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Lactic acid bacterial exopolysaccharides (EPS) are used in the food industry to improve the stability and rheological properties of fermented dairy products. β-Lactoglobulin (BLG), the dominant whey protein in bovine milk, is well known to bind small molecules such as fatty acids, vitamins, and flavors, and to interact with neutral and anionic polysaccharides used in food and pharmaceuticals. While sparse data are available on the affinity of EPS-milk protein interactions, structural information on BLG-EPS complexes, including the EPS binding sites, is completely lacking. Here, binding sites on BLG variant A (BLGA), for oligosaccharides prepared by mild acid hydrolysis of two EPS produced by Streptococcus thermophilus LY03 and Lactobacillus delbrueckii ssp. bulgaricus CNRZ 1187, respectively, are identified by NMR spectroscopy and supplemented by isothermal titration calorimetry (ITC) and molecular docking of complexes. Evidence of two binding sites (site 1 and site 2) on the surface of BLGA is achieved for both oligosaccharides (LY03-OS and 1187-OS) through NMR chemical shift perturbations, revealing multivalency of BLGA for EPS. The affinities of LY03-OS and 1187-OS for BLGA gave K D values in the mM range obtained by both NMR (pH 2.65) and ITC (pH 4.0). Molecular docking suggested that the BLGA and EPS complexes depend on hydrogen bonds and hydrophobic interactions. The findings provide insights into how BLGA engages structurally different EPS-derived oligosaccharides, which may facilitate the design of BLG-EPS complexation, of relevance for formulation of dairy products and improve understanding of BLGA coacervation.

Enzymes are central in producing compounds from grains that are desirable in modern food and beverages. Some of these enzymes occur naturally in grains, while others are exogenous, often of microbial origin, and either supplied as agents for pretreatment and manufacture or introduced into the grains using genetic techniques. Enzymes can add value by utilising raw materials more efficiently, enhance the impact of food and beverages on human health and nutrition, serve to eliminate antinutrients, facilitate techno-functional performance and be inspiration in pioneering handling of industrial waste from crop grains. The review focuses on recent findings on grains, grain fractions, flour and extracted grain components with emphasis on starch, proteins and cell wall polysaccharides subjected to industrially relevant enzyme-catalyzed processing.

Gelation of starch is a process during short-term retrogradation. However, long-term retrogradation always leads to the quality deterioration of starch-based food. In this work, a new type of modified tapioca starch (MTS) gel with maintained short-term gelation strength and retarded long-term retrogradation was prepared using a novel recombinantly produced and characterized 4-α-glucanotransferase (TuαGT). In the resulting MTS, the exterior chains of the amylopectin part were elongated and the content of amylose was reduced because of the disproportionation activity of TuαGT. The retrogradation analysis demonstrated that the MTS possessed highly weakened long-term retrogradation characteristics as compared to the native starch. Most importantly, the strength of the gel formed by regelatinized MTS is very close to that of gelatinized native tapioca starch when storing below 30 °C. These findings provide a starting point for developing a novel method for the enzymatic modification of the starch-based gels.

Two proteases, named C- and T-like proteases, respectively, were purified from the culture supernatant of Amycolatopsis keratinophila subsp. keratinophila D2T grown on a keratinous slaughterhouse by-product of pig bristles and nails as sole nitrogen and carbon source. The two proteases belong to peptidase family S1 as identified by mass spectrometric peptide mapping, have low mutual sequence identity (25.8%) and differ in substrate specificity. T-like protease showed maximum activity at 40 °C and pH 8–9, and C-like protease at 60 °C and pH 8–10. Peptides released from the keratinous by-product were identified by mass spectrometry and indicated P1 specificity for arginine and lysine of T-like and alanine, valine and isoleucine of C-like protease as also supported by the activity of the two proteases towards synthetic peptide and amino acid substrates. The specific activities of the C- and T-like proteases and proteinase K on keratin azure and azokeratin were comparable. However, C- and T-like proteases showed 5–10-fold higher keratin/casein (K/C) activity ratios than that of another S1 and two keratin-degrading S8 peptidases used for comparison. The findings support that the range of peptidase families considered to contain keratinases should be expanded to include S1 peptidases. Furthermore, the results indicated the quite thermostable C-like protease to be a promising candidate for use in industrial degradation of keratinous slaughterhouse by-products.

Glycoside hydrolases and phosphorylases are two major classes of enzymes responsible for the cleavage of glycosidic bonds. Here we show that two GH84 O-GlcNAcase enzymes can be converted to efficient phosphorylases by a single point mutation. Noteworthy, the mutated enzymes are over 10-fold more active than naturally occurring glucosaminide phosphorylases. We rationalize this novel transformation using molecular dynamics and QM/MM metadynamics methods, showing that the mutation changes the electrostatic potential at the active site and reduces the energy barrier for phosphorolysis by 10 kcal·mol-1. In addition, the simulations unambiguously reveal the nature of the intermediate as a glucose oxazolinium ion, clarifying the debate on the nature of such a reaction intermediate in glycoside hydrolases operating via substrate-assisted catalysis.

Enzymatic modification is an effective method to inhibit starch retrogradation. However, lack of quantification of relationships between enzymatic modification and starch retrogradation makes the enzymatic improvement unpredictable. In this study, maltogenic α-amylase (MA) was used to treat wheat starch granules to restrain retrogradation, aiming to elucidate the mechanism of MA hydrolysis on wheat starch granules and to establish a quantitative relationship between the degree of hydrolysis (DH) and retrogradation. Scanning electron microscopy and small angle X-ray scattering results showed that MA hydrolyzed starch granules by a “surface pitting” mode simultaneously acting on crystalline and amorphous regions. Debranching and high performance anion exchange chromatography analysis of MA-treated wheat starch granules demonstrated that the amount of short branches with degree of polymerization＜9 increased and the proportion of medium and long branches decreased. Importantly, the extent of impaired short- and long-term retrogradation of MA-treated starch was clearly linearly correlated with the DH. This finding provides a quantitative method for predicting the degree of retrogradation improvement by enzymatic modification.

Glycogen-like glucan (GnG), a hyperbranched glucose polymer, has been receiving increasing attention to generate synthetic polymers and nanoparticles. Importantly, different branching patterns strongly influence the functionality of GnG. To uncover ways of obtaining different GnG branching patterns, a series of GnG with radius from 22.03 to 27.06 nm were synthesized using sucrose phosphorylase, α-glucan phosphorylase (GP), and branching enzyme (BE). Adjusting the relative activity ratio of GP and BE (GP/BE) made the molecular weight (MW) distribution of intermediate GnG products follow two different paths. At a low GP/BE, the GnG developed from "small to large" during the synthetic process, with the MW increasing from 6.15 × 106 to 1.21 × 107 g/mol, and possessed a compact structure. By contrast, a high GP/BE caused the "large to small" model, with the MW reduction of GnG from 1.62 × 107 to 1.21 × 107 g/mol, and created a loose external structure. The higher GP activity promoted the elongation of external chains and restrained chain transfer by the BE to the inner zone of GnG, which would modulate the loose-tight structure of GnG. These findings provide new useful insights into the construction of structurally well-defined nanoparticles.

Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on β-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner. αFFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with αFFase1, conversions of β-D-Frup-(2→1)-α-D-Fruf to α-D-Fruf-1,2':2,1'-β-D-Frup (diheterolevulosan II) and β-D-Fruf-(2→1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2':2,1'-β-D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of αFFase1 in complex with β-D-Fruf and β-D-Araf were determined at the resolutions of up to 1.76 Å. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of αFFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172).

l-Fucose is the most widely distributed l-hexose in marine and terrestrial environments, and presents a variety of functional roles. l-Fucose is the major monosaccharide in the polysaccharide fucoidan from cell walls of brown algae, and is found in human milk oligosaccharides and the Lewis blood group system, where it is important in cell signaling and immune response stimulation. Removal of fucose from these biomolecules is catalyzed by fucosidases belonging to different carbohydrate-active enzyme (CAZy) families. Fucosidases of glycoside hydrolase family 29 (GH29) release α-l-fucose from non-reducing ends of glycans and display activities targeting different substrate compositions and linkage types. While several GH29 fucosidases from terrestrial environments have been characterized, much less is known about marine members of GH29 and their substrate specificities, as only four marine GH29 enzymes were previously characterized. Here, five GH29 fucosidases originating from an uncultured fucoidan-degrading marine bacterium (Paraglaciecola sp.) were cloned and produced recombinantly in E. coli. All five enzymes (Fp231, Fp239, Fp240, Fp251, Fp284) hydrolyzed the synthetic substrate CNP-α-l-fucose. By screening each of these enzymes against up to 17 fucose-containing oligosaccharides Fp231 and Fp284 showed strict substrate specificities against the fucosyl-N-acetylglucosamine regioisomers Fuc(α1,4)GlcNAc and Fuc(α1,6)GlcNAc, respectively, the former representing a new specificity. Fp231 is a monomeric enzyme with pH and temperature optima at pH 5.6–6.0 and 25°C, hydrolyzing Fuc(α1,4)GlcNAc with kcat = 1.3 s−1 and Km = 660 μM. Altogether, the findings extend our knowledge about GH29 family members from the marine environment, which are so far largely unexplored.

In a conventional microorganism-mediated biological process for degradation of keratinous waste material the production of keratin-specific proteases (i.e., keratinases) and the hydrolysis of keratin-rich residual biomass both take place during the same stage of the bioprocess and, as a consequence, occur simultaneously under suboptimal conditions. In the present study the keratinolytic actinomycete Amycolatopsis keratiniphila D2 was successfully employed to biodegrade thermally pretreated porcine bristles at high solids loading (16% w/v) via a novel cultivation methodology. Indeed, the two-stage submerged fermentation process developed in this work enabled to efficiently recover, in a single unit operation, about 73% of the protein material contained in the keratinous biowaste structure, resulting in an overall accumulation of 89.3 g·L−1 protein-rich hydrolysate and a productivity of 427 mg crude soluble proteins per litre per hour. The obtained protein hydrolysate powder displayed a 2.2-fold increase in its in vitro pepsin digestibility (95%) with respect to the non-hydrolysed pretreated substrate (43%). In addition, the chromatogram obtained by size-exclusion chromatography analysis of the final product indicated that, among the identified fractions, those consisting of small peptides and free amino acids were the most abundantly present inside the analysed sample. Given these facts it is possible to conclude that the soluble proteins, peptides and free amino acids recovered through the newly designed two-stage bioextraction process could represent a viable alternative source of protein in animal feed formulation.

β-lactoglobulin (BLG) is a promiscuous protein in terms of ligand interactions, having several binding sites reported for hydrophobic biomolecules such as fatty acids, lipids, and vitamins as well as detergents. BLG also interacts with neutral and anionic oligo- and polysaccharides for which the binding sites remain to be identified. The multivalency offered by these carbohydrate ligands is expected to facilitate coacervation, an electrostatically driven liquid-liquid phase separation. Using heteronuclear single quantum coherence NMR spectroscopy and monitoring chemical shift perturbations, we observed specific binding sites of modest affinity for alginate oligosaccharides (AOSs) prepared by alginate lyase degradation. Two different AOS binding sites (site 1 and site 2) centered around K75 and K101 were identified for monomeric BLG isoform A (BLGA) at pH 2.65. In contrast, only site 1 around K75 was observed for dimeric BLGA at pH 4.0. The data suggest a pH-dependent mechanism whereby both the BLGA dimer-monomer equilibrium and electrostatic interactions are exploited. This variability allows for control of coacervation and particle formation of BLGA/alginate mixtures via directed polysaccharide bridging of AOS binding sites and has implication for molecular network formation. The results are valuable for design of polyelectrolyte-based BLG particles and coacervates for carrying nutraceuticals and modulating viscosity in dairy products by use of alginates.

The family GH77 contains 4-α-glucanotransferase acting on α-1,4-glucans, known as amylomaltase in prokaryotes and disproportionating enzyme in plants. A group of bacterial GH77 members, represented by amylomaltases from Escherichia coli and Corynebacterium glutamicum, possesses an N-terminal extension that forms a distinct immunoglobulin-like fold domain, of which no function has been identified. Here, in silico analysis of 100 selected sequences of N-terminal domain homologues disclosed several well-conserved residues, among which Tyr108 (E. coli amylomaltase numbering) may be involved in α-glucan binding. These N-terminal domains, therefore, may represent a new type of starch-binding domain and define a new CBM family. This hypothesis is supported by docking of maltooligosaccharides to the N-terminal domain in amylomaltases, representing the four clusters of the phylogenetic tree. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02787-8.

The functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5 epimerases determine the former by epimerizing β-D-mannuronic acid residues (M) into α-L-guluronic acid residues (G). The molecular weight is affected by alginate lyases, which cleave alginate chains through β-elimination. The reaction mechanisms for the epimerization and cleavage are similar and some enzymes can perform both. These dualistic enzymes share high sequence identity with mannuronan C-5 epimerases without lyase activity, and the mechanism behind their activity as well as the amino acids responsible for it are still unknown. In this study, we investigate mechanistic determinants of the bifunctional epimerase and lyase activity of AlgE7 from Azotobacter vinelandii. Based on sequence analyses, a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by NMR. Our results show that the lyase activity of AlgE7 is regulated by the type of ion present: Calcium promotes it, whereas NaCl reduces it. By using defined poly-M and poly-MG substrates, the preferred cleavage sites of AlgE7 were found to be M↓XM and G↓XM, where X can be either M or G. By studying AlgE7 mutants, R148 was identified as an important residue for the lyase activity, and the point mutant R148G resulted in an enzyme with only epimerase activity. Based on the results obtained in the present study we suggest a unified catalytic reaction mechanism for both epimerase and lyase activities where H154 functions as the catalytic base and Y149 as the catalytic acid.ImportancePost-harvest valorisation and upgrading of algal constituents is a promising strategy in the development of a sustainable bioeconomy based on algal biomass. In this respect, alginate epimerases and lyases are valuable enzymes for tailoring of the functional properties of alginate, a polysaccharide extracted from brown seaweed with numerous applications in food, medicine and material industries. By providing a better understanding of the reaction mechanism and of how the two enzyme reactions can be altered by changes in reaction conditions, this study opens for further applications of bacterial epimerases and lyases in enzymatic tailoring of alginate polymers.

