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Sample records for glycoside hydrolase activity

  1. Glycoside hydrolases having multiple hydrolase activities

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    Chen, Zhiwei; Friedland, Gregory D.; Chhabra, Swapnil R.; Chivian, Dylan C.; Simmons, Blake A

    2017-08-08

    Glycoside hydrolases having at least two different hydrolytic activities are provided. In one embodiment, an isolated recombinant hydrolase having at least two activities selected from a group including asparagine derivatives, glutamine derivatives, and histidine derivatives is provided. Further, a method of generating free sugars from a mixture comprising asparagine derivatives, glutamine derivatives, and histidine derivatives is provided.

  2. Variants of glycoside hydrolases

    Science.gov (United States)

    Teter, Sarah [Davis, CA; Ward, Connie [Hamilton, MT; Cherry, Joel [Davis, CA; Jones, Aubrey [Davis, CA; Harris, Paul [Carnation, WA; Yi, Jung [Sacramento, CA

    2011-04-26

    The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  3. Glycoside hydrolase activities of thermophilic bacterial consortia adapted to switchgrass.

    Science.gov (United States)

    Gladden, John M; Allgaier, Martin; Miller, Christopher S; Hazen, Terry C; VanderGheynst, Jean S; Hugenholtz, Philip; Simmons, Blake A; Singer, Steven W

    2011-08-15

    Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60°C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermus marinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80°C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions.

  4. Colloid-based multiplexed method for screening plant biomass-degrading glycoside hydrolase activities in microbial communities

    Energy Technology Data Exchange (ETDEWEB)

    Reindl, W.; Deng, K.; Gladden, J.M.; Cheng, G.; Wong, A.; Singer, S.W.; Singh, S.; Lee, J.-C.; Yao, J.-S.; Hazen, T.C.; Singh, A.K; Simmons, B.A.; Adams, P.D.; Northen, T.R.

    2011-05-01

    The enzymatic hydrolysis of long-chain polysaccharides is a crucial step in the conversion of biomass to lignocellulosic biofuels. The identification and characterization of optimal glycoside hydrolases is dependent on enzyme activity assays, however existing methods are limited in terms of compatibility with a broad range of reaction conditions, sample complexity, and especially multiplexity. The method we present is a multiplexed approach based on Nanostructure-Initiator Mass Spectrometry (NIMS) that allowed studying several glycolytic activities in parallel under diverse assay conditions. Although the substrate analogs carried a highly hydrophobic perfluorinated tag, assays could be performed in aqueous solutions due colloid formation of the substrate molecules. We first validated our method by analyzing known {beta}-glucosidase and {beta}-xylosidase activities in single and parallel assay setups, followed by the identification and characterization of yet unknown glycoside hydrolase activities in microbial communities.

  5. Glycoside Hydrolases from a targeted Compost Metagenome, activity-screening and functional characterization

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    Dougherty Michael J

    2012-07-01

    Full Text Available Abstract Background Metagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes. Discovery of new glycoside hydrolases with improved biocatalytic properties for the efficient conversion of lignocellulosic material to biofuels is a critical challenge in the development of economically viable routes from biomass to fuels and chemicals. Results Twenty-two putative ORFs (open reading frames were identified from a switchgrass-adapted compost community based on sequence homology to related gene families. These ORFs were expressed in E. coli and assayed for predicted activities. Seven of the ORFs were demonstrated to encode active enzymes, encompassing five classes of hemicellulases. Four enzymes were over expressed in vivo, purified to homogeneity and subjected to detailed biochemical characterization. Their pH optima ranged between 5.5 - 7.5 and they exhibit moderate thermostability up to ~60-70°C. Conclusions Seven active enzymes were identified from this set of ORFs comprising five different hemicellulose activities. These enzymes have been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering towards the goal of developing efficient enzyme cocktails for biomass degradation under diverse process conditions.

  6. The Structural Basis of Exopolygalacturonase Activity in a Family 28 Glycoside Hydrolase

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    Abbott,D.; Boraston, A.

    2007-01-01

    Family 28 glycoside hydrolases (polygalacturonases) are found in organisms across the plant, fungal and bacterial kingdoms, where they are central to diverse biological functions such as fruit ripening, biomass recycling and plant pathogenesis. The structures of several polygalacturonases have been reported; however, all of these enzymes utilize an endo-mode of digestion, which generates a spectrum of oligosaccharide products with varying degrees of polymerization. The structure of a complementary exo-acting polygalacturonase and an accompanying explanation of the molecular determinants for its specialized activity have been noticeably lacking. We present the structure of an exopolygalacturonase from Yersinia enterocolitica, YeGH28 in a native form (solved to 2.19 {angstrom} resolution) and a digalacturonic acid product complex (solved to 2.10 {angstrom} resolution). The activity of YeGH28 is due to inserted stretches of amino acid residues that transform the active site from the open-ended channel observed in the endopolygalacturonases to a closed pocket that restricts the enzyme to the exclusive attack of the non-reducing end of oligogalacturonide substrates. In addition, YeGH28 possesses a fused FN3 domain with unknown function, the first such structure described in pectin active enzymes.

  7. First glycoside hydrolase family 2 enzymes from Thermus antranikianii and Thermus brockianus with β-glucosidase activity

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    Carola eSchröder

    2015-06-01

    Full Text Available Two genes tagh2 and tbgh2 coding for enzymes with hydrolytic activity towards esculin were identified from the extreme thermophilic, aerobic bacteria Thermus antranikianii (Ta and T. brockianus (Tb. Shortened conserved domains predicted a membership of the enzymes of glycoside hydrolase (GH family 2. At present, β-galactosidase activity is found frequently in GH family 2 but β-glucosidase activity has not been reported in this family before. The enzymes TaGH2 and TbGH2 preferred hydrolysis of nitrophenol-linked β-D-glucopyranosides with specific activities of 3,966 U/mg and 660 U/mg, respectively. Residual activities of 40 % (TaGH2 and 51 % (TbGH2 towards 4-NP-β-D-galactopyranoside were observed. Furthermore, TaGH2 hydrolyzed cellobiose. TbGH2, however, showed no activity on cellobiose or lactose. The enzymes exhibited highest activity at 95 °C (TaGH2 and 90 °C (TbGH2 at pH 6.5. Both enzymes were extremely thermostable and thermal activation up to 250 % was observed at temperatures between 50 and 60 °C. Accordingly, the first thermoactive glycoside hydrolase family 2 enzymes with β glucosidase activity have been identified and characterized. The hydrolysis of cellobiose is a unique property of TaGH2 when compared to the enzymes of GH family 2.

  8. Evaluation of the glycoside hydrolase activity of a Brettanomyces strain on glycosides from sour cherry (Prunus cerasus L.) used in the production of special fruit beers.

    Science.gov (United States)

    Daenen, Luk; Sterckx, Femke; Delvaux, Freddy R; Verachtert, Hubert; Derdelinckx, Guy

    2008-11-01

    The glycoside hydrolase activity of Saccharomyces cerevisiae and Brettanomyces custersii was examined on sour cherry (Prunus cerasus L.) glycosides with bound volatile compounds. Refermentations by the beta-glucosidase-negative S. cerevisiae strains LD25 and LD40 of sour cherry juice-supplemented beer demonstrated only a moderate increase of volatiles. In contrast, the beta-glucosidase-positive B. custersii strain LD72 showed a more pronounced activity towards glycosides with aliphatic alcohols, aromatic compounds and terpenoid alcohols. Important contributors to sour cherry aroma such as benzaldehyde, linalool and eugenol were released during refermentation as shown by analytical tools. A gradually increasing release was observed during refermentations by B. custersii when whole sour cherries, sour cherry pulp or juice were supplemented in the beer. Refermentations with whole sour cherries and with sour cherry stones demonstrated an increased formation of benzyl compounds. Thus, amygdalin was partially hydrolysed, and a large part of the benzaldehyde formed was mainly reduced to benzyl alcohol and some further esterified to benzyl acetate. These findings demonstrate the importance and interesting role of certain Brettanomyces species in the production of fruit lambic beers such as 'Kriek'.

  9. Comparative Analysis of Glycoside Hydrolases Activities from Phylogenetically Diverse Marine Bacteria of the Genus Arenibacter

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    Valery Mikhailov

    2013-06-01

    Full Text Available A total of 16 marine strains belonging to the genus Arenibacter, recovered from diverse microbial communities associated with various marine habitats and collected from different locations, were evaluated in degradation of natural polysaccharides and chromogenic glycosides. Most strains were affiliated with five recognized species, and some presented three new species within the genus Arenibacter. No strains contained enzymes depolymerizing polysaccharides, but synthesized a wide spectrum of glycosidases. Highly active β-N-acetylglucosaminidases and α-N-acetylgalactosaminidases were the main glycosidases for all Arenibacter. The genes, encoding two new members of glycoside hydrolyses (GH families, 20 and 109, were isolated and characterized from the genomes of Arenibacter latericius. Molecular genetic analysis using glycosidase-specific primers shows the absence of GH27 and GH36 genes. A sequence comparison with functionally-characterized GH20 and GH109 enzymes shows that both sequences are closest to the enzymes of chitinolytic bacteria Vibrio furnissii and Cellulomonas fimi of marine and terrestrial origin, as well as human pathogen Elisabethkingia meningoseptica and simbionts Akkermansia muciniphila, gut and non-gut Bacteroides, respectively. These results revealed that the genus Arenibacter is a highly taxonomic diverse group of microorganisms, which can participate in degradation of natural polymers in marine environments depending on their niche and habitat adaptations. They are new prospective candidates for biotechnological applications due to their production of unique glycosidases.

  10. Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

    Science.gov (United States)

    Oyama, Takuji; Schmitz, George E; Dodd, Dylan; Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

  11. Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

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    Takuji Oyama

    Full Text Available CpMan5B is a glycoside hydrolase (GH family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196 in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

  12. Glycoside Hydrolases across Environmental Microbial Communities.

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    Renaud Berlemont

    2016-12-01

    Full Text Available Across many environments microbial glycoside hydrolases support the enzymatic processing of carbohydrates, a critical function in many ecosystems. Little is known about how the microbial composition of a community and the potential for carbohydrate processing relate to each other. Here, using 1,934 metagenomic datasets, we linked changes in community composition to variation of potential for carbohydrate processing across environments. We were able to show that each ecosystem-type displays a specific potential for carbohydrate utilization. Most of this potential was associated with just 77 bacterial genera. The GH content in bacterial genera is best described by their taxonomic affiliation. Across metagenomes, fluctuations of the microbial community structure and GH potential for carbohydrate utilization were correlated. Our analysis reveals that both deterministic and stochastic processes contribute to the assembly of complex microbial communities.

  13. Community dynamics and glycoside hydrolase activities of thermophilic bacterial consortia adapted to switchgrass

    Energy Technology Data Exchange (ETDEWEB)

    Gladden, J.M.; Allgaier, M.; Miller, C.S.; Hazen, T.C.; VanderGheynst, J.S.; Hugenholtz, P.; Simmons, B.A.; Singer, S.W.

    2011-05-01

    Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60 C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermus marinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80 C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions.

  14. Glycoside hydrolase gene transcription by Alicyclobacillus acidocaldarius during growth on wheat arabinoxylan and monosaccharides: a proposed xylan hydrolysis mechanism

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    Lee, Brady D.; Apel, William A.; Sheridan, Peter P.; DeVeaux, Linda C.

    2018-04-16

    Background Metabolism of carbon bound in wheat arabinoxylan (WAX) polysaccharides by bacteria requires a number of glycoside hydrolases active toward different bonds between sugars and other molecules. Alicyclobacillus acidocaldarius is a Gram-positive thermoacidophilic bacterium capable of growth on a variety of mono-, di-, oligo-, and polysaccharides. Nineteen proposed glycoside hydrolases have been annotated in the A. acidocaldarius Type Strain ATCC27009/DSM 446 genome. Results Molecular analysis using high-density oligonucleotide microarrays was performed on A. acidocaldarius strain ATCC27009 when growing on WAX. When a culture growing exponentially at the expense of arabinoxylan saccharides was challenged with glucose or xylose, most glycoside hydrolases were down-regulated. Interestingly, regulation was more intense when xylose was added to the culture than when glucose was added, a clear departure from classical carbon catabolite repression demonstrated by many Gram-positive bacteria. In silico analyses of the regulated glycoside hydrolases, along with the results from the microarray analyses, yielded a potential mechanism for arabinoxylan metabolism by A. acidocaldarius. Glycoside hydrolases expressed by this strain may have broad substrate specificity, and initial hydrolysis is catalyzed by an extracellular xylanase, while subsequent steps are likely performed inside the growing cell. Conclusions Glycoside hydrolases, for the most part, appear to be found in clusters, throughout the A. acidocaldarius genome. Not all of the glycoside hydrolase genes found at loci within these clusters were regulated during the experiment, indicating that a specific subset of the 19 glycoside hydrolase genes found in A. acidocaldarius were used during metabolism of WAX. While specific functions of the glycoside hydrolases was not tested as part of the research discussed, many of the glycoside hydrolases found in the A. acidocaldarius Type Strain appear to have a broader

  15. α-Amylase: an enzyme specificity found in various families of glycoside hydrolases

    DEFF Research Database (Denmark)

    Janeček, Štefan; Svensson, Birte; MacGregor, E. Ann

    2014-01-01

    of all carbohydrate-active enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α...... investigation because of an obvious, but unexpected, homology with inverting β-glucan-active hydrolases....

  16. A remote but significant sequence homology between glycoside hydrolase clan GH-H and glycoside hydrolase family GH 31

    DEFF Research Database (Denmark)

    Janecek, S.; Svensson, Birte; MacGregor, E.A.

    2007-01-01

    Although both the α-amylase super-family, i.e. the glycoside hydrolase (GH) clan GH-H (the GH families 13, 70 and 77), and family GH31 share some characteristics, their different catalytic machinery prevents classification of GH31 in clan GH-H. A significant but remote evolutionary relatedness is...

  17. Characterization and engineering of thermostable glycoside hydrolases

    NARCIS (Netherlands)

    Lieshout, van J.F.T.

    2007-01-01

    Glycosidehydrolasesform a class of enzymes that play an important role in sugar-converting processes. They are applied as biocatalyst in both the hydrolysis of natural polymers to mono- andoligo-saccharides, and the reverse hydrolysis or

  18. Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass.

    Science.gov (United States)

    Su, Xiaoyun; Zhang, Jing; Mackie, Roderick I; Cann, Isaac K O

    2012-01-01

    The glycoside hydrolases (GH) of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.

  19. Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass.

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    Xiaoyun Su

    Full Text Available The glycoside hydrolases (GH of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.

  20. Annotation and comparative analysis of the glycoside hydrolase genes in Brachypodium distachyon

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    Wu Jiajie

    2010-10-01

    Full Text Available Abstract Background Glycoside hydrolases cleave the bond between a carbohydrate and another carbohydrate, a protein, lipid or other moiety. Genes encoding glycoside hydrolases are found in a wide range of organisms, from archea to animals, and are relatively abundant in plant genomes. In plants, these enzymes are involved in diverse processes, including starch metabolism, defense, and cell-wall remodeling. Glycoside hydrolase genes have been previously cataloged for Oryza sativa (rice, the model dicotyledonous plant Arabidopsis thaliana, and the fast-growing tree Populus trichocarpa (poplar. To improve our understanding of glycoside hydrolases in plants generally and in grasses specifically, we annotated the glycoside hydrolase genes in the grasses Brachypodium distachyon (an emerging monocotyledonous model and Sorghum bicolor (sorghum. We then compared the glycoside hydrolases across species, at the levels of the whole genome and individual glycoside hydrolase families. Results We identified 356 glycoside hydrolase genes in Brachypodium and 404 in sorghum. The corresponding proteins fell into the same 34 families that are represented in rice, Arabidopsis, and poplar, helping to define a glycoside hydrolase family profile which may be common to flowering plants. For several glycoside hydrolase familes (GH5, GH13, GH18, GH19, GH28, and GH51, we present a detailed literature review together with an examination of the family structures. This analysis of individual families revealed both similarities and distinctions between monocots and eudicots, as well as between species. Shared evolutionary histories appear to be modified by lineage-specific expansions or deletions. Within GH families, the Brachypodium and sorghum proteins generally cluster with those from other monocots. Conclusions This work provides the foundation for further comparative and functional analyses of plant glycoside hydrolases. Defining the Brachypodium glycoside hydrolases sets

  1. The Immunoreactive Exo-1,3-β-Glucanase from the Pathogenic Oomycete Pythium insidiosum Is Temperature Regulated and Exhibits Glycoside Hydrolase Activity.

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    Angsana Keeratijarut

    Full Text Available The oomycete organism, Pythium insidiosum, is the etiologic agent of the life-threatening infectious disease called "pythiosis". Diagnosis and treatment of pythiosis is difficult and challenging. Novel methods for early diagnosis and effective treatment are urgently needed. Recently, we reported a 74-kDa immunodominant protein of P. insidiosum, which could be a diagnostic target, vaccine candidate, and virulence factor. The protein was identified as a putative exo-1,3-ß-glucanase (Exo1. This study reports on genetic, immunological, and biochemical characteristics of Exo1. The full-length exo1 coding sequence (2,229 bases was cloned. Phylogenetic analysis showed that exo1 is grouped with glucanase-encoding genes of other oomycetes, and is far different from glucanase-encoding genes of fungi. exo1 was up-regulated upon exposure to body temperature, and its gene product is predicted to contain BglC and X8 domains, which are involved in carbohydrate transport, binding, and metabolism. Based on its sequence, Exo1 belongs to the Glycoside Hydrolase family 5 (GH5. Exo1, expressed in E. coli, exhibited ß-glucanase and cellulase activities. Exo1 is a major intracellular immunoreactive protein that can trigger host immune responses during infection. Since GH5 enzyme-encoding genes are not present in human genomes, Exo1 could be a useful target for drug and vaccine development against this pathogen.

