vija,Maria Gromkova,Tiina Alamäe,Katrin Viigand,Triinu Visnapuu,Anneli Aasamets

doi:10.3390/molecules19068434

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.

Highlights

Levansucrases (EC 2.4.1.10) are bacterial extracellular enzymes that convert sucrose into β-2,6linked fructooligosaccharides (FOS) of varied chain length and high-molecular weight levan [1]
Twenty two of them were previously characterized by us. Among those are the inactive mutants of the catalytic triad Asp62Ala, Asp219Ala and Glu303Ala [23], mutants with significantly decreased polymerizing ability (Thr302Pro, Gln301Ala and substitution mutants of His321 with Arg, Leu, Lys or Ser) [21,23], mutants Trp61Ala, Trp61Asn, Arg304Cys and Arg304Ala which are strongly hampered in sucrose-splitting as well as polymerization abilities [23] and some others exhibiting moderate changes compared to the wild-type Lsc3
36 Lsc3 mutants with largely varied catalytic activity, we show here that sucrose-splitting activity of the levansucrases can be reliably measured in a high-throughput way, i.e., on microplates

Summary

Introduction

Levansucrases (EC 2.4.1.10) are bacterial extracellular enzymes that convert sucrose into β-2,6linked fructooligosaccharides (FOS) of varied chain length and high-molecular weight levan [1]. These enzymes are present in many plant-related bacteria such as Pseudomonas syringae [2,3,4], Gluconacetobacter diazotrophicus [5], Zymomonas mobilis [6] and Erwinia amylovora [7,8,9], and in Bacillus subtilis, B. megaterium [10,11] and several lactic acid bacteria such as Lactobacillus sanfranciscensis, L. reuteri and Leuconostoc mesenteroides [12,13,14]. A recent paper by Marsh and coworkers states that water kefir grains originating from different regions of the world contain Z. mobilis as main bacterial component [19]

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Conclusion