Alginates, serving as hydrocolloids in the food and pharma industries, form particles at pH < 4.5 with positively charged proteins, such as β-lactoglobulin (β-Lg). Alginates are linear anionic polysaccharides composed of 1,4-linked β-d-mannuronate (M) and α-l-guluronate (G) residues. The impact of M and G contents and pH is investigated to correlate with the formation and size of β-Lg alginate complexes under relevant ionic strength. It is concluded, using three alginates of M/G ratios 0.6, 1.1, and 1.8 and similar molecular mass, that β-Lg binding capacity is higher at pH 4.0 than at pH 2.65 and for high M content. By contrast, the largest particles are obtained at pH 2.65 and with high G content. At pH 4.0 and 2.65, the stoichiometry was 28-48 and 3-10 β-Lg molecules bound per alginate, respectively, increasing with higher M content. The findings will contribute to the design of formation of the desired alginate-protein particles in the acidic pH range.

International audience; Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis. Here we describe a straightforward strategy that involves rational rapid in silico analysis of protein sequences. The method pinpoints 6‒12 single mutant candidates to improve transglycosylation yields. Requiring very little prior knowledge of the target enzyme other than its sequence, the method is generic and procures catalysts for the formation of glycosidic bonds involving various D/L-, α/β-pyranosides or furanosides, and exo- and endoaction. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.

Glycosyltransferases (GTs) catalyze the formation of glycosidic bonds in carbohydrates and glycoconjugates, with various outcomes depending not only on the acceptor molecules they bind but also on the type of glycosidic bond they form (C−O, C−N, C−S, or C−C). Here we show that the glucosyltransferase UGT1 from the indigo plant Polygonum tinctorium catalyzes either N-, O-, or S-glycosylation with similar rates. We solve the structure of the enzyme in complex with its donor and acceptor substrates and elucidate the molecular basis of N-, O-, and S-specificities using experimental mutagenesis and QM/MM simulations, revealing distinct mechanisms for N-, O-, and S-glycosylation. We also show that the active site can be engineered to increase or favor one of the three glycosylation activities over another. These results will foster the design of more active and specific enzyme variants for production of glycosides.

The industrial thermostable Bacillus licheniformis α-amylase (BLA) has wide applications, including in household detergents, and efforts to improve its performance are continuously ongoing. BLA during the industrial production is deamidated and glycated resulting in multiple forms with different isoelectric points. Forty modified positions were identified by tandem mass spectrometric peptide mapping of BLA forms separated by isoelectric focusing. These modified 12 asparagine, 9 glutamine, 8 arginine and 11 lysine residues are mostly situated on the enzyme surface and several belong to regions involved in stability, activity and carbohydrate binding. Eight residues presumed to interact with starch at the active site and surface binding sites (SBSs) were subjected to mutational analysis. Five mutants mimicking deamidation (N→D, Q→E) at the substrate binding cleft showed moderate to no effect on thermostability and k cat and K M for maltoheptaose and amylose. Notably, the mutations improved laundry wash efficiency in detergents at pH 8.5 and 10.0. Replacing three reducing sugar reactive side chains (K→M, R→L) at a distant substrate binding region and two SBSs enhanced wash performance especially in liquid detergent at pH 8.5, slightly improved enzymatic activity and maintained thermostability. Wash performance was most improved (5-fold) for the N265D mutant near substrate binding subsite +3.

The effects of different thawing procedures on the properties of frozen casein concentrates (CCs) produced by microfiltration were investigated and compared with fresh CC. Samples (5% protein) were thawed directly (30 °C, 30 min) or stored (4 °C) for 1 or 3 d, and subsequently kept at 30 °C for 30 min or 5 h. No significant differences were found in particle size, zeta potential or rennet coagulation for CC diluted with water or permeate from ultrafiltration. The viscosity of frozen CC diluted with water was lower for samples thawed at 30 °C for 30 min than for fresh CC, but increased with prolonged thawing procedures (4 °C for 3 d). Using 4 °C for 3 d followed by 5 h at 30 °C resulted in earlier gelation and increased storage modulus (G’) during acidification, which was related to the increased free Ca2+ activity and decreased pH.

We present an enzyme engineering approach based solely on amino-acids sequence to convert glycoside hydrolases into transglycosylases. We demonstrate its effectiveness on enzymes form five different glycoside hydrolase families, synthesizing various oligosaccharides containing different α-/β-pyranosides or furanosides in one-step with high yields.; Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccha-9 rides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible, but is 10 hampered by the innate hydrolytic activity of these enzymes. Protein engineering 11 methods are applicable, though usually require prior structural knowledge of the 12 target enzyme and the use of powerful computing methods, and/or relies on extensive 13 screening methodologies. Here we describe a straightforward strategy that involves 14 rapid in silico analysis of protein sequences. The method pinpoints a small number 15 (

The maltooligosaccharide (MOS) utilization locus in Lactobacillus acidophilus NCFM, a model for human small-intestine lactobacilli, encodes three glycoside hydrolases (GHs): a putative maltogenic α-amylase of family 13 subfamily 20 (LaGH13_20), a maltose phosphorylase of GH65 (LaGH65) and a family 13 subfamily 31 member (LaGH13_31B), annotated as a 1,6-α-glucosidase. Here, we reveal that LaGH13_31B is a 1,4-α-glucosyltransferase that disproportionates MOS of degree of polymerization (DP) ≥2, with preference for maltotriose. Kinetic analyses of the three GHs encoded by the MOS locus, revealed that the substrate preference of LaGH13_31B towards maltotriose, complements the about 40-fold lower k cat of LaGH13_20 towards this substrate, thereby enhancing the conversion of odd-numbered MOS to maltose. The concerted action of LaGH13_20 and LaGH13_31B confers the efficient conversion of MOS to maltose that is phosphorolysed by LaGH65. Structural analyses revealed the presence of a flexible elongated loop, which is unique for a previously unexplored clade of GH13_31 represented by LaGH13_31B. The identified loop insertion harbours a conserved aromatic residue that modulates the activity and substrate affinity of the enzyme, thereby offering a functional signature of this clade, which segregates from 1,6-α-glucosidases and sucrose isomerases previously described within GH13_31. Genomic analyses revealed that the LaGH13_31B gene is conserved in the MOS utilization loci of lactobacilli, including acidophilus cluster members that dominate the human small intestine. IMPORTANCE The degradation of starch in the small intestine generates short linear and branched α-glucans. The latter are poorly digestible by humans, rendering them available to the gut microbiota e.g lactobacilli adapted to the small intestine and considered as beneficial to health. This study unveils a previously unknown scheme of maltooligosaccharide (MOS) catabolism, via the concerted activity of an 1,4-α-glucosyltransferase together with a classical hydrolase and a phosphorylase. The intriguing involvement of a glucosyltransferase is likely to allow fine-tuning the regulation of MOS catabolism for optimal harnessing of this key metabolic resource in the human small intestine. The study extends the suite of specificities, which have been identified in GH13_31 and highlights amino acid signatures underpinning the evolution of 1,4-α-glucosyl transferases that have been recruited in the MOS catabolism pathway in lactobacilli.

