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The marine Bacteroidota Zobellia galactanivorans has a polysaccharide utilization locus dedicated to the catabolism of the red algal cell wall galactan carrageenan and its unique and industrially important α-3,6-anhydro-D-galactose (ADG) monosaccharide. Here we present the first analysis of the specific molecular interactions that the exo-(α-1,3)-3,6-anhydro-D-galactosidase ZgGH129 uses to cope with the strict steric restrictions imposed by its bicyclic ADG substrate - which is ring flipped relative to D-galactose. Crystallographic snapshots of key catalytic states obtained with the natural substrate and novel chemical tools designed to mimic species along the reaction coordinate, together with quantum mechanics/molecular mechanics (QM/MM) metadynamics methods and kinetic studies, demonstrate a retaining mechanism where the second step is rate limiting. The conformational landscape of the constrained 3,6-anhydro-D-galactopyranose ring proceeds through enzyme glycosylation B 1,4 →[E 4 ] ≠ →E 4 / 1 C 4 and deglycosylation E 4 / 1 C 4 →[E 4 ] ≠ →B 1,4 itineraries limited to the Southern Hemisphere of the Cremer-Pople sphere. These results demonstrate the conformational changes throughout catalysis in a non-standard, sterically restrained, bicyclic monosaccharide, and provide a molecular framework for mechanism-based inhibitor design for anhydro-type carbohydrate-processing enzymes and for future applications involving carrageenan degradation. In addition, our study provides a rare example of distinct niche-based conformational itineraries within the same carbohydrate-active enzyme family., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The biological function of terminal galactose on glycoprotein is an open field of research. Although progress had being made on enzymes that can remove the terminal galactose on glycoproteins, there is a lack of report on galactosidases that can work directly on living cells. In this study, a unique beta 1,4 galactosidase was isolated from Elizabethkingia meningoseptica (Em). It exhibited favorable stability at various temperatures (4-37 °C) and pH (5-8) levels and can remove β-1, 4 linked galactoses directly from glycoproteins. Using Alanine scanning, we found that two acidic residues (Glu-468, and Glu-531) in the predicted active pocket are critical for galactosidase activity. In addition, we also demonstrated that it could cleave galactose residues present on living cell surface. As this enzyme has a potential application for living cell glycan editing, we named it emGalaseE or glycan-editing galactosidase I (csgeGalaseI). In summary, our findings lay the groundwork for further investigation by presenting a simple and effective approach for the removal of galactose moieties from cell surface., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the manuscript entitled., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

β-Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal β-D-galactose residues in β1,3-, β1,4- or β1,6-linkage. Although this family of exoglycosidases has been extensively studied in vertebrates, plants, yeast, and bacteria, little information is available for mollusks. Mollusks are a diverse and highly successful group of animals that play many different roles in their ecosystems, including filter feeders and detritivores. Here, the first β-galactosidase from the Pacific oyster, Crassostrea gigas was discovered, biochemically characterized, and compared to our previously characterized slug enzyme from Arion vulgaris (UniProt Ref. Nr.: A0A0B7AQJ9). Overall, the mussel enzyme showed similar biochemical parameters to the snail enzyme. The enzyme from C. gigas was most active in an acidic environment (pH 3.5) and at a reaction temperature of 50 °C. Optimal storage conditions were up to 37 °C. In contrast to the enzyme from A. vulgaris , the supplementation of cations (Ni 2+ , Co 2+ , Mn 2+ , Mg 2+ , Ca 2+ , Cu 2+ , Ba 2+ ) increased the activity of the enzyme from C. gigas . Substrate specificity studies of the β-galactosidases from the mussel, C. gigas, and the slug, A. vulgaris , revealed activity towards terminal β1,3- and β1,4-linked galactose residues for both enzymes. Using the same substrates in labeled and unlabeled form, we were able to detect the effect of labeling on the β-galactosidase activity using MALDI-TOF MS, HPTLC, and HPLC. While lactose was cleaved by the enzymes in an unlabeled or labeled state, galacto-N-biose was not cleaved as soon as a 2-amino benzoic acid label was added. In this study we present the biochemical characterization of the first recombinantly expressed β-galactosidase from the Pacific oyster, C. gigas , and we compare different analytical methods for the determination of β-galactosidase activity using the enzyme from C. gigas and A. vulgaris .

Lacto-N-tetraose (LNT) is one of the most important components of human milk oligosaccharides, which has various beneficial health effects. β-Galactosidase is an important enzyme used in dairy processing. The transglycosylation activity of β-galactosidases offers an attractive approach for LNT synthesis. In this study, we reported for the first time the biochemical characterization of a novel β-galactosidase (LzBgal35A) from Lacticaseibacillus zeae. LzBgal35A belongs to glycoside hydrolases (GH) family 35 and shared the highest identity of 59.9% with other reported GH 35 members. The enzyme was expressed as soluble protein in Escherichia coli. The purified LzBgal35A displayed optimal activity at pH 4.5 and 55°C. It was stable within the pH range of 3.5 to 7.0 and up to 60°C. Moreover, LzBgal35A could catalyze the synthesis of LNT via transferring the galactose residue from o-nitrophenyl-β-galactopyranoside to lacto-N-triose II. Under optimal conditions, the conversion rate of LNT reached 45.4% (6.4 g/L) within 2 h, which was by far the highest yield of LNT synthesized through a β-galactosidase-mediated transglycosylation reaction. This study demonstrated that LzBgal35A has great potential application in LNT synthesis., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Intestinal lactic acid bacteria can help alleviate lactose maldigestion by promoting lactose hydrolysis in the small intestine. This study shows that protein extracts from probiotic bacterium Lactiplantibacillus plantarum WCFS1 possess two metabolic pathways for lactose metabolism, involving β-galactosidase (β-gal) and 6Pβ-galactosidase (6Pβ-gal) activities. As L. plantarum WCFS1 genome lacks a putative 6Pβ-gal gene, the 11 GH1 family proteins, in which their 6Pβ-glucosidase (6Pβ-glc) activity was experimentally demonstrated,, were assayed for 6Pβ-gal activity. Among them, only Lp_3525 (Pbg9) also exhibited a high 6Pβ-gal activity. The sequence comparison of this dual 6Pβ-gal/6Pβ-glc GH1 protein to previously described dual GH1 proteins revealed that L. plantarum WCFS1 Lp_3525 belonged to a new group of dual 6Pβ-gal/6Pβ-glc GH1 proteins, as it possessed conserved residues and structural motifs mainly present in 6Pβ-glc GH1 proteins. Finally, Lp_3525 exhibited, under intestinal conditions, an adequate 6Pβ-gal activity with possible relevance for lactose maldigestion management.

