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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7476-x) contains supplementary material, which is available to authorized users.

Catechol glucosides act as donor/acceptor substrates of glucansucrase enzymes of Lactobacillus reuteri

“…S2 ). With sucrose as donor substrate, GtfA-ΔN, Gtf180-ΔN, and GtfMLI-ΔN also efficiently glucosylated the phenolic compounds resorcinol, pyrogallol, and ethyl gallate (data not shown) and used their mono-glucosides, resG1 (Devlamynck et al 2016 ), pyrG1 (Supplemental material, Table S1 and Fig. S3 ), and etgaG1 (Supplemental material, Table S1 and Fig.…”

“…α- d -Glc p -catechol (catG1) and α- d -Glc p -(1→4)-α- d -Glc p -catechol (cat 4 G2) synthesized by GtfA-ΔN (te Poele et al 2016 ), and α- d -Glc p -(1→3)-α- d -Glc p -catechol (cat 3 G2) and α- d -Glc p -(1→6)-α- d -Glc p -catechol (cat 6 G2) synthesized by Gtf180-ΔN (Devlamynck et al 2016 ), were isolated and purified using preparative normal-phase HPLC (NP-HPLC) as described previously (te Poele et al 2016 ). GtfA-ΔN enzyme activity on catG1 was measured at six different catG1 concentrations ranging from 3.1 to 100 mM using 0.125 mg/mL GtfA-ΔN.…”

“…Glucansucrase enzymes are interesting biocatalysts for the glycosylation of small organic molecules, because they are promiscuous towards a wide range of acceptor substrates (Monsan et al 2010 ; Leemhuis et al 2012 ). Using inexpensive sucrose as glycosyl donor, glucansucrase enzymes glucosylate, for instance, phenolic compounds such as catechol (Meulenbeld and Hartmans 2000 ; Devlamynck et al 2016 ; te Poele et al 2016 ) and hydroquinone (Seo et al 2009 ; Devlamynck et al 2016 ), primary alcohols (Seibel et al 2006 ; Devlamynck et al 2016 ), and l -ascorbic acid (Kim et al 2010 ). Glycosylation may change the physico-chemical and biological properties of molecules, enhance bioavailability of antibiotics, or improve stability of molecules against auto-oxidation (Desmet et al 2012 ).…”

Mutational Analysis of the Role of the Glucansucrase Gtf180-ΔN Active Site Residues in Product and Linkage Specificity with Lactose as Acceptor Substrate

“…Similar effects were observed in studies where maltose was used as acceptor substrate, 35 or even when non-carbohydrate compounds were used as acceptor substrates. 6 The crystal structure of GTF180-ΔN in complex with maltose revealed that N1029 is involved in a hydrogen bond network with the nonreducing end glucosyl moiety of maltose in subsite +1, making direct and indirect hydrogen bonds with its C3 and C4 hydroxyl groups. 18 Mutant N1029G, when it acts on lactose as an acceptor substrate, added an (α1→3)-linked Glc moiety to compounds F2–F5 of the GL34 mixture to synthesize G1–G4 (Scheme 1).…”

Current state and latest advances in the concept, production and functionality of prebiotic oligosaccharides

“…The design of enzymes with specific features aimed at new acceptor substrates, to better control regioselectivity and/or to increase the reaction yield contributes to enhance the glycodiversification and quality of the attained products [15,16]. Protein engineering approaches have led to major achievements to create novel enzymes [17], and to improve their performance (i.e., productivity, activity, and pH and thermo-stability) [18].…”