A variety of glucosaccharides composed of glucosyl residues can be classified into α- and β-type and have wide application in food and medicine areas. Among these glucosaccharides, β-type, such as cellulose and α-type, such as starch and starch derivatives, both contain 1 → 4 linkages and are well studied. Notably, in past decades also α1 → 6 glucosaccharides obtained increasing attention for unique physiochemical and biological properties. Especially in recent years, α1 → 6 glucosaccharides of different molecular weight distribution have been created and proved to be functional. However, compared to β- type and α1 → 4 glucosaccharides, only few articles provide a systematic overview of α1 → 6 glucosaccharides. This motivated, the present first comprehensive review on structure, function and synthesis of these α1 → 6 glucosaccharides, aiming both at improving understanding of traditional α1 → 6 glucosaccharides, such as isomaltose, isomaltooligosaccharides and dextrans, and to draw the attention to newly explored α1 → 6 glucosaccharides and their derivatives, such as cycloisomaltooligosaccharides, isomaltomegalosaccharides, and isomalto/malto-polysaccharides.

Diabetes is considered one of the biggest health crises in 21st century. Controlling carbohydrate digestibility by inhibiting starch digestive enzyme (α-amylase and α-glucosidase) activities is an efficient strategy to control postprandial hyperglycemia. Over the last decade, synthetic inhibitors are most effective pharmaceutical approach for the management of type 2 diabetes mellitus (T2DM). With the increasing global prevalence of diabetes, identification of alternative enzyme inhibitors with potentially less negative side effects becomes imperative. Compounds of natural sources (such as dietary component) are more desirable due to the advantages in safety, diversity, and economy compared with the synthetic inhibitors. Food-derived non-starch polysaccharides, peptides, and lipids compounds have been determined to inhibit α-amylase and α-glucosidase activities. This review presents updated overview on classification, inhibitory effects, mechanism, and potential application of these inhibitors in foods. The abundant inhibitors in a variety of natural sources give more choices for patients with different requirements, and can guide design of the recipes to prophylactic treatment of T2DM.

In the marine environment agar degradation is assured by bacteria that contain large agarolytic systems with enzymes acting in various endo- and exo-modes. Agarase A (AgaA) is an endo-glycoside hydrolase of family 16 considered to initiate degradation of agarose. Agaro-oligosaccharide binding at a unique surface binding site (SBS) in AgaA from Zobellia galactanivorans was investigated by computational methods in conjunction with a structure/sequence guided approach of site-directed mutagenesis probed by surface plasmon resonance binding analysis of agaro-oligosaccharides of DP 4-10. The crystal structure has shown that agaro-octaose interacts via H-bonds and aromatic stacking along 7 subsites (L through R) of the SBS in the inactive catalytic nucleophile mutant AgaA-E147S. D271 is centrally located in the extended SBS where it forms H-bonds to galactose and 3,6-anhydrogalactose residues of agaro-octaose at subsites O and P. We propose D271 is a key residue in ligand binding to the SBS. Thus AgaA-E147S/D271A gave slightly decreasing KD values from 625 ± 118 to 468 ± 13 μM for agaro-hexaose, -octaose and -decaose, which represent 3-4-fold reduced affinity compared to AgaA-E147S. Molecular dynamics simulations and interaction analyses of AgaA-E147S/D271A indicated disruption of an extended H-bond network supporting that D271 is critical for the functional SBS. Notably, neither AgaA-E147S/W87A nor AgaA-E147S/W277A, designed to eliminate stacking with galactose residues at subsites O and Q, respectively, were produced in soluble form. W87 and W277 may thus control correct folding and structural integrity of AgaA. This article is protected by copyright. All rights reserved.

Sirtuins, a group of NAD+-dependent deacylases-have emerged as key in the connection between NAD+ metabolism and aging. This class of enzymes hydrolyze a range of ε- N-acyllysine PTMs and determining the repertoire of catalyzed deacylation reactions is of high importance to fully elucidate the roles of a given sirtuin. Here we have identified and produced two potential sirtuins from the probiotic bacterium Lactobacillus acidophilus NCFM and screening more than 80 different substrates, covering 26 acyl groups on five peptide scaffolds, demonstrated that one of the investigated proteins-Sir2La-is a bona fide NAD+-dependent sirtuin, catalyzing hydrolysis of acetyl-, propionyl-, and butyryllysine. Further substantiating the identity of Sir2La as a sirtuin, known sirtuin inhibitors nicotinamide and suramin as well as a thioacetyllysine compound inhibit the deacylase activity in a concentration-dependent manner. Based on steady-state kinetics Sir2La showed a slight preference for propionyllysine (Kpro) over acetyllysine (Kac). For non-fluorogenic peptide substrates the preference is driven by a remarkably low KM (280 nM vs 700 nM, for Kpro and Kac, respectively) whereas kcat was similar (21 X 10-3 s-1). Moreover, while NAD+ is a prerequisite for Sir2La-mediated deacylation, Sir2La has very high KM for NAD+ compared to the expected levels of the dinucleotide in L. acidophilus. Sir2La is the first sirtuin from Lactobacillales and of the Gram-positive bacterial subclass of sirtuins to be functionally characterized. The ability to hydrolyze propionyl- and butyryllysine emphasizes the relevance of further exploring the role of other short-chain acyl moieties as PTMs.

Protein cross-links are formed in regulated biochemical processes in many biological systems, but they are also generated inadvertently via the reactions of exogenous or endogenous oxidants. Site-specific identification and characterization of such cross-links is challenging, and the goal was, therefore, to develop mass-spectrometry-based approaches tailored for proteins subjected to oxidative challenges that also are applicable for the analysis of complex samples. Using trypsin-mediated 18O isotopic labeling, different types of data acquisition workflows, and designated database software tools, we successfully identified tyrosine–tyrosine, tyrosine–tryptophan, tyrosine–lysine, and histidine–lysine cross-links in proteins subjected to sensitizer-mediated photo-oxidation with rose bengal or chemical oxidation with peroxyl radicals generated from the water-soluble compound 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). Subsequently, AAPH was also applied to a protein extract from the Gram-positive ba...

Although the physiological consequences of plant growth under saline conditions have been well described, understanding the core mechanisms conferring plant salt adaptation has only started. We target the root plasma membrane proteomes of two barley varieties, cvs. Steptoe and Morex, with contrasting salinity tolerance. In total, 588 plasma membrane proteins were identified by mass spectrometry, of which 182 were either cultivar or salinity stress responsive. Three candidate proteins with increased abundance in the tolerant cv. Morex were involved either in sterol binding (a GTPase-activating protein for the adenosine diphosphate ribosylation factor [ZIGA2], and a membrane steroid binding protein [MSBP]) or in phospholipid synthesis (phosphoethanolamine methyltransferase [PEAMT]). Overexpression of barley MSBP conferred salinity tolerance to yeast cells, whereas the knock-out of the heterologous AtMSBP1 increased salt sensitivity in Arabidopsis. Atmsbp1 plants showed a reduced number of lateral roots under salinity, and root-tip-specific expression of barley MSBP in Atmsbp1 complemented this phenotype. In barley, an increased abundance of MSBP correlates with reduced root length and lateral root formation as well as increased levels of auxin under salinity being stronger in the tolerant cv. Morex. Hence, we concluded the involvement of MSBP in phytohormone-directed adaptation of root architecture in response to salinity.

