Unprecedented Diversity of the Glycoside Hydrolase Family 70: A Comprehensive Analysis of Sequence, Structure, and Function.

The glycoside hydrolase family 70 (GH70) contains bacterial extracellular multidomain enzymes, synthesizing α-glucans from sucrose or starch-like substrates. A few dozen have been biochemically characterized, while crystal structures cover only the core domains and lack significant parts of auxiliary domains. Here we present a systematic overview of GH70 enzymes and their 3D structural organization and bacterial origin.

Kinetics and products of Thermotoga maritima β-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 β-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many β-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields.

Tailor-Made α-Glucans by Engineering the Processivity of α-Glucanotransferases via Tunnel-Cleft Active Center Interconversions.

The function of polysaccharides is intimately associated with their size, which is largely determined by the processivity of transferases responsible for their synthesis. A tunnel active center architecture has been recognized as a key factor that governs processivity of several glycoside hydrolases (GHs), e.g.

Insights into the Structure-Function Relationship of GH70 GtfB α-Glucanotransferases from the Crystal Structure and Molecular Dynamic Simulation of a Newly Characterized N1 GtfB Enzyme.

α-Glucanotransferases of the CAZy family GH70 convert starch-derived donors to industrially important α-glucans. Here, we describe characteristics of a novel GtfB-type 4,6-α-glucanotransferase of high enzyme activity (60.8 U mg) from N1 (LrN1 GtfB), which produces surprisingly large quantities of soluble protein in heterologous expression (173 mg pure protein per L of culture) and synthesizes the reuteran-like α-glucan with (α1 → 6) linkages in linear chains and branch points.

Biological Relevance of Goat Milk Oligosaccharides to Infant Health.

Milk is often regarded as the gold standard for the nourishment of all mammalian offspring. The World Health Organization (WHO) recommends exclusive breastfeeding for the first 6 months of the life of the infant, followed by a slow introduction of complementary foods to the breastfeeding routine for a period of approximately 2 years, whenever this is possible ( ; WHO, 2003). One of the most abundant components in all mammals' milk, which is associated with important health benefits, is the oligosaccharides.

Biochemical characterization of glycoside hydrolase family 31 α-glucosidases from Myceliophthora thermophila for α-glucooligosaccharide synthesis.

The transglucosidase activity of GH31 α-glucosidases is employed to catalyze the synthesis of prebiotic isomaltooligosaccharides (IMOs) using the malt syrup prepared from starch as substrate. Continuous mining for new GH31 α-glucosidases with high stability and efficient transglucosidase activity is critical for enhancing the supply and quality of IMO preparations. In the present study, two α-glucosidases (MT31α1 and MT31α2) from Myceliophthora thermophila were explored for biochemical characterization.

Designing starch derivatives with desired structures and functional properties via rearrangements of glycosidic linkages by starch-active transglycosylases.

Modification of starch by transglycosylases from glycoside hydrolase families has attracted much attention recently; these enzymes can produce starch derivatives with novel properties, i.e. processability and functionality, employing highly efficient and safe methods.

Crystal Structure of 4,6-α-Glucanotransferase GtfC-ΔC from Thermophilic 12AMOR1: Starch Transglycosylation in Non-Permuted GH70 Enzymes.

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 12AMOR1, and the first one representing a non-permuted GH70 enzyme, reveals high structural similarity in the core domains with most GtfBs, featuring a similar tunneled active site.

Enzymatic depolymerization of alginate by two novel thermostable alginate lyases from .

Alginate (alginic acid) is a linear polysaccharide, wherein (1→4)-linked β-D-mannuronic acid and its C5 epimer, α-L-guluronic acid, are arranged in varying sequences. Alginate lyases catalyze the depolymerization of alginate, thereby cleaving the (1→4) glycosidic linkages between the monomers by a β-elimination mechanism, to yield unsaturated 4-deoxy-L--hex-4-enopyranosyluronic acid (Δ) at the non-reducing end of resulting oligosaccharides (α-L- configuration) or, depending on the enzyme, the unsaturated monosaccharide itself. In solution, the released free unsaturated monomer product is further hydrated in a spontaneous (keto-enol tautomerization) process to form two cyclic stereoisomers.