Galactosidases are industrially important enzymes that hydrolyze galactosidic bonds in carbohydrates. Identifying new galactosidases with distinct functional characteristics is of paramount importance. In this study, we report the finding of a novel β-galactosidase PoβGal35A from the fungus Penicillium oxalicum. PoβGal35A belongs to the glycoside hydrolase family 35 (GH35), functions optimally at 70 °C and pH 5.0, and exhibits a specific high activity (191 ± 6.2 U/mg) towards pNPβgal. Ca 2+ , Fe 3+ and Ba 2+ ions enhance the activity of the enzyme, whereas Cu 2+ and Hg 2+ significantly reduce it. This enzyme releases galactose from β-1,3-galactan, β-1,4-galactan, β-1,6-galactan, as well as arabinogalactan from larchwood (LWAG). In addition, PoβGal35A acts synergistically with arabinosidase to degrade LWAG. These results suggest that PoβGal35A is a high activity exo-β-1,3/4/6-galactanase that can be used to establish glycan blocks in glycoconjugates, and thus provides a new tool for biotechnological applications., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Agar is a galactan and a major component of the red algal cell wall. Agar is metabolized only by specific microorganisms. The final step of the β-agarolytic pathway is mediated by α-neoagarooligosaccharide hydrolase (α-NAOSH), which cleaves neoagarobiose to D-galactose and 3,6-anhydro-α-L-galactose. In the present study, two α-NAOSHs, SCO3481 and SCO3479, were identified in Streptomyces coelicolor A3(2). SCO3481 (370 amino acids, 41.12 kDa) and SCO3479 (995 amino acids, 108.8 kDa) catalyzed the hydrolysis of the α-(1,3) glycosidic bonds of neoagarobiose, neoagarotetraose, and neoagarohexaose at the nonreducing ends, releasing 3,6-anhydro-α-L-galactose. Both were intracellular proteins without any signal peptides for secretion. Similar to all α-NAOSHs reported to date, SCO3481 belonged to the glycosyl hydrolase (GH) 117 family and formed dimers. On the other hand, SCO3479 was a large monomeric α-NAOSH belonging to the GH2 family with a β-galactosidase domain. SCO3479 also clearly showed β-galactosidase activity toward lactose and artificial substrates, but SCO3481 did not. The optimum conditions for α-NAOSH were pH 6.0 and 25 °C for SCO3481, and pH 6.0 and 30 °C for SCO3479. Enzymatic activity was enhanced by Co 2+ for SCO3481 and Mg 2+ for SCO3479. The β-galactosidase activity of SCO3479 was maximum at pH 7.0 and 50 °C and was increased by Mg 2+ . Many differences were evident in the kinetic parameters of each enzyme. Although SCO3481 is typical of the GH117 family, SCO3479 is a novel α-NAOSH that was first reported in the GH2 family. SCO3479, a unique bifunctional enzyme with α-NAOSH and β-galactosidase activities, has many advantages for industrial applications. KEY POINTS: • SCO3481 is a dimeric α-neoagarooligosaccharide hydrolase belonging to GH117. • SCO3479 is a monomeric α-neoagarooligosaccharide hydrolase belonging to GH2. • SCO3479 is a novel and unique bifunctional enzyme that also acts as a β-galactosidase., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: In type 2 diabetes, insulin resistance is observed, and β-cells are incapable of responding to glycemia demands, leading to hyperglycemia. Although the nature of β-cells dysfunction in this disease is not fully understood, a link between the induction of pancreatic β-cell premature senescence and its metabolic implications has been proposed. This study aimed to understand the relationship between diabetes and pancreatic senescence, particularly at the beginning of the disease., Methods: C57Bl/6 J mice were fed two different diets, a normal diet and a high-fat diet, for 16 weeks. Pancreatic histomorphology analysis, insulin quantification, inflammation parameters, and senescence biomarkers for the experimental animals were assessed at weeks 12 and 16., Results: The results proved that diabetes onset occurred at week 16 in the High Fat Diet group, supported by glycaemia, weight and blood lipid levels. Increased β-cells size and number accompanied by increased insulin expression were observed. Also, an inflammatory status of the diabetic group was noted by increased levels of systemic IL-1β and increased pancreatic fibrosis. Finally, the expression of galactosidase-beta 1 (GLB1) was significantly increased in pancreatic β-cells., Conclusion: The study findings indicate that senescence, as revealed by an increase in GLB1 expression, is a key factor in the initial stage of diabetes., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Alpha-galactosidase seed imbibition protein (VvSIP) isolated from Vitis vinifera is up-regulated upon salt stress and mediates osmotic stress responses in a tolerant grapevine cultivar. So far, little is known about the putative role of this stress-responsive gene. In the present study, VvSIP function was investigated in model tobacco plants via Agrobacterium-mediated genetic transformation. Our results showed that overexpression of VvSIP exhibited increased tolerance to salinity at germination and late vegetative stage in transgenic Nicotiana benthamiana compared to the nontransgenic plants based on the measurement of the germination rate and biomass production. High salt concentrations of 200 and 400 mM NaCl in greenhouse-grown pot assay resulted in better relative water content, higher leaf osmotic potential, and leaf water potential in transgenic lines when compared to the wild-type (WT) plants. These physiological changes attributed to efficient osmotic adjustment improved plant performance and tolerance to salinity compared to the WT. Moreover, the VvSIP-expressing lines SIP1 and SIP2 showed elevated amounts of chlorophyll with lower malondialdehyde content indicating a reduced lipid peroxidation required to maintain membrane stability. When subjected to high salinity conditions, the transgenic tobacco VvSIP exhibited higher soluble sugar content, which may suggest an enhancement of the carbohydrate metabolism. Our findings indicate that the VvSIP is involved in plant salt tolerance by functioning as a positive regulator of osmotic adjustment and sugar metabolism, both of which are responsible for stress mitigation. Such a candidate gene is highly suitable to alleviate environmental stresses and thus could be a promising candidate for crop improvement., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Carbohydrate-active enzymes are important components of the polysaccharide metabolism system in marine bacteria. Carrageenase is indispensable for forming carrageenan catalytic pathways. Here, two GH16_13 carrageenases showed likely hydrolysis activities toward different types of carrageenans (e.g., κ-, hybrid β/κ, hybrid α/ι, and hybrid λ), which indicates that a novel pathway is present in the marine bacterium Flavobacterium algicola to use κ-carrageenan (KC), ι-carrageenan (IC), and λ-carrageenan (LC). A comparative study described the different features with another reported pathway based on the specific carrageenans (κ, ι, and λ) and expanded the carrageenan metabolic versatility in F. algicola . A further comparative genomic analysis of carrageenan-degrading bacteria indicated different distributions of carrageenan metabolism-related genes in marine bacteria. The crucial core genes encoding the GH127 α-3,6-anhydro-d-galactosidase (ADAG) and 3,6-anhydro-d-galactose (d-AHG)-utilized cluster have been conserved during evolution. This analysis further revealed the horizontal gene transfer (HGT) phenomenon of the carrageenan polysaccharide utilization loci (CarPUL) from Bacteroidetes to other bacterial phyla, as well as the versatility of carrageenan catalytic activities in marine bacteria through different metabolic pathways. IMPORTANCE Based on the premise that the specific carrageenan-based pathway involved in carrageenan use by Flavobacterium algicola has been identified, another pathway was further analyzed, and it involved two GH16_13 carrageenases. Among all the characterized carrageenases, the members of GH16_13 accounted for only a small portion. Here, the functional analysis of two GH16_13 carrageenases suggested their hydrolysis effects on different types of carrageenans (e.g., κ, hybrid β/κ, hybrid α/ι-, and hybrid λ-), which led to the identification of another pathway. Further exploration enabled us to elucidate the novel pathway that metabolizes KC and IC in F. algicola successfully. The coexistence of these two pathways may provide improved survivability by F. algicola in the marine environment.

Objective: To establish an optimized method for the isolation and purification of astrocytes from the neural tissues of young and aged rats. Then, the morphological and functional differences of astrocytes between young and aged rats were compared to explore the functional changes of astrocytes after aging and its possible mechanism in the aging process. Methods: Young (2 months old) and aged (20 months old) SD rats were used. Astrocytes in brain and spinal cord tissue were purified by 50% - 35% percoll density gradient centrifugation. Each group of cells was set up with three duplicate wells. After 72 h of culture, Glial fibrillary acidic protein (GFAP) which was astrocyte specific marker were detected by immunofluorescence to evaluate the morphological characteristics. Cell senescence markers (p16 and p21) and β- Galactosidase were detected by qPCR and staining respectively. The expressions of pro-inflammatory cytokines (IL-1β, TNF-α) and anti-inflammatory cytokines were detected by qPCR. Results: Using 50%-35% percoll gradient separation, astrocytes were obtained with large number, good activity and purity of more than 95%, which could be used in subsequent experiments. Compared with the astrocytes in the nerve tissue of young rats, the astrocytes in the nervous tissue of the aged rats had fewer protrusions and tended to be activated in cell morphology; the positive rate of β -galactosidase staining was increased significantly and the expressions of p16 and p21 were increased (P<0.01). The expressions of pro-inflammatory cytokines (IL-1β, TNF-α) were increased (P<0.05), and the expression of anti-inflammatory cytokine (IL-10) was decreased (P<0.05) in astrocytes of the aged rats nervous tissue. Conclusion: The percoll gradient of 50% - 35% could be used as a method for separation, purification and primary culture of astrocytes. With the increase of age, astrocytes undergo cellular senescence, showing a pro-inflammatory phenotype, promoting inflammaging of the nervous system, which may be one of the mechanisms of nervous system aging and neurodegenerative diseases.