Alginate is an anionic polysaccharide capable of forming insoluble particles with proteins. Hence, alginate has potential as a protein carrier. However, the role of physical properties of the polysaccharide, such as degree of polymerization (DPn) and mannuronic/guluronic acid ratio, remains to be fully explored. Particle formation of a high and a low molar mass alginate (ALG) with β-lactoglobulin (BLG) at pH 2-8 depends on the average DPn (HMW-ALG: 1.59·103; LMW-ALG: 0.23·103) and the mannuronic/guluronic acid ratio (1.0; 0.6) as supported by using ManA6 and GulA6 as models. Dynamic light scattering (DLS) showed that particles of BLG with either of the two ALGs have essentially the same hydrodynamic diameter (D H) at pH 3 and 2, while at pH 4 particles of LMW-ALG/BLG have larger D H than of HMW-ALG/BLG. At pH 5-8 no significant particle formation was observed. ManA6 did not form insoluble particles at pH 2-8, while GulA6 formed insoluble particles, albeit only at pH 4. K d was approximately 10-fold higher for LMW-ALG/BLG than HMW-ALG/BLG and 3 orders of magnitude higher for an alginate trisaccharide/BLG complexation as determined by isothermal titration calorimetry (ITC). The alginate trisaccharide did not form insoluble particles with BLG at pH 3 and 4, though interaction still occurred. δH app and molar stoichiometry of BLG in the complexes with the two ALGs differed by a factor of 7, as did their DPn, which thus affected the interaction strength, but not the BLG content. At pH 4 the BLG content doubled in the particle due to BLG dimerization. The findings emphasize the importance of DPn, mannuronic/guluronic acid ratio and pH in formulations containing alginate/whey protein particles.

Transglutaminase (TG) catalyzes formation of covalent bonds between lysine and glutamine side chains and has applications in manipulation of food structure. Physical properties of a whey protein mixture (SPC) denatured either at elevated pH or by heat-treatment and followed by TG catalyzed crosslinking, have been characterised using dynamic light scattering, size exclusion chromatography, flourescence spectroscopy and atomic force microscopy. The degree of enzymatic crosslinking appeared higher for pH- than for heat-denatured SPC. The hydrophobic surface properties depended on the treatment, thus heating caused the largest exposure of the hydrophobic core of SPC proteins, which was decreased by crosslinking. The particle size of the treated SPC samples increased upon crosslinking by TG. Moreover, the particle morphology depended on the type of denaturing treatment, thus heat-treated SPC contained fibrillar structures, while pH-denatured SPC remained globular as documented by using atomic force microscopy. Finally, the in vitro digestability of the different SPC samples was assessed under simulated gastric and intestinal conditions. Notably heat-treatment was found to lower the gastric digestion rate and enzymatic crosslinking reduced both the gastric and the intestinal rate of digestion. These characteristics of the various SPC samples provide a useful basis for design of isoenergic model foods applicable in animal and human studies on how food structure affects satiety.

Interactions of exopolysaccharides and proteins are of great importance in food science, but complicated to analyze and quantify at the molecular level. A surface plasmon resonance procedure was established to characterize binding of seven structure-determined, branched hetero-exopolysaccharides (HePSs) of 0.14–4.9 MDa from lactic acid bacteria to different milk proteins (β-casein, κ-casein, native and heat-treated β-lactoglobulin) at pH 4.0–5.0. Maximum binding capacity (RUmax) and apparent affinity (KA,app) were HePS- and protein-dependent and varied for example 10- and 600-fold, respectively, in the complexation with native β-lactoglobulin at pH 4.0. Highest RUmax and KA,app were obtained with heat-treated β-lactoglobulin and β-casein, respectively. Overall, RUmax and KA,app decreased 6- and 20-fold, respectively, with increasing pH from 4.0 to 5.0. KA,app was influenced by ionic strength and temperature, indicating that polar interactions stabilize HePS–protein complexes. HePS size as well as oligosaccharide repeat structure, conferring chain flexibility and hydrogen bonding potential, influence the KA,app.

Free thiol-containing proteins are suggested to work as antioxidants in beer, but the majority of thiols in wort are present in their oxidized form as disulfides and are therefore not active as antioxidants. Thioredoxin, a disulfide-reducing protein, is released into the wort from some yeast strains during fermentation. The capacity of the thioredoxin enzyme system (thioredoxin, thioredoxin reductase, NADPH) to reduce oxidized thiols in boiled wort under fermentation-like conditions was studied. Free thiols were quantitated in boiled wort samples by derivatization with ThioGlo1 and fluorescence detection of thiol-derivatives. When boiled wort was incubated with all components of the thioredoxin system at pH 7.0 and 25 °C for 60 min under anaerobic conditions, the free thiol concentration increased from 25 to 224 μM. At pH values similar to wort (pH 5.7) and beer (pH 4.5), the thioredoxin system was also capable of increasing the free thiol concentration, although with lower efficiency to 187 and 170 μM, respectively. The presence of sulfite, an important antioxidant in beer secreted by the yeast during fermentation, was found to inactivate thioredoxin by sulfitolysis. Reduction of oxidized thiols by the thioredoxin system was therefore only found to be efficient in the absence of sulfite.

Oxidation generates multiple diverse post-translational modifications resulting in changes in protein structure and function associated with a wide range of diseases. Of these modifications, carbonylations have often been used as hallmarks of oxidative damage. However, accumulating evidence supports the hypothesis that other oxidation products may be quantitatively more important under physiological conditions. To address this issue, we have developed a holistic mass spectrometry-based approach for the simultaneous identification, localization, and quantification of a broad range of oxidative modifications based on so-called "dependent peptides". The strategy involves unrestricted database searches with rigorous filtering focusing on oxidative modifications. The approach was applied to bovine serum albumin and human serum proteins subjected to metal ion-catalyzed oxidation, resulting in the identification of a wide range of different oxidative modifications. The most common modification in the oxidized samples is hydroxylation, but carbonylation, decarboxylation, and dihydroxylation are also abundant, while carbonylation showed the largest increase in abundance relative to nonoxidized samples. Site-specific localization of modified residues reveals several "oxidation hotspots" showing high levels of modification occupancy, including specific histidine, tryptophan, methionine, glutamate, and aspartate residues. The majority of the modifications, however, occur at low occupancy levels on a diversity of side chains.