  2. Bioprospecting metagenomics of decaying wood: mining for new glycoside hydrolases

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    Li Luen-Luen

    2011-08-01

    Full Text Available Abstract Background To efficiently deconstruct recalcitrant plant biomass to fermentable sugars in industrial processes, biocatalysts of higher performance and lower cost are required. The genetic diversity found in the metagenomes of natural microbial biomass decay communities may harbor such enzymes. Our goal was to discover and characterize new glycoside hydrolases (GHases from microbial biomass decay communities, especially those from unknown or never previously cultivated microorganisms. Results From the metagenome sequences of an anaerobic microbial community actively decaying poplar biomass, we identified approximately 4,000 GHase homologs. Based on homology to GHase families/activities of interest and the quality of the sequences, candidates were selected for full-length cloning and subsequent expression. As an alternative strategy, a metagenome expression library was constructed and screened for GHase activities. These combined efforts resulted in the cloning of four novel GHases that could be successfully expressed in Escherichia coli. Further characterization showed that two enzymes showed significant activity on p-nitrophenyl-α-L-arabinofuranoside, one enzyme had significant activity against p-nitrophenyl-β-D-glucopyranoside, and one enzyme showed significant activity against p-nitrophenyl-β-D-xylopyranoside. Enzymes were also tested in the presence of ionic liquids. Conclusions Metagenomics provides a good resource for mining novel biomass degrading enzymes and for screening of cellulolytic enzyme activities. The four GHases that were cloned may have potential application for deconstruction of biomass pretreated with ionic liquids, as they remain active in the presence of up to 20% ionic liquid (except for 1-ethyl-3-methylimidazolium diethyl phosphate. Alternatively, ionic liquids might be used to immobilize or stabilize these enzymes for minimal solvent processing of biomass.

  3. Novel glycoside hydrolases from thermophilic fungi

    DEFF Research Database (Denmark)

    2012-01-01

    The present invention relates to isolated polypeptides having cellulolytic activity or hemicellulolytic activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of produ...

  4. Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Ruzanski, Christian

    2014-01-01

    Surface binding sites (SBSs) interact with carbohydrates outside of the enzyme active site. They are frequently situated on catalytic domains and are distinct from carbohydrate binding modules (CBMs). SBSs are found in a variety of enzymes and often seen in crystal structures. Notably about half ...

  5. Conformational changes in a hyperthermostable glycoside hydrolase: enzymatic activity is a consequence of the loop dynamics and protonation balance.

    Directory of Open Access Journals (Sweden)

    Leandro C de Oliveira

    Full Text Available Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5 was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105. In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features.

  6. Discovery of α-L-arabinopyranosidases from human gut microbiome expands the diversity within glycoside hydrolase family 42

    DEFF Research Database (Denmark)

    Viborg, Alexander Holm; Katayama, Takane; Arakawa, Takatoshi

    2017-01-01

    Enzymes of the glycoside hydrolase family 42 (GH42) are widespread in bacteria of the human gut microbiome and play fundamental roles in the decomposition of both milk and plant oligosaccharides. All GH42 enzymes characterized so far have β-galactosidase activity. Here, we report the existence...

  7. Oxidoreductive Cellulose Depolymerization by the Enzymes Cellobiose Dehydrogenase and Glycoside Hydrolase 61▿†

    Science.gov (United States)

    Langston, James A.; Shaghasi, Tarana; Abbate, Eric; Xu, Feng; Vlasenko, Elena; Sweeney, Matt D.

    2011-01-01

    Several members of the glycoside hydrolase 61 (GH61) family of proteins have recently been shown to dramatically increase the breakdown of lignocellulosic biomass by microbial hydrolytic cellulases. However, purified GH61 proteins have neither demonstrable direct hydrolase activity on various polysaccharide or lignacious components of biomass nor an apparent hydrolase active site. Cellobiose dehydrogenase (CDH) is a secreted flavocytochrome produced by many cellulose-degrading fungi with no well-understood biological function. Here we demonstrate that the binary combination of Thermoascus aurantiacus GH61A (TaGH61A) and Humicola insolens CDH (HiCDH) cleaves cellulose into soluble, oxidized oligosaccharides. TaGH61A-HiCDH activity on cellulose is shown to be nonredundant with the activities of canonical endocellulase and exocellulase enzymes in microcrystalline cellulose cleavage, and while the combination of TaGH61A and HiCDH cleaves highly crystalline bacterial cellulose, it does not cleave soluble cellodextrins. GH61 and CDH proteins are coexpressed and secreted by the thermophilic ascomycete Thielavia terrestris in response to environmental cellulose, and the combined activities of T. terrestris GH61 and T. terrestris CDH are shown to synergize with T. terrestris cellulose hydrolases in the breakdown of cellulose. The action of GH61 and CDH on cellulose may constitute an important, but overlooked, biological oxidoreductive system that functions in microbial lignocellulose degradation and has applications in industrial biomass utilization. PMID:21821740

  8. Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community

    Energy Technology Data Exchange (ETDEWEB)

    Allgaier, M.; Reddy, A.; Park, J. I.; Ivanova, N.; D' haeseleer, P.; Lowry, S.; Sapra, R.; Hazen, T.C.; Simmons, B.A.; VanderGheynst, J. S.; Hugenholtz, P.

    2009-11-15

    Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, {approx}10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

  9. Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community.

    Directory of Open Access Journals (Sweden)

    Martin Allgaier

    Full Text Available Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, approximately 10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 degrees C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

  10. Differential recognition and hydrolysis of host carbohydrate antigens by Streptococcus pneumoniae family 98 glycoside hydrolases.

    Science.gov (United States)

    Higgins, Melanie A; Whitworth, Garrett E; El Warry, Nahida; Randriantsoa, Mialy; Samain, Eric; Burke, Robert D; Vocadlo, David J; Boraston, Alisdair B

    2009-09-18

    The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-beta-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-beta-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

  11. Targeted Discovery of Glycoside Hydrolases from a Switchgrass-Adapted Compost Community

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, Amitha; Allgaier, Martin; Park, Joshua I.; Ivanoval, Natalia; Dhaeseleer, Patrik; Lowry, Steve; Sapra, Rajat; Hazen, Terry C.; Simmons, Blake A.; VanderGheynst, Jean S.; Hugenholtz, Philip

    2011-05-11

    Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Smallsubunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, ,10percent were putative cellulasesmostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50uC and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

  12. COMPARATIVE MODELLING AND LIGAND BINDING SITE PREDICTION OF A FAMILY 43 GLYCOSIDE HYDROLASE FROM Clostridium thermocellum

    Directory of Open Access Journals (Sweden)

    Shadab Ahmed

    2012-06-01

    Full Text Available The phylogenetic analysis of Clostridium thermocellum family 43 glycoside hydrolase (CtGH43 showed close evolutionary relation with carbohydrate binding family 6 proteins from C. cellulolyticum, C. papyrosolvens, C. cellulyticum, and A. cellulyticum. Comparative modeling of CtGH43 was performed based on crystal structures with PDB IDs 3C7F, 1YIF, 1YRZ, 2EXH and 1WL7. The structure having lowest MODELLER objective function was selected. The three-dimensional structure revealed typical 5-fold beta–propeller architecture. Energy minimization and validation of predicted model with VERIFY 3D indicated acceptability of the proposed atomic structure. The Ramachandran plot analysis by RAMPAGE confirmed that family 43 glycoside hydrolase (CtGH43 contains little or negligible segments of helices. It also showed that out of 301 residues, 267 (89.3% were in most favoured region, 23 (7.7% were in allowed region and 9 (3.0% were in outlier region. IUPred analysis of CtGH43 showed no disordered region. Active site analysis showed presence of two Asp and one Glu, assumed to form a catalytic triad. This study gives us information about three-dimensional structure and reaffirms the fact that it has the similar core 5-fold beta–propeller architecture and so probably has the same inverting mechanism of action with the formation of above mentioned catalytic triad for catalysis of polysaccharides.

  13. Crystal structure of glycoside hydrolase family 127 β-L-arabinofuranosidase from Bifidobacterium longum

    Energy Technology Data Exchange (ETDEWEB)

    Ito, Tasuku; Saikawa, Kyo [Department of Biotechnology, The University of Tokyo, Tokyo (Japan); Kim, Seonah [National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO (United States); Fujita, Kiyotaka [Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima (Japan); Ishiwata, Akihiro [Synthetic Cellular Chemistry Laboratory, RIKEN (Japan); Kaeothip, Sophon [ERATO Glycotrilogy Project, JST, Wako, Saitama (Japan); Arakawa, Takatoshi; Wakagi, Takayoshi [Department of Biotechnology, The University of Tokyo, Tokyo (Japan); Beckham, Gregg T., E-mail: Gregg.Beckham@nrel.gov [National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO (United States); Ito, Yukishige [Synthetic Cellular Chemistry Laboratory, RIKEN (Japan); ERATO Glycotrilogy Project, JST, Wako, Saitama (Japan); Fushinobu, Shinya, E-mail: asfushi@mail.ecc.u-tokyo.ac.jp [Department of Biotechnology, The University of Tokyo, Tokyo (Japan)

    2014-04-25

    Graphical abstract: - Highlights: • HypBA1 β-L-arabinofuranosidase belongs to glycoside hydrolase family 127. • Crystal structure of HypBA1 was determined. • HypBA1 consists of a catalytic barrel and two additional β-sandwich domains. • The active site contains a Zn{sup 2+} coordinated by glutamate and three cysteines. • A possible reaction mechanism involving cysteine as the nucleophile is proposed. - Abstract: Enzymes acting on β-linked arabinofuranosides have been unknown until recently, in spite of wide distribution of β-L-arabinofuranosyl oligosaccharides in plant cells. Recently, a β-L-arabinofuranosidase from the glycoside hydrolase family 127 (HypBA1) was discovered in the newly characterized degradation system of hydroxyproline-linked β-L-arabinooligosaccharides in the bacterium Bifidobacterium longum. Here, we report the crystal structure of HypBA1 in the ligand-free and β-L-arabinofuranose complex forms. The structure of HypBA1 consists of a catalytic barrel domain and two additional β-sandwich domains, with one β-sandwich domain involved in the formation of a dimer. Interestingly, there is an unprecedented metal-binding motif with Zn{sup 2+} coordinated by glutamate and three cysteines in the active site. The glutamate residue is located far from the anomeric carbon of the β-L-arabinofuranose ligand, but one cysteine residue is appropriately located for nucleophilic attack for glycosidic bond cleavage. The residues around the active site are highly conserved among GH127 members. Based on biochemical experiments and quantum mechanical calculations, a possible reaction mechanism involving cysteine as the nucleophile is proposed.

  14. Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase.

    Science.gov (United States)

    Honda, Yuji; Fushinobu, Shinya; Hidaka, Masafumi; Wakagi, Takayoshi; Shoun, Hirofumi; Taniguchi, Hajime; Kitaoka, Motomitsu

    2008-04-01

    The tyrosine residue Y198 is known to support a nucleophilic water molecule with the general base residue, D263, in the reducing-end xylose-releasing exo-oligoxylanase (Rex). A mutation in the tyrosine residue changing it into phenylalanine caused a drastic decrease in the hydrolytic activity and a small increase in the F(-) releasing activity from alpha-xylobiosyl fluoride in the presence of xylose. In contrast, mutations at D263 resulted in the decreased F(-) releasing activity. As a result of the high F(-) releasing activity and low hydrolytic activity, Y198F of Rex accumulates a large amount of product during the glycosynthase reaction. We propose a novel method for producing a glycosynthase from an inverting glycoside hydrolase by mutating a residue that holds the nucleophilic water molecule with the general base residue while keeping the general base residue intact.

  15. Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

    International Nuclear Information System (INIS)

    Germane, Katherine L.; Servinsky, Matthew D.; Gerlach, Elliot S.; Sund, Christian J.; Hurley, Margaret M.

    2015-01-01

    The crystal structure of the protein product of the C. acetobutylicum ATCC 824 gene CA-C0359 is structurally similar to YteR, an unsaturated rhamnogalacturonyl hydrolase from B. subtilis strain 168. Substrate modeling and electrostatic studies of the active site of the structure of CA-C0359 suggests that the protein can now be considered to be part of CAZy glycoside hydrolase family 105. Clostridium acetobutylicum ATCC 824 gene CA-C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA-C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry (http://scripts.iucr.org/cgi-bin/cr.cgi?rm)) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA-C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate

  16. Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

    Energy Technology Data Exchange (ETDEWEB)

    Germane, Katherine L., E-mail: katherine.germane.civ@mail.mil [Oak Ridge Associated Universities, 4692 Millennium Drive, Suite 101, Belcamp, MD 21017 (United States); Servinsky, Matthew D. [US Army Research Laboratory, 2800 Powder Mill Road, Adelphi, MD 20783 (United States); Gerlach, Elliot S. [Federal Staffing Resources, 2200 Somerville Road, Annapolis, MD 21401 (United States); Sund, Christian J. [US Army Research Laboratory, 2800 Powder Mill Road, Adelphi, MD 20783 (United States); Hurley, Margaret M., E-mail: katherine.germane.civ@mail.mil [US Army Research Laboratory, 4600 Deer Creek Loop, Aberdeen Proving Ground, MD 21005 (United States); Oak Ridge Associated Universities, 4692 Millennium Drive, Suite 101, Belcamp, MD 21017 (United States)

    2015-07-29

    The crystal structure of the protein product of the C. acetobutylicum ATCC 824 gene CA-C0359 is structurally similar to YteR, an unsaturated rhamnogalacturonyl hydrolase from B. subtilis strain 168. Substrate modeling and electrostatic studies of the active site of the structure of CA-C0359 suggests that the protein can now be considered to be part of CAZy glycoside hydrolase family 105. Clostridium acetobutylicum ATCC 824 gene CA-C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA-C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry (http://scripts.iucr.org/cgi-bin/cr.cgi?rm)) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA-C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate

  17. Identification, structure, and function of a novel type VI secretion peptidoglycan glycoside hydrolase effector-immunity pair.

    Science.gov (United States)

    Whitney, John C; Chou, Seemay; Russell, Alistair B; Biboy, Jacob; Gardiner, Taylor E; Ferrin, Michael A; Brittnacher, Mitchell; Vollmer, Waldemar; Mougous, Joseph D

    2013-09-13

    Bacteria employ type VI secretion systems (T6SSs) to facilitate interactions with prokaryotic and eukaryotic cells. Despite the widespread identification of T6SSs among Gram-negative bacteria, the number of experimentally validated substrate effector proteins mediating these interactions remains small. Here, employing an informatics approach, we define novel families of T6S peptidoglycan glycoside hydrolase effectors. Consistent with the known intercellular self-intoxication exhibited by the T6S pathway, we observe that each effector gene is located adjacent to a hypothetical open reading frame encoding a putative periplasmically localized immunity determinant. To validate our sequence-based approach, we functionally investigate a representative family member from the soil-dwelling bacterium Pseudomonas protegens. We demonstrate that this protein is secreted in a T6SS-dependent manner and that it confers a fitness advantage in growth competition assays with Pseudomonas putida. In addition, we determined the 1.4 Å x-ray crystal structure of this effector in complex with its cognate immunity protein. The structure reveals the effector shares highest overall structural similarity to a glycoside hydrolase family associated with peptidoglycan N-acetylglucosaminidase activity, suggesting that T6S peptidoglycan glycoside hydrolase effector families may comprise significant enzymatic diversity. Our structural analyses also demonstrate that self-intoxication is prevented by the immunity protein through direct occlusion of the effector active site. This work significantly expands our current understanding of T6S effector diversity.

  18. Identification, Structure, and Function of a Novel Type VI Secretion Peptidoglycan Glycoside Hydrolase Effector-Immunity Pair*

    Science.gov (United States)

    Whitney, John C.; Chou, Seemay; Russell, Alistair B.; Biboy, Jacob; Gardiner, Taylor E.; Ferrin, Michael A.; Brittnacher, Mitchell; Vollmer, Waldemar; Mougous, Joseph D.

    2013-01-01

    Bacteria employ type VI secretion systems (T6SSs) to facilitate interactions with prokaryotic and eukaryotic cells. Despite the widespread identification of T6SSs among Gram-negative bacteria, the number of experimentally validated substrate effector proteins mediating these interactions remains small. Here, employing an informatics approach, we define novel families of T6S peptidoglycan glycoside hydrolase effectors. Consistent with the known intercellular self-intoxication exhibited by the T6S pathway, we observe that each effector gene is located adjacent to a hypothetical open reading frame encoding a putative periplasmically localized immunity determinant. To validate our sequence-based approach, we functionally investigate a representative family member from the soil-dwelling bacterium Pseudomonas protegens. We demonstrate that this protein is secreted in a T6SS-dependent manner and that it confers a fitness advantage in growth competition assays with Pseudomonas putida. In addition, we determined the 1.4 Å x-ray crystal structure of this effector in complex with its cognate immunity protein. The structure reveals the effector shares highest overall structural similarity to a glycoside hydrolase family associated with peptidoglycan N-acetylglucosaminidase activity, suggesting that T6S peptidoglycan glycoside hydrolase effector families may comprise significant enzymatic diversity. Our structural analyses also demonstrate that self-intoxication is prevented by the immunity protein through direct occlusion of the effector active site. This work significantly expands our current understanding of T6S effector diversity. PMID:23878199

  19. Steady state kinetic analysis of substrate specificity of glycoside hydrolases from families 13 and 38

    DEFF Research Database (Denmark)

    Nielsen, Jonas Willum

    Glycosidases are widespread in nature, where they perform a diverse range of functions. The glycoside hydrolase (GH) family 38, α-mannosidase II enzymes play a crucial role in mammalian cells, in the maturation of N-glycosylated proteins in the Golgi apparatus and in catabolism in cytosol...

  20. Synthesis of novel bioactive lactose-derived oligosaccharides by microbial glycoside hydrolases

    Science.gov (United States)

    Díez-Municio, Marina; Herrero, Miguel; Olano, Agustín; Moreno, F Javier

    2014-01-01

    Prebiotic oligosaccharides are increasingly demanded within the Food Science domain because of the interesting healthy properties that these compounds may induce to the organism, thanks to their beneficial intestinal microbiota growth promotion ability. In this regard, the development of new efficient, convenient and affordable methods to obtain this class of compounds might expand even further their use as functional ingredients. This review presents an overview on the most recent interesting approaches to synthesize lactose-derived oligosaccharides with potential prebiotic activity paying special focus on the microbial glycoside hydrolases that can be effectively employed to obtain these prebiotic compounds. The most notable advantages of using lactose-derived carbohydrates such as lactosucrose, galactooligosaccharides from lactulose, lactulosucrose and 2-α-glucosyl-lactose are also described and commented. PMID:24690139

  1. Identification of the C-Terminal GH5 Domain from CbCel9B/Man5A as the First Glycoside Hydrolase with Thermal Activation Property from a Multimodular Bifunctional Enzyme.