Human dermal fibroblasts (HDF) were obtained by skin punch biopsy from a 51-year old man with suspected Fabry disease (FD), carrying the hemizygous c.376A > G variant in the α-galactosidase A gene (GLA). Cultured HDF were reprogrammed to induced pluripotent stem cells (iPSC) using a non-modified RNA-based transfection protocol. GLA-S126G-iPSC exhibit typical embryonic stem cell-like morphology, normal karyotype, expression of all tested pluripotency markers, and three germ layer differentiation potential. We provide a novel patient-specific cell line that can be used to investigate a genetic variation of yet unknown significance., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Thermophilic Methanothermobacter spp. are used as model microbes to study the physiology and biochemistry of the conversion of molecular hydrogen and carbon dioxide into methane (i.e., hydrogenotrophic methanogenesis). Yet, a genetic system for these model microbes was missing despite intensive work for four decades. Here, we report the successful implementation of genetic tools for Methanothermobacter thermautotrophicus ΔH. We developed shuttle vectors that replicated in Escherichia coli and M. thermautotrophicus ΔH. For M. thermautotrophicus ΔH, a thermostable neomycin resistance cassette served as the selectable marker for positive selection with neomycin, and the cryptic plasmid pME2001 from Methanothermobacter marburgensis served as the replicon. The shuttle-vector DNA was transferred from E. coli into M. thermautotrophicus ΔH via interdomain conjugation. After the successful validation of DNA transfer and positive selection in M. thermautotrophicus ΔH, we demonstrated heterologous gene expression of a thermostable β-galactosidase-encoding gene ( bgaB ) from Geobacillus stearothermophilus under the expression control of four distinct synthetic and native promoters. In quantitative in-vitro enzyme activity assay, we found significantly different β-galactosidase activity with these distinct promoters. With a formate dehydrogenase operon-encoding shuttle vector, we allowed growth of M. thermautotrophicus ΔH on formate as the sole growth substrate, while this was not possible for the empty-vector control. IMPORTANCE The world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change. The power-to-gas platform is utilized to store renewable electric power and decarbonize the natural gas grid. The microbe Methanothermobacter thermautotrophicus is already applied as the industrial biocatalyst for the biological methanation step in large-scale power-to-gas processes. To improve the biocatalyst in a targeted fashion, genetic engineering is required. With our shuttle-vector system for heterologous gene expression in M. thermautotrophicus , we set the cornerstone to engineer the microbe for optimized methane production but also for production of high-value platform chemicals in power-to-x processes.

Much evidence suggests a role for human milk oligosaccharides (HMOs) in establishing the infant microbiota in the large intestine, but the response of particular bacteria to individual HMOs is not well known. Here twelve bacterial strains belonging to the genera Bifidobacterium, Enterococcus, Limosilactobacillus, Lactobacillus, Lacticaseibacillus, Staphylococcus and Streptococcus were isolated from infant faeces and their growth was analyzed in the presence of the major HMOs, 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 2',3-difucosyllactose (DFL), lacto-N-tetraose (LNT) and lacto-N-neo-tetraose (LNnT), present in human milk. Only the isolated Bifidobacterium strains demonstrated the capability to utilize these HMOs as carbon sources. Bifidobacterium infantis Y538 efficiently consumed all tested HMOs. Contrarily, Bifidobacterium dentium strains Y510 and Y521 just metabolized LNT and LNnT. Both tetra-saccharides are hydrolyzed into galactose and lacto-N-triose (LNTII) by B. dentium. Interestingly, this species consumed only the galactose moiety during growth on LNT or LNnT, and excreted the LNTII moiety. Two β-galactosidases were characterized from B. dentium Y510, Bdg42A showed the highest activity towards LNT, hydrolyzing it into galactose and LNTII, and Bdg2A towards lactose, degrading efficiently also 6'-galactopyranosyl-N-acetylglucosamine, N-acetyl-lactosamine and LNnT. The work presented here supports the hypothesis that HMOs are mainly metabolized by Bifidobacterium species in the infant gut., (© 2021. The Author(s).)

The transfer of foreign synthetic messenger RNA (mRNA) into cells is essential for mRNA-based protein-replacement therapies. Prophylactic mRNA COVID-19 vaccines commonly utilize nanotechnology to deliver mRNA encoding SARS-CoV-2 vaccine antigens, thereby triggering the body's immune response and preventing infections. In this study, a new combinatorial library of symmetric lipid-like compounds is constructed, and among which a lead compound is selected to prepare lipid-like nanoassemblies (LLNs) for intracellular delivery of mRNA. After multiround optimization, the mRNA formulated into core-shell-structured LLNs exhibits more than three orders of magnitude higher resistance to serum than the unprotected mRNA, and leads to sustained and high-level protein expression in mammalian cells. A single intravenous injection of LLNs into mice achieves over 95% mRNA translation in the spleen, without causing significant hematological and histological changes. Delivery of in-vitro-transcribed mRNA that encodes high-affinity truncated ACE2 variants (tACE2v mRNA) through LLNs induces elevated expression and secretion of tACE2v decoys, which is able to effectively block the binding of the receptor-binding domain of the SARS-CoV-2 to the human ACE2 receptor. The robust neutralization activity in vitro suggests that intracellular delivery of mRNA encoding ACE2 receptor mimics via LLNs may represent a potential intervention strategy for COVID-19., (© 2021 Wiley-VCH GmbH.)

The composition and structure of gut microbiota plays an important role in obesity induced by a high-fat diet (HFD) and related metabolic syndrome (MetS). Previous studies have shown that galacto-oligosaccharides (GOSs) have an effective anti-obesity effect. In this study, we aimed to investigate the effect of enzymatically synthesized α-galacto-oligosaccharides (ES-α-GOSs) on MetS and gut microbiota dysbiosis in HFD-fed mice, and to further investigate whether the attenuation of MetS is associated with the modulation of gut microbiota. Our results indicated that ES-α-GOS could notably ameliorate obesity-related MetS, including hyperlipidemia, insulin resistance and mild inflammation. The subsequent analysis of gut microbiota further showed that ES-α-GOS supplements can significantly modulate the overall composition of the gut microbiota and reverse the gut microbiota disorder caused by HFD feeding. Moreover, Spearman correlation analysis showed that 40 key bacteria reversed by ES-α-GOS were highly associated with metabolic parameters. These results suggested that ES-α-GOSs could serve as a potential candidate for preventing obesity-induced MetS in association with the modulation of gut microbiota.

