Oligosaccharide Metabolism and Lipoteichoic Acid Production in Lactobacillus gasseri and Lactobacillus paragasseri

Lactobacillus gasseri and Lactobacillus paragasseri are human commensal lactobacilli that are candidates for probiotic application. Knowledge of their oligosaccharide metabolic properties is valuable for synbiotic application. The present study characterized oligosaccharide metabolic systems and their impact on lipoteichoic acid (LTA) production in the two organisms, i.e., L. gasseri JCM 1131T and L. paragasseri JCM 11657. The two strains grew well in medium with glucose but poorly in medium with raffinose, and growth rates in medium with kestose differed between the strains. Oligosaccharide metabolism markedly influenced their LTA production, and apparent molecular size of LTA in electrophoresis recovered from cells cultured with glucose and kestose differed from that from cells cultured with raffinose in the strains. On the other hand, more than 15-fold more LTA was observed in the L. gasseri cells cultured with raffinose when compared with glucose or kestose after incubation for 15 h. Transcriptome analysis identified glycoside hydrolase family 32 enzyme as a potential kestose hydrolysis enzyme in the two strains. Transcriptomic levels of multiple genes in the dlt operon, involved in D-alanine substitution of LTA, were lower in cells cultured with raffinose than in those cultured with kestose or glucose. This suggested that the different sizes of LTA observed among the carbohydrates tested were partly due to different levels of alanylation of LTA. The present study indicates that available oligosaccharide has the impact on the LTA production of the industrially important lactobacilli, which might influence their probiotic properties.

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Pectin digestion in herbivorous beetles: Impact of pseudoenzymes exceeds that of their active counterparts

10.1101/462531 ◽

2018 ◽

Author(s):

Roy Kirsch ◽

Grit Kunert ◽

Heiko Vogel ◽

Yannick Pauchet

Keyword(s):

Plant Cell Wall ◽

Functional Characterization ◽

Gene Families ◽

Glycoside Hydrolase Family ◽

Cell Wall Degrading Enzymes ◽

Catalytic Function ◽

Catalytically Active ◽

Alternative Function ◽

The Impact ◽

Hydrolase Family

AbstractMany protein families harbor pseudoenzymes that have lost the catalytic function of their enzymatically active counterparts. Assigning alternative function and importance to these proteins is challenging [1]. Because the evolution towards pseudoenzymes is driven by gene duplication, they often accumulate in multigene families. Plant cell wall-degrading enzymes (PCWDEs) are prominent examples of expanded gene families. The pectolytic glycoside hydrolase family 28 (GH28) allows herbivorous insects to break down the PCW polysaccharide pectin. GH28 in the Phytophaga clade of beetles contains many active enzymes but also many inactive counterparts. Using functional characterization, gene silencing, global transcriptome analyses and recordings of life history traits, we found that not only catalytically active but also inactive GH28 proteins are part of the same pectin-digesting pathway. The robustness and plasticity of this pathway and thus its importance for the beetle is supported by extremely high steady-state expression levels and counter-regulatory mechanisms. Unexpectedly, the impact of pseudoenzymes on the pectin-digesting pathway in Phytophaga beetles exceeds even the influence of their active counterparts, such as a lowered efficiency of food-to-energy conversion and a prolongation of the developmental period.

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Xylanases of glycoside hydrolase family 30 – An overview

Biotechnology Advances ◽

10.1016/j.biotechadv.2021.107704 ◽

2021 ◽

Vol 47 ◽

pp. 107704

Author(s):

Vladimír Puchart ◽

Katarína Šuchová ◽

Peter Biely

Keyword(s):

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Hydrolase Family ◽

Glycoside Hydrolase Family 30

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Modeled 3D-Structures of Proteobacterial Transglycosylases from Glycoside Hydrolase Family 17 Give Insight in Ligand Interactions Explaining Differences in Transglycosylation Products

Applied Sciences ◽

10.3390/app11094048 ◽

2021 ◽

Vol 11 (9) ◽

pp. 4048

Author(s):

Javier A. Linares-Pastén ◽

Lilja Björk Jonsdottir ◽

Gudmundur O. Hreggvidsson ◽

Olafur H. Fridjonsson ◽

Hildegard Watzlawick ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Ligand Docking ◽

