Publications by authors named "Birte Svensson"

207 Publications

Wheat ATIs: Characteristics and Role in Human Disease.

Front Nutr 2021 28;8:667370. Epub 2021 May 28.

Laboratory of Food Chemistry, Wageningen University and Research, Wageningen, Netherlands.

Amylase/trypsin-inhibitors (ATIs) comprise about 2-4% of the total wheat grain proteins and may contribute to natural defense against pests and pathogens. However, they are currently among the most widely studied wheat components because of their proposed role in adverse reactions to wheat consumption in humans. ATIs have long been known to contribute to IgE-mediated allergy (notably Bakers' asthma), but interest has increased since 2012 when they were shown to be able to trigger the innate immune system, with attention focused on their role in coeliac disease which affects about 1% of the population and, more recently, in non-coeliac wheat sensitivity which may affect up to 10% of the population. This has led to studies of their structure, inhibitory properties, genetics, control of expression, behavior during processing, effects on human adverse reactions to wheat and, most recently, strategies to modify their expression in the plant using gene editing. We therefore present an integrated account of this range of research, identifying inconsistencies, and gaps in our knowledge and identifying future research needs.   This paper is the outcome of an invited international ATI expert meeting held in Amsterdam, February 3-5 2020.
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http://dx.doi.org/10.3389/fnut.2021.667370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192694PMC
May 2021

A putative novel starch-binding domain revealed by in silico analysis of the N-terminal domain in bacterial amylomaltases from the family GH77.

3 Biotech 2021 May 21;11(5):229. Epub 2021 Apr 21.

Department of Biology, Faculty of Natural Sciences, University of SS. Cyril and Methodius, Nám. J. Herdu 2, 91701 Trnava, Slovakia.

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 and , 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 ( 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.
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http://dx.doi.org/10.1007/s13205-021-02787-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8060381PMC
May 2021

Quantitative Label-Free Comparison of the Metabolic Protein Fraction in Old and Modern Italian Wheat Genotypes by a Shotgun Approach.

Molecules 2021 Apr 29;26(9). Epub 2021 Apr 29.

Laboratory of Organic Mass Spectrometry, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

Wheat represents one of the most important cereals for mankind. However, since wheat proteins are also the causative agent of several adverse reactions, during the last decades, consumers have shown an increasing interest in the old wheat genotypes, which are generally perceived as more "natural" and healthier than the modern ones. Comparison of nutritional value for modern and old wheat genotypes is still controversial, and to evaluate the real impact of these foods on human health comparative experiments involving old and modern genotypes are desirable. The nutritional quality of grain is correlated with its proteomic composition that depends on the interplay between the genetic characteristics of the plant and external factors related to the environment. We report here the label-free shotgun quantitative comparison of the metabolic protein fractions of two old Sicilian landraces (Russello and Timilia) and the modern variety Simeto, from the 2010-2011 and 2011-2012 growing seasons. The overall results show that Timilia presents the major differences with respect to the other two genotypes investigated. These differences may be related to different defense mechanisms and some other peculiar properties of these genotypes. On the other hand, our results confirm previous results leading to the conclusion that with respect to a nutritional value evaluation, there is a substantial equivalence between old and modern wheat genotypes. Data are available via ProteomeXchange with identifier .
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http://dx.doi.org/10.3390/molecules26092596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8124627PMC
April 2021

Rational Enzyme Design without Structural Knowledge: A Sequence-Based Approach for Efficient Generation of Transglycosylases.

Chemistry 2021 Apr 29. Epub 2021 Apr 29.

Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, building 224, DK-2800, Kongens Lyngby, Denmark.

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, a straightforward strategy that involves rational rapid in silico analysis of protein sequences is described. 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 or endo action. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.
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http://dx.doi.org/10.1002/chem.202100110DOI Listing
April 2021

Binding Sites for Oligosaccharide Repeats from Lactic Acid Bacteria Exopolysaccharides on Bovine β-Lactoglobulin Identified by NMR Spectroscopy.

ACS Omega 2021 Apr 23;6(13):9039-9052. Epub 2021 Mar 23.

Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark.

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 LY03 and 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 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.
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http://dx.doi.org/10.1021/acsomega.1c00060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8028130PMC
April 2021

Functional diversity of three tandem C-terminal carbohydrate-binding modules of a β-mannanase.