    Directory of Open Access Journals (Sweden)

    Rong Wang

    Full Text Available Caldicellulosiruptor bescii encodes at least six unique multimodular glycoside hydrolases crucial for plant cell wall polysaccharides degradation, with each having two catalytic domains separated by two to three carbohydrate binding modules. Among the six enzymes, three have one N- or C-terminal GH5 domain with identical amino acid sequences. Despite a few reports on some of these multimodular enzymes, little is known about how the conserved GH5 domains behave, which are believed to be important due to the gene duplication. We thus cloned a representative GH5 domain from the C-terminus of a multimodular protein, i.e. the bifunctional cellulase/mannanase CbCel9B/Man5A which has been reported, and expressed it in Escherichia coli. Without any appending CBMs, the recombinant CbMan5A was still able to hydrolyze a variety of mannan substrates with different backbone linkages or side-chain decorations. While CbMan5A displayed the same pH optimum as CbCel9B/Man5A, it had an increased optimal temperature (90°C and moreover, was activated by heating at 70°C and 80°C, a property not ever reported for the full-length protein. The turnover numbers of CbMan5A on mannan substrates were, however, lower than those of CbCel9B/Man5A. These data suggested that evolution of CbMan5A and the other domains into a single polypeptide is not a simple assembly; rather, the behavior of one module may be affected by the other ones in the full-length enzyme. The differential scanning calorimetry analysis further indicated that heating CbMan5A was not a simple transition state process. To the best knowledge of the authors, CbMan5A is the first glycoside hydrolase with thermal activation property identified from a multimodular bifunctional enzyme.

  2. Identification and characterization of some Aspergillus pectinolytic glycoside hydrolases

    NARCIS (Netherlands)

    Zandleven, J.S.

    2006-01-01

    Keywords: Aspergillusniger , Arabidopsis thaliana , homogalacturonan, rhamnogalacturonan, xylogalacturonan, xylogalacturonan hydrolase, exo-polygalacturonasePectinases are used for many food

  3. Friend or foe? Evolutionary history of glycoside hydrolase family 32 genes encoding for sucrolytic activity in fungi and its implications for plant-fungal symbioses

    Directory of Open Access Journals (Sweden)

    James Timothy Y

    2009-06-01

    Full Text Available Abstract Background Many fungi are obligate biotrophs of plants, growing in live plant tissues, gaining direct access to recently photosynthesized carbon. Photosynthate within plants is transported from source to sink tissues as sucrose, which is hydrolyzed by plant glycosyl hydrolase family 32 enzymes (GH32 into its constituent monosaccharides to meet plant cellular demands. A number of plant pathogenic fungi also use GH32 enzymes to access plant-derived sucrose, but less is known about the sucrose utilization ability of mutualistic and commensal plant biotrophic fungi, such as mycorrhizal and endophytic fungi. The aim of this study was to explore the distribution and abundance of GH32 genes in fungi to understand how sucrose utilization is structured within and among major ecological guilds and evolutionary lineages. Using bioinformatic and PCR-based analyses, we tested for GH32 gene presence in all available fungal genomes and an additional 149 species representing a broad phylogenetic and ecological range of biotrophic fungi. Results We detected 9 lineages of GH32 genes in fungi, 4 of which we describe for the first time. GH32 gene number in fungal genomes ranged from 0–12. Ancestral state reconstruction of GH32 gene abundance showed a strong correlation with nutritional mode, and gene family expansion was observed in several clades of pathogenic filamentous Ascomycota species. GH32 gene number was negatively correlated with animal pathogenicity and positively correlated with plant biotrophy, with the notable exception of mycorrhizal taxa. Few mycorrhizal species were found to have GH32 genes as compared to other guilds of plant-associated fungi, such as pathogens, endophytes and lichen-forming fungi. GH32 genes were also more prevalent in the Ascomycota than in the Basidiomycota. Conclusion We found a strong signature of both ecological strategy and phylogeny on GH32 gene number in fungi. These data suggest that plant biotrophic fungi

  4. Draft genome sequence of Streptomyces sp. strain F1, a potential source for glycoside hydrolases isolated from Brazilian soil

    Directory of Open Access Journals (Sweden)

    Ricardo Rodrigues de Melo

    Full Text Available ABSTRACT Here, we show the draft genome sequence of Streptomyces sp. F1, a strain isolated from soil with great potential for secretion of hydrolytic enzymes used to deconstruct cellulosic biomass. The draft genome assembly of Streptomyces sp. strain F1 has 69 contigs with a total genome size of 8,142,296 bp and G + C 72.65%. Preliminary genome analysis identified 175 proteins as Carbohydrate-Active Enzymes, being 85 glycoside hydrolases organized in 33 distinct families. This draft genome information provides new insights on the key genes encoding hydrolytic enzymes involved in biomass deconstruction employed by soil bacteria.

  5. Construction of a rice glycoside hydrolase phylogenomic database and identification of targets for biofuel research

    Directory of Open Access Journals (Sweden)

    Rita eSharma

    2013-08-01

    Full Text Available Glycoside hydrolases (GH catalyze the hydrolysis of glycosidic bonds in cell wall polymers and can have major effects on cell wall architecture. Taking advantage of the massive datasets available in public databases, we have constructed a rice phylogenomic database of GHs (http://ricephylogenomics.ucdavis.edu/cellwalls/gh/. This database integrates multiple data types including the structural features, orthologous relationships, mutant availability and gene expression patterns for each GH family in a phylogenomic context. The rice genome encodes 437 GH genes classified into 34 families. Based on pairwise comparison with eight dicot and four monocot genomes, we identified 138 GH genes that are highly diverged between monocots and dicots, 57 of which have diverged further in rice as compared with four monocot genomes scanned in this study. Chromosomal localization and expression analysis suggest a role for both whole-genome and localized gene duplications in expansion and diversification of GH families in rice. We examined the meta-profiles of expression patterns of GH genes in twenty different anatomical tissues of rice. Transcripts of 51 genes exhibit tissue or developmental stage-preferential expression, whereas, seventeen other genes preferentially accumulate in actively growing tissues. When queried in RiceNet, a probabilistic functional gene network that facilitates functional gene predictions, nine out of seventeen genes form a regulatory network with the well-characterized genes involved in biosynthesis of cell wall polymers including cellulose synthase and cellulose synthase-like genes of rice. Two-thirds of the GH genes in rice are up regulated in response to biotic and abiotic stress treatments indicating a role in stress adaptation. Our analyses identify potential GH targets for cell wall modification.

  6. Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

    Directory of Open Access Journals (Sweden)

    Caroline da Silva Moraes

    2014-08-01

    Full Text Available The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females or blood feeders (females only, and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18 and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes.

  7. Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

    Science.gov (United States)

    Moraes, Caroline da Silva; Diaz-Albiter, Hector M.; Faria, Maiara do Valle; Sant'Anna, Maurício R. V.; Dillon, Rod J.; Genta, Fernando A.

    2014-01-01

    The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females) or blood feeders (females only), and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases, and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18, and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes. PMID:25140153

  8. Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

    Science.gov (United States)

    Liberato, Marcelo V.; Silveira, Rodrigo L.; Prates, Érica T.; de Araujo, Evandro A.; Pellegrini, Vanessa O. A.; Camilo, Cesar M.; Kadowaki, Marco A.; Neto, Mario De O.; Popov, Alexander; Skaf, Munir S.; Polikarpov, Igor

    2016-04-01

    Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.

  9. Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

    Science.gov (United States)

    Wan, Qun; Parks, Jerry M.; Hanson, B. Leif; Fisher, Suzanne Zoe; Ostermann, Andreas; Schrader, Tobias E.; Graham, David E.; Coates, Leighton; Langan, Paul; Kovalevsky, Andrey

    2015-01-01

    Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen. PMID:26392527

  10. Synergistic function of four novel thermostable glycoside hydrolases from a long-term enriched thermophilic methanogenic digester

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    Meng eWang

    2015-05-01

    Full Text Available In biofuel production from lignocellulose, low thermostability and product inhibition strongly restrict the enzyme activities and production process. Application of multiple thermostable glycoside hydrolases, forming an enzyme cocktail, can result in a synergistic action and therefore improve production efficiency and reduce operational costs. Therefore, increasing enzyme thermostabilities and compatibility are important for the biofuel industry. In this study, we reported the screening, cloning and biochemical characterization of four novel thermostable lignocellulose hydrolases from a metagenomic library of a long-term dry thermophilic methanogenic digester community, which were highly compatible with optimal conditions and specific activities. The optimal temperatures of the four enzymes, β-xylosidase, xylanase, β-glucosidase, and cellulase ranged from 60°C to 75°C, and over 80% residual activities were observed after 2 h incubation at 50°C. Mixtures of these hydrolases retained high residual synergistic activities after incubation with cellulose, xylan, and steam-exploded corncob at 50°C for 72 h. In addition, about 55% dry weight of steam-exploded corncob was hydrolyzed to glucose and xylose by the synergistic action of the four enzymes at 50°C for 48 h. This work suggested that since different enzymes from a same ecosystem could be more compatible, screening enzymes from a long-term enriching community could be a favorable strategy.

  11. Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of a family 101 glycoside hydrolase from Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Gregg, Katie J.; Boraston, Alisdair B.

    2009-01-01

    The catalytic module of a family 101 glycoside hydrolase from S. pneumoniae was cloned, recombinantly produced and crystallized. Streptococcus pneumoniae is a serious human pathogen that is responsible for a wide range of diseases including pneumonia, meningitis, septicaemia and otitis media. The full virulence of this bacterium is reliant on carbohydrate processing and metabolism, as revealed by biochemical and genetic studies. One carbohydrate-processing enzyme is a family 101 glycoside hydrolase (SpGH101) that is responsible for catalyzing the liberation of galactosyl β1,3-N-acetyl-d-galactosamine (Galβ1,3GalNAc) α-linked to serine or threonine residues of mucin-type glycoproteins. The 124 kDa catalytic module of this enzyme (SpGH101CM) was cloned and overproduced in Escherichia coli and purified. Crystals were obtained in space group P2 1 and diffracted to 2.0 Å resolution, with unit-cell parameters a = 81.86, b = 88.91, c = 88.77 Å, β = 112.46°. SpGH101CM also qualitatively displayed good activity towards the synthetic substrate p-nitrophenyl-2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl) -α-d-galactopyranoside, which is consistent with the classification of this enzyme as an endo-α-N-acetylgalactosaminidase

  12. Protein features as determinants of wild-type glycoside hydrolase thermostability

    DEFF Research Database (Denmark)

    Geertz-Hansen, Henrik Marcus; Kiemer, Lars; Nielsen, Morten

    2017-01-01

    -silico methods guiding the discovery process would be of high value. To develop such an in-silico method and provide the data foundation of it, we determined the melting temperatures of 602 fungal glycoside hydrolases from the families GH5, 6, 7, 10, 11, 43 and AA9 (formerly GH61). We, then used sequence...... and homology modeled structure information of these enzymes to develop the ThermoP melting temperature prediction method. Futhermore, in the context of thermostability, we determined the relative importance of 160 molecular features, such as amino acid frequencies and spatial interactions, and exemplified...

  13. Structural and biochemical characterization of novel bacterial α-galactosidases belonging to glycoside hydrolase family 31.

    Science.gov (United States)

    Miyazaki, Takatsugu; Ishizaki, Yuichi; Ichikawa, Megumi; Nishikawa, Atsushi; Tonozuka, Takashi

    2015-07-01

    Glycoside hydrolase family 31 (GH31) proteins have been reportedly identified as exo-α-glycosidases with activity for α-glucosides and α-xylosides. We focused on a GH31 subfamily, which contains proteins with low sequence identity (Pedobacter heparinus and Pedobacter saltans. The enzymes unexpectedly exhibited α-galactosidase activity, but were not active on α-glucosides and α-xylosides. The crystal structures of one of the enzymes, PsGal31A, in unliganded form and in complexes with D-galactose or L-fucose and the catalytic nucleophile mutant in unliganded form and in complex with p-nitrophenyl-α-D-galactopyranoside, were determined at 1.85-2.30 Å (1 Å=0.1 nm) resolution. The overall structure of PsGal31A contains four domains and the catalytic domain adopts a (β/α)8-barrel fold that resembles the structures of other GH31 enzymes. Two catalytic aspartic acid residues are structurally conserved in the enzymes, whereas most residues forming the active site differ from those of GH31 α-glucosidases and α-xylosidases. PsGal31A forms a dimer via a unique loop that is not conserved in other reported GH31 enzymes; this loop is involved in its aglycone specificity and in binding L-fucose. Considering potential genes for α-L-fucosidases and carbohydrate-related proteins within the vicinity of Pedobacter Gal31, the identified Gal31 enzymes are likely to function in a novel sugar degradation system. This is the first report of α-galactosidases which belong to GH31 family. © 2015 Authors; published by Portland Press Limited.

  14. Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Hiras, Jennifer; Wu, Yu-Wei; Deng, Kai; Nicora, Carrie D.; Aldrich, Joshua T.; Frey, Dario; Kolinko, Sebastian; Robinson, Errol W.; Jacobs, Jon M.; Adams, Paul D.; Northen, Trent R.; Simmons, Blake A.; Singer, Steven W.

    2016-08-23

    ABSTRACT

    Glycoside hydrolases (GHs) are key enzymes in the depolymerization of plant-derived cellulose, a process central to the global carbon cycle and the conversion of plant biomass to fuels and chemicals. A limited number of GH families hydrolyze crystalline cellulose, often by a processive mechanism along the cellulose chain. During cultivation of thermophilic cellulolytic microbial communities, substantial differences were observed in the crystalline cellulose saccharification activities of supernatants recovered from divergent lineages. Comparative community proteomics identified a set of cellulases from a population closely related to actinobacteriumThermobispora bisporathat were highly abundant in the most active consortium. Among the cellulases fromT. bispora, the abundance of a GH family 12 (GH12) protein correlated most closely with the changes in crystalline cellulose hydrolysis activity. This result was surprising since GH12 proteins have been predominantly characterized as enzymes active on soluble polysaccharide substrates. Heterologous expression and biochemical characterization of the suite ofT. bisporahydrolytic cellulases confirmed that the GH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that it hydrolyzes cellulose chains by a predominantly random mechanism. This work suggests that the role of GH12 proteins in crystalline cellulose hydrolysis by cellulolytic microbes should be reconsidered.

    IMPORTANCECellulose is the most abundant organic polymer on earth, and its enzymatic hydrolysis is a key reaction in the global carbon cycle and the conversion of plant biomass to biofuels. The glycoside hydrolases that depolymerize crystalline cellulose have been primarily characterized from isolates. In this study, we demonstrate that adapting microbial consortia from compost to grow on crystalline cellulose

  15. Insight into Dominant Cellulolytic Bacteria from Two Biogas Digesters and Their Glycoside Hydrolase Genes

    Science.gov (United States)

    Zhang, Jun; Zhang, Lei; Geng, Alei; Liu, Fanghua; Zhao, Guoping; Wang, Shengyue; Zhou, Zhihua; Yan, Xing

    2015-01-01

    Diverse cellulolytic bacteria are essential for maintaining high lignocellulose degradation ability in biogas digesters. However, little was known about functional genes and gene clusters of dominant cellulolytic bacteria in biogas digesters. This is the foundation to understand lignocellulose degradation mechanisms of biogas digesters and apply these gene resource for optimizing biofuel production. A combination of metagenomic and 16S rRNA gene clone library methods was used to investigate the dominant cellulolytic bacteria and their glycoside hydrolase (GH) genes in two biogas digesters. The 16S rRNA gene analysis revealed that the dominant cellulolytic bacteria were strains closely related to Clostridium straminisolvens and an uncultured cellulolytic bacterium designated BG-1. To recover GH genes from cellulolytic bacteria in general, and BG-1 in particular, a refined assembly approach developed in this study was used to assemble GH genes from metagenomic reads; 163 GH-containing contigs ≥ 1 kb in length were obtained. Six recovered GH5 genes that were expressed in E. coli demonstrated multiple lignocellulase activities and one had high mannanase activity (1255 U/mg). Eleven fosmid clones harboring the recovered GH-containing contigs were sequenced and assembled into 10 fosmid contigs. The composition of GH genes in the 163 assembled metagenomic contigs and 10 fosmid contigs indicated that diverse GHs and lignocellulose degradation mechanisms were present in the biogas digesters. In particular, a small portion of BG-1 genome information was recovered by PhyloPythiaS analysis. The lignocellulase gene clusters in BG-1 suggested that it might use a possible novel lignocellulose degradation mechanism to efficiently degrade lignocellulose. Dominant cellulolytic bacteria of biogas digester possess diverse GH genes, not only in sequences but also in their functions, which may be applied for production of biofuel in the future. PMID:26070087

  16. 4,3-α-Glucanotransferase, a novel reaction specificity in glycoside hydrolase family 70 and clan GH-H

    NARCIS (Netherlands)

    Gangoiti Muñecas, Joana; van Leeuwen, Sander S; Gerwig, Gerrit J; Duboux, Stéphane; Vafiadi, Christina; Pijning, Tjaard; Dijkhuizen, Lubbert

    2017-01-01

    Lactic acid bacteria possess a diversity of glucansucrase (GS) enzymes that belong to glycoside hydrolase family 70 (GH70) and convert sucrose into α-glucan polysaccharides with (α1 → 2)-, (α1 → 3)-, (α1 → 4)- and/or (α1 → 6)-glycosidic bonds. In recent years 3 novel subfamilies of GH70 enzymes,

  17. Crystal Structure of α-1,4-Glucan Lyase, a Unique Glycoside Hydrolase Family Member with a Novel Catalytic Mechanism

    NARCIS (Netherlands)

    Rozeboom, Henriëtte J.; Yu, Shukun; Madrid, Susan; Kalk, Kor H.; Zhang, Ran; Dijkstra, Bauke W.

    2013-01-01

    α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-D-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades

  18. Biotechnological potential of novel glycoside hydrolase family 70 enzymes synthesizing α-glucans from starch and sucrose

    NARCIS (Netherlands)

    Gangoiti, Joana; Pijning, Tjaard; Dijkhuizen, Lubbert

    Transglucosidases belonging to the glycoside hydrolase (GH) family 70 are promising enzymatic tools for the synthesis of α-glucans with defined structures from renewable sucrose and starch substrates. Depending on the GH70 enzyme specificity, α-glucans with different structures and physicochemical

  19. Draft genome sequence of Streptomyces sp. strain F1, a potential source for glycoside hydrolases isolated from Brazilian soil.