Glycoside Hydrolase Family ◽

Ligand Interactions ◽

Tim Barrel ◽

Branching Point ◽

The Difference ◽

Dynamics Simulations ◽

And Function ◽

Hydrolase Family

The structures of glycoside hydrolase family 17 (GH17) catalytic modules from modular proteins in the ndvB loci in Pseudomonas aeruginosa (Glt1), P. putida (Glt3) and Bradyrhizobium diazoefficiens (previously B. japonicum) (Glt20) were modeled to shed light on reported differences between these homologous transglycosylases concerning substrate size, preferred cleavage site (from reducing end (Glt20: DP2 product) or non-reducing end (Glt1, Glt3: DP4 products)), branching (Glt20) and linkage formed (1,3-linkage in Glt1, Glt3 and 1,6-linkage in Glt20). Hybrid models were built and stability of the resulting TIM-barrel structures was supported by molecular dynamics simulations. Catalytic amino acids were identified by superimposition of GH17 structures, and function was verified by mutagenesis using Glt20 as template (i.e., E120 and E209). Ligand docking revealed six putative subsites (−4, −3, −2, −1, +1 and +2), and the conserved interacting residues suggest substrate binding in the same orientation in all three transglycosylases, despite release of the donor oligosaccharide product from either the reducing (Glt20) or non-reducing end (Glt1, Gl3). Subsites +1 and +2 are most conserved and the difference in release is likely due to changes in loop structures, leading to loss of hydrogen bonds in Glt20. Substrate docking in Glt20 indicate that presence of covalently bound donor in glycone subsites −4 to −1 creates space to accommodate acceptor oligosaccharide in alternative subsites in the catalytic cleft, promoting a branching point and formation of a 1,6-linkage. The minimum donor size of DP5, can be explained assuming preferred binding of DP4 substrates in subsite −4 to −1, preventing catalysis.

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Effect of Lipopolysaccharides (LPS) and Lipoteichoic acid (LTA) on the Inflammatory Response in Rumen Epithelial Cells (REC) and the Impact of LPS on Claw Explants

Animals ◽

10.3390/ani11072058 ◽

2021 ◽

Vol 11 (7) ◽

pp. 2058

Author(s):

Nicole Reisinger ◽

Dominik Wendner ◽

Nora Schauerhuber ◽

Elisabeth Mayer

Keyword(s):

Epithelial Cells ◽

Inflammatory Response ◽

Structural Integrity ◽

Animal Health ◽

Lipoteichoic Acid ◽

Local Inflammatory Response ◽

Tissue Integrity ◽

Separation Force ◽

The Impact ◽

Rumen Epithelial Cells

Endotoxins play a crucial role in ruminant health due to their deleterious effects on animal health. The study aimed to evaluate whether LPS and LTA can induce an inflammatory response in rumen epithelial cells. For this purpose, epithelial cells isolated from rumen tissue (RECs) were stimulated with LPS and LTA for 1, 2, 4, and 24 h. Thereafter, the expression of selected genes of the LPS and LTA pathway and inflammatory response were evaluated. Furthermore, it was assessed whether LPS affects inflammatory response and structural integrity of claw explants. Therefore, claw explants were incubated with LPS for 4 h to assess the expression of selected genes and for 24 h to evaluate tissue integrity via separation force. LPS strongly affected the expression of genes related to inflammation (NFkB, TNF-α, IL1B, IL6, CXCL8, MMP9) in RECs. LTA induced a delayed and weaker inflammatory response than LPS. In claw explants, LPS affected tissue integrity, as there was a concentration-dependent decrease of separation force. Incubation time had a strong effect on inflammatory genes in claw explants. Our data suggest that endotoxins can induce a local inflammatory response in the rumen epithelium. Furthermore, translocation of LPS might negatively impact claw health.

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The thermostable 4,6-α-glucanotransferase of Bacillus coagulans DSM 1 synthesizes isomaltooligosaccharides

Amylase ◽

10.1515/amylase-2021-0002 ◽

2021 ◽

Vol 5 (1) ◽

pp. 13-22

Author(s):

Gang Xiang ◽

Piet L. Buwalda ◽

Marc J.E.C van der Maarel ◽

Hans Leemhuis

Keyword(s):

Bacillus Coagulans ◽

Glycoside Hydrolase Family ◽

Rat Intestine ◽

Glycaemic Response ◽

Food Ingredient ◽

Starch Conversion ◽

Identification And Characterization ◽

Hydrolase Family

Abstract The 4,6-α-glucanotransferases of the glycoside hydrolase family 70 can convert starch into isomaltooligosaccharides (IMOs). However, no thermostable 4,6-α-glucanotransferases have been reported to date, limiting their applicability in the starch conversion industry. Here we report the identification and characterization of a thermostable 4,6-α-glucanotransferase from Bacillus coagulans DSM 1. The gene was cloned and the recombinant protein, called BcGtfC, was produced in Escherichia coli. BcGtfC is stable up to 66 °C in the presence of substrate. It converts debranched starch into an IMO product with a high percentage of α-1,6-glycosidic linkages and a relatively high molecular weight compared to commercially available IMOs. Importantly, the product is only partly and very slowly digested by rat intestine powder, suggesting that the IMO will provide a low glycaemic response in vivo when applied as food ingredient. Thus, BcGtfC is a thermostable 4,6-α-glucanotransferase suitable for the industrial production of slowly digestible IMOs from starch.