J Biol Chem 2021 Apr 7:100638. Epub 2021 Apr 7.

Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.

Carbohydrate active enzymes, such as those involved in plant cell wall and storage polysaccharide biosynthesis and deconstruction, often contain repeating non-catalytic carbohydrate binding modules (CBMs) to compensate for low-affinity binding typical of protein-carbohydrate interactions. The bacterium Saccharophagus degradans produces an endo-β-mannanase of glycoside hydrolase family 5 subfamily 8 with three phylogenetically distinct family 10 CBMs located C-terminally from the catalytic domain (SdGH5_8-CBM10x3). However, the functional roles and cooperativity of these CBM domains in polysaccharide binding is not clear. To learn more we studied the full-length enzyme, three stepwise CBM10 truncations, and green fluorescent protein fusions of the individual CBM10s and all three domains together by pull-down assays, affinity gel electrophoresis, and activity assays. Only the C-terminal CBM10-3 was found to bind strongly to microcrystalline cellulose (dissociation constant, K = 1.48 μM). CBM10-3 and CBM10-2 bound galactomannan with similar affinity (K = 0.2-0.4 mg/ml), but CBM10-1 had 20-fold lower affinity for this substrate. CBM10 truncations barely affected specific activity on carob galactomannan and konjac glucomannan. Full-length SdGH5_8-CBM10x3 was two-fold more active on the highly galactose-decorated viscous guar gum galactomannan and crystalline ivory nut mannan at high enzyme concentrations, but the specific activity was 4- to 9-fold reduced at low enzyme and substrate concentrations compared to the enzyme lacking CBM10-2 and -3. Comparison of activity and binding data for the different enzyme forms indicates unproductive and productive polysaccharide binding to occur. We conclude that the C-terminal-most CBM10-3 secures firm binding, with contribution from CBM10-2, which with CBM10-1 also provides spatial flexibility.
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http://dx.doi.org/10.1016/j.jbc.2021.100638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8121702PMC
April 2021

Impact of Alginate Mannuronic-Guluronic Acid Contents and pH on Protein Binding Capacity and Complex Size.

Biomacromolecules 2021 02 8;22(2):649-660. Epub 2021 Jan 8.

Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kongens Lyngby Denmark.

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.
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http://dx.doi.org/10.1021/acs.biomac.0c01485DOI Listing
February 2021

An 1,4-α-Glucosyltransferase Defines a New Maltodextrin Catabolism Scheme in Lactobacillus acidophilus.

Appl Environ Microbiol 2020 07 20;86(15). Epub 2020 Jul 20.

Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark

The maltooligosaccharide (MOS) utilization locus in NCFM, a model for human small-intestine lactobacilli, encodes three glycoside hydrolases (GHs): a putative maltogenic α-amylase of family 13, subfamily 20 (GH13_20), a maltose phosphorylase of GH65 (GH65), and a family 13, subfamily 31, member (GH13_31B), annotated as a 1,6-α-glucosidase. Here, we reveal that GH13_31B is a 1,4-α-glucosyltransferase that disproportionates MOS with a degree of polymerization of ≥2, with a preference for maltotriose. Kinetic analyses of the three GHs encoded by the MOS locus revealed that the substrate preference of GH13_31B toward maltotriose complements the ~40-fold lower of GH13_20 toward this substrate, thereby enhancing the conversion of odd-numbered MOS to maltose. The concerted action of GH13_20 and GH13_31B confers the efficient conversion of MOS to maltose that is phosphorolyzed by GH65. Structural analyses revealed the presence of a flexible elongated loop that is unique for a previously unexplored clade of GH13_31, represented by GH13_31B. The identified loop insertion harbors 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 GH13_31B gene is conserved in the MOS utilization loci of lactobacilli, including acidophilus cluster members that dominate the human small intestine. 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 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 likely allows the fine-tuning of 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 that 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.
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http://dx.doi.org/10.1128/AEM.00661-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376546PMC
July 2020

Thermophilic 4-α-Glucanotransferase from Retards the Long-Term Retrogradation but Maintains the Short-Term Gelation Strength of Tapioca Starch.

J Agric Food Chem 2020 May 8;68(20):5658-5667. Epub 2020 May 8.

Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.

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.
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http://dx.doi.org/10.1021/acs.jafc.0c00927DOI Listing
May 2020

Azo dying of α-keratin material improves microbial keratinase screening and standardization.