    Science.gov (United States)

    Melo, Ricardo Rodrigues de; Persinoti, Gabriela Felix; Paixão, Douglas Antonio Alvaredo; Squina, Fábio Márcio; Ruller, Roberto; Sato, Helia Harumi

    Here, we show the draft genome sequence of Streptomyces sp. F1, a strain isolated from soil with great potential for secretion of hydrolytic enzymes used to deconstruct cellulosic biomass. The draft genome assembly of Streptomyces sp. strain F1 has 69 contigs with a total genome size of 8,142,296bp and G+C 72.65%. Preliminary genome analysis identified 175 proteins as Carbohydrate-Active Enzymes, being 85 glycoside hydrolases organized in 33 distinct families. This draft genome information provides new insights on the key genes encoding hydrolytic enzymes involved in biomass deconstruction employed by soil bacteria. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  20. Insight into glycoside hydrolases for debranched xylan degradation from extremely thermophilic bacterium Caldicellulosiruptor lactoaceticus.

    Directory of Open Access Journals (Sweden)

    Xiaojing Jia

    Full Text Available Caldicellulosiruptor lactoaceticus 6A, an anaerobic and extremely thermophilic bacterium, uses natural xylan as carbon source. The encoded genes of C. lactoaceticus 6A for glycoside hydrolase (GH provide a platform for xylan degradation. The GH family 10 xylanase (Xyn10A and GH67 α-glucuronidase (Agu67A from C. lactoaceticus 6A were heterologously expressed, purified and characterized. Both Xyn10A and Agu67A are predicted as intracellular enzymes as no signal peptides identified. Xyn10A and Agu67A had molecular weight of 47.0 kDa and 80.0 kDa respectively as determined by SDS-PAGE, while both appeared as homodimer when analyzed by gel filtration. Xyn10A displayed the highest activity at 80 °C and pH 6.5, as 75 °C and pH 6.5 for Agu67A. Xyn10A had good stability at 75 °C, 80 °C, and pH 4.5-8.5, respectively, and was sensitive to various metal ions and reagents. Xyn10A possessed hydrolytic activity towards xylo-oligosaccharides (XOs and beechwood xylan. At optimum conditions, the specific activity of Xyn10A was 44.6 IU/mg with beechwood xylan as substrate, and liberated branched XOs, xylobiose, and xylose. Agu67A was active on branched XOs with methyl-glucuronic acids (MeGlcA sub-chains, and primarily generated XOs equivalents and MeGlcA. The specific activity of Agu67A was 1.3 IU/mg with aldobiouronic acid as substrate. The synergistic action of Xyn10A and Agu67A was observed with MeGlcA branched XOs and xylan as substrates, both backbone and branched chain of substrates were degraded, and liberated xylose, xylobiose, and MeGlcA. The synergism of Xyn10A and Agu67A provided not only a thermophilic method for natural xylan degradation, but also insight into the mechanisms for xylan utilization of C. lactoaceticus.

  1. Use of nanostructure initiator mass spectrometry (NIMS to deduce selectivity of reaction in glycoside hydrolases

    Directory of Open Access Journals (Sweden)

    Kai eDeng

    2015-10-01

    Full Text Available Chemically synthesized nanostructure-initiator mass spectrometry (NIMS probes derivatized with tetrasaccharides were used to study the reactivity of representative Clostridium thermocellum β-glucosidase, endoglucanases and cellobiohydrolase. Diagnostic patterns for reactions of these different classes of enzymes were observed. Results show sequential removal of glucose by the β-glucosidase and a progressive increase in specificity of reaction from endoglucanases to cellobiohydrolase. Time-dependent reactions of these polysaccharide-selective enzymes were modeled by numerical integration, which provides a quantitative basis to make functional distinctions among a continuum of naturally evolved catalytic properties. Consequently, our method, which combines automated protein translation with high-sensitivity and time-dependent detection of multiple products, provides a new approach to annotate glycoside hydrolase phylogenetic trees with functional measurements.

  2. Chitosanases from Family 46 of Glycoside Hydrolases: From Proteins to Phenotypes

    Directory of Open Access Journals (Sweden)

    Pascal Viens

    2015-10-01

    Full Text Available Chitosanases, enzymes that catalyze the endo-hydrolysis of glycolytic links in chitosan, are the subject of numerous studies as biotechnological tools to generate low molecular weight chitosan (LMWC or chitosan oligosaccharides (CHOS from native, high molecular weight chitosan. Glycoside hydrolases belonging to family GH46 are among the best-studied chitosanases, with four crystallography-derived structures available and more than forty enzymes studied at the biochemical level. They were also subjected to numerous site-directed mutagenesis studies, unraveling the molecular mechanisms of hydrolysis. This review is focused on the taxonomic distribution of GH46 proteins, their multi-modular character, the structure-function relationships and their biological functions in the host organisms.

  3. The use of neutron scattering to determine the functional structure of glycoside hydrolase.

    Science.gov (United States)

    Nakamura, Akihiko; Ishida, Takuya; Samejima, Masahiro; Igarashi, Kiyohiko

    2016-10-01

    Neutron diffraction provides different information from X-ray diffraction, because neutrons are scattered by atomic nuclei, whereas X-rays are scattered by electrons. One of the key advantages of neutron crystallography is the ability to visualize hydrogen and deuterium atoms, making it possible to observe the protonation state of amino acid residues, hydrogen bonds, networks of water molecules and proton relay pathways in enzymes. But, because of technical difficulties, less than 100 enzyme structures have been evaluated by neutron crystallography to date. In this review, we discuss the advantages and disadvantages of neutron crystallography as a tool to investigate the functional structure of glycoside hydrolases, with some examples. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Immobilization of Glycoside Hydrolase Families GH1, GH13, and GH70: State of the Art and Perspectives

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    Natália G. Graebin

    2016-08-01

    Full Text Available Glycoside hydrolases (GH are enzymes capable to hydrolyze the glycosidic bond between two carbohydrates or even between a carbohydrate and a non-carbohydrate moiety. Because of the increasing interest for industrial applications of these enzymes, the immobilization of GH has become an important development in order to improve its activity, stability, as well as the possibility of its reuse in batch reactions and in continuous processes. In this review, we focus on the broad aspects of immobilization of enzymes from the specific GH families. A brief introduction on methods of enzyme immobilization is presented, discussing some advantages and drawbacks of this technology. We then review the state of the art of enzyme immobilization of families GH1, GH13, and GH70, with special attention on the enzymes β-glucosidase, α-amylase, cyclodextrin glycosyltransferase, and dextransucrase. In each case, the immobilization protocols are evaluated considering their positive and negative aspects. Finally, the perspectives on new immobilization methods are briefly presented.

  5. New insights into plant glycoside hydrolase family 32 in Agave species.

    Science.gov (United States)

    Avila de Dios, Emmanuel; Gomez Vargas, Alan D; Damián Santos, Maura L; Simpson, June

    2015-01-01

    In order to optimize the use of agaves for commercial applications, an understanding of fructan metabolism in these species at the molecular and genetic level is essential. Based on transcriptome data, this report describes the identification and molecular characterization of cDNAs and deduced amino acid sequences for genes encoding fructosyltransferases, invertases and fructan exohydrolases (FEH) (enzymes belonging to plant glycoside hydrolase family 32) from four different agave species (A. tequilana, A. deserti, A. victoriae-reginae, and A. striata). Conserved amino acid sequences and a hypervariable domain allowed classification of distinct isoforms for each enzyme type. Notably however neither 1-FFT nor 6-SFT encoding cDNAs were identified. In silico analysis revealed that distinct isoforms for certain enzymes found in a single species, showed different levels and tissue specific patterns of expression whereas in other cases expression patterns were conserved both within the species and between different species. Relatively high levels of in silico expression for specific isoforms of both invertases and fructosyltransferases were observed in floral tissues in comparison to vegetative tissues such as leaves and stems and this pattern was confirmed by Quantitative Real Time PCR using RNA obtained from floral and leaf tissue of A. tequilana. Thin layer chromatography confirmed the presence of fructans with degree of polymerization (DP) greater than DP three in both immature buds and fully opened flowers also obtained from A. tequilana.

  6. New insights into plant glycoside hydrolase family 32 in Agave species

    Directory of Open Access Journals (Sweden)

    Emmanuel eAvila-de Dios

    2015-08-01

    Full Text Available In order to optimize the use of agaves for commercial applications, an understanding of fructan metabolism in these species at the molecular and genetic level is essential. Based on transcriptome data, this report describes the identification and molecular characterization of cDNAs and deduced amino acid sequences for genes encoding fructosyltransferases, invertases and fructan exohydrolases (enzymes belonging to plant glycoside hydrolase family 32 from four different agave species (A. tequilana, A. deserti, A. victoriae-reginae and A. striata. Conserved amino acid sequences and a hypervariable domain allowed classification of distinct isoforms for each enzyme type. Notably however neither 1-FFT nor 6-SFT encoding cDNAs were identified. In silico analysis revealed that distinct isoforms for certain enzymes found in a single species, showed different levels and tissue specific patterns of expression whereas in other cases expression patterns were conserved both within the species and between different species. Relatively high levels of in silico expression for specific isoforms of both invertases and fructosyltransferases were observed in floral tissues in comparison to vegetative tissues such as leaves and stems and this pattern was confirmed by Quantitative Real Time PCR using RNA obtained from floral and leaf tissue of A. tequilana. Thin layer chromatography confirmed the presence of fructans with degree of polymerization (DP greater than DP three in both immature buds and fully opened flowers also obtained from A. tequilana.

  7. The response to selection in Glycoside Hydrolase Family 13 structures: A comparative quantitative genetics approach.

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    Jose Sergio Hleap

    Full Text Available The Glycoside Hydrolase Family 13 (GH13 is both evolutionarily diverse and relevant to many industrial applications. Its members hydrolyze starch into smaller carbohydrates and members of the family have been bioengineered to improve catalytic function under industrial environments. We introduce a framework to analyze the response to selection of GH13 protein structures given some phylogenetic and simulated dynamic information. We find that the TIM-barrel (a conserved protein fold consisting of eight α-helices and eight parallel β-strands that alternate along the peptide backbone, common to all amylases is not selectable since it is under purifying selection. We also show a method to rank important residues with higher inferred response to selection. These residues can be altered to effect change in properties. In this work, we define fitness as inferred thermodynamic stability. We show that under the developed framework, residues 112Y, 122K, 124D, 125W, and 126P are good candidates to increase the stability of the truncated α-amylase protein from Geobacillus thermoleovorans (PDB code: 4E2O; α-1,4-glucan-4-glucanohydrolase; EC 3.2.1.1. Overall, this paper demonstrates the feasibility of a framework for the analysis of protein structures for any other fitness landscape.

  8. Analysis of Domain Architecture and Phylogenetics of Family 2 Glycoside Hydrolases (GH2.

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    David Talens-Perales

    Full Text Available In this work we report a detailed analysis of the topology and phylogenetics of family 2 glycoside hydrolases (GH2. We distinguish five topologies or domain architectures based on the presence and distribution of protein domains defined in Pfam and Interpro databases. All of them share a central TIM barrel (catalytic module with two β-sandwich domains (non-catalytic at the N-terminal end, but differ in the occurrence and nature of additional non-catalytic modules at the C-terminal region. Phylogenetic analysis was based on the sequence of the Pfam Glyco_hydro_2_C catalytic module present in most GH2 proteins. Our results led us to propose a model in which evolutionary diversity of GH2 enzymes is driven by the addition of different non-catalytic domains at the C-terminal region. This model accounts for the divergence of β-galactosidases from β-glucuronidases, the diversification of β-galactosidases with different transglycosylation specificities, and the emergence of bicistronic β-galactosidases. This study also allows the identification of groups of functionally uncharacterized protein sequences with potential biotechnological interest.

  9. Mechanism-based fluorescent labeling of beta-galactosidases. An efficient method in proteomics for glycoside hydrolases.

    Science.gov (United States)

    Kurogochi, Masaki; Nishimura, Shin-Ichiro; Lee, Yuan Chuan

    2004-10-22

    -259 is one of the nucleophiles in the active site. The present method can be readily extended to other glycosidases and should greatly aid the high throughput proteomics of many glycoside hydrolases showing both retaining- and inverting-type mechanisms.

  10. Engineering of family-5 glycoside hydrolase (Cel5A from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.

    Directory of Open Access Journals (Sweden)

    Amar A Telke

    Full Text Available Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.

  11. Comprehensive functional characterization of the glycoside hydrolase family 3 enzymes from Cellvibrio japonicus reveals unique metabolic roles in biomass saccharification.

    Science.gov (United States)

    Nelson, Cassandra E; Attia, Mohamed A; Rogowski, Artur; Morland, Carl; Brumer, Harry; Gardner, Jeffrey G

    2017-12-01

    Lignocellulose degradation is central to the carbon cycle and renewable biotechnologies. The xyloglucan (XyG), β(1→3)/β(1→4) mixed-linkage glucan (MLG) and β(1→3) glucan components of lignocellulose represent significant carbohydrate energy sources for saprophytic microorganisms. The bacterium Cellvibrio japonicus has a robust capacity for plant polysaccharide degradation, due to a genome encoding a large contingent of Carbohydrate-Active enZymes (CAZymes), many of whose specific functions remain unknown. Using a comprehensive genetic and biochemical approach, we have delineated the physiological roles of the four C. japonicus glycoside hydrolase family 3 (GH3) members on diverse β-glucans. Despite high protein sequence similarity and partially overlapping activity profiles on disaccharides, these β-glucosidases are not functionally equivalent. Bgl3A has a major role in MLG and sophorose utilization, and supports β(1→3) glucan utilization, while Bgl3B underpins cellulose utilization and supports MLG utilization. Bgl3C drives β(1→3) glucan utilization. Finally, Bgl3D is the crucial β-glucosidase for XyG utilization. This study not only sheds the light on the metabolic machinery of C. japonicus, but also expands the repertoire of characterized CAZymes for future deployment in biotechnological applications. In particular, the precise functional analysis provided here serves as a reference for informed bioinformatics on the genomes of other Cellvibrio and related species. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. Comprehensive functional characterization of the Glycoside Hydrolase Family 3 enzymes from Cellvibrio japonicus reveals unique metabolic roles in biomass saccharification

    International Nuclear Information System (INIS)

    Nelson, Cassandra E.; Attia, Mohamed A.; Rogowski, Artur; Morland, Carl; Brumer, Harry; Gardner, Jeffrey G.

    2017-01-01

    Here, lignocellulose degradation is central to the carbon cycle and renewable biotechnologies. The xyloglucan (XyG), β(1!3)/β(1!4) mixed-linkage glucan (MLG), and β(1!3) glucan components of lignocellulose represent significant carbohydrate energy sources for saprophytic microorganisms. The bacterium Cellvibrio japonicus has a robust capacity for plant polysaccharide degradation, due to a genome encoding a large contingent of Carbohydrate-Active Enzymes (CAZymes), many of whose specific functions remain unknown. Using a comprehensive genetic and biochemical approach we have delineated the physiological roles of the four C. japonicus Glycoside Hydrolase Family 3 (GH3) members on diverse β-glucans. Despite high protein sequence similarity and partially overlapping activity profiles on disaccharides, these β-glucosidases are not functionally equivalent. Bgl3A has a major role in MLG and sophorose utilization, and supports β(1!3) glucan utilization, while Bgl3B underpins cellulose utilization and supports MLG utilization. Bgl3C drives β(1!3) glucan utilization. Finally, Bgl3D is the crucial β-glucosidase for XyG utilization. This study not only sheds the light on the metabolic machinery of C. japonicus, but also expands the repertoire of characterized CAZymes for future deployment in biotechnological applications. In particular, the precise functional analysis provided here serves as a reference for informed bioinformatics on the genomes of other Cellvibrio and related species.

  13. Cloning, recombinant production, crystallization and preliminary X-ray diffraction studies of a family 84 glycoside hydrolase from Clostridium perfringens

    International Nuclear Information System (INIS)

    Ficko-Blean, Elizabeth; Boraston, Alisdair B.

    2005-01-01

    Crystallization of a family 84 glycoside hydrolase, a putative virulence factor, secreted by C. perfringens is reported. Clostridium perfringens is a ubiquitous environmental organism that is capable of causing a variety of diseases in mammals, including gas gangrene and necrotic enteritis in humans. The activity of a secreted hyaluronidase, attributed to the NagH protein, contributes to the pathogenicity of this organism. The family 84 catalytic module of one of the three homologues of NagH found in C. perfringens (ATCC 13124) has been cloned. The 69 kDa catalytic module of NagJ, here called GH84C, was overproduced in Escherichia coli and purified by immobilized metal-affinity chromatography (IMAC). Crystals belonging to space group I222 or I2 1 2 1 2 1 with unit-cell parameters a = 130.39, b = 150.05, c = 155.43 Å were obtained that diffracted to 2.1 Å. Selenomethionyl crystals have also been produced, leading to the possibility of solving the phase problem by MAD using synchrotron radiation

  14. Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of β-1,3-Glucan Quaternary Structure.

    Science.gov (United States)

    Pluvinage, Benjamin; Fillo, Alexander; Massel, Patricia; Boraston, Alisdair B

    2017-09-05

    Family 81 glycoside hydrolases (GHs), which are known to cleave β-1,3-glucans, are found in archaea, bacteria, eukaryotes, and viruses. Here we examine the structural and functional features of the GH81 catalytic module, BhGH81, from the Bacillus halodurans protein BH0236 to probe the molecular basis of β-1,3-glucan recognition and cleavage. BhGH81 displayed activity on laminarin, curdlan, and pachyman, but not scleroglucan; the enzyme also cleaved β-1,3-glucooligosaccharides as small as β-1,3-glucotriose. The crystal structures of BhGH81 in complex with various β-1,3-glucooligosaccharides revealed distorted sugars in the -1 catalytic subsite and an arrangement consistent with an inverting catalytic mechanism having a proposed conformational itinerary of 2 S 0 → 2,5 B ‡ → 5 S 1 . Notably, the architecture of the catalytic site, location of an adjacent ancillary β-1,3-glucan binding site, and the surface properties of the enzyme indicate the likely ability to recognize the double and/or triple-helical quaternary structures adopted by β-1,3-glucans. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. GH97 is a new family of glycoside hydrolases, which is related to the α-galactosidase superfamily

    Directory of Open Access Journals (Sweden)

    Naumoff Daniil G

    2005-08-01

    Full Text Available Abstract Background As a rule, about 1% of genes in a given genome encode glycoside hydrolases and their homologues. On the basis of sequence similarity they have been grouped into more than ninety GH families during the last 15 years. The GH97 family has been established very recently and initially included only 18 bacterial proteins. However, the evolutionary relationship of the genes encoding proteins of this family remains unclear, as well as their distribution among main groups of the living organisms. Results The extensive search of the current databases allowed us to double the number of GH97 family proteins. Five subfamilies were distinguished on the basis of pairwise sequence comparison and phylogenetic analysis. Iterative sequence analysis revealed the relationship of the GH97 family with the GH27, GH31, and GH36 families of glycosidases, which belong to the α-galactosidase superfamily, as well as a more distant relationship with some other glycosidase families (GH13 and GH20. Conclusion The results of this study show an unexpected sequence similarity of GH97 family proteins with glycoside hydrolases from several other families, that have (β/α8-barrel fold of the catalytic domain and a retaining mechanism of the glycoside bond hydrolysis. These data suggest a common evolutionary origin of glycosidases representing different families and clans.