Adhesion to intestinal mucosa is a crucial property for probiotic bacteria. Adhesion is thought to increase host-bacterial interactions, thus potentially enabling health benefits to the host. Molecular events connected with adhesion and surface proteome changes were investigated for the probiotic Lactobacillus acidophilus NCFM cultured with established or emerging prebiotic carbohydrates as carbon source and in the presence of mucin, the glycoprotein of the epithelial mucus layer. Variation in adhesion to HT29-cells and mucin was associated with carbon source and mucin-induced subproteome abundancy differences. Specifically, while growth on fructooligosaccharides (FOS) only stimulated adhesion to intestinal HT-29 cells, cellobiose and polydextrose in addition increased adhesion to mucin. Adhesion to HT-29 cells increased by about 2-fold for bacteria grown on mucin-supplemented glucose. Comparative 2DE-MS surface proteome analysis showed different proteins in energy metabolism appearing on the surface, suggesting they exert moonlighting functions. Mucin-supplemented bacteria had relative abundance of pyruvate kinase and fructose-bisphosphate aldolase increased by about 2-fold while six spots with 3.2–2.1 fold reduced relative abundance comprised elongation factor G, phosphoglycerate kinase, BipAEFTU family GTP-binding protein, ribonucleoside triphosphate reductase, adenylosuccinate synthetase, 30S ribosomal protein S1, and manganese-dependent inorganic pyrophosphatase. Surface proteome of cellobiose- compared to glucose-grown L. acidophilus NCFM had phosphate starvation inducible protein stress-related, thermostable pullulanase, and elongation factor G increasing 4.4–2.4 fold, while GAPDH, elongation factor Ts, and pyruvate kinase were reduced by 2.0–1.5 fold in relative abundance. Addition of recombinant L. acidophilus NCFM elongation factor G and pyruvate kinase to a coated mucin layer significantly suppressed subsequent adhesion of the bacterium. Biological significance Human diet is important for intestinal health and food components, especially non-digestible carbohydrates can beneficially modify the microbiota. In the present study, effects of emerging and established prebiotic carbohydrates on the probiotic potential of Lactobacillus acidophilus NCFM were investigated by testing adhesion to a mucin layer and intestinal cells, and comparing this with changes in abundancy of surface proteins thought to be important for host interactions. Increased adhesion was observed following culturing of the bacterium with fructooligosaccharides, cellobiose or polydextrose, as well as mucin-supplemented glucose as carbon source. Enhanced adhesion ability can prolong bacterial residence in GIT yielding positive health effects. Higher relative abundance of certain surface proteins under various conditions (i.e. grown on cellobiose or mucin-supplemented glucose) suggested involvement of these proteins in adhesion, as confirmed by competition in case of two recombinantly produced moonlighting proteins. Combination of Lactobacillus acidophilus NCFM with different carbohydrates revealed potential bacterial determinants of synbiotic interactions, including stimulation of adhesion.

Barley limit dextrinase (HvLD) of glycoside hydrolase family 13 is the sole enzyme hydrolysing α-1,6-glucosidic linkages from starch in the germinating seed. Surprisingly, HvLD shows 150- and 7-fold higher activity towards pullulan and β-limit dextrin, respectively, than amylopectin. This is investigated by mutational analysis of residues in the N-terminal CBM-21-like domain (Ser14Arg, His108Arg, Ser14Arg/His108Arg) and at the outer subsites +2 (Phe553Gly) and +3 (Phe620Ala, Asp621Ala, Phe620Ala/Asp621Ala) of the active site. The Ser14 and His108 mutants mimic natural LD variants from sorghum and rice with elevated enzymatic activity. Although situated about 40 A from the active site, the single mutants had 15–40% catalytic efficiency compared to wild type for the three polysaccharides and the double mutant retained 27% activity for β-limit dextrin and 64% for pullulan and amylopectin. These three mutants hydrolysed 4,6-O-benzylidene-4-nitrophenyl-63-α- d -maltotriosyl-maltotriose (BPNPG3G3) with 51–109% of wild-type activity. The results highlight that the N-terminal CBM21-like domain plays a role in activity. Phe553 and the highly conserved Trp512 sandwich a substrate main chain glucosyl residue at subsite +2 of the active site, while substrate contacts of Phe620 and Asp621 at subsite +3 are less prominent. Phe553Gly showed 47% and 25% activity on pullulan and BPNPG3G3, respectively having a main role at subsite +2. By contrast at subsite +3, Asp621Ala increased activity on pullulan by 2.4-fold, while Phe620Ala/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbours preference determinants for the branched substrates amylopectin and β-limit dextrin.

The term "starch-binding domain" (SBD) has been applied to a domain within an amylolytic enzyme that gave the enzyme the ability to bind onto raw, i.e. thermally untreated, granular starch. An SBD is a special case of a carbohydrate-binding domain, which in general, is a structurally and functionally independent protein module exhibiting no enzymatic activity but possessing potential to target the catalytic domain to the carbohydrate substrate to accommodate it and process it at the active site. As so-called families, SBDs together with other carbohydrate-binding modules (CBMs) have become an integral part of the CAZy database (http://www.cazy.org/). The first two well-described SBDs, i.e. the C-terminal Aspergillus-type and the N-terminal Rhizopus-type have been assigned the families CBM20 and CBM21, respectively. Currently, among the 85 established CBM families in CAZy, fifteen can be considered as families having SBD functional characteristics: CBM20, 21, 25, 26, 34, 41, 45, 48, 53, 58, 68, 69, 74, 82 and 83. All known SBDs, with the exception of the extra long CBM74, were recognized as a module consisting of approximately 100 residues, adopting a β-sandwich fold and possessing at least one carbohydrate-binding site. The present review aims to deliver and describe: (i) the SBD identification in different amylolytic and related enzymes (e.g., CAZy GH families) as well as in other relevant enzymes and proteins (e.g., laforin, the β-subunit of AMPK, and others); (ii) information on the position in the polypeptide chain and the number of SBD copies and their CBM family affiliation (if appropriate); (iii) structure/function studies of SBDs with a special focus on solved tertiary structures, in particular, as complexes with α-glucan ligands; and (iv) the evolutionary relationships of SBDs in a tree common to all SBD CBM families (except for the extra long CBM74). Finally, some special cases and novel potential SBDs are also introduced.