Microb Biotechnol 2020 07 28;13(4):984-996. Epub 2020 Feb 28.

Section of Microbiology, University of Copenhagen, DK-2100, Copenhagen, Denmark.

Microbial conversion through enzymatic reactions has received a lot of attention as a cost-effective and environmentally friendly way to recover amino acids and short peptides from keratin materials. However, accurate assessment of microbial keratinase activity is not straightforward, and current available methods lack sensitivity and standardization. Here, we suggest an optimized Azokeratin assay, with substrate generated directly from azo-dyed raw keratin material. We introduced supernatant filtration in the protocol for optimal stopping of keratinase reactions instead of the widely used trichloroacetic acid (TCA), as it generated biases and impacted the sensitivity. We furthermore suggest a method for standardization of keratinase activity signals using proteinase K, a well-known keratinase, as a reference enabling reproducibility between studies. Lastly, we evaluated our developed method with several bacterial isolates through benchmarking against a commercial assay (Keratin Azure). Under different setups, the Azokeratin method was more sensitive than commonly used Keratin Azure-based assays (3-fold). We argue that this method could be applied with any type of keratin substrate, enabling more robust and sensitive results which can be used for further comparison with other studies, thus representing an important progress within the field of microbial keratin degradation.
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http://dx.doi.org/10.1111/1751-7915.13541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264887PMC
July 2020

Community-intrinsic properties enhance keratin degradation from bacterial consortia.

PLoS One 2020 31;15(1):e0228108. Epub 2020 Jan 31.

Section for Microbiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark.

Although organic matter may accumulate sometimes (e.g. lignocellulose in peat bog), most natural biodegradation processes are completed until full mineralization. Such transformations are often achieved by the concerted action of communities of interacting microbes, involving different species each performing specific tasks. These interactions can give rise to novel "community-intrinsic" properties, through e.g. activation of so-called "silent genetic pathways" or synergistic interplay between microbial activities and functions. Here we studied the microbial community-based degradation of keratin, a recalcitrant biological material, by four soil isolates, which have previously been shown to display synergistic interactions during biofilm formation; Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus. We observed enhanced keratin weight loss in cultures with X. retroflexus, both in dual and four-species co-cultures, as compared to expected keratin degradation by X. retroflexus alone. Additional community intrinsic properties included accelerated keratin degradation rates and increased biofilm formation on keratin particles. Comparison of secretome profiles of X. retroflexus mono-cultures to co-cultures revealed that certain proteases (e.g. serine protease S08) were significantly more abundant in mono-cultures, whereas co-cultures had an increased abundance of proteins related to maintaining the redox environment, e.g. glutathione peroxidase. Hence, one of the mechanisms related to the community intrinsic properties, leading to enhanced degradation from co-cultures, might be related to a switch from sulfitolytic to proteolytic functions between mono- and co-cultures, respectively.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228108PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994199PMC
May 2020

Two novel S1 peptidases from Amycolatopsis keratinophila subsp. keratinophila D2 degrading keratinous slaughterhouse by-products.

Appl Microbiol Biotechnol 2020 Mar 27;104(6):2513-2522. Epub 2020 Jan 27.

Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads Building 224, 2800, Kgs. Lyngby, Denmark.

Two proteases, named C- and T-like proteases, respectively, were purified from the culture supernatant of Amycolatopsis keratinophila subsp. keratinophila D2 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.
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http://dx.doi.org/10.1007/s00253-020-10380-xDOI Listing
March 2020

Identification and Characterization of a β--Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium S66.

Int J Mol Sci 2020 Jan 9;21(2). Epub 2020 Jan 9.

Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark.

