Gluco-oligomers initially formed by the reuteransucrase enzyme of Lactobacillus reuteri 121 incubated with sucrose and malto-oligosaccharides

<p>The probiotic bacterium Lactobacillus reuteri 121 produces a complex, branched (1 -> 4, 1 -> 6)-alpha-d-glucan as extracellular polysaccharide (reuteran) from sucrose (Suc), using a single glucansucrase/glucosyltransferase (GTFA) enzyme (reuteransucrase). To gain insight into the reaction/product specificity of the GTFA enzyme and the mechanism of reuteran formation, incubations with Suc and/or a series of malto-oligosaccharides (MOSs) (degree of polymerization (DP2-DP6)) were followed in time. The structures of the initially formed products, isolated via high-performance anion-exchange chromatography, were analyzed by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and 1D/2D H-1/C-13 NMR spectroscopy. Incubations with Suc only, acting as both donor and acceptor, resulted in elongation of Suc with glucose (Glc) units via alternating (alpha 1 -> 4) and (alpha 1 -> 6) linkages, yielding linear gluco-oligosaccharides up to at least DP similar to 12. Simultaneously with the ensemble of oligosaccharides, polymeric material was formed early on, suggesting that alternan fragments longer than DP similar to 12 have higher affinity with the GTFA enzyme and are quickly extended, yielding high-molecular-mass branched reuteran (4 x 10(7) Da). MOSs (DP2-DP6) in the absence of Suc turned out to be poor substrates. Incubations of GTFA with Suc plus MOSs as substrates resulted in preferential elongation of MOSs (acceptors) with Glc units from Suc (donor). This apparently reflects the higher affinity of GTFA for MOSs compared with Suc. In accordance with the GTFA specificity, most prominent products were oligosaccharides with an (alpha 1 -> 4)/(alpha 1 -> 6) alternating structure.</p>

Residue Leu(940) Has a Crucial Role in the Linkage and Reaction Specificity of the Glucansucrase GTF180 of the Probiotic Bacterium Lactobacillus reuteri 180

Highly conserved GH70 family glucansucrases are able to catalyze the synthesis of α-glucans with different structure from sucrose. The structural determinants of glucansucrase specificity have remained unclear. Residue L940 in domain B of GTF180, the glucansucrase of the probiotic bacterium Lactobacillus reuteri 180, was shown to vary in different glucansucrases and is close to the +1 glucosyl unit in the crystal structure of GTF180-ΔN in complex with maltose. Herein, we show that mutations in L940 of wild-type GTF180-ΔN all caused an increased percentage of (α1→6) linkages and a decreased percentage of (α1→3) linkages in the products. α-Glucans with potential different physico-chemical properties [containing 67% to 100% of (α1→6) linkages] were produced by GTF180 and its L940 mutants. Mutant L940W was unable to form (α1→3) linkages and synthesized a smaller and linear glucan polysaccharide with only (α1→6) linkages. Docking studies revealed that the introduction of the large aromatic amino acid residue tryptophan at position 940 partially blocked the binding groove, preventing the isomalto-oligosaccharide acceptor to bind in an favorable orientation for the formation of (α1→3) linkages. Our data showed that the reaction specificity of GTF180 mutant was shifted either to increased polysaccharide synthesis (L940A, L940S, L940E and L940F) or increased oligosaccharide synthesis (L940W). The L940W mutant is capable of producing a large amount of isomalto-oligosaccharides using released glucose from sucrose as acceptors. Thus, residue L940 in domain B is crucial for linkage and reaction specificity of GTF180. This study provides clear and novel insights into the structure-function relationships of glucansucrase enzymes.</p

Structural analysis of rebaudioside A derivatives obtained by Lactobacillus reuteri 180 glucansucrase-catalyzed trans-α-glucosylation

The wild-type Gtf180-ΔN glucansucrase enzyme from Lactobacillus reuteri 180 was found to catalyze the α-glucosylation of the steviol glycoside rebaudioside A, using sucrose as glucosyl donor in a transglucosylation process. Structural analysis of the formed products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that rebaudioside A is specifically α-d-glucosylated at the steviol C-19 β-d-glucosyl moiety (55% conversion). The main product is a mono-(α1 → 6)-glucosylated derivative (RebA-G1). A series of minor products, up to the incorporation of eight glucose residues, comprise elongations of RebA-G1 with mainly alternating (α1 → 3)- and (α1 → 6)-linked glucopyranose residues. These studies were carried out in the context of a program directed to the improvement of the taste of steviol glycosides via enzymatic modification of their naturally occurring carbohydrate moieties

Enzymatic depolymerization of alginate by two novel thermostable alginate lyases from Rhodothermus marinus

