title: Glucansucrase Gtf180-ΔN of Lactobacillus reuteri 180: enzyme and reaction engineering for improved glycosylation of non-carbohydrate molecules.
abstract: Glucansucrases have a broad acceptor substrate specificity and receive increased attention as biocatalysts for the glycosylation of small non-carbohydrate molecules using sucrose as donor substrate. However, the main glucansucrase-catalyzed reaction results in synthesis of α-glucan polysaccharides from sucrose, and this strongly impedes the efficient glycosylation of non-carbohydrate molecules and complicates downstream processing of glucosylated products. This paper reports that suppressing α-glucan synthesis by mutational engineering of the Gtf180-ΔN enzyme of Lactobacillus reuteri 180 results in the construction of more efficient glycosylation biocatalysts. Gtf180-ΔN mutants (L938F, L981A, and N1029M) with an impaired α-glucan synthesis displayed a substantial increase in monoglycosylation yields for several phenolic and alcoholic compounds. Kinetic analysis revealed that these mutants possess a higher affinity for the model acceptor substrate catechol but a lower affinity for its mono-α-D-glucoside product, explaining the improved monoglycosylation yields. Analysis of the available high resolution 3D crystal structure of the Gtf180-ΔN protein provided a clear understanding of how mutagenesis of residues L938, L981, and N1029 impaired α-glucan synthesis, thus yielding mutants with an improved glycosylation potential.
authors.affiliation: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.|Centre for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000, Ghent, Belgium.
authors.forename: Tim
authors.initials: T
authors.lastname: Devlamynck
authors.affiliation: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
authors.forename: Evelien M
authors.initials: EM
authors.lastname: Te Poele
authors.affiliation: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
authors.forename: Xiangfeng
authors.initials: X
authors.lastname: Meng
authors.affiliation: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
authors.forename: Sander S
authors.initials: SS
authors.lastname: van Leeuwen
authors.affiliation: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands. l.dijkhuizen@rug.nl.
authors.forename: Lubbert
authors.initials: L
authors.lastname: Dijkhuizen
mesh_terms: D055162:Biocatalysis; D018360:Crystallography, X-Ray; D005936:Glucans / Q000378:metabolism*; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D052200:Limosilactobacillus reuteri / Q000201:enzymology*; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism
chemical_list: D005936:Glucans; D013395:Sucrose; D016695:Glycosyltransferases; C033837:alternansucrase
keywords: Acceptor reaction; Catechol; Enzyme engineering; Glucansucrase; Glycosylation; Lactobacillus reuteri