Alginate is a linear polysaccharide from brown algae consisting of 1,4-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) arranged in M, G, and mixed MG blocks. Alginate was assumed to be indigestible in humans, but bacteria isolated from fecal samples can utilize alginate. Moreover, genomes of some human gut microbiome–associated bacteria encode putative alginate-degrading enzymes. Here, we genome-mined a polysaccharide lyase family 6 alginate lyase from the gut bacterium Bacteroides cellulosilyticus (BcelPL6). The structure of recombinant BcelPL6 was solved by X-ray crystallography to 1.3 Å resolution, revealing a single-domain, monomeric parallel β-helix containing a 10-step asparagine ladder characteristic of alginate-converting parallel β-helix enzymes. Substitutions of the conserved catalytic site residues Lys-249, Arg-270, and His-271 resulted in activity loss. However, imidazole restored the activity of BcelPL6-H271N to 2.5% that of the native enzyme. Molecular docking oriented tetra-mannuronic acid for syn attack correlated with M specificity. Using biochemical analyses, we found that BcelPL6 initially releases unsaturated oligosaccharides of a degree of polymerization of 2–7 from alginate and polyM, which were further degraded to di- and trisaccharides. Unlike other PL6 members, BcelPL6 had low activity on polyMG and none on polyG. Surprisingly, polyG increased BcelPL6 activity on alginate 7-fold. LC–electrospray ionization–MS quantification of products and lack of activity on NaBH4-reduced octa-mannuronic acid indicated that BcelPL6 is an endolyase that further degrades the oligosaccharide products with an intact reducing end. We anticipate that our results advance predictions of the specificity and mode of action of PL6 enzymes. Author’s Choice—Final version open access under the terms of the Creative Commons CC-BY license

Bifidobacteria are exposed to substantial amounts of dietary β-galactosides. Distinctive preferences for growth on different β-galactosides are observed within Bifidobacterium members, but the basis of these preferences remains unclear. We previously described the first β-(1,6)/(1,3)-galactosidase from Bifidobacterium animalis subsp. lactis Bl-04. This enzyme is relatively promiscuous, exhibiting only 5-fold higher efficiency on the preferred β-(1,6)-galactobiose than the β-(1,4) isomer. Here, we characterize the solute-binding protein (Bal6GBP) that governs the specificity of the ABC transporter encoded by the same β-galactoside-utilization locus. We observed that although Bal6GBP recognizes both β-(1,6)- and β-(1,4)-galactobiose, Bal6GBP has a 1630-fold higher selectivity for the former, reflected in dramatic differences in growth, with several hours lag on less preferred β-(1,4)- and β-(1,3)-galactobiose. Experiments performed in the presence of varying proportions of β-(1,4)/ β-(1,6)-galactobioses indicated that the preferred substrate was preferentially depleted from the culture supernatant. This established that the poor growth on the non-preferred β-(1,4) was due to inefficient uptake. We solved the structure of Bal6GBP in complex with β-(1,6)-galactobiose at 1.39 Å resolution, revealing the structural basis of this strict selectivity. Moreover, we observed a close evolutionary relationship with the human milk disaccharide lacto-N-biose-binding protein from Bifidobacterium longum, indicating that the recognition of the non-reducing galactosyl is essentially conserved, whereas the adjacent position is diversified to fit different glycosidic linkages and monosaccharide residues. These findings indicate that oligosaccharide uptake has a pivotal role in governing selectivity for distinct growth substrates and have uncovered evolutionary trajectories that shape the diversification of sugar-uptake proteins within Bifidobacterium.

Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wastewater treatment sludge (wts) Fae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's K(d) for xylohexaose is 14.8 μm and that it does not bind to starch mimics, β-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a β-d-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 Å resolution) and wtsFae1B (1.98 Å) revealed that both are folded proteins comprising structurally-conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1–CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.

Various redox compounds are known to influence the structure of the gluten network in bread dough, and hence its strength. The cereal thioredoxin system (NTS), composed of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (NTR) and thioredoxin (Trx), is a major reducing enzymatic system that is involved in seed formation and germination. NTS is a particularly interesting tool for food processing due to its heat stability and its broad range of protein substrates. We show here that barley NTS is capable of remodeling the gluten network and weakening bread dough. Furthermore, functional wheat Trx that is present in the dough can be recruited by the addition of recombinant barley NTR, resulting in dough weakening. These results confirm the potential of NTS, especially NTR, as a useful tool in baking for weakening strong doughs, or in flat product baking.

Non-fat acidified milk model systems were constructed from frozen casein and whey protein concentrates produced from skim milk using membrane filtration, and combined with commercial whey protein ingredients, i.e. nano-particulated whey protein (NWP), micro-particulated whey protein (MWP), and whey protein concentrate (WPC). Model systems were characterised in terms of particle size distribution and fractionation, surface hydrophobicity and accessible thiol groups, rheological behaviour, water holding capacity and graininess. Samples containing NWP exhibited higher surface hydrophobicity and increase in accessible thiol groups, shorter gelation time, higher gelation pH and G′, but increasing particle size and number of grains, when compared with addition of MWP and WPC. Addition of MWP resulted in weak gels with a less connected protein network and decreased number of grains. Mixtures of NWP and MWP (1:1) had rheological properties closer to those seen for MWP. Systems with WPC differentiated themselves with a large quantity of small aggregates.

The structural and mechanical properties of thin films generated from two types of mucins, namely, bovine submaxillary mucin (BSM) and porcine gastric mucin (PGM) in aqueous environment were investigated with several bulk and surface analytical techniques. Both mucins generated hydrated films on hydrophobic polydimethylsiloxane (PDMS) surfaces from spontaneous adsorption arising from their amphiphilic characteristic. However, BSM formed more elastic films than PGM at neutral pH condition. This structural difference was manifested from the initial film formation processes to the responses to shear stresses applied to the films. Acidification of environmental pH led to strengthening the elastic character of BSM films with increased adsorbed mass, whereas an opposite trend was observed for PGM films. We propose that this contrast originates from that negatively charged motifs are present for both the central and terminal regions of BSM molecule, whereas a similar magnitude of negative charges is localized at the termini of PGM molecule. Given that hydrophobic motifs acting as an anchor are also localized in the terminal region, electrostatic repulsion between anchoring units of PGM molecules on a nonpolar PDMS surface leads to weakening of the mechanical integrity of the films.

The molecular details and impact of oligosaccharide uptake by distinct human gut microbiota (HGM) are currently not well understood. Non-digestible dietary galacto- and gluco--(1,6)-oligosaccharides from legumes and starch, respectively, are preferentially fermented by mainly bifidobacteria and lactobacilli in the human gut. Here we show that the solute binding protein (BlG16BP) associated with an ATP binding cassette (ABC) transporter from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 binds -(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide composition with preference for the trisaccharides raffinose and panose. This preference is also reflected in the -(1,6)-galactoside uptake profile of the bacterium. Structures of BlG16BP in complex with raffinose and panose revealed the basis for the remarkable ligand binding plasticity of BlG16BP, which recognizes the non-reducing -(1,6)-diglycoside in its ligands. BlG16BP homologues occur predominantly in bifidobacteria and a few Firmicutes but lack in other HGMs. Among seven bifidobacterial taxa, only those possessing this transporter displayed growth on -(1,6)-glycosides. Competition assays revealed that the dominant HGM commensal Bacteroides ovatus was out-competed by B. animalis subsp. lactis Bl-04 in mixed cultures growing on raffinose, the preferred ligand for the BlG16BP. By comparison, B. ovatus mono-cultures grew very efficiently on this trisaccharide. These findings suggest that the ABC-mediated uptake of raffinose provides an important competitive advantage, particularly against dominant Bacteroides that lack glycan-specific ABC-transporters. This novel insight highlights the role of glycan transport in defining the metabolic specialization of gut bacteria.