Alginate (alginic acid) is a linear polysaccharide, wherein (1→4)-linked β-D-mannuronic acid and its C5 epimer, α-L-guluronic acid, are arranged in varying sequences. Alginate lyases catalyze the depolymerization of alginate, thereby cleaving the (1→4) glycosidic linkages between the monomers by a β-elimination mechanism, to yield unsaturated 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid (Δ) at the non-reducing end of resulting oligosaccharides (α-L-erythro configuration) or, depending on the enzyme, the unsaturated monosaccharide itself. In solution, the released free unsaturated monomer product is further hydrated in a spontaneous (keto-enol tautomerization) process to form two cyclic stereoisomers. In this study, two alginate lyase genes, designated alyRm3 and alyRm4, from the marine thermophilic bacterium Rhodothermus marinus (strain MAT378), were cloned and expressed in Escherichia coli. The recombinant enzymes were characterized, and their substrate specificity and product structures determined. AlyRm3 (PL39) and AlyRm4 (PL17) are among the most thermophilic and thermostable alginate lyases described to date with temperature optimum of activity at ∼75 and 81°C, respectively. The pH optimum of activity of AlyRm3 is ∼5.5 and AlyRm4 at pH 6.5. Detailed NMR analysis of the incubation products demonstrated that AlyRm3 is an endolytic lyase, while AlyRm4 is an exolytic lyase, cleaving monomers from the non-reducing end of oligo/poly-alginates

Trans-α-glucosylation of stevioside by the mutant glucansucrase enzyme Gtf180-ΔN-Q1140E improves its taste profile

The adverse health effects of sucrose overconsumption, typical for diets in developed countries, necessitate use of low-calorie sweeteners. Following approval by the European Commission (2011), steviol glycosides are increasingly used as high-intensity sweeteners in food. Stevioside is the most prevalent steviol glycoside in Stevia rebaudiana plant leaves, but it has found limited applications in food products due to its lingering bitterness. Enzymatic glucosylation is a strategy to reduce stevioside bitterness, but reported glucosylation reactions suffer from low productivities. Here we present the optimized and efficient alpha-glucosylation of stevioside using the mutant glucansucrase Gtf180-Delta N-Q1140E and sucrose as donor substrate. Structures of novel products were elucidated by NMR spectroscopy, mass spectrometry and methylation analysis; stevioside was mainly glucosylated at the steviol C-19 glucosyl moiety. Sensory analysis of the alpha-glucosylated stevioside products by a trained panel revealed a significant reduction in bitterness compared to stevioside, resulting in significant improvement of edulcorant/organoleptic properties

High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays

Structural characterization of linear isomalto-/malto-oligomer products synthesized by the novel GTFB 4,6-α-glucanotransferase enzyme from Lactobacillus reuteri 121

Recently, a novel glucansucrase (GS)-like gene (gtfB) was isolated from the probiotic bacterium Lactobacillus reuteri 121 and expressed in Escherichia coli. The purified recombinant GTFB enzyme was characterized and turned out to be inactive with sucrose, the natural GS substrate. Instead, GTFB acted on malto-oligosaccharides (MOSs), thereby yielding elongated gluco-oligomers/polymers containing besides (α1→4) also (α1→6) glycosidic linkages, and it was classified as a 4,6-α-glucanotransferase. To gain more insight into its reaction specificity, incubations of the GTFB enzyme with a series of MOSs and their corresponding alditols [degree of polymerization, DP2(-ol)–DP7 (-ol)] were carried out, and (purified) products were structurally analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry and one-/two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy. With each of the tested malto-oligomers, the GTFB enzyme yielded series of novel linear isomalto-/malto-oligomers, in the case of DP7 up to DP >35.

Glucansucrase (mutant) enzymes from Lactobacillus reuteri 180 efficiently transglucosylate Stevia component rebaudioside A, resulting in a superior taste

Steviol glycosides from the leaves of the plant Stevia rebaudiana are high-potency natural sweeteners but suffer from a lingering bitterness. The Lactobacillus reuteri 180 wild-type glucansucrase Gtf180-ΔN, and in particular its Q1140E-mutant, efficiently α-glucosylated rebaudioside A (RebA), using sucrose as donor substrate. Structural analysis of the products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that both enzymes exclusively glucosylate the Glc(β1→C-19 residue of RebA, with the initial formation of an (α1→6) linkage. Docking of RebA in the active site of the enzyme revealed that only the steviol C-19 β-D-glucosyl moiety is available for glucosylation. Response surface methodology was applied to optimize the Gtf180-ΔN-Q1140E-catalyzed α-glucosylation of RebA, resulting in a highly productive process with a RebA conversion of 95% and a production of 115 g/L α-glucosylated products within 3 h. Development of a fed-batch reaction allowed further suppression of α-glucan synthesis which improved the product yield to 270 g/L. Sensory analysis by a trained panel revealed that glucosylated RebA products show a significant reduction in bitterness, resulting in a superior taste profile compared to RebA. The Gtf180-ΔN-Q1140E glucansucrase mutant enzyme thus is an efficient biocatalyst for generating α-glucosylated RebA variants with improved edulcorant/organoleptic properties