title: Levanase from Bacillus subtilis hydrolyses β-2,6 fructosyl bonds in bacterial levans and in grass fructans.
abstract: 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 3 U mg(-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.
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenhagen, Denmark. Electronic address: susanne.langgaard@gmail.com.
authors.forename: Susanne L
authors.initials: SL
authors.lastname: Jensen
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenhagen, Denmark. Electronic address: mikkelbechdiemer@gmail.com.
authors.forename: Mikkel B
authors.initials: MB
authors.lastname: Diemer
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenhagen, Denmark. Electronic address: mariatlundmark@gmail.com.
authors.forename: Maria
authors.initials: M
authors.lastname: Lundmark
authors.affiliation: Department of Food Science, University of Copenhagen, Denmark. Electronic address: fhl@food.ku.dk.
authors.forename: Flemming H
authors.initials: FH
authors.lastname: Larsen
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenhagen, Denmark. Electronic address: abl@plen.ku.dk.
authors.forename: Andreas
authors.initials: A
authors.lastname: Blennow
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenhagen, Denmark. Electronic address: hkm@plen.ku.dk.
authors.forename: Helle K
authors.initials: HK
authors.lastname: Mogensen
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenhagen, Denmark. Electronic address: thni@plen.ku.dk.
authors.forename: Tom H
authors.initials: TH
authors.lastname: Nielsen
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D066241:Carbon-13 Magnetic Resonance Spectroscopy; D004789:Enzyme Activation; D005630:Fructans / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006109:Poaceae / Q000201:enzymology*; D011993:Recombinant Fusion Proteins; D013379:Substrate Specificity
chemical_list: D005630:Fructans; D011993:Recombinant Fusion Proteins; C072599:levan; D006026:Glycoside Hydrolases; C021512:levanase
keywords: Fructan; Levanase