1,3-α-3,6-anhydro-L-galactosidase (α-neoagarooligosaccharide hydrolase) catalyzes the last step of agar degradation by hydrolyzing neoagarobiose into monomers, D-galactose, and 3,6-anhydro-Lgalactose, which is important for the bioindustrial application of algal biomass. Ahg943, from the agarolytic marine bacterium Gayadomonas joobiniege G7, is composed of 423 amino acids (47.96 kDa), including a 22-amino acid signal peptide. It was found to have 67% identity with the α-neoagarooligosaccharide hydrolase Zg AhgA, from Zobellia galactanivorans , but low identity (< 40%) with the other α-neoagarooligosaccharide hydrolases reported. The recombinant Ahg943 (rAhg943, 47.89 kDa), purified from Escherichia coli , was estimated to be a monomer upon gel filtration chromatography, making it quite distinct from other α-neoagarooligosaccharide hydrolases. The rAhg943 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into D-galactose, neoagarotriose, and neoagaropentaose, respectively, with a common product, 3,6- anhydro-L-galactose, indicating that it is an exo-acting α-neoagarooligosaccharide hydrolase that releases 3,6-anhydro-L-galactose by hydrolyzing α-1,3 glycosidic bonds from the nonreducing ends of neoagarooligosaccharides. The optimum pH and temperature of Ahg943 activity were 6.0 and 20°C, respectively. In particular, rAhg943 could maintain enzyme activity at 10°C (71% of the maximum). Complete inhibition of rAhg943 activity by 0.5 mM EDTA was restored and even, remarkably, enhanced by Ca 2+ ions. rAhg943 activity was at maximum at 0.5 M NaCl and maintained above 73% of the maximum at 3M NaCl. K m and V max of rAhg943 toward neoagarobiose were 9.7 mg/ml and 250 μM/min (3 U/mg), respectively. Therefore, Ahg943 is a unique α-neoagarooligosaccharide hydrolase that has cold- and high-salt-adapted features, and possibly exists as a monomer.

Flavobacteriia are important degraders in the marine carbon cycle, due to their ability to efficiently degrade complex algal polysaccharides. A novel exo-(α-1,3)-3,6-anhydro-D-galactosidase activity was recently discovered from a marine Flavobacteriia (Zobellia galactanivorans Dsij T ) on red algal carrageenan oligosaccharides. The enzyme activity is encoded by a gene found in the first described carrageenan-specific polysaccharide utilization locus (CarPUL) that codes for a family 129 glycoside hydrolase (GH129). The GH129 family is a CAZy family that is strictly partitioned into two niche-based clades: clade 1 contains human host bacterial enzymes and clade 2 contains marine bacterial enzymes. Clade 2 includes the GH129 exo-(α-1,3)-3,6-anhydro-D-galactosidase from Z. galactanivorans (ZgGH129). Despite the discovery of the unique activity for ZgGH129, finer details on the natural substrate specificity for this enzyme are lacking. Examination of enzyme activity on natural carrageenan oligomers using mass spectrometry demonstrated that ZgGH129 hydrolyses terminal 3,6-anhydro-D-galactose from unsulfated non-reducing end neo-β-carrabiose motifs. Due to the lack of chromogenic substrates to examine exo-(α-1,3)-3,6-anhydro-D-galactosidase activity, a novel substrate was synthesised to facilitate the first kinetic characterisation of an exo-(α-1,3)-3,6-anhydro-D-galactosidase, allowing determination of pH and temperature optimums and Michaelis-Menten steady state kinetic data., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid β-galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent β-d-galactosidase inhibitor 4- epi -isofagomine. Structure-activity relationships were investigated by X-ray crystallography as well as by alterations in the cyclopentane moiety such as deoxygenation and replacement by fluorine of a "strategic" hydroxyl group. New compounds have revealed highly promising activities with a range of β-galactosidase-compromised human cell lines and may serve as leads towards new pharmacological chaperones for G M1 -gangliosidosis and Morquio B disease.

Aspergillus oryzae β-galactosidase was immobilized by aggregation and crosslinking, obtaining catalysts (CLAGs) well-endowed for lactulose synthesis. Type and concentration of the precipitating agent were determinants of immobilization yield, specific activity and thermal stability. CLAGs with specific activities of 64,007, 48,374 and 44,560 IU H g -1 were obtained using 50% v/v methanol, ethanol and propanol as precipitating agents respectively, with immobilization yields over 90%. Lactulose synthesis was conducted at 50 °C, pH 4.5, 50% w/w total sugars, 200 IU H g -1 of enzyme and fructose/lactose molar ratio of 8 in batch and repeated-batch operation. Lactulose yields were 0.19 g g -1 and 0.24 g g -1 for fructose to lactose molar ratios of 4 mol mol -1 and 8 mol mol -1 while selectivities were 3.3 mol mol -1 and 6.6 mol mol -1 respectively for CLAGs obtained by ethanol and propanol precipitation. Based on these results, both CLAGs were selected for the synthesis in repeated-batch mode. The cumulative mass of lactulose in repeated-batch was higher with CLAGs produced by ethanol and propanol precipitation than with the free enzyme. 86 and 93 repeated-batches could have been respectively performed with those CLAGs considering a catalyst replacement criterion of 50% of residual activity, as determined by simulation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 ( Bal 6GBP) that governs the specificity of the ABC transporter encoded by the same β-galactoside utilization locus. We observed that although Bal 6GBP recognizes both β-(1,6)- and β-(1,4)-galactobiose, Bal 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 Bal 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- N -biose-binding protein from Bifidobacterium longum , 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 Bifidobacterium ., (© 2019 Theilmann et al.)

Agar, a major polysaccharide of red algal cells, is degraded by β-agarases into neoagarobiose, which is further hydrolyzed into the monomers, D-galactose and 3,6-anhydro-L-galactose, by 1,3-α-3,6-anhydro-L-galactosidases including α-1,3-L-neoagarooligasaccharide hydrolase (α-NAOSH). A novel cold-adapted alkaline α-NAOSH, Ahg558, consisting of 359 amino acids (40.8 kDa) was identified from Gayadomonas joobiniege G7. It was annotated as a glycosyl hydrolase family 43 based on genomic sequence analysis, showing 84% and 74% identities with the characterized α-NAOSHs from Agarivorans gilvus WH0801 and Saccharophagus degradans 2-40, respectively. The recombinant Ahg558 (rAhg558) purified from Escherichia coli formed dimers and cleaved α-1,3 glycosidic bonds at the non-reducing ends of the neoagarobiose, neoagarotetraose, and neoagarohexaose, which was confirmed by thin-layer chromatography and mass spectrometry. The optimum pH and temperature for rAhg558 activity were 9.0 and 30 °C, respectively. Unusually, it retained over 93% activity in a broad range of temperatures between 0 and 40 °C and over 73% in a broad range of pH between pH 6.0 and pH 9.0, indicating it is a unique cold-adapted alkaline exo-acting α-NAOSH. Its enzymatic activity was dependent on Mn 2+ ions. K m and V max values toward neoagarobiose were 2.6 mg/mL (8.01 mM) and 133.33 U/mg, respectively.