β--Acetylhexosaminidases are glycoside hydrolases (GHs) acting on -acetylated carbohydrates and glycoproteins with the release of -acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of -acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydrolysis, thus representing an alternative to chemical oligosaccharide synthesis. Two putative GH20 β--acetylhexosaminidases, Nah20A and Nah20B, encoded by the marine bacterium S66, are distantly related to previously characterized enzymes. Remarkably, Nah20A was located by phylogenetic analysis outside clusters of other studied β--acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant Nah20A showing optimum activity at pH 6.0 and 50 °C, hydrolysis of GlcNAc β-1,4 and β-1,3 linkages in chitobiose (GlcNAc) and GlcNAc-1,3-β-Gal-1,4-β-Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of Nah20A for -nitrophenyl-GlcNAc and -nitrophenyl-GalNAc were highly similar: / being 341 and 344 mM s, respectively. Nah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). Nah20A catalyzed the formation of LNT2, the non-reducing trisaccharide β-Gal-1,4-β-Glc-1,1-β-GlcNAc, and in low amounts the β-1,2- or β-1,3-linked trisaccharide β-Gal-1,4(β-GlcNAc)-1,-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. Nah20A is the first characterized member of a distinct subgroup within GH20 β--acetylhexosaminidases.
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http://dx.doi.org/10.3390/ijms21020417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7014002PMC
January 2020

A Single Point Mutation Converts GH84 -GlcNAc Hydrolases into Phosphorylases: Experimental and Theoretical Evidence.

J Am Chem Soc 2020 02 24;142(5):2120-2124. Epub 2020 Jan 24.

Departament de Quı́mica Inorgànica i Orgànica (Secció de Quı́mica Orgànica) and Institut de Quı́mica Teòrica i Computacional (IQTCUB) , Universitat de Barcelona , Martí i Franquès 1 , 08028 Barcelona , Spain.

Glycoside hydrolases and phosphorylases are two major classes of enzymes responsible for the cleavage of glycosidic bonds. Here we show that two GH84 -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. 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.
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http://dx.doi.org/10.1021/jacs.9b09655DOI Listing
February 2020

Dataset of the metabolic and CM-like protein fractions in old and modern wheat Italian genotypes.

Data Brief 2019 Dec 1;27:104730. Epub 2019 Nov 1.

Laboratory of Organic Mass Spectrometry, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125, Catania, Italy.

The present work reports the first comprehensive proteomic profiling and qualitative comparison of metabolic and Chloroform-Methanol (CM)-like protein fractions extracted from mature kernels of two old Sicilian durum wheat landraces, and , and , an improved durum wheat variety widespread in Italy and other Mediterranean countries and chosen as representative of the most widely commercial cultivars. The data are discussed in the related research article "Qualitative proteomic comparison of metabolic and CM-like protein fractions in old and modern wheat Italian genotypes by a shotgun approach" [1]. The results of this work could be used for investigations to understand the relationship between protein profiles of old and modern wheat genotypes and their potential benefits for human consumption.
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http://dx.doi.org/10.1016/j.dib.2019.104730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859223PMC
December 2019

Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase.

Biochim Biophys Acta Proteins Proteom 2020 01 30;1868(1):140294. Epub 2019 Oct 30.

Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, building 224, DK 2800 Kgs. Lyngby, Denmark. Electronic address:

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 Å 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-6-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.
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http://dx.doi.org/10.1016/j.bbapap.2019.140294DOI Listing
January 2020

Qualitative proteomic comparison of metabolic and CM-like protein fractions in old and modern wheat Italian genotypes by a shotgun approach.

J Proteomics 2020 01 16;211:103530. Epub 2019 Oct 16.

Laboratory of Organic Mass Spectrometry, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

The close relationship between diet and health is generally recognized and the growing wellness and consciousness, especially in developed countries, have led to increasing interest for old wheat genotypes, based on perceived health benefits. Although nutritional comparison between old and modern wheat varieties is still controversial, it is generally accepted that old wheat genotypes remained unchanged over the last hundred years. By contrast, modern wheat genotypes are derived by modification of old wheats during the so-called "Green-Revolution" in the second half of the 20th century focusing on obtaining properties in terms of higher grain yield. The present work reports the first comprehensive proteomic profiling and qualitative comparison at the molecular level of metabolic and Chloroform-Methanol (CM)-like protein fractions extracted from mature kernels of two old Sicilian durum wheat landraces, Russello and Timilia Reste Bianche, and Simeto, an improved durum wheat variety widespread in Italy and other Mediterranean countries and chosen as representative of the most widely commercial cultivars. The results obtained reveal that metabolic and CM-like protein fractions of old and modern genotypes present remarkably high similarity with only minor differences. This leads to the conclusion that from a food and nutritional perspective there is a substantial equivalence of the protein composition of the old and modern cultivars. Data are available via ProteomeXchange with identifier PXD014449. BIOLOGICAL SIGNIFICANCE: In recent years consumers have shown growing interest in the old wheat genotypes, which are generally perceived more "natural" and healthier than modern ones. However, comparison of nutritional value for modern and old wheat varieties is still controversial suggesting further studies. In particular proteome analysis of old and modern wheat genotypes is currently ongoing with particular focus on gluten proteins, whereas the metabolic protein fraction has not yet been investigated. In the present study, we conducted a comprehensive proteomic profile and qualitative comparison at the molecular level of metabolic and Chloroform-Methanol (CM)-like protein fractions of the old Sicilian landraces Russello and Timilia Reste Bianche and the modern cultivar Simeto by applying a shotgun approach. The results reveal that the metabolic and CM-like protein fractions of old and modern genotypes are remarkably similar with only minor differences, leading to the conclusion that from a food and nutritional perspective there is a substantial equivalence of these cultivars. These results may contribute to improved understanding of the relationship between protein profiles of old wheat genotypes and their potential benefits for human consumption.
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http://dx.doi.org/10.1016/j.jprot.2019.103530DOI Listing
January 2020

