Rational designed mutagenesis of levansucrase from Bacillus licheniformis 8-37-0-1 for product specificity study

Applied Microbiology and Biotechnology
Chunjuan HeMin Xiao

Abstract

Levansucrases, which belong to the glycoside hydrolase family 68 (GH68), synthesize β (2-6)-linked fructan levan with sucrose as substrate. We described the use of a levansucrase (Bl_SacB) from Bacillus licheniformis 8-37-0-1 for catalysis of fructosyl transfer to obtain high levan yield previously. In the present study, six variants (Y246A, N251A, K372A, R369A, R369S, and R369K) were constructed through sequence alignment and structural analysis to explore the synthesis mechanism of Bl_SacB. The selected residues were predicted to localize to the substrate-entering channel of the active cavity and close to or remote from the catalytic triad. The products of these variants ranged from homopolymers levan to fructo-oligosaccharides (FOSs). The primary FOSs were identified through MS and NMR analyses as neolevan-type neokestose [β-D-Fru-(2-6)-α-D-Glc-(1-2)-β-D-Fru], levan-type 6-kestose [β-D-Fru-(2-6)-β-D-Fru-(2-1)-α-D-Glc], and inulin-type 1-kestose [β-D-Fru-(2-1)-β-D-Fru-(2-1)-α-D-Glc]. The mutation at Tyr246 located remote from the catalytic triad led to the production of short-chain oligosaccharides with degree of polymerization (DP) of up to 25. The replaced Arg369 located close to the catalytic triad resulted in either elimi...Continue Reading

References

Dec 29, 1999·FEMS Microbiology Letters·S P MarxW Hartmeier
Oct 3, 2002·Journal of Biochemistry·Hideshi YanaseKenji Okamoto
Jan 16, 2003·FEBS Letters·S A F T van HijumL Dijkhuizen
Oct 1, 2003·Nature Structural Biology·Guoyu Meng, Klaus Fütterer
May 5, 2005·The Biochemical Journal·Carlos Martínez-FleitesGideon J Davies
Oct 13, 2005·Journal of Agricultural and Food Chemistry·Ing-Lung ShihChien-Yan Hsieh
Jun 21, 2008·Biotechnology Advances·Carel A G M WeijersGerben M Visser
Jul 4, 2008·Protein Engineering, Design & Selection : PEDS·Maria Elena Ortiz-SotoAgustin López-Munguía
Jul 9, 2008·Archives of Microbiology·Florian W WaldherrRudi F Vogel
Feb 2, 2010·Journal of Physiology and Biochemistry·M Sabater-MolinaS Zamora
Apr 2, 2011·The Journal of Biological Chemistry·Christian P StrubeDirk W Heinz
May 15, 2012·International Journal of Biological Macromolecules·Imen DahechHafedh Mejdoub
Nov 26, 2013·Nucleic Acids Research·Vincent LombardBernard Henrissat
Nov 2, 2014·Applied Microbiology and Biotechnology·María Gimeno-PérezMaría Fernández-Lobato
May 8, 2015·Nature Protocols·Lawrence A KelleyMichael J E Sternberg
May 15, 2016·Biotechnology Advances·Ebru Toksoy ÖnerJoan Combie

❮ Previous
Next ❯

Citations

Oct 26, 2018·Applied Microbiology and Biotechnology·David Rodrigo-FrutosMaría Fernández-Lobato
Jan 1, 2019·Critical Reviews in Food Science and Nutrition·Wei XuWanmeng Mu
Aug 23, 2019·International Journal of Biological Macromolecules·Pongsakorn KanjanataninSurasak Chunsrivirot
Dec 12, 2020·The Journal of Biological Chemistry·Enrique Raga-CarbajalClarita Olvera
May 13, 2018·International Journal of Biological Macromolecules·Wei XuWanmeng Mu

❮ Previous
Next ❯

Datasets Mentioned

BETA
AGZ16261
CAA26513.1
ADF38395.1
49946
CBJ48143.1
AAB36606.1

Methods Mentioned

BETA
ELISA
NMR

Software Mentioned

PyMOL
Phyre
GraphPad Prism
ImageJ

Related Concepts

Related Feeds

Cardiac Glycosides

Cardiac glycosides are a diverse family of naturally derived compounds that bind to and inhibit na+/k+-atpase. Discover the latest research on cardiac glycosides heres.