Levanase from Bacillus subtilis hydrolyses β-2,6 fructosyl bonds in bacterial levans and in grass fructans

•Recombinant LevB has endo-activity and specifically hydrolyses (β-2,6) fructosyl bonds.•Ryegrass mixed linkage fructans hydrolysed by LevB produces a range of fructooligosaccharides.•LevB in combination with exoinulinase completely hydrolyses fructan and levan. A Levanase, LevB, from Bacillus subti...

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Published in:	International journal of biological macromolecules Vol. 85; pp. 514 - 521
Main Authors:	Jensen, Susanne L., Diemer, Mikkel B., Lundmark, Maria, Larsen, Flemming H., Blennow, Andreas, Mogensen, Helle K., Nielsen, Tom H
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 01-04-2016
Subjects:	Bacillus subtilis Bacillus subtilis - enzymology Carbon-13 Magnetic Resonance Spectroscopy Enzyme Activation Erwinia herbicola Escherichia coli Fructan Fructans - chemistry Glycoside Hydrolases - chemistry Glycoside Hydrolases - genetics Glycoside Hydrolases - isolation & purification Hydrogen-Ion Concentration Hydrolysis Levanase Lolium perenne Poaceae - enzymology Recombinant Fusion Proteins Substrate Specificity Levanase Fructan
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Summary:	•Recombinant LevB has endo-activity and specifically hydrolyses (β-2,6) fructosyl bonds.•Ryegrass mixed linkage fructans hydrolysed by LevB produces a range of fructooligosaccharides.•LevB in combination with exoinulinase completely hydrolyses fructan and levan. A Levanase, LevB, from Bacillus subtilis 168, was expressed as a His6-tagged protein in Escherichia coli. The enzyme was purified and characterised for its activity and substrate specificity. LevB has a pH optimum of 6.0-6.5 and a maximum observed specific activity of 3Umg−1 using levan from Erwinia herbicola as substrate. Hydrolysis products were analysed by HPAEC, TLC, and NMR using chicory root inulin, mixed linkage fructans purified from ryegrass (Lolium perenne) and levan from E. herbicola as substrates. This revealed that LevB is an endolevanase that selectively cleaves the (β-2,6) fructosyl bonds and does not hydrolyse inulin. Ryegrass fructans and bacterial levan was hydrolysed partially releasing oligosaccharides, but together with exoinulinase, LevB hydrolysed both ryegrass fructans and bacterial levan to near completion. We suggest that LevB can be used as a tool to achieve more structural information on complex fructans and to achieve complete degradation and quantification of mixed linkage fructans.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0141-8130 1879-0003
DOI:	10.1016/j.ijbiomac.2016.01.008