  16. The crystal structure of an inverting glycoside hydrolase family 9 exo-β-D-glucosaminidase and the design of glycosynthase.

    Science.gov (United States)

    Honda, Yuji; Arai, Sachiko; Suzuki, Kentaro; Kitaoka, Motomitsu; Fushinobu, Shinya

    2016-02-15

    Exo-β-D-glucosaminidase (EC 3.2.1.165) from Photobacterium profundum (PpGlcNase) is an inverting GH (glycoside hydrolase) belonging to family 9. We have determined the three-dimensional structure of PpGlcNase to describe the first structure-function relationship of an exo-type GH9 glycosidase. PpGlcNase has a narrow and straight active-site pocket, in contrast with the long glycan-binding cleft of a GH9 endoglucanase. This is because PpGlcNase has a long loop, which blocks the position corresponding to subsites -4 to -2 of the endoglucanase. The pocket shape of PpGlcNase explains its substrate preference for a β1,4-linkage at the non-reducing terminus. Asp(139), Asp(143) and Glu(555) in the active site were located near the β-O1 hydroxy group of GlcN (D-glucosamine), with Asp(139) and Asp(143) holding a nucleophilic water molecule for hydrolysis. The D139A, D143A and E555A mutants significantly decreased hydrolytic activity, indicating their essential role. Of these mutants, D139A exclusively exhibited glycosynthase activity using α-GlcN-F (α-D-glucosaminyl fluoride) and GlcN as substrates, to produce (GlcN)2. Using saturation mutagenesis at Asp(139), we obtained D139E as the best glycosynthase. Compared with the wild-type, the hydrolytic activity of D139E was significantly suppressed (strategy for creating an effective glycosynthase from inverting GHs. However, for GH9, where two acidic residues seem to share the catalytic base role, mutation of Asp(139) might inevitably reduce F(-)-release activity. © 2016 Authors; published by Portland Press Limited.

  17. Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird?s-Nest Fern

    OpenAIRE

    Wu, Guan-Long; Kuo, Tzu-Hao; Tsay, Tung-Tsuan; Tsai, Isheng J.; Chen, Peichen J.

    2016-01-01

    Five Aphelenchoides besseyi isolates collected from bird's-nest ferns or rice possess different parasitic capacities in bird's-nest fern. Two different glycoside hydrolase (GH) 45 genes were identified in the fern isolates, and only one was found in the rice isolates. A Abe GH5-1 gene containing an SCP-like family domain was found only in the fern isolates. Abe GH5-1 gene has five introns suggesting a eukaryotic origin. A maximum likelihood phylogeny revealed that Abe GH5-1 is part of the nem...

  18. Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts.

    Science.gov (United States)

    Daenen, L; Saison, D; Sterckx, F; Delvaux, F R; Verachtert, H; Derdelinckx, G

    2008-02-01

    The aim of this study was to select and examine Saccharomyces and Brettanomyces brewing yeasts for hydrolase activity towards glycosidically bound volatile compounds. A screening for glucoside hydrolase activity of 58 brewing yeasts belonging to the genera Saccharomyces and Brettanomyces was performed. The studied Saccharomyces brewing yeasts did not show 1,4-beta-glucosidase activity, but a strain dependent beta-glucanase activity was observed. Some Brettanomyces species did show 1,4-beta-glucosidase activity. The highest constitutive activity was found in Brettanomyces custersii. For the most interesting strains the substrate specificity was studied and their activity was evaluated in fermentation experiments with added hop glycosides. Fermentations with Br. custersii led to the highest release of aglycones. Pronounced exo-beta-glucanase activity in Saccharomyces brewing yeasts leads to a higher release of certain aglycones. Certain Brettanomyces brewing yeasts, however, are more interesting for hydrolysis of glycosidically bound volatiles of hops. The release of flavour active compounds from hop glycosides opens perspectives for the bioflavouring and product diversification of beverages like beer. The release can be enhanced by using Saccharomyces strains with high exo-beta-glucanase activity. Higher activities can be found in Brettanomyces species with beta-glucosidase activity.

  19. Characterization of a novel theme C glycoside hydrolase family 9 cellulase and its CBM-chimeric enzymes.

    Science.gov (United States)

    Duan, Cheng-Jie; Huang, Ming-Yue; Pang, Hao; Zhao, Jing; Wu, Chao-Xing; Feng, Jia-Xun

    2017-07-01

    In bacterial cellulase systems, glycoside hydrolase family 9 (GH9) cellulases are generally regarded as the major cellulose-degrading factors besides GH48 exoglucanase. In this study, umcel9A, which was cloned from uncultured microorganisms from compost, with the encoded protein being theme C GH9 cellulase, was heterologously expressed in Escherichia coli, and the biochemical properties of the purified enzyme were characterized. Hydrolysis of carboxylmethylcellulose (CMC) by Umcel9A led to the decreased viscosity of CMC solution and production of reducing sugars. Interestingly, cellobiose was the major product when cellulosic materials were hydrolyzed by Umcel9A. Six representative carbohydrate-binding modules (CBMs) from different CBM families (CBM1, CBM2, CBM3, CBM4, CBM10, and CBM72) were fused with Umcel9A at the natural terminal position, resulting in significant enhancement of the binding capacity of the chimeric enzymes toward four different insoluble celluloses as compared with that of Umcel9A. Catalytic activity of the chimeric enzymes against insoluble celluloses, including phosphoric acid-swollen cellulose (PASC), alkali-pretreated sugarcane bagasse (ASB), filter paper powder (FPP), and Avicel, was higher than that of Umcel9A, except for Umcel9A-CBM3. In these chimeric enzymes, CBM4-Umcel9A exhibited the highest activity toward the four tested insoluble celluloses and displayed 4.2-, 3.0-, 2.4-, and 6.6-fold enhanced activity toward PASC, ASB, FPP, and Avicel, respectively, when compared with that of Umcel9A. CBM4-Umcel9A also showed highest V max and catalytic efficiency (k cat /K M ) against PASC. Construction of chimeric enzymes may have potential applications in biocatalytic processes and provides insight into the evolution of the molecular architecture of catalytic module and CBM in GH9 cellulases.

  20. Diverse modes of galacto-specific carbohydrate recognition by a family 31 glycoside hydrolase from Clostridium perfringens.

    Directory of Open Access Journals (Sweden)

    Julie M Grondin

    Full Text Available Clostridium perfringens is a commensal member of the human gut microbiome and an opportunistic pathogen whose genome encodes a suite of putative large, multi-modular carbohydrate-active enzymes that appears to play a role in the interaction of the bacterium with mucin-based carbohydrates. Among the most complex of these is an enzyme that contains a presumed catalytic module belonging to glycoside hydrolase family 31 (GH31. This large enzyme, which based on its possession of a GH31 module is a predicted α-glucosidase, contains a variety of non-catalytic ancillary modules, including three CBM32 modules that to date have not been characterized. NMR-based experiments demonstrated a preference of each module for galacto-configured sugars, including the ability of all three CBM32s to recognize the common mucin monosaccharide GalNAc. X-ray crystal structures of the CpGH31 CBM32s, both in apo form and bound to GalNAc, revealed the finely-tuned molecular strategies employed by these sequentially variable CBM32s in coordinating a common ligand. The data highlight that sequence similarities to previously characterized CBMs alone are insufficient for identifying the molecular mechanism of ligand binding by individual CBMs. Furthermore, the overlapping ligand binding profiles of the three CBMs provide a fail-safe mechanism for the recognition of GalNAc among the dense eukaryotic carbohydrate networks of the colonic mucosa. These findings expand our understanding of ligand targeting by large, multi-modular carbohydrate-active enzymes, and offer unique insights into of the expanding ligand-binding preferences and binding site topologies observed in CBM32s.

  1. Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases.

    Science.gov (United States)

    Brulc, Jennifer M; Antonopoulos, Dionysios A; Miller, Margret E Berg; Wilson, Melissa K; Yannarell, Anthony C; Dinsdale, Elizabeth A; Edwards, Robert E; Frank, Edward D; Emerson, Joanne B; Wacklin, Pirjo; Coutinho, Pedro M; Henrissat, Bernard; Nelson, Karen E; White, Bryan A

    2009-02-10

    The complex microbiome of the rumen functions as an effective system for the conversion of plant cell wall biomass to microbial protein, short chain fatty acids, and gases. As such, it provides a unique genetic resource for plant cell wall degrading microbial enzymes that could be used in the production of biofuels. The rumen and gastrointestinal tract harbor a dense and complex microbiome. To gain a greater understanding of the ecology and metabolic potential of this microbiome, we used comparative metagenomics (phylotype analysis and SEED subsystems-based annotations) to examine randomly sampled pyrosequence data from 3 fiber-adherent microbiomes and 1 pooled liquid sample (a mixture of the liquid microbiome fractions from the same bovine rumens). Even though the 3 animals were fed the same diet, the community structure, predicted phylotype, and metabolic potentials in the rumen were markedly different with respect to nutrient utilization. A comparison of the glycoside hydrolase and cellulosome functional genes revealed that in the rumen microbiome, initial colonization of fiber appears to be by organisms possessing enzymes that attack the easily available side chains of complex plant polysaccharides and not the more recalcitrant main chains, especially cellulose. Furthermore, when compared with the termite hindgut microbiome, there are fundamental differences in the glycoside hydrolase content that appear to be diet driven for either the bovine rumen (forages and legumes) or the termite hindgut (wood).

  2. From Soil to Structure, a Novel Dimeric β-Glucosidase Belonging to Glycoside Hydrolase Family 3 Isolated from Compost Using Metagenomic Analysis

    Science.gov (United States)

    McAndrew, Ryan P.; Park, Joshua I.; Heins, Richard A.; Reindl, Wolfgang; Friedland, Gregory D.; D'haeseleer, Patrik; Northen, Trent; Sale, Kenneth L.; Simmons, Blake A.; Adams, Paul D.

    2013-01-01

    A recent metagenomic analysis sequenced a switchgrass-adapted compost community to identify enzymes from microorganisms that were specifically adapted to switchgrass under thermophilic conditions. These enzymes are being examined as part of the pretreatment process for the production of “second-generation” biofuels. Among the enzymes discovered was JMB19063, a novel three-domain β-glucosidase that belongs to the GH3 (glycoside hydrolase 3) family. Here, we report the structure of JMB19063 in complex with glucose and the catalytic variant D261N crystallized in the presence of cellopentaose. JMB19063 is first structure of a dimeric member of the GH3 family, and we demonstrate that dimerization is required for catalytic activity. Arg-587 and Phe-598 from the C-terminal domain of the opposing monomer are shown to interact with bound ligands in the D261N structure. Enzyme assays confirmed that these residues are absolutely essential for full catalytic activity. PMID:23580647

  3. Key aromatic residues at subsites +2 and +3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates

    DEFF Research Database (Denmark)

    Tagami, Takayoshi; Okuyama, Masayuki; Nakai, Hiroyuki

    2013-01-01

    Glycoside hydrolase family 31 α-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger α-glucosidase (ANG) is specific for short-chain substrates with the highest kcat/Km for maltotriose, while sugar beet α-glucosidase (SBG) prefers...

  4. Enzymatic synthesis of β-xylosyl-oligosaccharides by transxylosylation using two beta-xylosidases of glycoside hydrolase family 3 from Aspergillus nidulans FGSC A4

    DEFF Research Database (Denmark)

    Dilokpimol, Adiphol; Nakai, Hiroyuki; Gotfredsen, Charlotte Held

    2011-01-01

    Two beta-xylosidases of glycoside hydrolase family 3 (GH 3) from Aspergillus nidulans FGSC A4, BxlA and BxlB were produced recombinantly in Pichia pastoris and secreted to the culture supernatants in yields of 16 and 118 mg/L, respectively. BxlA showed about sixfold higher catalytic efficiency (k...

  5. Mining novel starch-converting Glycoside Hydrolase 70 enzymes from the Nestlé Culture Collection genome database : The Lactobacillus reuteri NCC 2613 GtfB

    NARCIS (Netherlands)

    Gangoiti, Joana; van Leeuwen, Sander S.; Meng, Xiangfeng; Duboux, Stéphane; Vafiadi, Christina; Pijning, Tjaard; Dijkhuizen, Lubbert

    2017-01-01

    The Glycoside hydrolase (GH) family 70 originally was established for glucansucrases of lactic acid bacteria (LAB) converting sucrose into α-glucan polymers. In recent years we have identified 3 subfamilies of GH70 enzymes (designated GtfB, GtfC and GtfD) as 4,6-α-glucanotransferases, cleaving

  6. The phosducin-like protein PhLP1 impacts regulation of glycoside hydrolases and light response in Trichoderma reesei

    Directory of Open Access Journals (Sweden)

    Tisch Doris

    2011-12-01

    Full Text Available Abstract Background In the biotechnological workhorse Trichoderma reesei (Hypocrea jecorina transcription of cellulase genes as well as efficiency of the secreted cellulase mixture are modulated by light. Components of the heterotrimeric G-protein pathway interact with light-dependent signals, rendering this pathway a key regulator of cellulase gene expression. Results As regulators of heterotrimeric G-protein signaling, class I phosducin-like proteins, are assumed to act as co-chaperones for G-protein beta-gamma folding and exert their function in response to light in higher eukaryotes. Our results revealed light responsive transcription of the T. reesei class I phosducin-like protein gene phlp1 and indicate a light dependent function of PhLP1 also in fungi. We showed the functions of PhLP1, GNB1 and GNG1 in the same pathway, with one major output being the regulation of transcription of glycoside hydrolase genes including cellulase genes in T. reesei. We found no direct correlation between the growth rate and global regulation of glycoside hydrolases, which suggests that regulation of growth does not occur only at the level of substrate degradation efficiency. Additionally, PhLP1, GNB1 and GNG1 are all important for proper regulation of light responsiveness during long term exposure. In their absence, the amount of light regulated genes increased from 2.7% in wild type to 14% in Δphlp1. Besides from the regulation of degradative enzymes, PhLP1 was also found to impact on the transcription of genes involved in sexual development, which was in accordance with decreased efficiency of fruiting body formation in Δphlp1. The lack of GNB1 drastically diminished ascospore discharge in T. reesei. Conclusions The heterotrimeric G-protein pathway is crucial for the interconnection of nutrient signaling and light response of T. reesei, with the class I phosducin-like protein PhLP1, GNB1 and GNG1 acting as important nodes, which influence light

  7. Anticancer Activity of Sea Cucumber Triterpene Glycosides

    Directory of Open Access Journals (Sweden)

    Dmitry L. Aminin

    2015-03-01

    Full Text Available Triterpene glycosides are characteristic secondary metabolites of sea cucumbers (Holothurioidea, Echinodermata. They have hemolytic, cytotoxic, antifungal, and other biological activities caused by membranotropic action. These natural products suppress the proliferation of various human tumor cell lines in vitro and, more importantly, intraperitoneal administration in rodents of solutions of some sea cucumber triterpene glycosides significantly reduces both tumor burden and metastasis. The anticancer molecular mechanisms include the induction of tumor cell apoptosis through the activation of intracellular caspase cell death pathways, arrest of the cell cycle at S or G2/M phases, influence on nuclear factors, NF-κB, and up-down regulation of certain cellular receptors and enzymes participating in cancerogenesis, such as EGFR (epidermal growth factor receptor, Akt (protein kinase B, ERK (extracellular signal-regulated kinases, FAK (focal adhesion kinase, MMP-9 (matrix metalloproteinase-9 and others. Administration of some glycosides leads to a reduction of cancer cell adhesion, suppression of cell migration and tube formation in those cells, suppression of angiogenesis, inhibition of cell proliferation, colony formation and tumor invasion. As a result, marked growth inhibition of tumors occurs in vitro and in vivo. Some holothurian triterpene glycosides have the potential to be used as P-gp mediated MDR reversal agents in combined therapy with standard cytostatics.

  8. Hydrolase activity in Jerusalem artichoke and chicory

    Energy Technology Data Exchange (ETDEWEB)

    Klaushofer, H.; Abraham, B.; Leichtfried, G.

    1988-03-01

    Post-harvest storage of chicory and Jerusalem artichoke and overwintering of Jerusalem artichoke in the soil cause a more or less pronounced shortening of the fructan chain, depending on the variety. The proportion of fructose in the total fructan thus shifts towards glucose. This reduction on the fructose/glucose ratio is undesirable if the intention is to obtain a sweetener of high fructose content. In this work an attempt was made, via the quantity of fructose formed after a 4(3)-hour reaction of a tuber (root) extract with inulin, to assign a characteristic value to the depolymerization tendency of the material in question. However, since the plant extract not only contains enzymes (hydrolase A and B) that shorten the fructan chains but the activity of fructosyltransferase (SST, FFT) and enzymes of microbial origin (inulinase II, invertase) must also be considered, the concept of 'hydrolase activity' used by the authors is essentially an expression of 'total activity'. The activity unit (EU) is defined as the ability to split of 1 ..mu..mol of fructose from (chicory) inulin per minute under experimental conditions. Values of 0.25 to 0.77 EU/g dry solids were found in Jerusalem artichoke. Considerable differences may occur between varieties from the same cultivated area and the same harvest period. With one and the same variety, the activity appears to be subject to marked yearly fluctuations, so that at present, because of hydrolase activity, nothing certain can be said about the depolymerization tendency of a variety.

  9. Streptococcus pneumoniae Endohexosaminidase D, Structural and Mechanistic Insight into Substrate-Assisted Catalysis in Family 85 Glycoside Hydrolases

    International Nuclear Information System (INIS)

    Abbott, D.; Macauley, M.; Vocadlo, D.; Boraston, A.