A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan.

J Biol Chem 2019 11 26;294(46):17339-17353. Epub 2019 Sep 26.

Section for Protein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kongens Lyngby, Denmark

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 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.
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http://dx.doi.org/10.1074/jbc.RA119.009523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873179PMC
November 2019

Starch-binding domains as CBM families-history, occurrence, structure, function and evolution.

Biotechnol Adv 2019 12 16;37(8):107451. Epub 2019 Sep 16.

Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark.

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.
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http://dx.doi.org/10.1016/j.biotechadv.2019.107451DOI Listing
December 2019

Structural and functional aspects of mannuronic acid-specific PL6 alginate lyase from the human gut microbe .

J Biol Chem 2019 11 17;294(47):17915-17930. Epub 2019 Sep 17.

Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark

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 (PL6). The structure of recombinant PL6 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 PL6-H271N to 2.5% that of the native enzyme. Molecular docking oriented tetra-mannuronic acid for attack correlated with M specificity. Using biochemical analyses, we found that PL6 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, PL6 had low activity on polyMG and none on polyG. Surprisingly, polyG increased PL6 activity on alginate 7-fold. LC-electrospray ionization-MS quantification of products and lack of activity on NaBH-reduced octa-mannuronic acid indicated that PL6 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.
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http://dx.doi.org/10.1074/jbc.RA119.010206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879350PMC
November 2019

Alginate Trisaccharide Binding Sites on the Surface of β-Lactoglobulin Identified by NMR Spectroscopy: Implications for Molecular Network Formation.

ACS Omega 2019 Apr 2;4(4):6165-6174. Epub 2019 Apr 2.

Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark.

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.
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http://dx.doi.org/10.1021/acsomega.8b03532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6647953PMC
April 2019

Expanding the citrullinome of synovial fibrinogen from rheumatoid arthritis patients.

J Proteomics 2019 09 10;208:103484. Epub 2019 Aug 10.

Department of Biotechnology and Biomedicine, Technical University of Denmark, Kgs. Lyngby, Denmark. Electronic address:

Citrullination is a post-translational protein modification, which is associated with inflammation in general and is thought to play an important pathogenic role in rheumatoid arthritis (RA). Here a mass spectrometry-based proteomics approach was applied to identify citrullination sites in synovial fluid fibrinogen from four RA patients. In general, high disease activity correlated with increased number of identified citrullination sites and higher relative citrulline occupancy. Altogether, 23 sites were identified, of which 9 have not been previously reported to be citrullinated in vivo. Citrullination at site α84, α123, α129, α547, α573, α591, β334 and γ134 was identified in more than one patient, and these positions were therefore regarded as hotspots. Following citrullination of fibrinogen in vitro using human recombinant peptidylarginine deiminase 2 (PAD2), a total of 46 citrullination sites were identified, including 6 hitherto unreported in vitro citrullination sites. Twenty-two out of the 23 citrullination sites identified in vivo were also detected in vitro, supporting the validity of the identifications. SIGNIFICANCE: This work provides information about previously uncharacterized citrullination sites in synovial fluid fibrinogen from rheumatoid arthritis patients. Detection of these novel citrullination sites may prove to have diagnostic or prognostic value in RA and enhance our understanding of the immune pathogenesis.
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http://dx.doi.org/10.1016/j.jprot.2019.103484DOI Listing
September 2019

Substrate preference of an ABC importer corresponds to selective growth on β-(1,6)-galactosides in subsp. .

