Regioselective thexyldimethylsilylation of (1➔3)-glucans - Does the linkage type matter.

α- and β-(1➔3)-linked polysaccharides dissolved in N,N-dimethyl acetamide (DMA) were subjected to conversion with thexyldimethylchlorosilane (TDMS-Cl) in presence of pyridine as base. A degree of substitution of TDMS groups (DS) between 0.7 and 1.

A chemo-enzymatic pathway to expand cellooligosaccharide chemical space through amine bond introduction.

Enzymatic functionalization of oligosaccharides is a useful and environmentally friendly way to expand their structural chemical space and access to a wider range of applications in the health, food, feed, cosmetics and other sectors. In this work, we first tested the laccase/TEMPO system to generate oxidized forms of cellobiose and methyl β-D-cellobiose, and obtained high yields of novel anionic disaccharides (>60 %) at pH 6.0.

Expanding the Enzymatic Polymerization Landscape by Lipid Mesophase Soft Nanoconfinement.

Soft nanoconfinement can increase chemical reactivity in nature and has therefore led to considerable interest in transferring this universal feature to artificial biological systems. However, little is known about the underlying principles of soft nanoconfinement responsible for the enhancement of biochemical reactions. Herein we demonstrate how enzymatic polymerization can be expanded, optimized, and engineered when carried out under soft nanoconfinement mediated by lipidic mesophases.

Phosphorylated polysaccharides: Applications, natural abundance, and new-to-nature structures generated by chemical and enzymatic functionalisation.

Polysaccharides are foreseen as serious candidates for the future generation of polymers, as they are biosourced and biodegradable materials. Their functionalisation is an attractive way to modify their properties, thereby increasing their range of applications. Introduction of phosphate groups in polysaccharide chains for the stimulation of the immune system was first described in the nineteen seventies.

Computer-aided engineering of a branching sucrase for the glucodiversification of a tetrasaccharide precursor of S. flexneri antigenic oligosaccharides.

Enzyme engineering approaches have allowed to extend the collection of enzymatic tools available for synthetic purposes. However, controlling the regioselectivity of the reaction remains challenging, in particular when dealing with carbohydrates bearing numerous reactive hydroxyl groups as substrates. Here, we used a computer-aided design framework to engineer the active site of a sucrose-active [Formula: see text]-transglucosylase for the 1,2-cis-glucosylation of a lightly protected chemically synthesized tetrasaccharide, a common precursor for the synthesis of serotype-specific S.

Bacterial α-Glucan and Branching Sucrases from GH70 Family: Discovery, Structure-Function Relationship Studies and Engineering.

Glucansucrases and branching sucrases are classified in the family 70 of glycoside hydrolases. They are produced by lactic acid bacteria occupying very diverse ecological niches (soil, buccal cavity, sourdough, intestine, dairy products, etc.).

Redirecting substrate regioselectivity using engineered ΔN-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens.

The (chemo-)enzymatic synthesis of oligosaccharides has been hampered by the lack of appropriate enzymatic tools with requisite regio- and stereo-specificities. Engineering of carbohydrate-active enzymes, in particular targeting the enzyme active site, has notably led to catalysts with altered regioselectivity of the glycosylation reaction thereby enabling to extend the repertoire of enzymes for carbohydrate synthesis. Using a collection of 22 mutants of ΔN-GBD-CD2 branching sucrase, an enzyme from the Glycoside Hydrolase family 70, containing between one and three mutations in the active site, and a lightly protected chemically synthesized tetrasaccharide as an acceptor substrate, we showed that altered glycosylation product specificities could be achieved compared to the parental enzyme.

Natural and engineered transglycosylases: Green tools for the enzyme-based synthesis of glycoproducts.

An increasing number of transglycosylase-based processes provide access to oligosaccharides or glycoconjugates, some of them reaching performance levels compatible with industrial developments. Nevertheless, the full potential of transglycosylases has not been explored because of the challenges in transforming a glycoside hydrolase into an efficient transglycosylase. Advances in studying enzyme structure/function relationships, screening enzyme activity, and generating synthetic libraries guided by computational protein design or machine learning methods should considerably accelerate the development of these catalysts.

Convergent Chemoenzymatic Strategy to Deliver a Diversity of Serotype-Specific O-Antigen Segments from a Unique Lightly Protected Tetrasaccharide Core.

Progress in glycoscience is strongly dependent on the availability of broadly diverse tailor-made, well-defined, and often complex oligosaccharides. Herein, going beyond natural resources and aiming to circumvent chemical boundaries in glycochemistry, we tackle the development of an chemoenzymatic strategy holding great potential to answer the need for molecular diversity characterizing microbial cell-surface carbohydrates. The concept is exemplified in the context of , a major cause of diarrhoeal disease.

A specific oligosaccharide-binding site in the alternansucrase catalytic domain mediates alternan elongation.

Microbial α-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) from NRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASRΔ2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR.

Processivity of dextransucrases synthesizing very-high-molar-mass dextran is mediated by sugar-binding pockets in domain V.

The dextransucrase DSR-OK from the Gram-positive bacterium DSM17330 produces a dextran of the highest molar mass reported to date (∼10 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding.

Futile Encounter Engineering of the DSR-M Dextransucrase Modifies the Resulting Polymer Length.

The factors that define the resulting polymer length of distributive polymerases are poorly understood. Here, starting from the crystal structure of the dextransucrase DSR-M in complex with an isomaltotetraose, we define different anchoring points for the incoming acceptor. Mutation of one of these, Trp624, decreases the catalytic rate of the enzyme but equally skews the size distribution of the resulting dextran chains toward shorter chains.

Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides.

Combined with chemical synthesis, the use of glycoenzyme biocatalysts has shown great synthetic potential over recent decades owing to their remarkable versatility in terms of substrates and regio- and stereoselectivity that allow structurally controlled synthesis of carbohydrates and glycoconjugates. Nonetheless, the lack of appropriate enzymatic tools with requisite properties in the natural diversity has hampered extensive exploration of enzyme-based synthetic routes to access relevant bioactive oligosaccharides, such as cell-surface glycans or prebiotics. With the remarkable progress in enzyme engineering, it has become possible to improve catalytic efficiency and physico-chemical properties of enzymes but also considerably extend the repertoire of accessible catalytic reactions and tailor novel substrate specificities.

Engineering a branching sucrase for flavonoid glucoside diversification.

Enzymatic glycosylation of flavonoids is an efficient mean to protect aglycons against degradation while enhancing their solubility, life time and, by extension, their bioavailability which is critical for most of their applications in health care. To generate a valuable enzymatic platform for flavonoid glucosylation, an α-1,2 branching sucrase belonging to the family 70 of glycoside-hydrolases was selected as template and subsequently engineered. Two libraries of variants targeting pair-wise mutations inferred by molecular docking simulations were generated and screened for quercetin glucosylation using sucrose as a glucosyl donor.

Macromolecular structure and film properties of enzymatically-engineered high molar mass dextrans.

New α(1→2) or α(1→3) branched dextrans with high molar masses and controlled architecture were synthesized using a dextransucrase and branching sucrases. Their molecular structure, solubility, conformation, film-forming ability, as well as their thermal and mechanical properties were determined. These new dextrans present structures with low densities from 9,500 to 14,000gm in HO/DMSO medium, their molar mass, size and dispersity increase with increasing branching degree (weight-average molar mass up to 10gmol and radius of gyration around 500nm).

A dextran with unique rheological properties produced by the dextransucrase from Oenococcus kitaharae DSM 17330.

A gene encoding a novel dextransucrase was identified in the genome of Oenococcus kitaharae DSM17330 and cloned into E. coli. With a kcat of 691s and a half-life time of 111h at 30°C, the resulting recombinant enzyme -named DSR-OK- stands as one of the most efficient and stable dextransucrase characterized to date.

The stability of an mRNA is influenced by its concentration: a potential physical mechanism to regulate gene expression.

Changing mRNA stability is a major post-transcriptional way of controlling gene expression, particularly in newly encountered conditions. As the concentration of mRNA is the result of an equilibrium between transcription and degradation, it is generally assumed that at constant transcription, any change in mRNA concentration is the consequence of mRNA stabilization or destabilization. However, the literature reports many cases of opposite variations in mRNA concentration and stability in bacteria.

Engineering of anp efficient mutant of Neisseria polysaccharea amylosucrase for the synthesis of controlled size maltooligosaccharides.

Amylosucrase from Neisseria polysaccharea naturally catalyzes the synthesis of α-1,4 glucans from sucrose. The product profile is quite polydisperse, ranging from soluble chains called maltooligosaccharides to high-molecular weight insoluble amylose. This enzyme was recently subjected to engineering of its active site to enable recognition of non-natural acceptor substrates.

Novel product specificity toward erlose and panose exhibited by multisite engineered mutants of amylosucrase.

A computer-aided engineering approach recently enabled to deeply reshape the active site of N. polysaccharea amylosucrase for recognition of non-natural acceptor substrates. Libraries of variants were constructed and screened on sucrose allowing the identification of 17 mutants able to synthesize molecules from sole sucrose, which are not synthesized by the parental wild-type enzyme.

Evaluation of dough rheological properties and bread texture of pearl millet-wheat flour mix.

This study was undertaken with the objective of formulating composite bread using pearl millet (Pennisetum glaucum) and wheat (Triticum aestivum) flours . Rheological and bread making properties of composite flours were evaluated. Mixolab results revealed torque increased and dough stability time decreased upon incorporation of pearl millet flour in wheat flour.

GH13 amylosucrases and GH70 branching sucrases, atypical enzymes in their respective families.

Amylosucrases and branching sucrases are α-retaining transglucosylases found in the glycoside-hydrolase families 13 and 70, respectively, of the clan GH-H. These enzymes display unique activities in their respective families. Using sucrose as substrate and without mediation of nucleotide-activated sugars, amylosucrase catalyzes the formation of an α-(1 → 4) linked glucan that resembles amylose.

Characterization of the First α-(1→3) Branching Sucrases of the GH70 Family.

Leuconostoc citreumNRRL B-742 has been known for years to produce a highly α-(1→3)-branched dextran for which the synthesis had never been elucidated. In this work a gene coding for a putative α-transglucosylase of the GH70 family was identified in the reported genome of this bacteria and functionally characterized. From sucrose alone, the corresponding recombinant protein, named BRS-B, mainly catalyzed sucrose hydrolysis and leucrose synthesis.

Overview of the glucansucrase equipment of Leuconostoc citreum LBAE-E16 and LBAE-C11, two strains isolated from sourdough.

The whole set of putative glucansucrases from Leuconostoc citreum LBAE-E16 and LBAE-C11 was retrieved from the draft genome sequence of these two sourdough strains previously suggested as alternan producers. Four and five putative glycoside hydrolase family 70 (GH70) encoding genes were identified in the genome sequence of strain C11 and E16, respectively. Some putative genes have high sequence identity to known Leuconostoc dextransucrases.

Inventory of the GH70 enzymes encoded by Leuconostoc citreum NRRL B-1299 - identification of three novel α-transglucosylases.

Leuconostoc citreum NRRL B-1299 has long been known to produce α-glucans containing both α-(1→6) and α-(1→2) linkages, which are synthesized by α-transglucosylases of the GH70 family. We sequenced the genome of Leuconostoc citreum NRRL B-1299 to identify the full inventory of GH70 enzymes in this strain. Three new genes (brsA, dsrM and dsrDP) putatively encoding GH70 enzymes were identified.

Complete Genome Sequence of Leuconostoc citreum Strain NRRL B-742.

Leuconostoc citreum belongs to the group of lactic acid bacteria and plays an important role in fermented foods of plant origin. Here, we report the complete genome of the Leuconostoc citreum strain NRRL B-742, isolated in 1954 for its capacity to produce dextran.