    2009-01-01

    Endo-?-d-glucosaminidases from family 85 of glycoside hydrolases (GH85 endohexosaminidases) act to cleave the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. Endohexosaminidase D (Endo-D), produced by Streptococcus pneumoniae, is believed to contribute to the virulence of this organism by playing a role in the deglycosylation of IgG antibodies. Endohexosaminidases have received significant attention for this reason and, moreover, because they are powerful tools for chemoenzymatic synthesis of proteins having defined glycoforms. Here we describe mechanistic and structural studies of the catalytic domain (SpGH85) of Endo-D that provide compelling support for GH85 enzymes using a catalytic mechanism involving substrate-assisted catalysis. Furthermore, the structure of SpGH85 in complex with the mechanism-based competitive inhibitor NAG-thiazoline (Kd = 28 ?m) provides a coherent rationale for previous mutagenesis studies of Endo-D and other related GH85 enzymes. We also find GH85, GH56, and GH18 enzymes have a similar configuration of catalytic residues. Notably, GH85 enzymes have an asparagine in place of the aspartate residue found in these other families of glycosidases. We propose that this residue, as the imidic acid tautomer, acts analogously to the key catalytic aspartate of GH56 and GH18 enzymes. This topographically conserved arrangement of the asparagine residue and a conserved glutamic acid, coupled with previous kinetic studies, suggests these enzymes may use an unusual proton shuttle to coordinate effective general acid and base catalysis to aid cleavage of the glycosidic bond. These results collectively provide a blueprint that may be used to facilitate protein engineering of these enzymes to improve their function as biocatalysts for synthesizing glycoproteins having defined glycoforms and also may serve as a guide for generating inhibitors of GH85 enzymes.

  10. Functional characterization and target discovery of glycoside hydrolases from the digestome of the lower termite Coptotermes gestroi

    Directory of Open Access Journals (Sweden)

    Franco Cairo João Paulo L

    2011-11-01

    Full Text Available Abstract Background Lignocellulosic materials have been moved towards the forefront of the biofuel industry as a sustainable resource. However, saccharification and the production of bioproducts derived from plant cell wall biomass are complex and lengthy processes. The understanding of termite gut biology and feeding strategies may improve the current state of biomass conversion technology and bioproduct production. Results The study herein shows comprehensive functional characterization of crude body extracts from Coptotermes gestroi along with global proteomic analysis of the termite's digestome, targeting the identification of glycoside hydrolases and accessory proteins responsible for plant biomass conversion. The crude protein extract from C. gestroi was enzymatically efficient over a broad pH range on a series of natural polysaccharides, formed by glucose-, xylose-, mannan- and/or arabinose-containing polymers, linked by various types of glycosidic bonds, as well as ramification types. Our proteomic approach successfully identified a large number of relevant polypeptides in the C. gestroi digestome. A total of 55 different proteins were identified and classified into 29 CAZy families. Based on the total number of peptides identified, the majority of components found in the C. gestroi digestome were cellulose-degrading enzymes. Xylanolytic enzymes, mannan- hydrolytic enzymes, pectinases and starch-degrading and debranching enzymes were also identified. Our strategy enabled validation of liquid chromatography with tandem mass spectrometry recognized proteins, by enzymatic functional assays and by following the degradation products of specific 8-amino-1,3,6-pyrenetrisulfonic acid labeled oligosaccharides through capillary zone electrophoresis. Conclusions Here we describe the first global study on the enzymatic repertoire involved in plant polysaccharide degradation by the lower termite C. gestroi. The biochemical characterization of whole

  11. Purification and enzymatic characterization of secretory glycoside hydrolase family 3 (GH3) aryl β-glucosidases screened from Aspergillus oryzae genome.

    Science.gov (United States)

    Kudo, Kanako; Watanabe, Akira; Ujiie, Seiryu; Shintani, Takahiro; Gomi, Katsuya

    2015-12-01

    By a global search of the genome database of Aspergillus oryzae, we found 23 genes encoding putative β-glucosidases, among which 10 genes with a signal peptide belonging to glycoside hydrolase family 3 (GH3) were overexpressed in A. oryzae using the improved glaA gene promoter. Consequently, crude enzyme preparations from three strains, each harboring the genes AO090038000223 (bglA), AO090103000127 (bglF), and AO090003001511 (bglJ), showed a substrate preference toward p-nitrophenyl-β-d-glucopyranoside (pNPGlc) and thus were purified to homogeneity and enzymatically characterized. All the purified enzymes (BglA, BglF, and BglJ) preferentially hydrolyzed aryl β-glycosides, including pNPGlc, rather than cellobiose, and these enzymes were proven to be aryl β-glucosidases. Although the specific activity of BglF toward all the substrates tested was significantly low, BglA and BglJ showed appreciably high activities toward pNPGlc and arbutin. The kinetic parameters of BglA and BglJ for pNPGlc suggested that both the enzymes had relatively higher hydrolytic activity toward pNPGlc among the fungal β-glucosidases reported. The thermal and pH stabilities of BglA were higher than those of BglJ, and BglA was particularly stable in a wide pH range (pH 4.5-10). In contrast, BglJ was the most heat- and alkaline-labile among the three β-glucosidases. Furthermore, BglA was more tolerant to ethanol than BglJ; as a result, it showed much higher hydrolytic activity toward isoflavone glycosides in the presence of ethanol than BglJ. This study suggested that the mining of novel β-glucosidases exhibiting higher activity from microbial genome sequences is of great use for the production of beneficial compounds such as isoflavone aglycones. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. A metagenome-derived thermostable β-glucanase with an unusual module architecture which defines the new glycoside hydrolase family GH148.

    Science.gov (United States)

    Angelov, Angel; Pham, Vu Thuy Trang; Übelacker, Maria; Brady, Silja; Leis, Benedikt; Pill, Nicole; Brolle, Judith; Mechelke, Matthias; Moerch, Matthias; Henrissat, Bernard; Liebl, Wolfgang

    2017-12-11

    The discovery of novel and robust enzymes for the breakdown of plant biomass bears tremendous potential for the development of sustainable production processes in the rapidly evolving new bioeconomy. By functional screening of a metagenomic library from a volcano soil sample a novel thermostable endo-β-glucanase (EngU) which is unusual with regard to its module architecture and cleavage specificity was identified. Various recombinant EngU variants were characterized. Assignment of EngU to an existing glycoside hydrolase (GH) family was not possible. Two regions of EngU showed weak sequence similarity to proteins of the GH clan GH-A, and acidic residues crucial for catalytic activity of EngU were identified by mutation. Unusual, a carbohydrate-binding module (CBM4) which displayed binding affinity for β-glucan, lichenin and carboxymethyl-cellulose was found as an insertion between these two regions. EngU hydrolyzed β-1,4 linkages in carboxymethyl-cellulose, but displayed its highest activity with mixed linkage (β-1,3-/β-1,4-) glucans such as barley β-glucan and lichenin, where in contrast to characterized lichenases cleavage occurred predominantly at the β-1,3 linkages of C4-substituted glucose residues. EngU and numerous related enzymes with previously unknown function represent a new GH family of biomass-degrading enzymes within the GH-A clan. The name assigned to the new GH family is GH148.

  13. Structure of a bacterial glycoside hydrolase family 63 enzyme in complex with its glycosynthase product, and insights into the substrate specificity.

    Science.gov (United States)

    Miyazaki, Takatsugu; Ichikawa, Megumi; Yokoi, Gaku; Kitaoka, Motomitsu; Mori, Haruhide; Kitano, Yoshikazu; Nishikawa, Atsushi; Tonozuka, Takashi

    2013-09-01

    Proteins belonging to glycoside hydrolase family 63 (GH63) are found in bacteria, archaea and eukaryotes. Although the eukaryotic GH63 proteins have been identified as processing α-glucosidase I, the substrate specificities of the bacterial and archaeal GH63 proteins are not clear. Here, we converted a bacterial GH63 enzyme, Escherichia coli YgjK, to a glycosynthase to probe its substrate specificity. Two mutants of YgjK (E727A and D324N) were constructed, and both mutants showed glycosynthase activity. The reactions of E727A with β-D-glucosyl fluoride and monosaccharides showed that the largest amount of glycosynthase product accumulated when galactose was employed as an acceptor molecule. The crystal structure of E727A complexed with the reaction product indicated that the disaccharide bound at the active site was 2-O-α-D-glucopyranosyl-α-D-galactopyranose (Glc12Gal). A comparison of the structures of E727A-Glc12Gal and D324N-melibiose showed that there were two main types of conformation: the open and closed forms. The structure of YgjK adopted the closed form when subsite -1 was occupied by glucose. These results suggest that sugars containing the Glc12Gal structure are the most likely candidates for natural substrates of YgjK. © 2013 FEBS.

  14. A Trapped Covalent Intermediate of a Glycoside Hydrolase on the Pathway to Transglycosylation. Insights from Experiments and Quantum Mechanics/Molecular Mechanics Simulations.

    Science.gov (United States)

    Raich, Lluís; Borodkin, Vladimir; Fang, Wenxia; Castro-López, Jorge; van Aalten, Daan M F; Hurtado-Guerrero, Ramón; Rovira, Carme

    2016-03-16

    The conversion of glycoside hydrolases (GHs) into transglycosylases (TGs), i.e., from enzymes that hydrolyze carbohydrates to enzymes that synthesize them, represents a promising solution for the large-scale synthesis of complex carbohydrates for biotechnological purposes. However, the lack of knowledge about the molecular details of transglycosylation hampers the rational design of TGs. Here we present the first crystallographic structure of a natural glycosyl-enzyme intermediate (GEI) of Saccharomyces cerevisiae Gas2 in complex with an acceptor substrate and demonstrate, by means of quantum mechanics/molecular mechanics metadynamics simulations, that it is tuned for transglycosylation (ΔG(⧧) = 12 kcal/mol). The 2-OH···nucleophile interaction is found to be essential for catalysis: its removal raises the free energy barrier significantly (11 and 16 kcal/mol for glycosylation and transglycosylation, respectively) and alters the conformational itinerary of the substrate (from (4)C1 → [(4)E](⧧) → (1,4)B/(4)E to (4)C1 → [(4)H3](⧧) → (4)C1). Our results suggest that changes in the interactions involving the 2-position could have an impact on the transglycosylation activity of several GHs.

  15. Multiple rewards from a treasure trove of novel glycoside hydrolase and polysaccharide lyase structures: new folds, mechanistic details, and evolutionary relationships.

    Science.gov (United States)

    Fushinobu, Shinya; Alves, Victor D; Coutinho, Pedro M

    2013-10-01

    Recent progress in three-dimensional structure analyses of glycoside hydrolases (GHs) and polysaccharide lyases (PLs), the historically relevant enzyme classes involved in the cleavage of glycosidic bonds of carbohydrates and glycoconjugates, is reviewed. To date, about 80% and 95% of the GH and PL families, respectively, have a representative crystal structure. New structures have been determined for enzymes acting on plant cell wall polysaccharides, sphingolipids, blood group antigens, milk oligosaccharides, N-glycans, oral biofilms and dietary seaweeds. Some GH enzymes have very unique catalytic residues such as the Asp-His dyad. New methods such as high-speed atomic force microscopy and computational simulation have opened up a path to investigate both the dynamics and the detailed molecular interactions displayed by these enzymes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-α-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers.

    Science.gov (United States)

    Bai, Yuxiang; van der Kaaij, Rachel Maria; Leemhuis, Hans; Pijning, Tjaard; van Leeuwen, Sander Sebastiaan; Jin, Zhengyu; Dijkhuizen, Lubbert

    2015-10-01

    4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkages but mostly (relatively long) linear chains with α1→6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-ΔN (approximately 75-fold compared to full-length wild type GTFB) in Escherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-α-GTase enzymes. The data show that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. Glycoside hydrolase family 13 α-glucosidases encoded by Bifidobacterium breve UCC2003; A comparative analysis of function, structure and phylogeny.

    Science.gov (United States)

    Kelly, Emer D; Bottacini, Francesca; O'Callaghan, John; Motherway, Mary O'Connell; O'Connell, Kerry Joan; Stanton, Catherine; van Sinderen, Douwe

    2016-05-02

    Bifidobacterium breve is a noted inhabitant and one of the first colonizers of the human gastro intestinal tract (GIT). The ability of this bacterium to persist in the GIT is reflected by the abundance of carbohydrate-active enzymes that are encoded by its genome. One such family of enzymes is represented by the α-glucosidases, of which three, Agl1, Agl2 and MelD, have previously been identified and characterized in the prototype B. breve strain UCC2003. In this report, we describe an additional B. breve UCC2003-encoded α-glucosidase, along with a B. breve UCC2003-encoded α-glucosidase-like protein, designated here as Agl3 and Agl4, respectively, which together with the three previously described enzymes belong to glycoside hydrolase (GH) family 13. Agl3 was shown to exhibit hydrolytic specificity towards the α-(1→6) linkage present in palatinose; the α-(1→3) linkage present in turanose; the α-(1→4) linkages found in maltotriose and maltose; and to a lesser degree, the α-(1→2) linkage found in sucrose and kojibiose; and the α-(1→5) linkage found in leucrose. Surprisingly, based on the substrates analyzed, Agl4 did not exhibit biologically relevant α-glucosidic activity. With the presence of four functionally active GH13 α-glucosidases, B. breve UCC2003 is capable of hydrolyzing all α-glucosidic linkages that can be expected in glycan substrates in the lower GIT. This abundance of α-glucosidases provides B. breve UCC2003 with an adaptive ability and metabolic versatility befitting the transient nature of growth substrates in the GIT. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

    Science.gov (United States)

    John, George; Nagarajan, Subbiah; Chapman, Kent; Faure, Lionel; Koulen, Peter

    2016-10-25

    A method for enhancing amidohydrolase activity of Fatty Acid Amide Hydrolase (FAAH) is disclosed. The method comprising administering a phenoxyacylethanolamide that causes the enhanced activity. The enhanced activity can have numerous effects on biological organisms including, for example, enhancing the growth of certain seedlings. The subject matter disclosed herein relates to enhancers of amidohydrolase activity.

  19. Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

    Science.gov (United States)

    John, George; Nagarajan, Subbiah; Chapman, Kent; Faure, Lionel; Koulen, Peter

    2017-12-26

    A method for enhancing amidohydrolase activity of Fatty Acid Amide Hydrolase (FAAH) is disclosed. The method comprising administering a phenoxyacyl-ethanolamide that causes the enhanced activity. The enhanced activity can have numerous effects on biological organisms including, for example, enhancing the growth of certain seedlings.

  20. The gram-negative bacterium Azotobacter chroococcum NCIMB 8003 employs a new glycoside hydrolase family 70 4,6-α-glucanotransferase enzyme (GtfD) to synthesize a reuteran like polymer from maltodextrins and starch

    NARCIS (Netherlands)

    Gangoiti, Joana; van Leeuwen, Sander S; Vafiadi, Christina; Dijkhuizen, Lubbert

    BACKGROUND: Originally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize α-glucan polymers from sucrose. Recently we have identified 2 novel subfamilies of GH70 enzymes represented by the Lactobacillus reuteri 121 GtfB and the

  1. The Exiguobacterium sibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes.

    Science.gov (United States)

    Gangoiti, Joana; Pijning, Tjaard; Dijkhuizen, Lubbert

    2016-01-15

    The glycoside hydrolase 70 (GH70) family originally was established for glucansucrase enzymes found solely in lactic acid bacteria synthesizing α-glucan polysaccharides from sucrose (e.g., GtfA). In recent years, we have characterized GtfB and related Lactobacillus enzymes as 4,6-α-glucanotransferase enzymes. These GtfB-type enzymes constitute the first GH70 subfamily of enzymes that are unable to act on sucrose as a substrate but are active with maltodextrins and starch, cleave α1→4 linkages, and synthesize linear α1→6-glucan chains. The GtfB disproportionating type of activity results in the conversion of malto-oligosaccharides into isomalto/malto-polysaccharides with a relatively high percentage of α1→6 linkages. This paper reports the identification of the members of a second GH70 subfamily (designated GtfC enzymes) and the characterization of the Exiguobacterium sibiricum 255-15 GtfC enzyme, which is also inactive with sucrose and displays 4,6-α-glucanotransferase activity with malto-oligosaccharides. GtfC differs from GtfB in synthesizing isomalto/malto-oligosaccharides. Biochemically, the GtfB- and GtfC-type enzymes are related, but phylogenetically, they clearly constitute different GH70 subfamilies, displaying only 30% sequence identity. Whereas the GtfB-type enzyme largely has the same domain order as glucansucrases (with α-amylase domains A, B, and C plus domains IV and V), this GtfC-type enzyme differs in the order of these domains and completely lacks domain V. In GtfC, the sequence of conserved regions I to IV of clan GH-H is identical to that in GH13 (I-II-III-IV) but different from that in GH70 (II-III-IV-I because of a circular permutation of the (β/α)8 barrel. The GtfC 4,6-α-glucanotransferase enzymes thus represent structurally and functionally very interesting evolutionary intermediates between α-amylase and glucansucrase enzymes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  2. The Differential Proteome of the Probiotic Lactobacillus acidophilus NCFM Grown on the Potential Prebiotic Cellobiose Shows Upregulation of Two beta-Glycoside Hydrolases

    DEFF Research Database (Denmark)

    van Zanten, Gabriella Christina; Sparding, Nadja; Majumder, Avishek

    2015-01-01

    Probiotics, prebiotics, and combinations there of, that is, synbiotics, are known to exert beneficial health effects in humans; however interactions between pro-and prebiotics remain poorly understood at the molecular level. The present study describes changes in abundance of different proteins...... of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) when grown on the potential prebiotic cellobiose as compared to glucose. Cytosolic cell extract proteomes after harvest at late exponential phase of NCFM grown on cellobiose or glucose were analyzed by two dimensional difference gel electrophoresis....... Many of these proteins were associated with energy metabolism, including the cellobiose related glycoside hydrolases phospho-β-glucosidase (LBA0881) and phospho-β-galactosidase II (LBA0726). The data provide insight into the utilization of the candidate prebiotic cellobiose by the probiotic bacterium...

