Host genetic regulation of human gut microbial structural variation.

Although the impact of host genetics on gut microbial diversity and the abundance of specific taxa is well established1-6, little is known about how host genetics regulates the genetic diversity of gut microorganisms. Here we conducted a meta-analysis of associations between human genetic variation and gut microbial structural variation in 9,015 individuals from four Dutch cohorts. Strikingly, the presence rate of a structural variation segment in Faecalibacterium prausnitzii that harbours an N-acetylgalactosamine (GalNAc) utilization gene cluster is higher in individuals who secrete the type A oligosaccharide antigen terminating in GalNAc, a feature that is jointly determined by human ABO and FUT2 genotypes, and we could replicate this association in a Tanzanian cohort. In vitro experiments demonstrated that GalNAc can be used as the sole carbohydrate source for F. prausnitzii strains that carry the GalNAc-metabolizing pathway. Further in silico and in vitro studies demonstrated that other ABO-associated species can also utilize GalNAc, particularly Collinsella aerofaciens. The GalNAc utilization genes are also associated with the host's cardiometabolic health, particularly in individuals with mucosal A-antigen. Together, the findings of our study demonstrate that genetic associations across the human genome and bacterial metagenome can provide functional insights into the reciprocal host-microbiome relationship.

Kinetics and products of Thermotoga maritima ß-glucosidase with lactose and cellobiose.

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother's milk. They are synthesized by ß-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many ß-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable ß-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the ß-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly ß(1 → 3) and ß(1 → 6) elongating activity, but also some ß(1 → 4) elongation was observed. The ß-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose. KEY POINTS: • ß-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield. • Thermotoga maritima ß-glucosidase has high activity and high thermostability. • Thermotoga maritima ß-glucosidase GOS contains mainly (ß1-3) and (ß1-6) linkages.

Sialic acid, the secret gift for the brain.

The human brain grows rapidly in early life which requires adequate nutrition. Human milk provides optimal nutrition for the developing brain, and breastfeeding significantly improves the cognition development of infants. These benefits have been largely attributed to human milk oligosaccharides (HMOS), associated with sialic acid (Sia). Subsequently, sialylated HMOS present a vital source of exogenous Sia to infants. Sialic acid is a key molecule essential for proper development of gangliosides, and therefore critical in brain development and function. Recent pre-clinical studies suggest dietary supplementation with Sia or sialylated oligosaccharides enhances intelligence and cognition performance in early and later life. Furthermore, emerging evidence suggests the involvement of Sia in brain homeostasis and disbalance correlates with common pathologies such as Alzheimer's disease (AD). Therefore, this review will discuss early brain health and development and the role of Sia in this process. Additionally, studies associating breastfeeding and specific HMOS to benefits in cognitive development are critically assessed. Furthermore, the review will assess studies implying the potential role of HMOS and microbiota in brain development via the gut-brain axis. Finally, the review will summarize recent advances regarding the role of Sia in neurodegenerative disease in later life and potential roles of dietary Sia sources.

Characterization of the (Engineered) Branching Sucrase GtfZ-CD2 from Apilactobacillus kunkeei for Efficient Glucosylation of Benzenediol Compounds.

Branching sucrases, a subfamily of Glycoside Hydrolase family (GH70), display transglycosidase activity using sucrose as donor substrate to catalyze glucosylation reaction in the presence of suitable acceptor substrates. In this study, the (α1→3) branching sucrase GtfZ-CD2 from Apilactobacillus kunkeei DSM 12361 was demonstrated to glucosylate benzenediol compounds (i.e., catechol, resorcinol, and hydroquinone) to form monoglucoside and diglucoside products. The production and yield of catechol glucosylated products were significantly higher than that of resorcinol and hydroquinone, revealing a preference for adjacent aromatic hydroxyl groups in glucosylation. Amino residues around acceptor substrate binding subsite +1 were targeted for semirational mutagenesis, yielding GtfZ-CD2 variants with improved resorcinol and hydroquinone glucosylation. Mutant L1560Y with improved hydroquinone mono-glucosylated product synthesis allowed enzymatic conversion of hydroquinone into α-arbutin. This study thus revealed the high potential of GH70 branching sucrases for glucosylating noncarbohydrate molecules. IMPORTANCE Glycosylation represents one of the most important ways to expand the diversity of natural products and improve their physico-chemical properties. Aromatic polyphenol compounds widely found in plants are reported to exhibit various remarkable biological activities; however, they generally suffer from low solubility and stability, which can be improved by glycosylation. Our present study on the glucosylation of benzenediol compounds by GH70 branching sucrase GtfZ-CD2 and its semirational engineering to improve the glucosylation efficiency provides insight into the mechanism of acceptor substrates binding and its glucosylation selectivity. The results demonstrate the potential of using branching sucrase as an effective enzymatic glucosylation tool.

Variations in N-linked glycosylation of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) whey protein: Intercow differences and dietary effects.

In bovine milk serum, the whey proteins with the highest N-glycan contribution are lactoferrin, IgG, and glycosylation-dependent cellular adhesion molecule 1 (GlyCAM-1); GlyCAM-1 is the dominant N-linked glycoprotein in bovine whey protein products. Whey proteins are base ingredients in a range of food products, including infant formulas. Glycan monosaccharide composition and variation thereof may affect functionality, such as the interaction of glycans with the immune system via recognition receptors. It is therefore highly relevant to understand whether and how the glycosylation of whey proteins (and their functionality) can be modulated. We recently showed that the glycoprofile of GlyCAM-1 varies between cows and during early lactation, whereas the glycoprofile of lactoferrin was highly constant. In the current study, we evaluated intercow differences and the effects of macronutrient supply on the N-linked glycosylation profiles of the major whey proteins in milk samples of Holstein-Friesian cows. Overall, approximately 60% of the N-glycan pool in milk protein was sialylated, or fucosylated, or both; GlyCAM-1 contributed approximately 78% of the total number of glycans in the overall whey protein N-linked glycan pool. The degree of fucosylation ranged from 44.8 to 73.3% between cows, and this variation was mainly attributed to the glycans of GlyCAM-1. Dietary supplementation with fat or protein did not influence the overall milk serum glycoprofile. Postruminal infusion of palm olein, glucose, and essential AA resulted in shifts in the degree of GlyCAM-1 fucosylation within individual cows, ranging in some cases from 50 to 71% difference in degree of fucosylation, regardless of treatment. Overall, these data demonstrate that the glycosylation, and particularly fucosylation, of GlyCAM-1 was variable, although these shifts appear to be related more to individual cow variation than to nutrient supply. To our knowledge, this is the first report of variation in glycosylation of a milk glycoprotein in mature, noncolostral milk. The functional implications of variable GlyCAM-1 fucosylation remain to be investigated.

Gut bacterial deamination of residual levodopa medication for Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by both motor and non-motor symptoms. Gastrointestinal tract dysfunction is one of the non-motor features, where constipation is reported as the most common gastrointestinal symptom. Aromatic bacterial metabolites are attracting considerable attention due to their impact on gut homeostasis and host's physiology. In particular, Clostridium sporogenes is a key contributor to the production of these bioactive metabolites in the human gut. RESULTS: Here, we show that C. sporogenes deaminates levodopa, the main treatment in Parkinson's disease, and identify the aromatic aminotransferase responsible for the initiation of the deamination pathway. The deaminated metabolite from levodopa, 3-(3,4-dihydroxyphenyl)propionic acid, elicits an inhibitory effect on ileal motility in an ex vivo model. We detected 3-(3,4-dihydroxyphenyl)propionic acid in fecal samples of Parkinson's disease patients on levodopa medication and found that this metabolite is actively produced by the gut microbiota in those stool samples. CONCLUSIONS: Levodopa is deaminated by the gut bacterium C. sporogenes producing a metabolite that inhibits ileal motility ex vivo. Overall, this study underpins the importance of the metabolic pathways of the gut microbiome involved in drug metabolism not only to preserve drug effectiveness, but also to avoid potential side effects of bacterial breakdown products of the unabsorbed residue of medication.

Structural and functional characterization of a family GH53 ß-1,4-galactanase from Bacteroides thetaiotaomicron that facilitates degradation of prebiotic galactooligosaccharides.

Galactooligosaccharides (GOS) are prebiotic compounds synthesized from lactose using bacterial enzymes and are known to stimulate growth of beneficial bifidobacteria in the human colon. Bacteroides thetaiotaomicron is a prominent human colon commensal bacterial species that hydrolyzes GOS using an extracellular Glycosyl Hydrolase (GH) family GH53 endo-galactanase enzyme (BTGH53), releasing galactose-based products for growth. Here we dissect the molecular basis for GOS activity of this B. thetaiotaomicron GH53 endo-galactanase. Elucidation of its X-ray crystal structure revealed that BTGH53 has a relatively open active site cleft which was not observed with the bacterial enzyme from Bacillus licheniformis (BLGAL). BTGH53 acted on GOS with degree of polymerization ≤3 and therefore more closely resembles activity of fungal GH53 enzymes (e.g. Aspergillus aculeatus AAGAL and Meripileus giganteus MGGAL). Probiotic lactobacilli that lack galactan utilization systems constitute a group of bacteria with relevance for a healthy (infant) gut. The strains tested were unable to use GOS ≥ DP3. However, they completely consumed GOS in the presence of BTGH53, resulting in clear stimulation of their extent of growth. The extracellular BTGH53 enzyme thus may play an important role in carbohydrate metabolism in complex microbial environments such as the human colon. It also may find application for the development of synergistic synbiotics.

Stimulatory effects of novel glucosylated lactose derivatives GL34 on growth of selected gut bacteria.

Previously we structurally characterized five glucosylated lactose derivatives (F1-F5) with a degree of polymerization (DP) of 3-4 (GL34), products of Lactobacillus reuteri glucansucrases, with lactose and sucrose as substrates. Here, we show that these GL34 compounds are largely resistant to the hydrolytic activities of common carbohydrate-degrading enzymes. Also, the ability of single strains of gut bacteria, bifidobacteria, lactobacilli, and commensal bacteria, to ferment the GL34 compounds was studied. Bifidobacteria clearly grew better on the GL34 mixture than lactobacilli and commensal bacteria. Lactobacilli and the commensal bacteria Escherichia coli Nissle and Bacteroides thetaiotaomicron only degraded the F2 compound α-D-Glcp-(1 → 2)-[ß-D-Galp-(1 → 4)-]D-Glcp, constituting around 30% w/w of GL34. Bifidobacteria digested more than one compound from the GL34 mixture, varying with the specific strain tested. Bifidobacterium adolescentis was most effective, completely degrading four of the five GL34 compounds, leaving only one minor constituent. GL34 thus represents a novel oligosaccharide mixture with (potential) synbiotic properties towards B. adolescentis, synthesized from cheap and abundantly available lactose and sucrose.

Biochemical characterization of a GH70 protein from Lactobacillus kunkeei DSM 12361 with two catalytic domains involving branching sucrase activity.

The fructophilic bacterium Lactobacillus kunkeei has promising applications as probiotics promoting the health of both honey bees and humans. Here, we report the synthesis of a highly branched dextran by L. kunkeei DSM 12361 and biochemical characterization of a GH70 enzyme (GtfZ). Sequence analysis revealed that GtfZ harbors two separate catalytic cores (CD1 and CD2), predicted to have glucansucrase and branching sucrase specificity, respectively. GtfZ-CD1 was not characterized biochemically due to its unsuccessful expression. With only sucrose as substrate, GtfZ-CD2 was found to mainly catalyze sucrose hydrolysis and leucrose synthesis. When dextran was available as acceptor substrate, GtfZ-CD2 displayed an efficient transglycosidase activity with sucrose as donor substrate. Kinetic analysis showed that the GtfZ-CD2-catalyzed transglycosylation reaction follows a Ping Pong Bi Bi mechanism, indicating the in-turn binding of donor and acceptor substrates in the active site. Structural characterization of the products revealed that GtfZ-CD2 catalyzes the synthesis of single glucosyl (α1 → 3) linked branches onto dextran, resulting in the production of highly branched comb-like α-glucan products. These (α1 → 3) branches can be formed on adjacent positions, as shown when isomaltotriose was used as acceptor substrate. Homology modeling of the GtfZ-CD1 and GtfZ-CD2 protein structure strongly suggests that amino acid differences in conserved motifs II, III, and IV in the catalytic domain contribute to product specificity. Our present study highlights the ability of beneficial lactic acid bacteria to produce structurally complex α-glucans and provides novel insights into the molecular mechanism of an (α1 → 3) branching sucrase.

Biochemical Characterization of the Functional Roles of Residues in the Active Site of the ß-Galactosidase from Bacillus circulans ATCC 31382.

The ß-galactosidase enzyme from Bacillus circulans ATCC 31382 BgaD is widely used in the food industry to produce prebiotic galactooligosaccharides (GOS). Recently, the crystal structure of a C-terminally truncated version of the enzyme (BgaD-D) has been elucidated. The roles of active site amino acid residues in ß-galactosidase enzyme reaction and product specificity have remained unknown. On the basis of a structural alignment of the ß-galactosidase enzymes BgaD-D from B. circulans and BgaA from Streptococcus pneumoniae, and the complex of BgaA with LacNAc, we identified eight active site amino acid residues (Arg185, Asp481, Lys487, Tyr511, Trp570, Trp593, Glu601, and Phe616) in BgaD-D. This study reports an investigation of the functional roles of these residues, using site-directed mutagenesis, and a detailed biochemical characterization and product profile analysis of the mutants obtained. The data show that these residues are involved in binding and positioning of the substrate and thus determine the BgaD-D activity and product linkage specificity. This study provides detailed insights into the structure-function relationships of the B. circulans BgaD-D enzyme, especially regarding GOS product linkage specificity, allowing the rational mutation of ß-galactosidase enzymes to produce specific mixtures of GOS structures.

Engineering of the Bacillus circulans ß-Galactosidase Product Specificity.

Microbial ß-galactosidase enzymes are widely used as biocatalysts in industry to produce prebiotic galactooligosaccharides (GOS) from lactose. GOS mixtures are used as beneficial additives in infant formula to mimic the prebiotic effects of human milk oligosaccharides (hMOS). The structural variety in GOS mixtures is significantly lower than in hMOS. Since this structural complexity is considered as the basis for the multiple biological functions of hMOS, it is important to broaden the variety of GOS structures. In this study, residue R484 near +1 subsite of the C-terminally truncated ß-galactosidase from Bacillus circulans (BgaD-D) was subjected to site saturation mutagenesis. Especially the R484S and R484H mutant enzymes displayed significantly altered enzyme specificity, leading to a new type of GOS mixture with altered structures and linkage types. The GOS mixtures produced by these mutant enzymes contained 14 structures that were not present in the wild-type enzyme GOS mixture; 10 of these are completely new structures. The GOS produced by these mutant enzymes contained a combination of (ß1 → 3) and (ß1 → 4) linkages, while the wild-type enzyme has a clear preference toward (ß1 → 4) linkages. The yield of the trisaccharide ß-d-Galp-(1 → 3)-ß-d-Galp-(1 → 4)-d-Glcp produced by mutants R484S and R484H increased 50 times compared to that of the wild-type enzyme. These results indicate that residue R484 is crucial for the linkage specificity of BgaD-D. This is the first study showing that ß-galactosidase enzyme engineering results in an altered GOS linkage specificity and product mixture. The more diverse GOS mixtures produced by these engineered enzymes may find industrial applications.

Discovery of a Xylooligosaccharide Oxidase from Myceliophthora thermophila C1.

By inspection of the predicted proteome of the fungus Myceliophthora thermophila C1 for vanillyl-alcohol oxidase (VAO)-type flavoprotein oxidases, a putative oligosaccharide oxidase was identified. By homologous expression and subsequent purification, the respective protein could be obtained. The protein was found to contain a bicovalently bound FAD cofactor. By screening a large number of carbohydrates, several mono- and oligosaccharides could be identified as substrates. The enzyme exhibits a strong substrate preference toward xylooligosaccharides; hence it is named xylooligosaccharide oxidase (XylO). Chemical analyses of the product formed upon oxidation of xylobiose revealed that the oxidation occurs at C1, yielding xylobionate as product. By elucidation of several XylO crystal structures (in complex with a substrate mimic, xylose, and xylobiose), the residues that tune the unique substrate specificity and regioselectivity could be identified. The discovery of this novel oligosaccharide oxidase reveals that the VAO-type flavoprotein family harbors oxidases tuned for specific oligosaccharides. The unique substrate profile of XylO hints at a role in the degradation of xylan-derived oligosaccharides by the fungus M. thermophila C1.

The Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 employs a new glycoside hydrolase family 70 4,6-α-glucanotransferase enzyme (GtfD) to synthesize a reuteran like polymer from maltodextrins and starch.

BACKGROUND: Originally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize α-glucan polymers from sucrose. Recently we have identified 2 novel subfamilies of GH70 enzymes represented by the Lactobacillus reuteri 121 GtfB and the Exiguobacterium sibiricum 255-15 GtfC enzymes. Both enzymes catalyze the cleavage of (α1→4) linkages in maltodextrin/starch and the synthesis of consecutive (α1→6) linkages. Here we describe a novel GH70 enzyme from the nitrogen-fixing Gram-negative bacterium Azotobacter chroococcum, designated as GtfD. METHODS: The purified recombinant GtfD enzyme was biochemically characterized using the amylose-staining assay and its products were identified using profiling chromatographic techniques (TLC and HPAEC-PAD). Glucans produced by the GtfD enzyme were analyzed by HPSEC-MALLS-RI, methylation analysis, 1D/2D [1]H/[13]C NMR spectroscopy and enzymatic degradation studies. RESULTS: The A. chroococcum GtfD is closely related to GtfC enzymes, sharing the same non-permuted domain organization also found in GH13 enzymes and displaying 4,6-α-glucanotransferase activity. However, the GtfD enzyme is unable to synthesize consecutive (α1→6) glucosidic bonds. Instead, it forms a high molecular mass and branched α-glucan with alternating (α1→4) and (α1→6) linkages from amylose/starch, highly similar to the reuteran polymer synthesized by the L. reuteri GtfA glucansucrase from sucrose. CONCLUSIONS: In view of its origin and specificity, the GtfD enzyme represents a unique evolutionary intermediate between family GH13 (α-amylase) and GH70 (glucansucrase) enzymes. GENERAL SIGNIFICANCE: This study expands the natural repertoire of starch-converting enzymes providing the first characterization of an enzyme that converts starch into a reuteran-like α-glucan polymer, regarded as a health promoting food ingredient.

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

Previously, we have shown that the glucansucrase GtfA-ΔN enzyme of Lactobacillus reuteri 121, incubated with sucrose, efficiently glucosylated catechol and we structurally characterized catechol glucosides with up to five glucosyl units attached (te Poele et al. in Bioconjug Chem 27:937-946, 2016). In the present study, we observed that upon prolonged incubation of GtfA-ΔN with 50 mM catechol and 1000 mM sucrose, all catechol had become completely glucosylated and then started to reappear. Following depletion of sucrose, this glucansucrase GtfA-ΔN used both α-D-Glcp-catechol and α-D-Glcp-(1→4)-α-D-Glcp-catechol as donor substrates and transferred a glucose unit to other catechol glycoside molecules or to sugar oligomers. In the absence of sucrose, GtfA-ΔN used α-D-Glcp-catechol both as donor and acceptor substrate to synthesize catechol glucosides with 2 to 10 glucose units attached and formed gluco-oligosaccharides up to a degree of polymerization of 4. Also two other glucansucrases tested, Gtf180-ΔN from L. reuteri 180 and GtfML1-ΔN from L. reuteri ML1, used α-D-Glcp-catechol and di-glucosyl-catechol as donor/acceptor substrate to synthesize both catechol glucosides and gluco-oligosaccharides. With sucrose as donor substrate, the three glucansucrase enzymes also efficiently glucosylated the phenolic compounds pyrogallol, resorcinol, and ethyl gallate; also these mono-glucosides were used as donor/acceptor substrates.

Structure-function relationships of family GH70 glucansucrase and 4,6-α-glucanotransferase enzymes, and their evolutionary relationships with family GH13 enzymes.

Lactic acid bacteria (LAB) are known to produce large amounts of α-glucan exopolysaccharides. Family GH70 glucansucrase (GS) enzymes catalyze the synthesis of these α-glucans from sucrose. The elucidation of the crystal structures of representative GS enzymes has advanced our understanding of their reaction mechanism, especially structural features determining their linkage specificity. In addition, with the increase of genome sequencing, more and more GS enzymes are identified and characterized. Together, such knowledge may promote the synthesis of α-glucans with desired structures and properties from sucrose. In the meantime, two new GH70 subfamilies (GTFB- and GTFC-like) have been identified as 4,6-α-glucanotransferases (4,6-α-GTs) that represent novel evolutionary intermediates between the family GH13 and "classical GH70 enzymes". These enzymes are not active on sucrose; instead, they use (α1 â†’ 4) glucans (i.e. malto-oligosaccharides and starch) as substrates to synthesize novel α-glucans by introducing linear chains of (α1 â†’ 6) linkages. All these GH70 enzymes are very interesting biocatalysts and hold strong potential for applications in the food, medicine and cosmetic industries. In this review, we summarize the microbiological distribution and the structure-function relationships of family GH70 enzymes, introduce the two newly identified GH70 subfamilies, and discuss evolutionary relationships between family GH70 and GH13 enzymes.

Glucosylation of Catechol with the GTFA Glucansucrase Enzyme from Lactobacillus reuteri and Sucrose as Donor Substrate.

Lactic acid bacteria use glucansucrase enzymes for synthesis of gluco-oligosaccharides and polysaccharides (α-glucans) from sucrose. Depending on the glucansucrase enzyme, specific α-glucosidic linkages are introduced. GTFA-ΔN (N-terminally truncated glucosyltransferase A) is a glucansucrase enzyme of Lactobacillus reuteri 121 that synthesizes the reuteran polysaccharide with (α1 → 4) and (α1 → 6) glycosidic linkages. Glucansucrases also catalyze glucosylation of various alternative acceptor substrates. At present it is unclear whether the linkage specificity of these enzymes is the same in oligo/polysaccharide synthesis and in glucosylation of alternative acceptor substrates. Our results show that GTFA-ΔN glucosylates catechol into products with up to at least 5 glucosyl units attached. These catechol glucosides were isolated and structurally characterized using 1D/2D (1)H NMR spectroscopy. They contained 1 to 5 glucose units with different (α1 → 4) and (α1 → 6) glycosidic linkage combinations. Interestingly, a branched catechol glucoside was also formed along with a catechol glucoside with 2 successive (α1 → 6) glycosidic linkages, products that are absent when only sucrose is used as both glycosyl donor and acceptor substrate.

Glucansucrase Gtf180-ΔN of Lactobacillus reuteri 180: enzyme and reaction engineering for improved glycosylation of non-carbohydrate molecules.

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.

A GH57 4-α-glucanotransferase of hyperthermophilic origin with potential for alkyl glycoside production.

4-α-Glucanotransferase (GTase) enzymes (EC 2.4.1.25) modulate the size of α-glucans by cleaving and reforming α-1,4 glycosidic bonds in α-glucans, an essential process in starch and glycogen metabolism in plants and microorganisms. The glycoside hydrolase family 57 enzyme (GTase57) studied in the current work catalyzes both disproportionation and cyclization reactions. Amylose was converted into cyclic amylose (with a minimum size of 17 glucose monomers) as well as to a spectrum of maltodextrins, but in contrast to glycoside hydrolase family 13 cyclodextrin glucanotransferases (CGTases), no production of cyclodextrins (C6-C8) was observed. GTase57 also effectively produced alkyl-glycosides with long α-glucan chains from dodecyl-ß-D-maltoside and starch, demonstrating the potential of the enzyme to produce novel variants of surfactants. Importantly, the GTase57 has excellent thermostability with a maximal activity at 95 °C and an activity half-life of 150 min at 90 °C which is highly advantageous in this manufacturing process suggesting that enzymes from this relatively uncharacterized family, GH57, can be powerful biocatalysts for the production of large head group glucosides from soluble starch.

Rapid milk group classification by 1H NMR analysis of Le and H epitopes in human milk oligosaccharide donor samples.

Human milk oligosaccharides (HMOs) are a major constituent of human breast milk and play an important role in reducing the risk of infections in infants. The structures of these HMOs show similarities with blood group antigens in protein glycosylation, in particular in relation to fucosylation in Lewis blood group-type epitopes, matching the maternal pattern. Previously, based on the Secretor and Lewis blood group system, four milk groups have been defined, i.e. Lewis-positive Secretors, Lewis-positive non-Secretors, Lewis-negative Secretors and Lewis-negative non-Secretors. Here, a rapid one-dimensional (1)H nuclear magnetic resonance (NMR) analysis method is presented that identifies the presence/absence of (α1-2)-, (α1-3)- and (α1-4)-linked fucose residues in HMO samples, affording the essential information to attribute different HMO samples to a specific milk group. The developed method is based on the NMR structural-reporter-group concept earlier established for glycoprotein glycans. Further evaluation of the data obtained from the analysis of 36 HMO samples shows that within each of the four milk groups the relative levels of the different fucosylation epitopes can greatly vary. The data also allow a separation of the Lewis-positive Secretor milk group into two sub-groups.

Galactosyl-lactose sialylation using Trypanosoma cruzi trans-sialidase as the biocatalyst and bovine κ-casein-derived glycomacropeptide as the donor substrate.

trans-Sialidase (TS) enzymes catalyze the transfer of sialyl (Sia) residues from Sia(α2-3)Gal(ß1-x)-glycans (sialo-glycans) to Gal(ß1-x)-glycans (asialo-glycans). Aiming to apply this concept for the sialylation of linear and branched (Gal)nGlc oligosaccharide mixtures (GOS) using bovine κ-casein-derived glycomacropeptide (GMP) as the sialic acid donor, a kinetic study has been carried out with three components of GOS, i.e., 3'-galactosyl-lactose (ß3'-GL), 4'-galactosyl-lactose (ß4'-GL), and 6'-galactosyl-lactose (ß6'-GL). This prebiotic GOS is prepared from lactose by incubation with suitable ß-galactosidases, whereas GMP is a side-stream product of the dairy industry. The trans-sialidase from Trypanosoma cruzi (TcTS) was expressed in Escherichia coli and purified. Its temperature and pH optima were determined to be 25°C and pH 5.0, respectively. GMP [sialic acid content, 3.6% (wt/wt); N-acetylneuraminic acid (Neu5Ac), >99%; (α2-3)-linked Neu5Ac, 59%] was found to be an efficient sialyl donor, and up to 95% of the (α2-3)-linked Neu5Ac could be transferred to lactose when a 10-fold excess of this acceptor substrate was used. The products of the TcTS-catalyzed sialylation of ß3'-GL, ß4'-GL, and ß6'-GL, using GMP as the sialic acid donor, were purified, and their structures were elucidated by nuclear magnetic resonance spectroscopy. Monosialylated ß3'-GL and ß4'-GL contained Neu5Ac connected to the terminal Gal residue; however, in the case of ß6'-GL, TcTS was shown to sialylate the 3 position of both the internal and terminal Gal moieties, yielding two different monosialylated products and a disialylated structure. Kinetic analyses showed that TcTS had higher affinity for the GL substrates than lactose, while the Vmax and kcat values were higher in the case of lactose.