To date, most studies of lactose utilization have focused on the genetic diversity of lactic acid bacteria or its influence on product quality, but phenotypic evaluation has rarely been based on metabolic characteristics. In the present study, we investigated the growth, acid production, β-galactosidase, and 6-phospho-β-galactosidase activities of 16 Lactococcus lactis strains obtained from various habitats with lactose as the sole carbon source. The 15 L. lactis strains obtained from various habitats exhibited significant differences in growth and acid production characteristics in the de Man, Rogosa, and Sharpe-lactose broth, and 4 strains consumed more lactose when cultured in skim milk than the type strain ATCC 19435. Among these strains, DQHXNQ38-12 mainly produced acetoin and diacetyl when cultured in skim milk, whereas the strains 15M2 and 5G2 produced high levels of acid and formed curd rapidly. We concluded that the use of lactose is necessary for strain adaptation to the dairy niche. Comprehensive studies of lactose use and the fermentation characteristics of L. lactis are of significant importance., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Human milk oligosaccharides (HMOs) function as prebiotics for beneficial bacteria in the developing gut, often dominated by Bifidobacterium spp. To understand the relationship between bifidobacteria utilizing HMOs and how the metabolites that are produced could affect the host, we analyzed the metabolism of HMO 2'-fucosyllactose (2'-FL) in Bifidobacterium longum subsp. infantis Bi-26. RNA-seq and metabolite analysis (NMR/GCMS) was performed on samples at early (A600 = 0.25), mid-log (0.5-0.7) and late-log phases (1.0-2.0) of growth. Transcriptomic analysis revealed many gene clusters including three novel ABC-type sugar transport clusters to be upregulated in Bi-26 involved in processing of 2'-FL along with metabolism of its monomers glucose, fucose and galactose. Metabolite data confirmed the production of formate, acetate, 1,2-propanediol, lactate and cleaving of fucose from 2'-FL. The formation of acetate, formate, and lactate showed how the cell uses metabolites during fermentation to produce higher levels of ATP (mid-log compared to other stages) or generate cofactors to balance redox. We concluded that 2'-FL metabolism is a complex process involving multiple gene clusters, that produce a more diverse metabolite profile compared to lactose. These results provide valuable insight on the mode-of-action of 2'-FL utilization by Bifidobacterium longum subsp. infantis Bi-26.

Our previous research showed that Pleurotus eryngii and Pleurotus ostreatus were effective fungi for pretreatment of industrial hemp stalks to improve enzymatic saccharification. The secretomes of these two fungi were analyzed to search for the effective enzyme cocktails degrading hemp lignin during the pretreatment process. In total, 169 and 155 proteins were identified in Pleurotus eryngii and Pleurotus ostreatus, respectively, and 50% of the proteins involved in lignocellulose degradation were CAZymes. Because most of the extracellular proteins secreted by fungi are glycosylated proteins, the N-linked glycosylation of enzymes could be mapped. In total, 27 and 24 N-glycosylated peptides were detected in Pleurotus eryngii and Pleurotus ostreatus secretomes, respectively. N-Glycosylated peptides of laccase, GH92, exoglucanase, phenol oxidase, α-galactosidase, carboxylic ester hydrolase, and pectin lyase were identified. Deglycosylation could decrease enzymatic saccharification of hemp stalks. The activities of laccase, α-galactosidase, and phenol oxidase and the thermal stability of laccase were reduced after deglycosylation.

β-d-Galactofuranose (Galf) is a component of polysaccharides and glycoconjugates. There are few reports about the involvement of galactofuranosyltransferases and galactofuranosidases (Galf-ases) in the synthesis and degradation of galactofuranose-containing glycans. The cell walls of filamentous fungi in the genus Aspergillus include galactofuranose-containing polysaccharides and glycoconjugates, such as O-glycans, N-glycans, and fungal-type galactomannan, which are important for cell wall integrity. In this study, we investigated the synthesis of p-nitrophenyl β-d-galactofuranoside and its disaccharides by chemo-enzymatic methods including use of galactosidase. The key step was selective removal of the concomitant pyranoside by enzymatic hydrolysis to purify p-nitrophenyl β-d-galactofuranoside, a promising substrate for β-d-galactofuranosidase from Streptomyces species., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Contemporary chemoenzymatic approaches can provide highly complex multi-antennary N-linked glycans. These procedures are, however, very demanding and typically involve as many as 100 chemical steps to prepare advanced intermediates that can be diversified by glycosyltransferases in a branch-selective manner to give asymmetrical structures commonly found in nature. Only highly specialized laboratories can perform such syntheses, which greatly hampers progress in glycoscience. Here we describe a biomimetic approach in which a readily available bi-antennary glycopeptide can be converted in ten or fewer chemical and enzymatic steps into multi-antennary N-glycans that at each arm can be uniquely extended by glycosyltransferases to give access to highly complex asymmetrically branched N-glycans. A key feature of our approach is the installation of additional branching points using recombinant MGAT4 and MGAT5 in combination with unnatural sugar donors. At an appropriate point in the enzymatic synthesis, the unnatural monosaccharides can be converted into their natural counterpart, allowing each arm to be elaborated into a unique appendage.

Naturally fermented tofu whey (NFTW) has been used as traditional tofu coagulant in China for hundreds of years. In this study, the microbial diversity in NFTW was firstly analyzed with high-throughput sequencing and its effect on chemical contents of tofu whey (TW) was investigated. Lactobacillus with 95.31% was the predominant genus in the microbial community of NFTW while Picha, Enterococcus, Bacillus and Acetobacter occupied about only 0.90%, 0.04%, 0.02% and 0.09%, respectively. Besides, Lactobacillus amylolyticus were determined to be one of the dominated species with metagenomic analysis and culture method. Lactobacillus with α-galactosidase activities played leading role in metabolizing the soybean oligosaccharides of TW to produce lactic acid. And acetic acid produced by genus of Acetobacter was another main organic acid attributed to the acidification of TW except lactic acid. Meanwhile, the bioconversion of isoflavone glucosides into aglycones could also be promoted by Lactobacillus with the help of β-glucosidase activity. Moreover, the production of equol in NFTW was confirmed, which might be jointly converted from daidzein by several strains. Therefore, our results indicated that Lactobacillus was the dominated microorganism and mainly affected the chemical changes of NFTW. This study help provide basic theory and technical references for the production of tofu and its derivative products (like sufu) with NFTW as coagulator., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The present study compares for the first time the effects of h-MoO3 and α-MoO3 against two fungal strains: Aspergillus niger and Aspergillus flavus. The h-MoO3 nanoparticles were more toxic to both fungi than α-MoO3. The toxic effects of h-MoO3 were more pronounced toward A. flavus, which presented a growth inhibition of 67.4% at 200 mg L-1. The presence of the nanoparticles affected drastically the hyphae morphology by triggering nuclear condensation and compromising the hyphae membrane. Further analysis of the volatile organic compounds (VOCs) produced by both fungi in the presence of the nanomaterials indicated important metabolic changes related to programmed cell death. These nanomaterials induced the production of specific antifungal VOCs, such as β-Elemene and t-Cadinol, by the fungi. The production of essential enzymes involved in fungal metabolism, such as acid phosphatase, naphthol-As-BI-phosphohydrolase, β-galactosidase, β-glucosidase and N-acetyl-β-glucosaminidase, reduced significantly in the presence of the nanomaterials. The changes in enzymatic production and VOCs corroborate the fact that these nanoparticles, especially h-MoO3, exert changes in the fungal metabolism, triggering apoptotic-like cell death responses in these fungi.

Agar is a major polysaccharide of red algal cells and is mainly decomposed into neoagarobiose by the co-operative effort of β-agarases. Neoagarobiose is hydrolyzed into monomers, D-galactose and 3,6-anhydro-L-galactose, via a microbial oxidative process. Therefore, the enzyme, 1,3-α-3,6-anhydro-L-galactosidase (α-neoagarobiose/neoagarooligosaccharide hydrolase) involved in the final step of the agarolytic pathway is crucial for bioindustrial application of agar. A novel cold-adapted α-neoagarooligosaccharide hydrolase, Ahg786, was identified and characterized from an agarolytic marine bacterium Gayadomonas joobiniege G7. Ahg786 comprises 400 amino acid residues (45.3 kDa), including a 25 amino acid signal peptide. Although it was annotated as a hypothetical protein from the genomic sequencing analysis, NCBI BLAST search showed 57, 58, and 59% identities with the characterized α-neoagarooligosaccharide hydrolases from Saccharophagus degradans 2-40, Zobellia galactanivorans, and Bacteroides plebeius, respectively. The signal peptide-deleted recombinant Ahg786 expressed and purified from Escherichia coli showed dimeric forms and hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into 3,6-anhydro-L-galactose and other compounds by cleaving α-1,3-glycosidic bonds from the non-reducing ends of neoagarooligosaccharides, as confirmed by thin-layer chromatography and mass spectrometry. The optimum pH and temperature for Ahg786 activity were 7.0 and 15 °C, respectively, indicative of its unique cold-adapted features. The enzymatic activity severely inhibited with 0.5 mM ethylenediaminetetraacetic acid was completely restored or remarkably enhanced by Mn 2+ in a concentration-dependent manner, suggestive of the dependence of the enzyme on Mn 2+ ions. K m and V max values for neoagarobiose were 4.5 mM and 1.33 U/mg, respectively.