  3. Apoptotic activities of cardenolide glycosides from Asclepias subulata.

    Science.gov (United States)

    Rascón-Valenzuela, L A; Velázquez, C; Garibay-Escobar, A; Vilegas, W; Medina-Juárez, L A; Gámez-Meza, N; Robles-Zepeda, R E

    2016-12-04

    Asclepias subulata Decne. (Apocynaceae) is a shrub occurring in Sonora-Arizona desert. The ethnic groups of Sonora, Mexico, Seris and Pimas, use this plant for the treatment of sore eyes, gastrointestinal disorders and cancer. To determine the cell death pathways that the cardenolide glycosides with antiproliferative activity found in the methanol extract of A. subulata are able to activate. The effect of cardenolide glycosides isolated of A. subulata on induction of apoptosis in cancer cells was evaluated through the measuring of several key events of apoptosis. A549 cells were treated for 12h with doses of 3.0, 0.2, 3.0 and 1.0µM of 12, 16-dihydroxicalotropin, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin, respectively. Apoptotic and necrotic cell levels were measured by double staining with annexin V-FITC/PI. Mitochondrial membrane depolarization was examined through JC-1 staining. Apoptosis cell death and the apoptosis pathways activated by cardenolide glycosides isolated of A. subulata were further characterized by the measurement of caspase-3, caspase-8 and caspase-9 activity. Apoptotic assays showed that the four cardenolide glycosides isolated of A. subulata induced apoptosis in A549 cells, which was evidencing by phosphatidylserine externalization in 18.2%, 17.0%, 23.9% and 22.0% for 12, 16-dihydroxicalotropin, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin, respectively, compared with 4.6% of control cells. Cell death was also associated with a decrease in mitochondrial membrane potential, which was more than 75% in the treated cultures respect to control. The activation of caspase-3 was observed in all cardenolide glycosides-treated cancer cells indicating the caspase-dependent apoptosis of A549 cells. Extrinsic and intrinsic apoptosis pathways were activated by cardenolide glycosides treatment at the doses tested. In this study was found that cardenolide glycosides, 12, 16-dihydroxicalotropin, calotropin

  4. The apo structure of sucrose hydrolase from Xanthomonas campestris pv. campestris shows an open active-site groove

    DEFF Research Database (Denmark)

    Champion, Elise; Remaud-Simeon, Magali; Skov, Lars Kobberøe

    2009-01-01

    Glycoside hydrolase family 13 (GH-13) mainly contains starch-degrading or starch-modifying enzymes. Sucrose hydrolases utilize sucrose instead of amylose as the primary glucosyl donor. Here, the catalytic properties and X-ray structure of sucrose hydrolase from Xanthomonas campestris pv. campestris...... of GH-13. Comparisons with structures of the highly similar sucrose hydrolase from X. axonopodis pv. glycines most notably showed that residues Arg516 and Asp138, which form a salt bridge in the X. axonopodis sucrose complex and define part of the subsite -1 glucosyl-binding determinants...

  5. Glycoside Hydrolase MoGls2 Controls Asexual/Sexual Development, Cell Wall Integrity and Infectious Growth in the Rice Blast Fungus.

    Directory of Open Access Journals (Sweden)

    Mengying Li

    Full Text Available N-linked glycosylation is a way of glycosylation for newly synthesized protein, which plays a key role in the maturation and transport of proteins. Glycoside hydrolases (GHs are essential in this process, and are involved in processing of N-linked glycoproteins or degradation of carbohydrate structures. Here, we identified and characterized MoGls2 in Magnaporthe oryzae, which is a yeast glucosidase II homolog Gls2 and is required for trimming the final glucose in N-linked glycans and normal cell wall synthesis. Target deletion of MoGLS2 in M. oryzae resulted in a reduced mycelial growth, an increased conidial production, delayed conidial germination and loss the ability of sexual reproduction. Pathogenicity assays revealed that the ΔMogls2 mutant showed significantly decreased in virulence and infectious growth. Further studies showed that the mutant was less sensitive to salt and osmotic stress, and increased sensitivity to cell wall stresses. Additionally, the ΔMogls2 mutant showed a defect in cell wall integrity. Our results indicate that MoGls2 is a key protein for the growth and development of M. oryzae, involving in the regulation of asexual/sexual development, stress response, cell wall integrity and infectious growth.

  6. The Differential Proteome of the Probiotic Lactobacillus acidophilus NCFM Grown on the Potential Prebiotic Cellobiose Shows Upregulation of Two β-Glycoside Hydrolases

    Directory of Open Access Journals (Sweden)

    Gabriella C. van Zanten

    2015-01-01

    Full Text Available Probiotics, prebiotics, and combinations thereof, that is, synbiotics, are known to exert beneficial health effects in humans; however interactions between pro- and prebiotics remain poorly understood at the molecular level. The present study describes changes in abundance of different proteins of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM when grown on the potential prebiotic cellobiose as compared to glucose. Cytosolic cell extract proteomes after harvest at late exponential phase of NCFM grown on cellobiose or glucose were analyzed by two dimensional difference gel electrophoresis (2D-DIGE in the acidic (pH 4–7 and the alkaline (pH 6–11 regions showing a total of 136 spots to change in abundance. Proteins were identified by MS or MS/MS from 81 of these spots representing 49 unique proteins and either increasing 1.5–13.9-fold or decreasing 1.5–7.8-fold in relative abundance. Many of these proteins were associated with energy metabolism, including the cellobiose related glycoside hydrolases phospho-β-glucosidase (LBA0881 and phospho-β-galactosidase II (LBA0726. The data provide insight into the utilization of the candidate prebiotic cellobiose by the probiotic bacterium NCFM. Several of the upregulated or downregulated identified proteins associated with utilization of cellobiose indicate the presence of carbon catabolite repression and regulation of enzymes involved in carbohydrate metabolism.

  7. Insights into the structure and function of fungal β-mannosidases from glycoside hydrolase family 2 based on multiple crystal structures of the Trichoderma harzianum enzyme.

    Science.gov (United States)

    Nascimento, Alessandro S; Muniz, Joao Renato C; Aparício, Ricardo; Golubev, Alexander M; Polikarpov, Igor

    2014-09-01

    Hemicellulose is an important part of the plant cell wall biomass, and is relevant to cellulosic ethanol technologies. β-Mannosidases are enzymes capable of cleaving nonreducing residues of β-d-mannose from β-d-mannosides and hemicellulose mannose-containing polysaccharides, such as mannans and galactomannans. β-Mannosidases are distributed between glycoside hydrolase (GH) families 1, 2, and 5, and only a handful of the enzymes have been structurally characterized to date. The only published X-ray structure of a GH family 2 mannosidase is that of the bacterial Bacteroides thetaiotaomicron enzyme. No structures of eukaryotic mannosidases of this family are currently available. To fill this gap, we set out to solve the structure of Trichoderma harzianum GH family 2 β-mannosidase and to refine it to 1.9-Å resolution. Structural comparisons of the T. harzianum GH2 β-mannosidase highlight similarities in its structural architecture with other members of GH family 2, reveal the molecular mechanism of β-mannoside binding and recognition, and shed light on its putative galactomannan-binding site. Coordinates and observed structure factor amplitudes have been deposited with the Protein Data Bank (4CVU and 4UOJ). The T. harzianum β-mannosidase 2A nucleotide sequence has GenBank accession number BankIt1712036 GeneMark.hmm KJ624918. © 2014 FEBS.

  8. Purification, crystallization and preliminary X-ray analysis of a thermostable glycoside hydrolase family 43 β-xylosidase from Geobacillus thermoleovorans IT-08

    International Nuclear Information System (INIS)

    Rohman, Ali; Oosterwijk, Niels van; Kralj, Slavko; Dijkhuizen, Lubbert; Dijkstra, Bauke W.; Puspaningsih, Ni Nyoman Tri

    2007-01-01

    The β-xylosidase was crystallized using PEG 6000 as precipitant. 5% PEG 6000 yielded bipyramid-shaped tetragonal crystals diffracting to 1.55 Å resolution, and 13% PEG 6000 gave rectangular monoclinic crystals diffracting to 1.80 Å resolution. The main enzymes involved in xylan-backbone hydrolysis are endo-1,4-β-xylanase and β-xylosidase. β-Xylosidase converts the xylo-oligosaccharides produced by endo-1,4-β-xylanase into xylose monomers. The β-xylosidase from the thermophilic Geobacillus thermoleovorans IT-08, a member of glycoside hydrolase family 43, was crystallized at room temperature using the hanging-drop vapour-diffusion method. Two crystal forms were observed. Bipyramid-shaped crystals belonging to space group P4 3 2 1 2, with unit-cell parameters a = b = 62.53, c = 277.4 Å diffracted to 1.55 Å resolution. The rectangular crystals belonged to space group P2 1 , with unit-cell parameters a = 57.94, b = 142.1, c = 153.9 Å, β = 90.5°, and diffracted to 1.80 Å resolution

  9. Structural features of Aspergillus niger β-galactosidase define its activity against glycoside linkages.

    Science.gov (United States)

    Rico-Díaz, Agustín; Ramírez-Escudero, Mercedes; Vizoso-Vázquez, Ángel; Cerdán, M Esperanza; Becerra, Manuel; Sanz-Aparicio, Julia

    2017-06-01

    β-Galactosidases are biotechnologically interesting enzymes that catalyze the hydrolysis or transgalactosylation of β-galactosides. Among them, the Aspergillus niger β-galactosidase (AnβGal) belongs to the glycoside hydrolase family 35 (GH35) and is widely used in the industry due to its high hydrolytic activity which is used to degrade lactose. We present here its three-dimensional structure in complex with different oligosaccharides, to illustrate the structural determinants of the broad specificity of the enzyme against different glycoside linkages. Remarkably, the residues Phe264, Tyr304, and Trp806 make a dynamic hydrophobic platform that accommodates the sugar at subsite +1 suggesting a main role on the recognition of structurally different substrates. Moreover, complexes with the trisaccharides show two potential subsites +2 depending on the substrate type. This feature and the peculiar shape of its wide cavity suggest that AnβGal might accommodate branched substrates from the complex net of polysaccharides composing the plant material in its natural environment. Relevant residues were selected and mutagenesis analyses were performed to evaluate their role in the catalytic performance and the hydrolase/transferase ratio of AnβGal. Thus, we generated mutants with improved transgalactosylation activity. In particular, the variant Y304F/Y355H/N357G/W806F displays a higher level of galacto-oligosaccharides production than the Aspergillus oryzae β-galactosidase, which is the preferred enzyme in the industry owing to its high transferase activity. Our results provide new knowledge on the determinants modulating specificity and the catalytic performance of fungal GH35 β-galactosidases. In turn, this fundamental background gives novel tools for the future improvement of these enzymes, which represent an interesting target for rational design. Structural data are available in PDB database under the accession numbers 5IFP (native form), 5IHR (in complex with 6

  10. New polyacetylenes glycoside from Eclipta prostrate with DGAT inhibitory activity.

    Science.gov (United States)

    Meng, Xiao; Li, Ban-Ban; Lin, Xin; Jiang, Yi-Yu; Zhang, Le; Li, Hao-Ze; Cui, Long

    2018-06-08

    One new polyacetylene glycoside eprostrata Ⅰ (1), together with seven known compounds (2-8), were isolated from Eclipta prostrata. Their structures were elucidated on the basis of spectroscopic and physico-chemical analyses. All the isolates were evaluated inhibitory activity on DGAT in an in vitro assay. Compounds 1-8 were found to exhibit inhibitory activity of DGAT1 with IC 50 values ranging from 74.4 ± 1.3 to 101.1 ± 1.1 μM.

  11. A comparative metagenome survey of the fecal microbiota of a breast- and a plant-fed Asian elephant reveals an unexpectedly high diversity of glycoside hydrolase family enzymes.

    Directory of Open Access Journals (Sweden)

    Nele Ilmberger

    Full Text Available A phylogenetic and metagenomic study of elephant feces samples (derived from a three-weeks-old and a six-years-old Asian elephant was conducted in order to describe the microbiota inhabiting this large land-living animal. The microbial diversity was examined via 16S rRNA gene analysis. We generated more than 44,000 GS-FLX+454 reads for each animal. For the baby elephant, 380 operational taxonomic units (OTUs were identified at 97% sequence identity level; in the six-years-old animal, close to 3,000 OTUs were identified, suggesting high microbial diversity in the older animal. In both animals most OTUs belonged to Bacteroidetes and Firmicutes. Additionally, for the baby elephant a high number of Proteobacteria was detected. A metagenomic sequencing approach using Illumina technology resulted in the generation of 1.1 Gbp assembled DNA in contigs with a maximum size of 0.6 Mbp. A KEGG pathway analysis suggested high metabolic diversity regarding the use of polymers and aromatic and non-aromatic compounds. In line with the high phylogenetic diversity, a surprising and not previously described biodiversity of glycoside hydrolase (GH genes was found. Enzymes of 84 GH families were detected. Polysaccharide utilization loci (PULs, which are found in Bacteroidetes, were highly abundant in the dataset; some of these comprised cellulase genes. Furthermore the highest coverage for GH5 and GH9 family enzymes was detected for Bacteroidetes, suggesting that bacteria of this phylum are mainly responsible for the degradation of cellulose in the Asian elephant. Altogether, this study delivers insight into the biomass conversion by one of the largest plant-fed and land-living animals.

  12. Flavonoid Glycosides from Siparuna gigantotepala Leaves and Their Antioxidant Activity.

    Science.gov (United States)

    Torres Castañeda, Harlen Gerardo; Colmenares Dulcey, Ana Julia; Isaza Martínez, José Hipólito

    2016-01-01

    Two new flavonol glycosides were isolated from the leaves of Siparuna gigantotepala. Their structures were determined to be kaempferol 3-O-β-xylopyranosyl-(1→2)-α-arabinofuranoside (1) and kaempferol 3,7-di-O-methyl-4'-O-α-rhamnopyranosyl-(1→2)-β-glucopyranoside (2). In addition, three known flavonol glycosides, rutin (3), kaempferol 3-O-rutinoside (4), and kaempferol 3,7-di-O-methyl-4'-O-rutinoside (5), and three flavonol aglycones, quercetin (6), kaempferol 3,7-dimethyl ether (7), and kaempferol 3,7,4'-trimethyl ether (8), were also isolated and are reported here for the first time in this species. The structures of compounds 1 and 2 were established on the basis of their LC-MS and one- and two-dimensional (1D)- and (2D)-NMR spectroscopic analyses, combined with acid methanolysis and silylation of sugar moieties for GC-MS. Evaluation of the antioxidant activity, conducted in the 96-well plate format, showed that the flavonoids isolated possess strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity and moderate oxygen radical absorption capacity.

  13. Glycosides in medicine: "The role of glycosidic residue in biological activity"

    Czech Academy of Sciences Publication Activity Database

    Křen, Vladimír; Martínková, Ludmila

    2001-01-01

    Roč. 8, - (2001), s. 1303-1328 ISSN 0929-8673 R&D Projects: GA ČR GA303/99/1382; GA ČR GA303/98/0414 Institutional research plan: CEZ:AV0Z5020903 Keywords : glycosides Subject RIV: EE - Microbiology, Virology Impact factor: 5.760, year: 2001

  14. Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature

    DEFF Research Database (Denmark)

    Busk, Peter Kamp; Lange, Mette; Pilgaard, Bo

    2014-01-01

    The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence....... In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important...

  15. Heterologous expression and characterization of a putative glycoside hydrolase family 43 arabinofuranosidase from Clostridium thermocellum B8

    NARCIS (Netherlands)

    Camargo, de Brenda R.; Claassens, Nico J.; Quirino, Betania Ferraz; Noronha, Eliane F.; Kengen, Servé W.M.

    2018-01-01

    An extensive list of putative cellulosomal enzymes from C. thermocellum is now available in the public databanks, however, most of these remain unvalidated with regard to their activity and expression control mechanisms. This is particularly true of those enzymes putatively involved in hemicellulose

  16. Cell wall proteome of sugarcane stems: comparison of a destructive and a non-destructive extraction method showed differences in glycoside hydrolases and peroxidases.

    Science.gov (United States)

    Calderan-Rodrigues, Maria Juliana; Jamet, Elisabeth; Douché, Thibaut; Bonassi, Maria Beatriz Rodrigues; Cataldi, Thaís Regiani; Fonseca, Juliana Guimarães; San Clemente, Hélène; Pont-Lezica, Rafael; Labate, Carlos Alberto

    2016-01-11

    Sugarcane has been used as the main crop for ethanol production for more than 40 years in Brazil. Recently, the production of bioethanol from bagasse and straw, also called second generation (2G) ethanol, became a reality with the first commercial plants started in the USA and Brazil. However, the industrial processes still need to be improved to generate a low cost fuel. One possibility is the remodeling of cell walls, by means of genetic improvement or transgenesis, in order to make the bagasse more accessible to hydrolytic enzymes. We aimed at characterizing the cell wall proteome of young sugarcane culms, to identify proteins involved in cell wall biogenesis. Proteins were extracted from the cell walls of 2-month-old culms using two protocols, non-destructive by vacuum infiltration vs destructive. The proteins were identified by mass spectrometry and bioinformatics. A predicted signal peptide was found in 84 different proteins, called cell wall proteins (CWPs). As expected, the non-destructive method showed a lower percentage of proteins predicted to be intracellular than the destructive one (33% vs 44%). About 19% of CWPs were identified with both methods, whilst the infiltration protocol could lead to the identification of 75% more CWPs. In both cases, the most populated protein functional classes were those of proteins related to lipid metabolism and oxido-reductases. Curiously, a single glycoside hydrolase (GH) was identified using the non-destructive method whereas 10 GHs were found with the destructive one. Quantitative data analysis allowed the identification of the most abundant proteins. The results highlighted the importance of using different protocols to extract proteins from cell walls to expand the coverage of the cell wall proteome. Ten GHs were indicated as possible targets for further studies in order to obtain cell walls less recalcitrant to deconstruction. Therefore, this work contributed to two goals: enlarge the coverage of the sugarcane

  17. Potential hypoglycaemic activity phenolic glycosides from Moringa oleifera seeds.

    Science.gov (United States)

    Wang, Fang; Zhong, Huan-Huan; Chen, Wei-Ke; Liu, Qing-Pu; Li, Cun-Yu; Zheng, Yun-Feng; Peng, Guo-Ping

    2017-08-01

    Moringa oleifera seed has remarkable curative effects on reducing blood pressure, blood sugar and enhancing human immunity. In this study, one novel phenolic glycoside (1) together with four known compounds 2-5 were isolated from the macroporous resin adsorption extract of M. oleifera seeds, and the compound 3 was reported for the first time from this plant. The structure of the new crystalline compound was determined on the basis of spectroscopic analyses including mass spectrometry, 1D and 2D NMR experiments. The hypoglycaemic activity of isolated compounds was investigated with HepG2 cell and STZ-induced mice. It was found that compound 1, 4 and 5 could promote the glucose consumption of insulin resistance cells and reduce blood glucose levels of STZ-induced mice. This study concludes that compound 1, 4 and 5 may be developed as new and safe hypoglycaemic drugs.

