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

Molecules

Karin Mardo²  Triinu Visnapuu²  Maria Gromkova²  Anneli Aasamets²  Katrin Viigand²  Heiki Vija¹ 

[1] National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn, Estonia; E-Mail:;Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia; E-Mails:

关键词: levansucrase;    fructooligosaccharides;    levan;    Pseudomonas syringae;    cell permeabilization;    Thermofluor;    prebiotics;   

DOI  :  10.3390/molecules19068434

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland.