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Polyphenols exhibit various beneficial biological activities and represent very promising candidates as active compounds for food industry. However, the low solubility, poor stability and low bioavailability of polyphenols have severely limited their industrial applications. Enzymatic glycosylation is an effective way to improve the physicochemical properties of polyphenols. As efficient transglucosidases, glycoside hydrolase family 70 (GH70) glucansucrases naturally catalyze the synthesis of polysaccharides and oligosaccharides from sucrose. Notably, GH70 glucansucrases show broad acceptor substrate promiscuity and catalyze the glucosylation of a wide range of non-carbohydrate hydroxyl group-containing molecules, including benzenediol, phenolic acids, flavonoids and steviol glycosides. Branching sucrase enzymes, a newly established subfamily of GH70, are shown to possess a broader acceptor substrate binding pocket that acts efficiently for glucosylation of larger size polyphenols such as flavonoids. Here we present a comprehensive review of glucosylation of polyphenols using GH70 glucansucrase and branching sucrases. Their catalytic efficiency, the regioselectivity of glucosylation and the structure of generated products are described for these reactions. Moreover, enzyme engineering is effective for improving their catalytic efficiency and product specificity. The combined information provides novel insights on the glucosylation of polyphenols by GH70 glucansucrases and branching sucrases, and may promote their applications.

Solid-liquid thermo-reversibility, lacking in raw starch is an important property for gels allowing repeated thermal processing in food. In this work, the Thermoproteus uzoniensis 4-α-glucanotransferase (TuαGT) was used to modify five different starches from potato, corn, rice, wheat, and pea and the thermo-reversibility of the modified starch gels was evaluated by rheological assay. Only modified potato starch (MPS) exhibited thermo-reversibility, which prompted us to perform detailed analyses of the structural and gel properties of this product. The enzymatic modification resulted in a marked decrease in amylopectin side chains with a degree of polymerization (DP) < 23, while the contents of side chains with DP 23–43 increased. These data indicated that TuαGT preferentially extended the outer chains of amylopectin using amylose as a donor, which resulted in 17% increase in the amylopectin molecular weight to 3.0 × 107 g/mol. Notably, MPS displayed a delayed retrogradation and improved stability in four freeze-thaw cycles (−70 °C and 25 °C) as compared to the native starch. These analyses supported a model to explain the TuαGT catalyzed conversion of potato starch to a thermo-reversible product. Importantly, the MPS thermo-reversible 6% (w/v) gel showed comparable rheological properties to a 1.5% (w/v) κ-carrageenan hydrocolloid, highlighting the potential of MPS as a substitute to κ-carrageenan and other hydrocolloids of emerging health concerns. This work offers a starch based thermo-reversible gel and a methodological framework to broadens the use of starch into novel application as a food ingredient.

GtfC-type 4,6-α-glucanotransferase (α-GT) enzymes from Glycoside Hydrolase Family 70 (GH70) are of interest for the modification of starch into low-glycemic index food ingredients. Compared to the related GH70 GtfB-type α-GTs, found exclusively in lactic acid bacteria (LAB), GtfCs occur in non-LAB, share low sequence identity, lack circular permutation of the catalytic domain, and feature a single-segment auxiliary domain IV and auxiliary C-terminal domains. Despite these differences, the first crystal structure of a GtfC, GbGtfC-ΔC from

GtfB-type α-glucanotransferase enzymes from glycoside hydrolase family 70 (GH70) convert starch substrates into α-glucans that are of interest as food ingredients with a low glycemic index. Characterization of several GtfBs showed that they differ in product- and substrate specificity, especially with regard to branching, but structural information is limited to a single GtfB, preferring mostly linear starches and featuring a tunneled binding groove. Here, we present the second crystal structure of a 4,6-α-glucanotransferase (Limosilactobacillus reuteri NCC 2613) and an improved homology model of a 4,3-α-glucanotransferase GtfB (L. fermentum NCC 2970) and show that they are able to convert both linear and branched starch substrates. Compared to the previously described GtfB structure, these two enzymes feature a much more open binding groove, reminiscent of and evolutionary closer to starch-converting GH13 α-amylases. Sequence analysis of 287 putative GtfBs suggests that only 20% of them are similarly "open" and thus suitable as broad-specificity starch-converting enzymes.

ScopeCost-effective microbial biosynthesis of 2′-fucosyllactose (2′-FL) allows its application in infant formula. The specific effects of 2′-FL on the gastrointestinal immune barrier are still largely unknown.Methods/resultsHere, we quantified and compared the effects of HMOs isolated from human milk, 2′-FL/lactose (Lac), and 2′-FL on the expression of the mucus associated genes MUC2, TFF3, RETLB and the Golgi sulfotransferases, CHST5, and GAL3ST5, in human goblet cells. We also determined whether these compounds have protective effects on A23187-induced barrier disruption of human T84 gut epithelial cells in vitro. The impact of isolated HMOs and 2′-FL/Lac on the mRNA expression of the mucus-related genes was minor while pure 2′-FL significantly induced GAL3ST2 and CHST5. Isolated HMOs, 2′-FL/Lac and 2′-FL all prevented A23187-induced barrier disruption in human T84 cells.ConclusionOur findings indicate that 2′-FL modulates the secretory function of goblet cells and protects gut epithelial cells.

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.

Human milk is considered the golden standard in infant nutrition. Free oligosaccharides in human milk provide important health benefits. These oligosaccharides function as prebiotics, immune modulators, and pathogen inhibitors and were found to improve barrier function in the gut. Infant formulas nowadays often contain prebiotics but lack the specific functions of human milk oligosaccharides (hMOS). Milk from domesticated animals also contains milk oligosaccharides but at much lower levels and with less diversity. Goat milk contains significantly more oligosaccharides (gMOS) than bovine (bMOS) or sheep (sMOS) milk and also has a larger diversity of structures. This review summarizes structural studies, revealing a diversity of up to 77 annotated gMOS structures with almost 40 structures fully characterized. Quantitative studies of goat milk oligosaccharides range from 60 to 350 mg/L in mature milk and from 200 to 650 mg/L in colostrum. These levels are clearly lower than in human milk (5–20 g/L) but higher than in other domesticated dairy animals, e.g., bovine (30–60 mg/L) and sheep (20–40 mg/L). Finally, the review focuses on demonstrated and potential functionalities of gMOS. Some studies have shown anti-inflammatory effects of mixtures enriched in gMOS. Goat MOS also display prebiotic potential, particularly in stimulating growth of bifidobacteria preferentially. Although functional studies of gMOS are still limited, several structures are also found in human milk and have known functions as immune modulators and pathogen inhibitors. In conclusion, goat milk constitutes a promising alternative source for milk oligosaccharides, which can be used in infant formula.

The N-linked glycoprofile of bovine whey is the combined result of individual protein glycoprofiles. In this work, we provide in-depth structural information on the glycan structures of known whey glycoproteins, namely lactoferrin, lactoperoxidase, α-lactalbumin, immunoglobulin-G (IgG) and glycosylation dependent cellular adhesion molecule 1 (GlyCAM-1, PP3). The majority (~95%) of N-glycans present in the overall whey glycoprofile were attributed to three proteins; Lactoferrin, IgG and GlyCAM-1. We identified specific signature glycans for these main proteins; Lactoferrin contributes oligomannose-type glycans, while IgG carries fucosylated di-antennary glycans with Gal-β(1,4)GlcNAc (LacNAc) motifs. GlyCAM-1 is the sole whey glycoprotein carrying tri- and tetra-antennary structures, with a high degree of fucosylation and sialylation. Signature glycans can be used to recognize individual proteins in the overall whey glycoprofile, as well as for protein concentration estimations. Application of the whey glycoprofile analysis to colostrum samples revealed dynamic protein concentration changes for IgG, lactoferrin and GlyCAM-1 over time.

GtfB-type α-glucanotransferase enzymes from glycoside hydrolase family 70 (GH70) convert starch substrates into α-glucans that are of interest as food ingredients with a low glycemic index. Characterization of several GtfBs showed that they differ in product- and substrate specificity, especially with regard to branching, but structural information is limited to a single GtfB, preferring mostly linear starches and featuring a tunneled binding groove. Here, we present the second crystal structure of a 4,6-α-glucanotransferase (Limosilactobacillus reuteri NCC 2613) and an improved homology model of a 4,3-α-glucanotransferase GtfB (L. fermentum NCC 2970) and show that they are able to convert both linear and branched starch substrates. Compared to the previously described GtfB structure, these two enzymes feature a much more open binding groove, reminiscent of and evolutionary closer to starch-converting GH13 α-amylases. Sequence analysis of 287 putative GtfBs suggests that only 20% of them are similarly “open” and thus suitable as broad-specificity starch-converting enzymes.

Starch-acting α-glucanotransferase enzymes are of great interest for applications in the food industry. In previous work, we have characterized various 4,6- and 4,3-α-glucanotransferases of the glycosyl hydrolase (GH) family 70 (subfamily GtfB), synthesizing linear or branched α-glucans. Thus far, GtfB enzymes have only been identified in mesophilic Lactobacilli. Database searches showed that related GtfC enzymes occur in Gram-positive bacteria of the genera Exiguobacterium, Bacillus, and Geobacillus, adapted to growth at more extreme temperatures. Here, we report characteristics of the Geobacillus sp. 12AMOR1 GtfC enzyme, with an optimal reaction temperature of 60 °C and a melting temperature of 68 °C, allowing starch conversions at relatively high temperatures. This thermostable 4,6-α-glucanotransferase has a novel product specificity, cleaving off predominantly maltose units from amylose, attaching them with an (α1 → 6)-linkage to acceptor substrates. In fact, this GtfC represents a novel maltogenic α-amylase. Detailed structural characterization of its starch-derived α-glucan products revealed that it yielded a unique polymer with alternating (α1 → 6)/(α1 → 4)-linked glucose units but without branches. Notably, this Geobacillus sp. 12AMOR1 GtfC enzyme showed clear antistaling effects in bread bakery products.

Starch-acting α-glucanotransferase enzymes are of great interest for applications in the food industry. In previous work, we have characterized various 4,6- and 4,3-α-glucanotransferases of the glycosyl hydrolase (GH) family 70 (subfamily GtfB), synthesizing linear or branched α-glucans. Thus far, GtfB enzymes have only been identified in mesophilic

The glycemic carbohydrates we consume are currently viewed in an unfavorable light in both the consumer and medical research worlds. In significant part, these carbohydrates, mainly starch and sucrose, are looked upon negatively due to their rapid and abrupt glucose delivery to the body which causes a high glycemic response. However, dietary carbohydrates which are digested and release glucose in a slow manner are recognized as providing health benefits. Slow digestion of glycemic carbohydrates can be caused by several factors, including food matrix effect which impedes α-amylase access to substrate, or partial inhibition by plant secondary metabolites such as phenolic compounds. Differences in digestion rate of these carbohydrates may also be due to their specific structures (e.g. variations in degree of branching and/or glycosidic linkages present). In recent years, much has been learned about the synthesis and digestion kinetics of novel α-glucans (i.e. small oligosaccharides or larger polysaccharides based on glucose units linked in different positions by α-bonds). It is the synthesis and digestion of such structures that is the subject of this review.

Various beta-galactosidase enzymes catalyze the trans-glycosylation reaction with lactose. The resulting galactooligosaccharide (GOS) mixtures are widely used in infant nutrition to stimulate growth of beneficial gut bacteria. GOS consists mainly of compounds with a degree of polymerization (DP) varying from 2-8 and with diverse glycosidic linkages. In recent years, we have elucidated in detail the composition of several commercial GOS mixtures in terms of DP and the structural identity of the individual compounds. In this work, 13 (single) probiotic strains of gut bacteria, belonging to 11 different species, were grown to stationary phase with a Vivinal GOS-derived sample purified to remove lactose and monosaccharides (pGOS). Growth among the probiotic strains varied strongly between 30 and 100% of OD600nm relative to positive controls with glucose. By identifying the components of the pGOS mixture that remain after growth, we showed that strains varied in their consumption of specific GOS compounds. All strains commonly used most of the GOS DP2 pool. Lactobacillus salivarius W57 also utilized the DP3 branched compound beta-D-Galp-(1 -> 4)-[beta-D-Galp-(1 -> 2)]-D-G1c. Bifidobacterial strains tended to use GOS with higher DP and branching than lactobacilli; Bifidobacterium breve DSM 20091, Lactobacillus acidophilus W37, and Bifidobacterium infantis DSM 20088 were exceptional in using 38, 36, and 35 compounds, respectively, out of the 40 different structures identified in pGOS. We correlated these bacterial GOS consumption profiles with their genomic information and were able to relate metabolic activity with the presence of genome-encoded transporters and carbohydrate-active enzymes. These detailed insights may support the design of synbiotic combinations pairing probiotic bacterial strains with GOS compounds that specifically stimulate their growth. Such synbiotic combinations may be of interest in food/feed and/or pharmacy/medicine applications.

Human milk oligosaccharides (hMOS) are associated with health benefits for newborns. We studied the composition of goat MOS (gMOS) from colostrum up to the 9th month of lactation to conceive an overview of the structures present and their fate. Potential correlations with factors such as age, parity, and lifetime milk production were examined. An effective method for gMOS extraction and ultra-high-performance liquid chromatography coupled to fluorescence detection (UPLC-FLD) analysis was established, following 2-aminobenzamide gMOS labeling. Considerable biological variability was highlighted among the 12 quantified gMOS and the 9 non-quantified structures in the individual milk samples. Most characteristic, 2'-fucosyllactose was present in 73.7% of the milk samples analyzed, suggesting the possibility of a secretor/non-secretor goat genotype, similar to humans. Contributing factors to the observed biological variability were goat age, parity, lifetime milk production, and the kids' sex. The results significantly contribute to the current understanding of (variations in) gMOS composition.

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.

We applied dynamic combinatorial chemistry (DCC) to find novel ligands of the bacterial virulence factor glucosyltransferase (GTF) 180. GTFs are the major producers of extracellular polysaccharides, which are important factors in the initiation and development of cariogenic dental biofilms. Following a structure‐based strategy, we designed a series of 36 glucose‐ and maltose‐based acylhydrazones as substrate mimics. Synthesis of the required mono‐ and disaccharide‐based aldehydes set the stage for DCC experiments. Analysis of the dynamic combinatorial libraries (DCLs) by UPLC‐MS revealed major amplification of four compounds in the presence of GTF180. Moreover, we found that derivatives of the glucose‐acceptor maltose at the C1‐hydroxy group act as glucose‐donors and are cleaved by GTF180. The synthesized hits display medium to low binding affinity (K D values of 0.4–10.0 mm) according to surface plasmon resonance. In addition, they were investigated for inhibitory activity in GTF‐activity assays. The early‐stage DCC study reveals that careful design of DCLs opens up easy access to a broad class of novel compounds that can be developed further as potential inhibitors., Delay decay: Structure‐based design in combination with acylhydrazone‐based dynamic combinatorial chemistry (DCC) afforded inhibitors of glucansucrase (GS), the main virulence factor responsible for dental caries. DCC offered a facile pathway for finding the first hits of GS in the form of glucose‐ and maltose‐based acylhydrazones, mimicking the GS substrate.

Previously, we have identified and characterized 4,6-α-glucanotransferase enzymes of the glycosyl hydrolase (GH) family 70 (GH70) that cleave (α1→4)-linkages in amylose and introduce (α1→6)-linkages in linear chains. The 4,6-α-glucanotransferase of Lactobacillus reuteri 121, for instance, converts amylose into an isomalto/malto-polysaccharide (IMMP) with 90% (α1→6)-linkages. Over the years, also, branching sucrase enzymes belonging to GH70 have been characterized. These enzymes use sucrose as a donor substrate to glucosylate dextran as an acceptor substrate, introducing single -(1→2,6)-α-d-Glcp-(1→6)- (Leuconostoc citreum enzyme) or -(1→3,6)-α-d-Glcp-(1→6)-branches (Leuconostoc citreum, Leuconostoc fallax, Lactobacillus kunkeei enzymes). In this work, we observed that the catalytic domain 2 of the L. kunkeei branching sucrase used not only dextran but also IMMP as the acceptor substrate, introducing -(1→3,6)-α-d-Glcp-(1→6)-branches. The products obtained have been structurally characterized in detail, revealing the addition of single (α1→3)-linked glucose units to IMMP (resulting in a comb-like structure). The in vitro digestibility of the various α-glucans was estimated with the glucose generation rate (GGR) assay that uses rat intestinal acetone powder to simulate the digestive enzymes in the upper intestine. Raw wheat starch is known to be a slowly digestible carbohydrate in mammals and was used as a benchmark control. Compared to raw wheat starch, IMMP and dextran showed reduced digestibility, with partially digestible and indigestible portions. Interestingly, the digestibility of the branching sucrase modified IMMP and dextran products considerably decreased with increasing percentages of (α1→3)-linkages present. The treatment of amylose with 4,6-α-glucanotransferase and branching sucrase/sucrose thus allowed for the synthesis of amylose/starch derived α-glucans with markedly reduced digestibility. These starch derived α-glucans may find applications in the food industry.

Toll-like receptor 8 (TLR-8) plays a role in the pathogenesis of autoimmune disorders and associated gastrointestinal symptoms that reduce quality of life of patients. Dietary interventions are becoming more accepted as mean to manage onset, progression, and treatment of a broad spectrum of inflammatory conditions. In this study, we assessed the impact of N-glycans derived from bovine lactoferrin (bLF) on the inhibition of TLR-8 activation. We investigated the effects of N-glycans in their native form, as well as in its partially demannosylated and partially desialylated form, on HEK293 cells expressing TLR-8, and in human monocyte-derived dendritic cells (MoDCs). We found that in HEK293 cells, N-glycans strongly inhibited the ssRNA40 induced TLR-8 activation but to a lesser extent the R848 induced TLR-8 activation. The impact was compared with a pharmaceutical agent, i.e., chloroquine (CQN), that is clinically applied to antagonize endosomal TLR- activation. Inhibitory effects of the N-glycans were not influenced by the partially demannosylated or partially desialylated N-glycans. As the difference in charge of the N-glycans did not influence the inhibition capacity of TLR-8, it is possible that the inhibition mediated by the N-glycans is a result of a direct interaction with the receptor rather than a result of pH changes in the endosome. The inhibition of TLR-8 in MoDCs resulted in a significant decrease of IL-6 when cells were treated with the unmodified (0.5-fold, p

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)-[β-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. Electronic supplementary material The online version of this article (10.1007/s00253-018-9473-8) contains supplementary material, which is available to authorized users.

A genetically-modified bacterium, for example of the class Actinobacteria, and the use of such a bacterium in the bioconversion of a steroidal substrate into a steroidal product of interest. A method of converting a steroidal substrate into a steroidal product of interest, wherein the method comprises: inoculating culture medium with genetically-modified bacteria according to any of Claims 1 to 28 and growing the bacterial culture until a target OD600 is reached; adding a steroidal substrate to the bacterial culture when the target OD600 is reached; culturing the bacterial culture so that the steroidal substrate is converted to the steroidal product of interest; and extracting and/or purifying the steroidal product of interest from the bacterial culture.

β-Galacto-oligosaccharides (GOS) are used commercially in infant nutrition, aiming to functionally replace human milk oligosaccharides (hMOS). Glucansucrases Gtf180-ΔN and GtfA-ΔN of Lactobacillus reuteri strains convert sucrose into α-glucans with (α1→6)/(α1→3) and (α1→4)/(α1→6) glucosidic linkages, respectively. Previously we reported that both glucansucrases glucosylate lactose, producing a minimum of 5 compounds (degree of polymerization 3-4) (GL34 mixture) with (α1→2/3/4) linkages. This GL34 mixture exhibited growth stimulatory effects on various probiotic bacteria. Aiming to obtain additional compounds mimicking hMOS in structure and function, we here studied glucosylation of 3 commercially available galactosyl-lactose GOS compounds. Both Gtf180-ΔN and GtfA-ΔN were unable to use 3'-galactosyl-lactose (β3'-GL), but used sucrose to add a single glucose moiety to 4'-galactosyl-lactose (β4'-GL) and 6'-galactosyl-lactose (β6'-GL). β6'-GL was elongated at its reducing glucosyl unit with an (α1→2)-linked moiety and at its non-reducing end with an (α1→4) linked moiety; β4'-GL was only elongated at its reducing end with an (α1→2) linked moiety. Glucansucrases Gtf180-ΔN and GtfA-ΔN thus can be used to produce galactosyl-lactose-derived oligosaccharides containing (α1→2) and (α→4) glucosidic linkages, potentially with valuable bioactive (prebiotic) properties.

Glucansucrase Gtf180-Delta N from Lactobacillus reuteri uses lactose as acceptor substrate to synthesize five glucosylated lactose molecules (F1-F5) with a degree of polymerization (DP) of 3-4 (GL34) and with (alpha 1 -> 2)/(alpha 1 -> 3)/(alpha 1 -> 4) glycosidic linkages. Q1140/W1065/N1029 mutations significantly changed the GL34 product ratios. Q1140 mutations clearly decreased F3 3'-glc-lac with an (alpha 1 -> 3) linkage and increased F4 4',2-glc-lac with (alpha 1 -> 4)/(alpha 1 -> 2) linkages. Formation of F2 2-glc-lac with an (alpha 1 -> 2) linkage and F4 was negatively affected in most W1065 and N1029 mutants, respectively. Mutant N1029G synthesized four new products with additional (alpha 1 -> 3)-linked glucosyl moieties (2xDP4 and 2xDP5). Sucrose/lactose strongly reduced Gtf180-Delta N hydrolytic activity and increased transferase activity of Gtf180-Delta N and mutant N1029G, in comparison to activity with sucrose alone. N1029/W1065/Q1140 thus are key determinants of Gtf180-Delta N linkage and product specificity in the acceptor reaction with lactose. Mutagenesis of key residues in Gtf180-Delta N may allow synthesis of tailor-made mixtures of novel lactose-derived oligosaccharides with potential applications as prebiotic compounds in food/feed and in pharmacy/medicine.

Oligosaccharides derived from lactulose (β-D-Galp-(1 → 4)-D-Fru) are drawing more and more attention nowadays because of their strong resistance to gut digestion, and the interest to discover novel prebiotics. Compared to galactooligosaccharides, currently known structures of lactulose oligosaccharides are very limited. In this study, the wild-type β-galactosidase BgaD-D of Bacillus circulans ATCC 31382, as well as the derived mutant R484H, were used to synthesize oligosaccharides from lactulose. In total, 9 oligosaccharide structures were identified by MALDI-TOF-MS and NMR spectroscopy analysis. Trisaccharide β-D-Galp-(1 → 4)-β-D-Galp-(1 → 4)-D-Fru was the major structure produced by the wild-type enzyme, while the R484H mutant showed a preference for synthesis of β-D-Galp-(1 → 3)-β-D-Galp-(1 → 4)-D-Fru. Our study greatly enriched the structural information about oligosaccharides derived from lactulose.

Rhodococcus genome sequence analysis has revealed a surprisingly large (and unexplored) potential for the production of secondary metabolites. Also, putative γ-butyrolactone gene clusters have been identified in some Rhodococci. These signalling molecules are known to regulate secondary metabolism in Streptomyces. This work provides evidence for synthesis of a γ-butyrolactone(-like) molecule by Rhodococci (RJB), the first report in the Rhodococcus genus. The Rhodococcus jostii RHA1 RJB molecule was detected by a reporter system based on the γ-butyrolactone receptor protein (ScbR) of Streptomyces coelicolor. This RJB is structurally identical to 6-dehydro SCB2, the predicted precursor of the S. coelicolor γ-butyrolactone SCB2. The R. jostii RHA1 key RJB biosynthesis gene was identified (gblA): Deletion of gblA resulted in complete loss of RJB synthesis whereas higher RJB levels were detected when gblA was overexpressed. Interaction of the RJB molecule with ScbR indicates that communication may occur between these two Actinomycete genera in their natural habitat. Furthermore, RJB may provide a highly relevant tool for awakening cryptic secondary metabolic gene clusters in Rhodococci. This study provides preliminary evidence that R. jostii RHA1 indeed synthesizes diffusible molecules with antimicrobial activity, but a possible role for RJB in this remains to be established.

β-Galactosidase enzymes are used in the dairy industry to convert lactose into galactooligosaccharides (GOS) that are added to infant formula to mimic the molecular sizes and prebiotic functions of human milk oligosaccharides. Here we report a detailed analysis of the clearly different GOS profiles of the commercial β-galactosidases from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae. Also the GOS yields of these enzymes differed, varying from 48.3% (B. circulans) to 34.9% (K. lactis), and 19.5% (A. oryzae). Their incubation with lactose plus the monosaccharides Gal or Glc resulted in altered GOS profiles. Experiments with (13)C6 labelled Gal and Glc showed that both monosaccharides act as acceptor substrates in the transgalactosylation reactions. The data shows that the lactose isomers β-d-Galp-(1→2)-d-Glcp, β-d-Galp-(1→3)-d-Glcp and β-d-Galp-(1→6)-d-Glcp are formed from acceptor reactions with free Glc and not by rearrangement of Glc in the active site.

Fibronectin type III (FNIII) domains were first identified in the eukaryotic plasma protein fibronectin, where they act as structural spacers or enable protein-protein interactions. Recently we characterized two large and multi-domain amylases in Microbacterium aurum B8.A that both carry multiple FNIII and carbohydrate binding modules (CBMs). The role of (multiple) FNIII domains in such carbohydrate acting enzymes is currently unclear. Four hypothetical functions are considered here: a substrate surface disruption domain, a carbohydrate binding module, as a stable linker, or enabling protein-protein interactions. We performed a phylogenetic analysis of all FNIII domains identified in proteins listed in the CAZy database. These data clearly show that the FNIII domains in eukaryotic and archaeal CAZy proteins are of bacterial origin and also provides examples of interkingdom gene transfer from Bacteria to Archaea and Eucarya. FNIII domains occur in a wide variety of CAZy enzymes acting on many different substrates, suggesting that they have a non-specific role in these proteins. While CBM domains are mostly found at protein termini, FNIII domains are commonly located between other protein domains. FNIII domains in carbohydrate acting enzymes thus may function mainly as stable linkers to allow optimal positioning and/or flexibility of the catalytic domain and other domains, such as CBM.

It has been reported previously that glycosylation of bovine lactoferrin changes over time. A detailed structural overview of these changes over the whole course of lactation, including predry period milk, is lacking. In this study, a high-throughput analysis method was applied to the glycoprofile of lactoferrin isolated from colostrum, mature, and predry period mature milk, which was analyzed over two subsequent lactation cycles for 8 cows from diverse genetic backgrounds. In addition, comparisons are made with commercial bovine lactoferrin samples. During the first 72 h, dynamic changes in lactoferrin glycosylation occurred. Shifts in the oligomannose distribution and the number of sialylated and fucosylated glycans were observed. In some cows, we observed (α2,3)-linked sialic acid in the earliest colostrum samples. The glycoprofiles appeared stable from 1 month after delivery, as well as between cows. In addition, the glycosylation profiles of commercial lactoferrins isolated from pooled mature milk were stable over the year. Lactoferrin glycosylation in the predry period resembles colostrum lactoferrin. The variations in lactoferrin glycosylation profiles, lactoferrin concentrations, and other milk parameters provide detailed information that potentially assists in unraveling the functions and biosynthesis regulation of lactoferrin glycosylation.

Galacto-oligosaccharides (GOS) have been reported to modulate the function of intestinal goblet cells and to improve mucus barrier function. However, GOS is available in many structurally different compositions and it is unknown how GOS structural diversity impacts this modulation of goblet cells. This study aims to investigate the effects of oligosaccharide content and glycosidic linkages of GOS on expression of genes associated with the secretory function of goblet cells. To investigate the effect of oligosaccharide content, LS174T cells were incubated with (β1 → 4)GOS of variable transgalactosylated oligosaccharides and lactose (Lac) composition. To investigate the effect of glycosidic linkages, we compared the effects of (β1 → 4)GOS with (β1 → 3)GOS, and with a mixture of α-linked oligosaccharides (lactose-derived oligosaccharides-LDO). The changes in mRNA expression of mucus-related genes were assessed by RT-PCR. GOS containing Lac significantly enhanced the expression of MUC2, TFF3 and RETNLB but not of Golgi sulfotransferases genes. In contrast, GOS without Lac did not impact these genes. Lac alone significantly enhanced MUC2, TFF3, RETNLB, CHST5, and GAL3ST2 genes suggesting that Lac might be responsible for goblet cell modulation in (β1 → 4)GOS preparations. (β1 → 3)GOS induced the expression of MUC2 and TFF3, and downregulated the RETNLB gene. Compared with the (β1 → 3) and GOS (β1 → 4)GOS, the α-linked LDO significantly upregulated the expression MUC2, TFF3, RETNLB and the Golgi sulfotransferases genes. We identify structural features of GOS that contribute to enhanced mucus integrity. Our study might lead to better GOS formulations for foods to prevent or treat different types of intestinal disorders.

Glycoproteins are biologically active proteins of which the attached glycans contribute to their biological functionality. Limited data is available on the functional properties of these N-glycans in isolation, without the protein core. Glycan release, typically performed with the PNGase F enzyme, is achieved on denatured proteins in the presence of detergents which are notoriously difficult to be completely removed. In this work we compared two methods aiming at recovering N-glycans in a high yield and at high purity from a PNGase F glycoprotein digest of bovine lactoferrin. Detergents were removed from the digest by two separate approaches. In the first approach, protein and glycans were precipitated with acetone and the detergent containing supernatant was discarded. In the second approach, detergent was removed by adsorption onto a polystyrene resin. Following detergent removal, the glycans were further purified by a sequence of solid phase extraction (SPE) steps. Both approaches for detergent removal yielded a final glycan purity above 85%. Recovery of the glycans from lactoferrin was, however, much lower when utilizing acetone precipitation versus the polystyrene resin; 52% versus 85% respectively. A more detailed analysis of the acetone precipitation step revealed a loss of shorter oligomannose structures specifically. A loss of glycans of lesser complexity (oligomannose and biantennary structures) was also observed for other glycoproteins (RNase B, porcine thyroglobulin, human lactoferrin). These results indicate that acetone precipitation, a commonly used step for small-scale glycan purification, is not suitable for all target glycoproteins. The polystyrene resin detergent removal step conserved the full N-glycan profile and could be applied to all mammalian glycoproteins tested. Using this optimized protocol, large-scale quantitative isolation of N-glycan structures was achieved with sufficient purity for functional studies.

Sialylated oligosaccharides contribute 12.6-21.9 % of total free oligosaccharides in human milk (hMOS). These acidic hMOS possess prebiotic properties and display anti-adhesive effects against pathogenic bacteria. Only limited amounts of sialylated hMOS are currently available. The aim of our work is to enzymatically synthesize sialylated oligosaccharides mimicking hMOS functionality. In this study we tested mixtures of glucosylated-lactose (GL34), galactosylated-lactulose (LGOS) and galacto-oligosaccharide (Vivinal GOS) molecules, as trans-sialylation acceptor substrates. The recombinant trans-sialidase enzyme from Trypanosoma cruzi (TcTS) was used for enzymatic decoration, transferring (α2→3)-linked sialic acid from donor substrates to the non-reducing terminal β-galactopyranosyl units of these acceptor substrates. The GL34 F2 2-glc-lac compound with an accessible terminal galactosyl residue was sialylated efficiently (conversion degree of 47.6 %). TcTS also sialylated at least five LGOS structures and eleven Vivinal GOS DP3-4 compounds. These newly synthesized sialylated oligosaccharides are interesting as potential hMOS-mimics for applications in biomedical and functional food products.

Galactooligosaccharides (GOS) are prebiotic carbohydrates that impart changes in the gut bacterial composition of formula-fed infants to more closely resemble that of breast-fed infants. Consuming human milk oligosaccharides (HMOs) provides specific bacterial strains with an advantage for colonizing the infant intestine. These same effects are seen in infants after GOS consumption, however GOS are very complex mixtures and the underlying molecular mechanisms of how GOS mimic HMOs are relatively unknown. Here we studied the effects of GOS utilization on a prominent gut symbiont, Bacteroides thetaiotaomicron, which has been previously shown to consume HMOs via mucin O-glycan degradation pathways. We show that several pathways for targeting O-mucin glycans are activated in B. thetaiotaomicron by GOS, as well as the galactan utilization sytem. Characterization of the endo-galactanase from this system identified activity on various longer GOS substrates while a subset of GOS compounds were identified as potential activators of mucin glycan metabolism in B. thetaiotaomicron. Our results show that GOS functions as an inducer of mucin-glycan pathways while providing a nutrient source in the form of β-(1 → 4)-galactan. These metabolic features of GOS mixtures may serve to explain the beneficial effects that are seen for GOS supplemented infant formula.

Recently, we have shown that glycoside hydrolases enzymes of family GH17 from proteobacteria (genera Pseudomonas, Azotobacter) catalyze elongation transfer reactions with laminari-oligosaccharides generating (β1→3) linkages preferably and to a lesser extent (β1→6) or (β1→4) linkages. In the present study, the cloning and characterization of the gene encoding the structurally very similar GH17 domain of the NdvB enzyme from Bradyrhizobium diazoefficiens, designated Glt20, as well as its catalytic properties are described. The Glt20 enzyme was strikingly different from the previously investigated bacterial GH17 enzymes, both regarding substrate specificity and product formation. The Azotobacter and Pseudomonas enzymes cleaved the donor laminari-oligosaccharide substrates three or four moieties from the non-reducing end, generating linear oligosaccharides. In contrast, the Glt20 enzyme cleaved donor laminari-oligosaccharide substrates two glucose moieties from the reducing end, releasing laminaribiose and transferring the remainder to laminari-oligosaccharide acceptor substrates creating only (β1→3)(β1→6) branching points. This enables Glt20 to transfer larger oligosaccharide chains than the other type of bacterial enzymes previously described, and helps explain the biologically significant formation of cyclic β-glucans in B. diazoefficiens.

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 Lombard et al. (2014) H/ Machius et al. (1995) 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 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.

Microbacterium aurum B8.A is a bacterium that originates from a potato starch-processing plant and employs a GH13 α-amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi-modular protein that contains a novel domain at its C-terminus (Domain 2). Deletion of Domain 2 from MaAmyA did not affect its ability to degrade starch granules but resulted in a strong reduction in granular pore size. Here, we separately expressed and purified this Domain 2 in Escherichia coli and determined its likely function in starch pore formation. Domain 2 independently binds amylose, amylopectin and granular starch but does not have any detectable catalytic (hydrolytic or oxidizing) activity on α-glucan substrates. Therefore we propose that this novel starch binding domain is a new carbohydrate binding module (CBM), the first representative of family CBM74, that assists MaAmyA in efficient pore formation in starch granules. Protein-sequence based BLAST searches revealed that CBM74 occurs widespread, but in Bacteria only, and is often associated with large and multi-domain α-amylases containing family CBM25 or CBM26 domains. CBM74 may specifically function in binding to granular starches to enhance the capability of α-amylase enzymes to degrade resistant starches. Interestingly, the majority of family CBM74 representatives are found in α-amylases originating from human gut associated Bifidobacteria where they may assist in raw starch degradation. The CBM74 domain thus may have a strong impact on the efficiency of resistant starch digestion in the mammalian gastrointestinal tract. This article is protected by copyright. All rights reserved.

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 → 4) glucans (i.e. malto-oligosaccharides and starch) as substrates to synthesize novel α-glucans by introducing linear chains of (α1 → 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. Electronic supplementary material The online version of this article (doi:10.1007/s00018-016-2245-7) contains supplementary material, which is available to authorized users.

Many β-galactosidase enzymes convert lactose into a mixture of galacto-oligosaccharides (GOS) when incubated under the right conditions. Recently, the composition of commercial Vivinal GOS produced by Bacillus circulans β-galactosidase was studied in much detail in another study by van Leeuwen et al. As a spin-off of this study, we used the developed analytical strategy for the evaluation of 6 anonymous commercial GOS products, in comparison with Vivinal GOS. These GOS products were first subjected to HPLC-SEC, calibrated HPAEC-PAD profiling (glucose units in relation to a malto-oligosaccharide ladder), and 1D (1)H NMR spectroscopy. For a more detailed analysis and support of the conclusions based on the initial analysis, the GOS products were separated into DP-pure subpools on Bio-Gel P-2 (MALDI-TOF-MS analysis), which were subjected to calibrated HPAEC-PAD profiling and (1)H NMR analysis. Unidentified peaks from different GOS products, not present in Vivinal GOS, were isolated for detailed structural characterization. In this way, the differences between the various GOS products in terms of DP distribution and type of glycosidic linkages were established. A total of 13 new GOS structures were characterized, adding structural-reporter-group signals and HPAEC-PAD based glucose unit G.U. values to the analytical toolbox. The newly characterized products enhance the quality of the database with GOS structures up to DP4. The combined data provide a firm basis for the rapid profiling of the GOS products of microbial β-galactosidase enzymes.

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.