Lactobacilli have several attributes that provide health benefits to the host. The aim of this study was to screen indigenous lactobacilli from human gut and fermented foods for such attributes as production of β- and α-galactosidase and also their ability to reduce serum cholesterol. Lactobacilli were cultured on MRS broth and β-galactosidase activity was determined using o-nitrophenyl-β-D-galactopyranoside (ONPG) as a substrate. Three isolates Lactobacillus fermentum GPI-3 and L. fermentum GPI-6 and Lactobacillus salivarius GPI-1(S) showed better β-galactosidase activity than the standard strains Lactobacillus rhamnosus GG (LGG) and Lactobacillus plantarum ATCC 8014. The isolates showed variability in assimilating cholesterol during growth. Several isolates showed excellent cholesterol-lowering ability compared to standard strains LGG and L. plantarum ATCC 8014. Isolate L. rhamnosus SCB being the highest acid producer (pH 4.38) also showed the highest cholesterol reduction compared to other strains including standard strains. The ability of these isolates to produce α-galactosidase was also studied and the maximum α-galactosidase activity was found in isolate L. salivarius GPI-1(S) followed by L. fermentum FA-5 and Lactobacillus helveticus FA-7. This study therefore reports Lactobacillus isolates that have superior probiotic properties when compared to the standard strains; hence, they could be considered as potential probiotic strains, which can provide health benefits to the Indian population.

To understand the action mechanism of yieldin (YLD) on the regulation of the yield threshold (Y), one of the critical parameters of cell wall extension, YLD was extracted from the cell walls of cowpea (Vigna unguiculata L.) hypocotyls and the hemagglutinin activity (HA) as well as the glycosidase activity of the protein was measured. Sedimentation assays using trypsinated rabbit erythrocytes showed that YLD possessed HA at pH 7. The digestion assays using 4-nitrophenyl (pNP) glycopyranosides as artificial substrates showed that YLD liberated galactose residues from pNP alpha-d-galactopyranoside mainly at pH 4.0, i.e. the pH level where Y was decreased at most. These results show that YLD is a bifunctional protein that switches between the HA and the galactosidase activities depending on the surrounding pH. Since D-galactose at concentration of 0.03 g l -1 perfectly inhibited the HA, YLD was suggested to associate with galactose residues. However, the galactose application ten times concentrated was necessary to inhibit both the galactosidase activity of YLD and the acid-induced shift of Y regulated by YLD. In addition, the specific inhibitor of alpha-d-galactosidase (deoxygalactonojirimycin) inhibited both the galactosidase activity of YLD and the shift of Y at the same concentration, but not the HA. On the basis of these results, it is suggested the galactosidase activity of YLD plays a central role in the mechanism of Y-regulation at acidic pH., (© 2017 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Problem: We investigated the effect of oxygen concentrations on cellular senescence and autophagy and examined the role of autophagy in human trophoblast cells., Method of Study: Human first-trimester trophoblast cells (Sw.71) were incubated under 21%, 5%, or 1% O 2 concentrations for 24 hours. We examined the extent of senescence caused using senescence-associated β-galactosidase (SA-β-Gal) and senescence-associated secretory phenotype (SASP) as markers. Moreover, we examined the role of autophagy in causing cellular senescence using an autophagy inhibitor (3-methyladenine, 3MA)., Results: Physiological normoxia (5% O 2 ) decreased SA-β-Gal-positive cells and SASP including interleukin-6 (IL-6) and IL-8 compared with cultured cells in 21% O 2 . Pathophysiological hypoxia (1% O 2 ) caused cytotoxicity, including extracellular release of ATP and lactate dehydrogenase, and decreased senescence phenotypes. 3MA-treated trophoblast cells significantly suppressed senescence markers (SA-β-Gal-positive cells and SASP secretion) in O 2 -independent manner., Conclusion: We conclude that O 2 concentration modulates cellular senescence phenotypes regulating autophagy in the human trophoblast cells. Moreover, inhibiting autophagy suppresses cellular senescence, suggesting that autophagy contributes to oxygen stress-induced cellular senescence., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: Endothelial dysfunction is central to the pathogenesis of many rheumatic diseases, typified by vascular inflammation and damage. Immunosuppressive drugs induce disease remission and lead to improved patient survival. However, there remains a higher incidence of cardiovascular disease in these patients even after adequate disease control. The purpose of this study was to determine the effect of mycophenolic acid (MPA), a commonly used immunosuppressive drug in rheumatology, on blood vessel or circulating endothelial colony forming cell number and function., Methods: We tested whether mycophenolic acid exerts an inhibitory effect on proliferation, clonogenic potential and vasculogenic function of endothelial colony forming cell. We also studied potential mechanisms involved in the observed effects., Results: Treatment with MPA decreased endothelial colony forming cell proliferation, clonogenic potential and vasculogenic function in a dose-dependent fashion. MPA increased senescence-associated β-galactosidase expression, p21 gene expression and p53 phosphorylation, indicative of activation of cellular senescence. Exogenous guanosine supplementation rescued diminished endothelial colony forming cell proliferation and indices of senescence, consistent with the known mechanism of action of MPA., Conclusion: Our findings show that clinically relevant doses of MPA have potent anti-angiogenic and pro-senescent effects on vascular precursor cells in vitro, thus indicating that treatment with MPA can potentially affect vascular repair and regeneration. This warrants further studies in vivo to determine how MPA therapy contributes to vascular dysfunction and increased cardiovascular disease seen in patients with inflammatory rheumatic disease.

Bacillus anthracis , the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is essential for bacterial growth and cell division. B. anthracis SCWP is comprised of trisaccharide repeats with the structure, [→4)-β-ManNAc-(1→4)-β-GlcNAc( O 3-α-Gal)-(1→6)-α-GlcNAc( O 3-α-Gal, O 4-β-Gal)-(1→] 6-12 The genes whose products promote the galactosylation of B. anthracis SCWP are not yet known. We show here that the expression of galE1 , encoding a UDP-glucose 4-epimerase necessary for the synthesis of UDP-galactose, is required for B. anthracis SCWP galactosylation. The galE1 mutant assembles surface (S) layer and S layer-associated proteins that associate with ketal-pyruvylated SCWP via their S layer homology domains similarly to wild-type B. anthracis , but the mutant displays a defect in γ-phage murein hydrolase binding to SCWP. Furthermore, deletion of galE1 diminishes the capsulation of B. anthracis with poly-d-γ-glutamic acid (PDGA) and causes a reduction in bacterial virulence. These data suggest that SCWP galactosylation is required for the physiologic assembly of the B. anthracis cell wall envelope and for the pathogenesis of anthrax disease. IMPORTANCE Unlike virulent Bacillus anthracis isolates, B. anthracis strain CDC684 synthesizes secondary cell wall polysaccharide (SCWP) trisaccharide repeats without galactosyl modification, exhibits diminished growth in vitro in broth cultures, and is severely attenuated in an animal model of anthrax. To examine whether SCWP galactosylation is a requirement for anthrax disease, we generated variants of B. anthracis strains Sterne 34F2 and Ames lacking UDP-glucose 4-epimerase by mutating the genes galE1 and galE2 We identified galE1 as necessary for SCWP galactosylation. Deletion of galE1 decreased the poly-d-γ-glutamic acid (PDGA) capsulation of the vegetative form of B. anthracis and increased the bacterial inoculum required to produce lethal disease in mice, indicating that SCWP galactosylation is indeed a determinant of anthrax disease., (Copyright © 2018 American Society for Microbiology.)