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Understanding the Polymerization Mechanism of Glycoside-Hydrolase Family 70 Glucansucrases

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)84038-3 ◽

2006 ◽

Vol 281 (42) ◽

pp. 31254-31267

Author(s):

Claire Moulis ◽

Gilles Joucla ◽

David Harrison ◽

Emeline Fabre ◽

Gabrielle Potocki-Veronese ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Polymerization Mechanism ◽

Hydrolase Family

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Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-l-fucosidases from GH29

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.005134 ◽

2018 ◽

Vol 293 (47) ◽

pp. 18296-18308 ◽

Cited By ~ 10

Author(s):

Chelsea Vickers ◽

Feng Liu ◽

Kento Abe ◽

Orly Salama-Alber ◽

Meredith Jenkins ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Biological Activities ◽

Bacterial Species ◽

Structural Data ◽

Side Chain ◽

Glycoside Hydrolase Family ◽

X Ray Crystallography ◽

Catalytic Mechanisms ◽

Thickening Agents ◽

Hydrolase Family

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas. Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-l-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans.

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A novel dextranase gene from the marine bacterium Bacillus aquimaris S5 and its expression and characteristics

FEMS Microbiology Letters ◽

10.1093/femsle/fnab007 ◽

2021 ◽

Vol 368 (3) ◽

Author(s):

Dongxue Dong ◽

Xuelian Wang ◽

Tian Deng ◽

Zhe Ning ◽

Xiaopeng Tian ◽

...

Keyword(s):

Dental Plaque ◽

Marine Bacterium ◽

3D Structure ◽

Great Promise ◽

Glycoside Hydrolase Family ◽

Alpha Helices ◽

Random Coils ◽

Pharmaceutical Industries ◽

Bacterium Bacillus ◽

Hydrolase Family

ABSTRACT Dextranase specifically hydrolyzes dextran and is used to produce functional isomalto-saccharide prebiotics. Moreover, dextranase is used as an additive in mouthwash to remove dental plaque. We cloned and expressed the dextranase gene of the marine bacterium Bacillus aquimaris S5. The length of the BaDex gene was 1788 bp, encoding 573 amino acids. Using bioinformatics to predict and analyze the amino acid sequence of BaDex, we found the isoelectric point and instability coefficient to be 4.55 and 29.22, respectively. The average hydrophilicity (GRAVY) was −0.662. The secondary structure of BaDex consisted of 145 alpha helices, accounting for 25.31% of the protein; 126 extended strands, accounting for 21.99%; and 282 random coils, accounting for 49.21%. The 3D structure of the BaDex protein was predicted and simulated using SWISS-MODEL, and BaDex was classified as a Glycoside Hydrolase Family 66 protein. The optimal temperature and pH for BaDex activity were 40°C and 6.0, respectively. The hydrolysates had excellent antioxidant activity, and 8 U/mL of BaDex could remove 80% of dental plaque in MBRC experiment. This recombinant protein thus has great promise for applications in the food and pharmaceutical industries.

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Characterization of Glycoside Hydrolase Family 5 Proteins in Schizosaccharomyces pombe

Eukaryotic Cell ◽

10.1128/ec.00187-10 ◽

2010 ◽

Vol 9 (11) ◽

pp. 1650-1660 ◽

Cited By ~ 11

Author(s):

Encarnación Dueñas-Santero ◽

Ana Belén Martín-Cuadrado ◽

Thierry Fontaine ◽

Jean-Paul Latgé ◽

Francisco del Rey ◽

...

Keyword(s):

Cell Wall ◽

Schizosaccharomyces Pombe ◽

Protein Characterization ◽

Wall Material ◽

Glycoside Hydrolase Family ◽

Content Type ◽

Hydrolysis Of ◽

Controlled Hydrolysis ◽

Hydrolase Family

ABSTRACT In yeast, enzymes with β-glucanase activity are thought to be necessary in morphogenetic events that require controlled hydrolysis of the cell wall. Comparison of the sequence of the Saccharomyces cerevisiae exo-β(1,3)-glucanase Exg1 with the Schizosaccharomyces pombe genome allowed the identification of three genes that were named exg1 + (locus SPBC1105.05), exg2 + (SPAC12B10.11), and exg3 + (SPBC2D10.05). The three proteins have different localizations: Exg1 is secreted to the periplasmic space, Exg2 is a membrane protein, and Exg3 is a cytoplasmic protein. Characterization of the biochemical activity of the proteins indicated that Exg1 and Exg3 are active only against β(1,6)-glucans while no activity was detected for Exg2. Interestingly, Exg1 cleaves the glucans with an endohydrolytic mode of action. exg1 + showed periodic expression during the cell cycle, with a maximum coinciding with the septation process, and its expression was dependent on the transcription factor Sep1. The Exg1 protein localizes to the septum region in a pattern that was different from that of the endo-β(1,3)-glucanase Eng1. Overexpression of Exg2 resulted in an increase in cell wall material at the poles and in the septum, but the putative catalytic activity of the protein was not required for this effect.

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