Whole cell and surface proteomes were analyzed together with adhesive properties of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) grown on the emerging prebiotic raffinose, exemplifying a synbiotic. Adhesion of NCFM to mucin and intestinal HT-29 cells increased three-fold after culture with raffinose versus glucose, as also visualized by scanning electron microscopy. Comparative proteomics using 2D-DIGE showed 43 unique proteins to change in relative abundance in whole cell lysates from NCFM grown on raffinose compared to glucose. Furthermore, 14 unique proteins in 18 spots of the surface subproteome underwent changes identified by differential 2DE, including elongation factor G, thermostable pullulanase, and phosphate starvation inducible stress-related protein increasing in a range of +2.1 - +4.7 fold. By contrast five known moonlighting proteins decreased in relative abundance by up to -2.4 fold. Enzymes involved in raffinose catabolism were elevated in the whole cell proteome; α-galactosidase (+13.9 fold); sucrose phosphorylase (+5.4 fold) together with metabolic enzymes from the Leloir pathway for galactose utilization and the glycolysis; β-galactosidase (+5.7 fold); galactose (+2.9/+3.1 fold) and fructose (+2.8 fold) kinases. The insights at the molecular and cellular levels contributed to the understanding of the interplay of a synbiotic composed of NCFM and raffinose with the host.

Starch is a major energy store in plants. It provides most of the calories in the human diet and, as a bulk commodity, it is used across broad industry sectors. Starch synthesis and degradation are not fully understood, owing to challenging biochemistry at the liquid/solid interface and relatively limited knowledge about the nature and control of starch degradation in plants. Increased societal and commercial demand for enhanced yield and quality in starch crops requires a better understanding of starch metabolism as a whole. Here we review recent advances in understanding the roles of carbohydrate-active enzymes in starch degradation in cereal grains through complementary chemical and molecular genetics. These approaches have allowed us to start dissecting aspects of starch degradation and the interplay with cell-wall polysaccharide hydrolysis during germination. With a view to improving and diversifying the properties and uses of cereal grains, it is possible that starch degradation may be amenable to manipulation through genetic or chemical intervention at the level of cell wall metabolism, rather than simply in the starch degradation pathway per se.

Ultrahigh temperature (UHT) processed and lactose-free milk are for export to Eastern markets. Free galactose and glucose in lactose-free milk increase occurrence of Maillard products and hence lower milk quality. Notably, enzymatic conversion of galactose into galacto-oligosaccharides (GOS) mitigates such undesirable reactions. As little is known about the effect of GOS on caseins, interactions between caseins in presence or absence of GOS or lactose were assessed by surface plasmon resonance analysis. Based on steady state binding constants (KD) soluble αS-casein showed one order of magnitude lower affinity than soluble β-casein and κ-casein for chip-immobilised αS- and β-casein. By contrast, αS-casein had higher affinity for immobilised κ-casein, followed by β- and κ-casein. The interaction between the caseins did not obey a 1:1 binding model and it was not possible to calculate a precise stoichiometry. Importantly, lactose and GOS exerted very modest or no effect on the various interaction between the caseins.

&beta, N-Acetylhexosaminidases are glycoside hydrolases (GHs) acting on N-acetylated carbohydrates and glycoproteins with the release of N-acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of N-acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydrolysis, thus representing an alternative to chemical oligosaccharide synthesis. Two putative GH20 &beta, N-acetylhexosaminidases, PhNah20A and PhNah20B, encoded by the marine bacterium Paraglaciecola hydrolytica S66T, are distantly related to previously characterized enzymes. Remarkably, PhNah20A was located by phylogenetic analysis outside clusters of other studied &beta, N-acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant PhNah20A showing optimum activity at pH 6.0 and 50 &deg, C, hydrolysis of GlcNAc &beta, 1,4 and &beta, 1,3 linkages in chitobiose (GlcNAc)2 and GlcNAc-1,3-&beta, Gal-1,4-&beta, Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of PhNah20A for p-nitrophenyl-GlcNAc and p-nitrophenyl-GalNAc were highly similar: kcat/KM being 341 and 344 mM&minus, 1 s&minus, 1, respectively. PhNah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). PhNah20A catalyzed the formation of LNT2, the non-reducing trisaccharide &beta, Glc-1,1-&beta, GlcNAc, and in low amounts the &beta, 1,2- or &beta, 1,3-linked trisaccharide &beta, Gal-1,4(&beta, GlcNAc)-1,x-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-&alpha, d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. PhNah20A is the first characterized member of a distinct subgroup within GH20 &beta, N-acetylhexosaminidases.

Biomacromolecules 19(7), 2905 - 2912 (2018). doi:10.1021/acs.biomac.8b00471, The dimeric structure of bovine β-lactoglobulin A (BLGA) at pH 4.0 was solved to 2.0 Å resolution. Fitting the BLGA pH 4.0 structure to SAXS data at low ionic strength (goodness of fit R-factor = 3.6%) verified the dimeric state in solution. Analysis of the monomer–dimer equilibrium at varying pH and ionic strength by SAXS and scattering modeling showed that BLGA is dimeric at pH 3.0 and 4.0, shifting toward a monomer at pH 2.2, 2.6, and 7.0 yielding monomer/dimer ratios of 80/20%, 50/50%, and 25/75%, respectively. BLGA remained a dimer at pH 3.0 and 4.0 in 50–150 mM NaCl, whereas the electrostatic shielding raised the dimer content at pH 2.2, 2.6, and 7.0, i.e., below and above the pI. Overall, the findings provide new insights into the molecular characteristics of BLGA relevant for dairy product formulations and for various biotechnological and pharmaceutical applications., Published by American Chemical Soc., Columbus, Ohio

Sirtuins—a group of NAD+-dependent deacylases—have emerged as key in the connection between NAD+metabolism and aging. This class of enzymes hydrolyze a range ofε-N-acyllysine PTMs and determining the repertoire of catalyzed deacylation reactions is of high importance to fully elucidate the roles of a given sirtuin. Here we have identified and produced two potential sirtuins from the probiotic bacteriumLactobacillus acidophilusNCFM and screening more than 80 different substrates, covering 26 acyl groups on five peptide scaffolds, showed that one of the investigated proteins—Sir2La—is abona fideNAD+-dependent sirtuin, catalyzing hydrolysis of acetyl‐, propionyl‐, and butyryllysine. Further substantiating the identity as a sirtuin, known sirtuin inhibitors nicotinamide and suramin as well as a thioacetyllysine compound inhibit the deacylase activity in a concentration-dependent manner. Based on steady-state kinetics Sir2La showed a slight preference for propionyllysine over acetyllysine and butyryllysine, driven both byKM(14 μMvs21 μM and 15 μM) andkcat(4.4·10−3s−1vs2.5·10−3s−1and 1.21·10−3s−1). Moreover, while NAD+is a prerequisite for Sir2La-mediated deacylation, Sir2La has very highKMfor NAD+compared to the expected levels of the dinucleotide inL. acidophilus. Sir2La is the first sirtuin from Lactobacillales and of the Gram-positive bacterial subclass of sirtuins to be functionally characterized. The ability to hydrolyze propionyl‐ and butyryllysine emphasizes the relevance of further exploring the role of other short-chain acyl moieties as PTMs.