Macroalgae contribute substantially to primary production in coastal ecosystems. Their biomass, mainly consisting of polysaccharides, is cycled into the environment by marine heterotrophic bacteria using largely uncharacterized mechanisms. Here we describe the complete catabolic pathway for carrageenans, major cell wall polysaccharides of red macroalgae, in the marine heterotrophic bacterium Zobellia galactanivorans. Carrageenan catabolism relies on a multifaceted carrageenan-induced regulon, including a non-canonical polysaccharide utilization locus (PUL) and genes distal to the PUL, including a susCD-like pair. The carrageenan utilization system is well conserved in marine Bacteroidetes but modified in other phyla of marine heterotrophic bacteria. The core system is completed by additional functions that might be assumed by non-orthologous genes in different species. This complex genetic structure may be the result of multiple evolutionary events including gene duplications and horizontal gene transfers. These results allow for an extension on the definition of bacterial PUL-mediated polysaccharide digestion.

Chronic obstructive pulmonary disease and emphysema are associated with increased elastin peptides (EP) production because of excessive breakdown of lung connective tissue. We recently reported that exposure of mice to EP elicited hallmark features of emphysema. EP effects are largely mediated through a receptor complex that includes the elastin-binding protein spliced-galactosidase (S-gal). In previous studies, we established a correlation between cytokine production and S-gal protein expression in EP-treated immune cells. In this study, we investigated the S-gal-dependent EP effects on T-helper (Th) and T-cytotoxic (Tc) responses during murine EP-triggered pulmonary inflammation. C57BL/6J mice were endotracheally instilled with the valine-glycine-valine-alanine-proline-glycine (VGVAPG) elastin peptide, and, 21 days after treatment, local and systemic T-lymphocyte phenotypes were analyzed at cytokine and transcription factor expression levels by multicolor flow cytometry. Exposure of mice to the VGVAPG peptide resulted in a significant increase in the proportion of the CD4 + and CD8 + T cells expressing the cytokines IFN-γ or IL-17a and the transcription factors T-box expressed in T cells or retinoic acid-related orphan receptor-γt (RORγt) without effects on IL-4 and Gata-binding protein 3 to DNA sequence [A/T]GATA[A/G] expression. These effects were maximized when each T-cell subpopulation was challenged ex vivo with EP, and they were inhibited in vivo when an analogous peptide antagonizing the EP/S-gal interactions was instilled together with the VGVAPG peptide. This study demonstrates that, during murine emphysema, EP-S-gal interactions contribute to a Th-1 and Th-17 proinflammatory T-cell response combined with a Tc-1 response. Our study also highlights the S-gal receptor as a putative pharmacological target to modulate such an immune response., (Copyright © 2017 the American Physiological Society.)

In a search for better comprehension of β-galactosidase function and specificity, we solved the crystal structures of the GH42 β-galactosidase BbgII from Bifidobacterium bifidum S17, a well-adapted probiotic microorganism from the human digestive tract, and its complex with d-α-galactose. BbgII is a three-domain molecule that forms barrel-shaped trimers in solution. BbgII interactions with d-α-galactose, a competitive inhibitor, showed a number of residues that are involved in the coordination of ligands. A combination of site-directed mutagenesis of these amino acid residues with enzymatic activity measurements confirmed that Glu161 and Glu320 are fundamental for catalysis and their substitution by alanines led to catalytically inactive mutants. Mutation Asn160Ala resulted in a two orders of magnitude decrease of the enzyme k cat without significant modification in its K m , whereas mutations Tyr289Phe and His371Phe simultaneously decreased k cat and increased K m values. Enzymatic activity of Glu368Ala mutant was too low to be detected. Our docking and molecular dynamics simulations showed that the enzyme recognizes and tightly binds substrates with β1→6 and β1→3 bonds, while binding of the substrates with β1→4 linkages is less favorable., Database: Structural data are available in the PDB under the accession numbers 4UZS and 4UCF., (© 2016 Federation of European Biochemical Societies.)

The use of prebiotics to escalate certain gut flora is a current aspect of research for effective gut ecology. In the present study we appraise the efficacy of gum odina obtained from the bark of Odina wodier (Anacardiaceae), which is not fully degraded (16%) in the upper GI tract and becomes available to the lower region, as a prebiotic. An in vitro prebiotic activity assay established a quantitative score to describe the extent to which gum odina supports the selective growth of probiotics with a maximum of 5.60 ± 0.11 for Lactobacillus plantarum MTCC 6160. The polysaccharide, upon fermentation, also liberates lactic acid (0.46 ± 0.003 mg ml(-1)) and acetic acid (1.03 ± 0.003 mg ml(-1)). In vivo studies revealed that natural gum selectively stimulates Lactobacillus sp., and eliminates enteric pathogens with a C.F.U. of 384.48 ± 0.11 and 40.56 ± 0.17 respectively on the 8(th) day. The changes in the level of β-galactosidase signify maturation of macrophages in the gut environment. It also boosts the immune system by increasing sIgA upon infection from the 5(th) day in the gut, when incorporated into the feed of mice. Moreover an increase in levels of IFNγ on the 5(th) day also manifest additional protection against various pathogen-induced primary and secondary infections. Thus, gum odina is a potential prebiotic which not only provides nutrition but also improves gut ecology.

We have described the design, synthesis, spectroscopy and biological applications of NI-βGal, a versatile fluorescent probe to detect E. coliβ-galactosidase in live cells and mice sensitively and directly, which holds great promise for its application in biomedical research such as gene therapy for cancer in the future.