J Biol Chem 2019 08 11;294(31):11701-11711. Epub 2019 Jun 11.

Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark

Bifidobacteria are exposed to substantial amounts of dietary β-galactosides. Distinctive preferences for growth on different β-galactosides are observed within members, but the basis of these preferences remains unclear. We previously described the first β-(1,6)/(1,3)-galactosidase from subsp. 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 (6GBP) that governs the specificity of the ABC transporter encoded by the same β-galactoside utilization locus. We observed that although 6GBP recognizes both β-(1,6)- and β-(1,4)-galactobiose, 6GBP 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 nonpreferred β-(1,4) was due to inefficient uptake. We solved the structure of 6GBP 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--biose-binding protein from , indicating that the recognition of the nonreducing 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 .
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http://dx.doi.org/10.1074/jbc.RA119.008843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682729PMC
August 2019

Quantitative Proteomics Analysis of Barley-Based Liquid Feed and the Effect of Protease Inhibitors and NADPH-Dependent Thioredoxin Reductase/Thioredoxin (NTR/Trx) System.

J Agric Food Chem 2019 Jun 28;67(22):6432-6444. Epub 2019 May 28.

Agricultural and Environmental Proteomics, Department of Systems Biology , Technical University of Denmark , Søltofts Plads, Building 224 , DK-2800 Kgs. Lyngby , Denmark.

Liquid feeding strategies have been devised with the aim of enhancing grain nutrient availability for livestock. It is characterized by a steeping/soaking period that softens the grains and initiates mobilization of seed storage reserves. The present study uses 2D gel-based proteomics to investigate the role of proteolysis and reduction by thioredoxins over a 48 h steeping period by monitoring protein abundance dynamics in barley-based liquid feed samples supplemented with either protease inhibitors or NADPH-dependent thioredoxin reductase/thioredoxin (NTR/Trx). Several full-length storage proteins were only identified in the water-extractable fraction of feed containing protease inhibitors, illustrating significant inhibition of proteolytic activities arising during the steeping period. Application of functional NTR/Trx to liquid feed reductively increased the solubility of known and potentially new Trx-target proteins, e.g., outer membrane protein X, and their susceptibility to proteolysis. Thus, the NTR/Trx system exhibits important potential as a feed additive to enhance nutrient digestibility in monogastric animals.
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http://dx.doi.org/10.1021/acs.jafc.9b01708DOI Listing
June 2019

An integrated strategy for the effective production of bristle protein hydrolysate by the keratinolytic filamentous bacterium Amycolatopsis keratiniphila D2.

Waste Manag 2019 Apr 5;89:94-102. Epub 2019 Apr 5.

Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads 229, DK-2800 Kgs. Lyngby, Denmark.

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 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.
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http://dx.doi.org/10.1016/j.wasman.2019.03.067DOI Listing
April 2019

New Insights into the Potential of Endogenous Redox Systems in Wheat Bread Dough.

Antioxidants (Basel) 2018 Dec 12;7(12). Epub 2018 Dec 12.

Enzyme and Protein Chemistry (EPC), Department of Systems Biology, Technical University of Denmark, Building 224, DK-2800 Kgs Lyngby, Denmark.

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.
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http://dx.doi.org/10.3390/antiox7120190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6316651PMC
December 2018

Functional Roles of Starch Binding Domains and Surface Binding Sites in Enzymes Involved in Starch Biosynthesis.

Front Plant Sci 2018 13;9:1652. Epub 2018 Nov 13.

Enzyme and Protein Chemistry, Department of Bioengineering and Biomedicine, Technical University of Denmark, Lyngby, Denmark.