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 DSM 12361 was demonstrated to glucosylate benzenediol compounds (i.e.

Mutations in Amino Acid Residues of 121 GtfB 4,6-α-Glucanotransferase that Affect Reaction and Product Specificity.

121 4,6-α-glucanotransferase (Lr121 4,6-α-GTase), belonging to the glycosyl hydrolase (GH) 70 GtfB subfamily, converts starch and maltodextrins into linear isomalto/malto polysaccharides (IMMPs) with consecutive (α1 → 6) linkages. The recent elucidation of its crystal structure allowed identification and analysis of further structural features that determine its reaction and product specificity. Herein, sequence alignments between GtfB enzymes with different product linkage specificities (4,6-α-GTase and 4,3-α-GTase) identified amino acid residues in GH70 homology motifs, which may be critical for reaction and product specificity.

Enzymatic glucosylation of polyphenols using glucansucrases and branching sucrases of glycoside hydrolase family 70.

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.

Insights into Broad-Specificity Starch Modification from the Crystal Structure of NCC 2613 4,6-α-Glucanotransferase GtfB.

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 ( NCC 2613) and an improved homology model of a 4,3-α-glucanotransferase GtfB ( NCC 2970) and show that they are able to convert both linear and branched starch substrates.

GtfC Enzyme of sp. 12AMOR1 Represents a Novel Thermostable Type of GH70 4,6-α-Glucanotransferase That Synthesizes a Linear Alternating (α1 → 6)/(α1 → 4) α-Glucan and Delays Bread Staling.

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 .

Extraction and Quantitative Analysis of Goat Milk Oligosaccharides: Composition, Variation, Associations, and 2'-FL Variability.

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.

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.

Goat Milk Oligosaccharides: Their Diversity, Quantity, and Functional Properties in Comparison to Human Milk Oligosaccharides.

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.

Structures, physico-chemical properties, production and (potential) applications of sucrose-derived α-d-glucans synthesized by glucansucrases.

Glycoside hydrolase family 70 (GH70) glucansucrases produce α-d-glucan polysaccharides (e.g. dextran), which have different linkage composition, branching degree and size distribution, and hold potential applications in food, cosmetic and medicine industry.

Inhibitory Effects of Dietary -Glycans From Bovine Lactoferrin on Toll-Like Receptor 8; Comparing Efficacy With Chloroquine.

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 -glycans derived from bovine lactoferrin (bLF) on the inhibition of TLR-8 activation.

In Depth Analysis of the Contribution of Specific Glycoproteins to the Overall Bovine Whey N-Linked Glycoprofile.

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 -glycans present in the overall whey glycoprofile were attributed to three proteins: lactoferrin, IgG, and GlyCAM-1.

The impact of oligosaccharide content, glycosidic linkages and lactose content of galacto-oligosaccharides (GOS) on the expression of mucus-related genes in goblet cells.

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.

Structural Comparison of Different Galacto-oligosaccharide Mixtures Formed by β-Galactosidases from Lactic Acid Bacteria and Bifidobacteria.

The LacLM-type β-galactosidase from DSM 20075 expressed in both (β-gal) and (β-gal) was tested for their potential to form galacto-oligosaccharides (GOS) from lactose. The Lh-GOS mixture formed by β-galactosidase from , together with three GOS mixtures produced using β-galactosidases of both the LacLM and the LacZ type from other lactic acid bacteria, namely, (Lr-GOS), (Lb-GOS), and (St-GOS), as well as two GOS mixtures (Br-GOS1 and Br-GOS2) produced using β-galactosidases (β-gal I and β-gal II) from , was analyzed and structurally compared with commercial GOS mixtures analyzed in previous work (Vivinal GOS, GOS I, GOS III, and GOS V) using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), high-performance size-exclusion chromatography with a refractive index (RI) detector (HPSEC-RI), and one-dimensional H NMR spectroscopy. β-Galactosidases from lactic acid bacteria and displayed a preference to form β-(1→6)- and β-(1→3)-linked GOS.

Dynamic Temporal Variations in Bovine Lactoferrin Glycan Structures.

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.