Candidate phylum Saccharibacteria (former TM7) are abundant and widespread in nature, but little is known about their ecophysiology and detailed phylogeny. In this study phylogeny, morphology and ecophysiology of Saccharibacteria were investigated in activated sludge from nine wastewater treatment plants (WWTPs) from Japan and Denmark using the full-cycle 16S rRNA approach in combination with microautoradiography (MAR) and fluorescence in situ hybridization (FISH). Phylogenetic analysis showed that Saccharibacteria from all WWTPs were evenly distributed within subdivision 1 and 3 and in a distinct phylogenetic clade. Three probes were designed for the distinct saccharibacterial groups, and revealed morphotypes representing thin filaments, thick filaments and rods/cocci. MAR-FISH results showed that most probe-defined Saccharibacteria utilized glucose under aerobic-, nitrate reducing- and anaerobic conditions. Some Saccharibacteria also utilized N-acetylglucosamine, oleic acid, amino acids and butyrate, which are not predicted from available genomes so far. In addition, some filamentous Saccharibacteria exhibited β-galactosidase and lipase activities determined using a combination of enzyme-labeled fluorescence and FISH (ELF-FISH). No uptake of acetate, propionate, pyruvate, glycerol and ethanol was observed. These results indicate that Saccharibacteria is a phylogenetically diverse group and play a role in the degradation of various organic compounds as well as sugar compounds under aerobic-, nitrate reducing- and anaerobic conditions., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The fragile nature of most enzymes is a major hindrance to their use in industrial processes. Herein, we describe a synthetic chemical strategy to produce hybrid organic/inorganic nanobiocatalysts; it exploits the self-assembly of silane building blocks at the surface of enzymes to grow an organosilica layer, of controlled thickness, that fully shields the enzyme. Remarkably, the enzyme triggers a rearrangement of this organosilica layer into a significantly soft structure. We demonstrate that this change in stiffness correlates with the biocatalytic turnover rate, and that the organosilica layer shields the enzyme in a soft environment with a markedly enhanced resistance to denaturing stresses., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A series of 1,5-dideoxy-1,5-imino-(l)-ribitol (DIR) derivatives carrying alkyl or functionalized alkyl groups were prepared and investigated as glycosidase inhibitors. These compounds were designed as simplified 4-epi-isofagomine (4-epi-IFG) mimics and were expected to behave as selective inhibitors of β-galactosidases. All compounds were indeed found to be highly selective for β-galactosidases versus α-glycosidases, as they generally did not inhibit coffee bean α-galactosidase or other α-glycosidases. Some compounds were also found to be inhibitors of almond β-glucosidase. The N-alkyl DIR derivatives were only modest inhibitors of bovine β-galactosidase, with IC50 values in the 30-700 μM range. Likewise, imino-L-ribitol substituted at the C1 position was found to be a weak inhibitor of this enzyme. In contrast, alkyl substitution at C5 resulted in enhanced β-galactosidase inhibitory activity by a factor of up to 1000, with at least six carbon atoms in the alkyl substituent. Remarkably, the 'pseudo-anomeric' configuration in this series does not appear to play a role. Human lysosomal β-galactosidase from leukocyte lysate was, however, poorly inhibited by all iminoribitol derivatives tested (IC50 values in the 100 μM range), while 4-epi-IFG was a good inhibitor of this enzyme. Two compounds were evaluated as pharmacological chaperones for a GM1-gangliosidosis cell line (R301Q mutation) and were found to enhance the mutant enzyme activity by factors up to 2.7-fold., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A hybrid sensor kinase termed RetS (regulator of exopolysaccharide and Type III secretion) controls expression of numerous genes in Pseudomonas aeruginosa. To investigate the function of RetS in P. fluorescens FD6, the retS gene was disrupted. Genetic inactivation of retS resulted in enhanced production of 2, 4-diacetylphloroglucinol, pyrrolnitrin, and pyoluteorin. The retS mutant also exhibited significant increase in phlA-lacZ, prnA-lacZ, and pltA-lacZ transcription levels, influencing expression levels of the small regulatory RNAs RsmX and RsmZ. In the gacSretS double mutant, all the phenotypic changes caused by the retS deletion were reversed to the level of gacS single mutant. Furthermore, the retS mutation drastically elevated biofilm formation and improved the colonization ability of strain FD6 on wheat rhizospheres. Based on these results, we proposed that RetS negatively controlled the production of antibiotics through the Gac/Rsm pathway in P. fluorescens FD6., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

The methodology adopted by Michaelis and Menten in 1913 is still routinely used to characterize the catalytic power and selectivity of enzymes. These kinetic measurements must be performed soon after the purified enzyme is mixed with a large excess of substrate. Other time scales and solution compositions are no less physiologically relevant, but fall outside the range of applicability of the classical formalism. Here we show that the complete picture of an enzyme's mode of function is critically obscured by the limited scope of conventional kinetic analysis, even in the simplest case of a single active site without inhibition. This picture is now unveiled in a mathematically closed form that remains valid over the reaction time for all combinations of enzyme/substrate concentrations and rate constants. Algebraic simplicity is maintained in the new formalism when stationary reaction phases are considered. By achieving this century-old objective, the otherwise hidden role of the reversible binding step is revealed and atypical kinetic profiles are explained. Most singular kinetic behaviors are identified in a critical region of conditions that coincide with typical cell conditions. Because it is not covered by the Michaelis-Menten model, the critical region has been missed until now by low- and high-throughput screenings of new drugs. New possibilities are therefore raised for novel and once-promising inhibitors to therapeutically target enzymes., (© 2015 FEBS.)

This study was aimed to evaluate the preventive role of phytic acid on lysosomal enzymes in isoproterenol (ISO)-induced myocardial infarction (MI) in male Wistar rats. Rats subcutaneously injected with ISO (85 mg/kg) at an interval of 24 h for two days showed a significant increase in the activities of lysosomal enzymes (glucuronidase, N-acetyl glucosaminidase, galactosidase, cathepsin-B and cathepsin-D) were increased significantly in serum and the heart of ISO-induced rats, but the activities of glucuronidase and cathepsin-D were decreased significantly in the lysosomal fraction of the heart. Pretreatment with phytic acid (25 and 50 mg/kg) daily for a period of 56 d positively altered activities of lysosomal hydrolases in ISO-induced rats. Thus, phytic acid possesses a cardioprotective effect in ISO-induced MI in rats.

The design of mechanism-based enzyme inactivators to generate chemical probes for biological research is an important challenge in carbohydrate chemistry. Here we describe the synthesis and biological evaluation of a novel carbocyclic mechanism-based inactivator of galactosidases (glycoside hydrolase families 27 and 36). Upon catalysis of this unnatural substrate, a transient non-classical carbocation forms within the enzyme active site. We show that the inactivation event, which proceeds via a bicyclobutonium ion intermediate, leads to a single alkylation event that occurs on the enzymatic nucleophile, an aspartic acid residue in this case. We also show that the catalytic proficiencies for enzymatic hydrolysis of substrates and inactivation by our bicyclo[4.1.0]heptyl analogue of galactose differ by only a factor of 20. This inactivator has the potential for further development as a useful biological research tool for both basic research and biotechnological applications.

Overproduction of heterologous proteins in bacterial systems often results in the formation of insoluble inclusion bodies (IBs), which is a major impediment in biochemical research and biotechnology. In principle, the activity of molecular chaperones could be employed to gain control over the IB formation and to improve the recombinant protein yields, but the potential of each of the major bacterial chaperones (DnaK/J, GroEL/ES, and ClpB) to process IBs has not been fully established yet. We investigated the formation of inclusion bodies (IBs) of two aggregation-prone proteins, VP1LAC and VP1GFP, overproduced in Escherichiacoli in the presence and absence of the chaperone ClpB. We found that both ClpB isoforms, ClpB95 and ClpB80 accumulated in E. coli cells during the production of IBs. The amount of IB proteins increased in the absence of ClpB. ClpB supported the resolubilization and reactivation of the aggregated VP1LAC and VP1GFP in E. coli cells. The IB disaggregation was optimal in the presence of both ClpB95 and ClpB80. Our results indicate an essential role of ClpB in controlling protein aggregation and inclusion body formation in bacteria., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Chagas disease (CD), a neglected tropical disease caused by Trypanosoma cruzi, remains a serious public health problem in several Latin American countries. The available chemotherapies for CD have limited efficacy and exhibit undesirable side effects. Aromatic diamidines and arylimidamides (AIAs) have shown broad-spectrum activity against intracellular parasites, including T. cruzi. Therefore, our aim was to evaluate the biological activity of eight novel AIAs (16DAP002, 16SAB079, 18SAB075, 23SMB022, 23SMB026, 23SMB054, 26SMB070, and 27SMB009) against experimental models of T. cruzi infection in vitro and in vivo. Our data show that none of the compounds induced a loss of cellular viability up to 32 μM. Two AIAs, 18SAB075 and 16DAP002, exhibited good in vitro activity against different parasite strains (Y and Tulahuen) and against the two relevant forms of the parasite for mammalian hosts. Due to the excellent selective indexes of 18SAB075, this AIA was moved to in vivo tests for acute toxicity and parasite efficacy; nontoxic doses (no-observed-adverse-effect level [NOAEL], 50 mg/kg) were employed in the tests for parasite efficacy. In experimental models of acute T. cruzi infection, 18SAB075 reduced parasitemia levels only up to 50% and led to 40% protection against mortality (at 5 mg/kg of body weight), being less effective than the reference drug, benznidazole., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)