Biosynthesis of starch is catalyzed by a cascade of enzymes. The activity of a large number of these enzymes depends on interaction with polymeric substrates via carbohydrate binding sites, which are situated outside of the catalytic site and its immediate surroundings including the substrate-binding crevice. Such secondary binding sites can belong to distinct starch binding domains (SBDs), classified as carbohydrate binding modules (CBMs), or be surface binding sites (SBSs) exposed on the surface of catalytic domains. Currently in the Carbohydrate-Active enZYmes (CAZy) database SBDs are found in 13 CBM families. Four of these families; CBM20, CBM45, CBM48, and CBM53 are represented in enzymes involved in starch biosynthesis, namely starch synthases, branching enzymes, isoamylases, glucan, water dikinases, and α-glucan phosphatases. A critical role of the SBD in activity has not been demonstrated for any of these enzymes. Among the well-characterized SBDs important for starch biosynthesis are three CBM53s of starch synthase III, which have modest affinity. SBSs, which are overall less widespread than SBDs, have been reported in some branching enzymes, isoamylases, synthases, phosphatases, and phosphorylases active in starch biosynthesis. SBSs appear to exert roles similar to CBMs. SBSs, however, have also been shown to modulate specificity for example by discriminating the length of chains transferred by branching enzymes. Notably, the difference in rate of occurrence between SBDs and SBSs may be due to lack of awareness of SBSs. Thus, SBSs as opposed to CBMs are not recognized at the protein sequence level, which hampers their identification. Moreover, only a few SBSs in enzymes involved in starch biosynthesis have been functionally characterized, typically by structure-guided site-directed mutagenesis. The glucan phosphatase Like SEX4 2 from has two SBSs with weak affinity for β-cyclodextrin, amylose and amylopectin, which were indicated by mutational analysis to be more important than the active site for initial substrate recognition. The present review provides an update on occurrence of functional SBDs and SBSs in enzymes involved in starch biosynthesis.
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http://dx.doi.org/10.3389/fpls.2018.01652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6243121PMC
November 2018

The exopolysaccharide properties and structures database: EPS-DB. Application to bacterial exopolysaccharides.

Carbohydr Polym 2019 Feb 28;205:565-570. Epub 2018 Oct 28.

Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark. Electronic address:

The EPS Database (EPS-DB) is a web-based, platform-independent database of bacterial exopolysaccharides (EPSs) providing access to detailed structural, taxonomic, growth conditions, functional properties, genetic, and bibliographic information for EPSs. It is freely available on the Internet as a website at http://www.epsdatabase.com. Several structural data representation schemes are used following the most commonly accepted formats. This guarantees full interoperability with other structural, experimental, and functional databases in the area of glycoscience. The scientific usage of EPS-DB throughout a user-friendly interface is presented with a subsection of the database exemplified by EPSs from lactic acid bacteria.
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http://dx.doi.org/10.1016/j.carbpol.2018.10.063DOI Listing
February 2019

Asp271 is critical for substrate interaction with the surface binding site in β-agarase a from Zobellia galactanivorans.

Proteins 2019 01 4;87(1):34-40. Epub 2018 Nov 4.

Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.

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 K values from 625 ± 118 to 468 ± 13 μM for agaro-hexaose, -octaose, and -decaose, which represent 3- to 4-fold reduced affinity compared with 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.
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http://dx.doi.org/10.1002/prot.25614DOI Listing
January 2019

Dietary Nutrients, Proteomes, and Adhesion of Probiotic Lactobacilli to Mucin and Host Epithelial Cells.

Microorganisms 2018 Aug 21;6(3). Epub 2018 Aug 21.

Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kgs Lyngby, Denmark.

The key role of diet and environment in human health receives increasing attention. Thus functional foods, probiotics, prebiotics, and synbiotics with beneficial effects on health and ability to prevent diseases are in focus. The efficacy of probiotic bacteria has been connected with their adherence to the host epithelium and residence in the gut. Several in vitro techniques are available for analyzing bacterial interactions with mucin and intestinal cells, simulating adhesion to the host in vivo. Proteomics has monitored and identified proteins of probiotic bacteria showing differential abundance elicited in vitro by exposure to food components, including potential prebiotics (e.g., certain carbohydrates, and plant polyphenols). While adhesion of probiotic bacteria influenced by various environmental factors relevant to the gastrointestinal tract has been measured previously, this was rarely correlated with changes in the bacterial proteome induced by dietary nutrients. The present mini-review deals with effects of selected emerging prebiotics, food components and ingredients on the adhesion of probiotic lactobacilli to mucin and gut epithelial cells and concomitant abundancy changes of specific bacterial proteins. Applying this in vitro synbiotics-like approach enabled identification of moonlighting and other surface-located proteins of NCFM that are possibly associated with the adhesive mechanism.
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http://dx.doi.org/10.3390/microorganisms6030090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6163540PMC
August 2018