  18. Soluble epoxide hydrolase inhibitory activity of anthraquinone components from Aloe.

    Science.gov (United States)

    Sun, Ya Nan; Kim, Jang Hoon; Li, Wei; Jo, A Reum; Yan, Xi Tao; Yang, Seo Young; Kim, Young Ho

    2015-10-15

    Aloe is a short-stemmed succulent herb widely used in traditional medicine to treat various diseases and as raw material in cosmetics and heath foods. In this study, we isolated and identified two new anthraquinone derivatives, aloinoside C (6) and aloinoside D (7), together with six known compounds from an aqueous dissolved Aloe exudate. Their structures were identified by spectroscopic analysis. The inhibitory effects of the isolated compounds on soluble epoxide hydrolase (sEH) were evaluated. Compounds 1-8 inhibited sEH activity potently, with IC50 values ranging from 4.1±0.6 to 41.1±4.2 μM. A kinetic analysis of compounds 1-8 revealed that the inhibitory actions of compounds 1, 6 and 8 were non-competitive, whereas those of compounds 2-5 and 7 were the mixed-type. Molecular docking increases our understanding of receptor-ligand binding of all compounds. These results demonstrate that compounds 1-8 from Aloe are potential sEH inhibitors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Functional analysis of the Glucan Degradation Locus (GDL) in Caldicellulosiruptor bescii reveals essential roles of component glycoside hydrolases in plant biomass deconstruction.

    Science.gov (United States)

    Conway, Jonathan M; McKinley, Bennett S; Seals, Nathaniel L; Hernandez, Diana; Khatibi, Piyum A; Poudel, Suresh; Giannone, Richard J; Hettich, Robert L; Williams-Rhaesa, Amanda M; Lipscomb, Gina L; Adams, Michael W W; Kelly, Robert M

    2017-10-06

    The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but one that can be exploited for conversion of lignocellulosic feedstocks into bio-based fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The Glucan Degradation Locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tāpirins), and putative post-translational modifying enzymes, in addition to multi-domain, multi-functional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation, as compared to fungal or cellulosomal systems. To examine the individual and collective in vivo roles of the glycolytic enzymes, the six GHs in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extent to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomasses (switchgrass or poplar) was examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically in vivo and accounted for 92% of naked microcellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture and not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization. Importance The efficient and extensive degradation of complex

  20. Glycosides from Stevia rebaudiana Bertoni Possess Insulin-Mimetic and Antioxidant Activities in Rat Cardiac Fibroblasts

    Directory of Open Access Journals (Sweden)

    Cecilia Prata

    2017-01-01

    Full Text Available Stevia rebaudiana Bertoni is a shrub having a high content of sweet diterpenoid glycosides in its leaves, mainly stevioside and rebaudioside A, which are used as noncaloric, natural sweeteners. The aim of this study was to deepen the knowledge about the insulin-mimetic effect exerted by four different mixtures of steviol glycosides, rich in stevioside and rebaudioside A, in neonatal rat cardiac fibroblasts. The potential antioxidant activity of these steviol glycosides was also assessed, as oxidative stress is associated with diabetes. Likewise the insulin effect, steviol glycosides caused an increase in glucose uptake into rat fibroblasts by activating the PI3K/Akt pathway, thus inducing Glut4 translocation to the plasma membrane. The presence of S961, an insulin antagonist, completely abolished these effects, allowing to hypothesize that steviol glycosides could act as ligands of the same receptor engaged by insulin. Moreover, steviol glycosides counteracted oxidative stress by increasing reduced glutathione intracellular levels and upregulating expression and activity of the two antioxidant enzymes superoxide dismutase and catalase. The present work unravels the insulin-mimetic effect and the antioxidant property exerted by steviol glycosides, suggesting their potential beneficial role in the cotreatment of diabetes and in health maintenance.

  1. Glycosides from Stevia rebaudiana Bertoni Possess Insulin-Mimetic and Antioxidant Activities in Rat Cardiac Fibroblasts

    Science.gov (United States)

    Prata, Cecilia; Zambonin, Laura; Rizzo, Benedetta; Vieceli Dalla Sega, Francesco

    2017-01-01

    Stevia rebaudiana Bertoni is a shrub having a high content of sweet diterpenoid glycosides in its leaves, mainly stevioside and rebaudioside A, which are used as noncaloric, natural sweeteners. The aim of this study was to deepen the knowledge about the insulin-mimetic effect exerted by four different mixtures of steviol glycosides, rich in stevioside and rebaudioside A, in neonatal rat cardiac fibroblasts. The potential antioxidant activity of these steviol glycosides was also assessed, as oxidative stress is associated with diabetes. Likewise the insulin effect, steviol glycosides caused an increase in glucose uptake into rat fibroblasts by activating the PI3K/Akt pathway, thus inducing Glut4 translocation to the plasma membrane. The presence of S961, an insulin antagonist, completely abolished these effects, allowing to hypothesize that steviol glycosides could act as ligands of the same receptor engaged by insulin. Moreover, steviol glycosides counteracted oxidative stress by increasing reduced glutathione intracellular levels and upregulating expression and activity of the two antioxidant enzymes superoxide dismutase and catalase. The present work unravels the insulin-mimetic effect and the antioxidant property exerted by steviol glycosides, suggesting their potential beneficial role in the cotreatment of diabetes and in health maintenance. PMID:28947927

  2. Antiproliferative activity of cardenolide glycosides from Asclepias subulata.

    Science.gov (United States)

    Rascón-Valenzuela, L; Velázquez, C; Garibay-Escobar, A; Medina-Juárez, L A; Vilegas, W; Robles-Zepeda, R E

    2015-08-02

    Asclepias subulata Decne. is a shrub occurring in Sonora-Arizona desert (Mexico-USA). The ethnic groups, Seris and Pimas, use this plant for the treatment of sore eyes, gastrointestinal disorders and cancer. To isolate the compounds responsible for antiproliferative activity of the methanol extract of A. subulata. A bioguided fractionation of methanol extract of A. subulata was performed using MTT assay to measure the antiproliferative activity of different compounds on three human cancer cell lines (A549, LS 180 and PC-3), one murine cancer cell line (RAW 264.7) and one human normal cell line (ARPE-19). The methanol extract was partitioned with hexane, ethyl acetate and ethanol. The active fractions, ethanol and residual, were fractioned by silica-column chromatography and active sub-fractions were separated using HPLC. The chemical structures of isolated compounds were elucidated with different chemical and spectroscopic methods. A new cardenolide glycoside, 12, 16-dihydroxycalotropin, and three known, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin, were isolated of active sub-fractions. All isolated compounds showed a strong antiproliferative activity in human cancer cells. Calotropin was the more active with IC50 values of 0.0013, 0.06 and 0.41 µM on A549, LS 180 and PC-3 cell lines, respectively; while 12, 16-dihydroxycalotropin reached values of 2.48, 5.62 and 11.70 µM, on the same cells; corotoxigenin 3-O-glucopyranoside had IC50 of 2.64, 3.15 and 6.62 µM and desglucouzarin showed values of 0.90, 6.57 and 6.62, µM. Doxorubicin, positive control, showed IC50 values of 1.78, 6.99 and 3.18 µM, respectively. The isolated compounds had a weak effect on murine cancer cells and human normal cells, exhibiting selectivity to human cancer cells. In this study, we found that 12, 16-dihydroxicalotropin, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin are responsible of antiproliferative properties of A. subulata, and that these

  3. Production and characterisation of glycoside hydrolases from GH3, GH5, GH10, GH11 and GH61 for chemo-enzymatic synthesis of xylo- and mannooligosaccharides

    DEFF Research Database (Denmark)

    Dilokpimol, Adiphol

    Produktion og karakterisering af glykosid hydrolaser fro GH3, GH5, GH10, GH11 og GH61 til chemo-enzymatisk syntese af xylo- og mannooligosakkarider Biprodukter fra hydrolyse af plantecellevægge er kilder til oligosakkarider, som potentielt kan fungere som prebiotika ved at stimulere væksten af...... omfatter karakterisering af de producerede enzymer samt cDNA kloning af formodet GH61 endo Produktion og karakterisering af glykosid hydrolaser fro GH3, GH5, GH10, GH11 og GH61 til chemo-enzymatisk syntese af xylo- og mannooligosakkarider Biprodukter fra hydrolyse af plantecellevægge er kilder til...

  4. Ethanol production with a flocculating mutant of Zymomonas mobilis and immobilized glycoside hydrolases. Ethanolgewinnung mit einer flockenden Mutante von Zymomonas mobilis und immobilisierten Glycosidhydrolasen

    Energy Technology Data Exchange (ETDEWEB)

    Tramm-Werner, S.

    1987-05-25

    A method to extend the substrate spectrum of Z. mobilis was developed. Higher ethanol yields were achieved by simultaneous use of hydrolases cross-linked with glutar aldehyde together with the flocculating Zymomonas cells (TW 602). Apart from the high product yields, the method is characterized by low susceptibility to infections.

  5. High-throughput screening for gene libraries expressing carbohydrate hydrolase activity

    NARCIS (Netherlands)

    Leemhuis, Hans; Euverink, Gert-Jan W.; Dijkhuizen, Lubbert

    2003-01-01

    A simple and fast method is described allowing screening of large number of Escherichia coli clones (4000 per day) for the presence of functional or improved carbohydrate hydrolase enzymes. The procedure is relatively cheap and has the advantage that carbohydrate degrading activity can be directly

  6. Production of enzymatically active recombinant full-length barley high pI alpha-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification

    DEFF Research Database (Denmark)

    Næsted, Henrik; Kramhøft, Birte; Lok, F.

    2006-01-01

    Recombinant barley high pI alpha-glucosidase was produced by high cell-density fermentation of Pichia pastoris expressing the cloned full-length gene. The gene was amplified from a genomic clone and exons (coding regions) were assembled by overlap PCR. The resulting cDNA was expressed under contr...... nM x s(-1), and 85 s(-1) using maltose as substrate. This work presents the first production of fully active recombinant alpha-glucosidase of glycoside hydrolase family 31 from higher plants. (c) 2005 Elsevier Inc. All rights reserved....

  7. Murein Hydrolase Activity in the Surface Layer of Lactobacillus acidophilus ATCC 4356▿

    OpenAIRE

    Prado Acosta, Mariano; Palomino, María Mercedes; Allievi, Mariana C.; Rivas, Carmen Sanchez; Ruzal, Sandra M.

    2008-01-01

    We describe a new enzymatic functionality for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356, namely, an endopeptidase activity against the cell wall of Salmonella enterica serovar Newport, assayed via zymograms and identified by Western blotting. Based on amino acid sequence comparisons, the hydrolase activity was predicted to be located at the C terminus. Subsequent cloning and expression of the C-terminal domain in Bacillus subtilis resulted in the functional verificati...

  8. Enzymatic synthesis of glycosides: from natural O- and N-glycosides to rare C- and S-glycosides

    Directory of Open Access Journals (Sweden)

    Jihen Ati

    2017-09-01

    Full Text Available Carbohydrate related enzymes, like glycosyltransferases and glycoside hydrolases, are nowadays more easily accessible and are thought to represent powerful and greener alternatives to conventional chemical glycosylation procedures. The knowledge of their corresponding mechanisms has already allowed the development of efficient biocatalysed syntheses of complex O-glycosides. These enzymes can also now be applied to the formation of rare or unnatural glycosidic linkages.

  9. Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase.

    Science.gov (United States)

    Placido, Antonio; Hai, Tran; Ferrer, Manuel; Chernikova, Tatyana N; Distaso, Marco; Armstrong, Dale; Yakunin, Alexander F; Toshchakov, Stepan V; Yakimov, Michail M; Kublanov, Ilya V; Golyshina, Olga V; Pesole, Graziano; Ceci, Luigi R; Golyshin, Peter N

    2015-12-01

    A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli. Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes, Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae, Armatimonadetes, and Planctomycetes. Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct α/β-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p-nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p-nitrophenol-α-(L)-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the α/β-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 °C. Furthermore, enzymes were active in organic solvents (e.g., >30% methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1.

  10. Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase

    Science.gov (United States)

    Legler, Patricia; Boisvert, Susanne; Compton, Jaimee; Millard, Charles

    2014-07-01

    We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine O?. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its kcat/Km for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. pNBE was tested as a surrogate scaffold for mammalian esterases. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1. We discuss the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases.

  11. A comparison of methods to determine tannin acyl hydrolase activity

    Directory of Open Access Journals (Sweden)

    Cristóbal Aguilar

    1999-01-01

    Full Text Available Six methods to determine the activity of tannase produced by Aspergillus niger Aa-20 on polyurethane foam by solid state fermentation, which included two titrimetric techniques, three spectrophotometric methods and one HPLC assay were tested and compared. All methods assayed enabled the measurement of extracellular tannase activity. However, only five were useful to evaluate intracellular tannase activity. Studies on the effect of pH on tannase extraction demonstrated that tannase activity was considerably under-estimated when its extraction was carried out at pH values below 5.5 and above 6.0. Results showed that the HPLC technique and the modified Bajpai and Patil methods presented several advantages in comparison to the other methods tested.Seis métodos para determinar a atividade de tannase produzida por Aspergillus niger O Aa-20 em espuma de polyuretano por fermentação em estado sólido foram estudados. Duas técnicas titulométricas , três métodos spectrofotométricos e um método por HPLC foram testados e comparados. Todos os métodos testados permitiram determinar a atividade da tannase produzida extracelularmente. Entretanto, somente cinco se mostraram úteis para avaliar a atividade da tannase produzida intracelularmente. Os estudos do efeito do pH na extração de tannase demonstraram que a atividade de tannase era consideravelmente subestimada quando sua extração foi executada em valores de pH inferiores a 5.5 e superior a pH 6.0. Os resultados demostraram que a técnica de HPLC o método Bajpai and Patil modificado apresentam várias vantagens em comparação aos outros métodos testados.

  12. Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

    Energy Technology Data Exchange (ETDEWEB)

    Ortega, Corrie; Anderson, Lindsey N.; Frando, Andrew; Sadler, Natalie C.; Brown, Robert W.; Smith, Richard D.; Wright, Aaron T.; Grundner, Christoph

    2016-02-01

    The transition between replication and non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenicity, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating, persistent populations is a priority for tuberculosis treatment, but only few drug targets in non-replicating Mtb are currently known. Here, we directly measure the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication by activity-based proteomics. We predict serine hydrolase activity for 78 proteins, including 27 proteins with previously unknown function, and identify 37 SHs that remain active even in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with large shifts in the activity of the majority of SHs. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.

  13. Phenolic Glycosides from Capsella bursa-pastoris (L. Medik and Their Anti-Inflammatory Activity

    Directory of Open Access Journals (Sweden)

    Joon Min Cha

    2017-06-01

    Full Text Available A new sesquilignan glycoside 1, together with seven known phenolic glycosides 2–8 were isolated from the aerial parts of Capsella bursa-pastoris. The chemical structure of the new compound 1 was elucidated by extensive nuclear magnetic resonance (NMR data (1H- and 13C-NMR, 1H-1H correlation spectroscopy (1H-1H COSY, heteronuclear single-quantum correlation (HSQC, heteronuclear multiple bond correlation (HMBC, and nuclear overhauser effect spectroscopy (NOESY and HR-FABMS analysis. The anti-inflammatory effects of 1–8 were evaluated in lipopolysaccharide (LPS-stimulated murine microglia BV-2 cells. Compounds 4 and 7 exhibited moderate inhibitory effects on nitric oxide production in LPS-activated BV-2 cells, with IC50 values of 17.80 and 27.91 µM, respectively.

  14. Cellular localization of peptide hydrolases in chicken embryo tissues and influence of gamma irradiation on their activity

    Energy Technology Data Exchange (ETDEWEB)

    Khristov, D; Marinopolski, G

    1975-01-01

    Studied was the influence of chicken embryo irradiation at 600 R and 1000 R gamma rays on the activity of tissue peptide hydrolases in mitochondrial-lysosomal, microsomal and supernatant (cell hyaloplasm) cell fractions. The investigation was performed 50 to 168 hours post irradiation. The wole tissue (of the whole embryo) was examined following irradiation of 4-day-old embryos whose liver, muscle and brain tissues were post irradiation examined on day 12 and 16 of incubation. Prior to treatment, the tissues were threfold rinsed with sucrose solution to eliminate proeinase inhibitors. Lysosome membranes were destroyed by adding 0.5 % desoxycholate. It was found that: Peptide hydrolase activity of mitochondrial-lysosomal cell fractions of tissues of whole 6-day chicken embryos is 4-5 times as high as that of cell hyaloplasm. Peptide hydrolase activity of mitochondrial-lysosomal fractions of liver tissues decreases on day 18 and 19 post incubation, while the same fraction of muscle and brain tissues shows high activity. Peptide hydrolase activity of microsomal fraction and of cell hyaloplasm rises during embryonal development and exceeds the activity of liver tissue mitochondrial fraction. Peptide hydrolase activity of mitochondrial-lysosomal fraction of tissue of whole 6-day-old embryos 50 hours post irradiation is higher than the activity of non-irradiated embryos. Later the activity of this fraction diminishes and on the 168 hr post irradiation it drops below the normal. Microsomal fraction and cell hyaloplasm activity likewise show deviation from the norm. Peptide hydrolase activity of mitochondrial-lysosomal fraction of liver, muscle and brain tissue of 14 and 18-day-old embryos is higher than the control 50 hours post irradiation and then declines. The activity of mitochondrial-lysosomal fraction of embryo brain tissue changes most strikingly on irradiation, while other brain cell fractions change less compared with liver and muscle fractions.

  15. Cellular localization of peptide hydrolases in chicken embryo tissues and influence of gamma irradiation on their activity

    International Nuclear Information System (INIS)

    Khristov, D.; Marinopolski, G.

    1975-01-01

    Studied was the influence of chicken embryo irradiation at 600 R and 1000 R gamma rays on the activity of tissue peptide hydrolases in mitochondrial-lysosomal, microsomal and supernatant (cell hyaloplasm) cell fractions. The investigation was performed 50 to 168 hours post irradiation. The wole tissue (of the whole embryo) was examined following irradiation of 4-day-old embryos whose liver, muscle and brain tissues were post irradiation examined on day 12 and 16 of incubation. Prior to treatment, the tissues were threfold rinsed with sucrose solution to eliminate proeinase inhibitors. Lysosome membranes were destroyed by adding 0.5 % desoxycholate. It was found that: Peptide hydrolase activity of mitochondrial-lysosomal cell fractions of tissues of whole 6-day chicken embryos is 4-5 times as high as that of cell hyaloplasm. Peptide hydrolase activity of mitochondrial-lysosomal fractions of liver tissues decreases on day 18 and 19 post incubation, while the same fraction of muscle and brain tissues shows high activity. Peptide hydrolase activity of microsomal fraction and of cell hyaloplasm rises during embryonal development and exceeds the activity of liver tissue mitochondrial fraction. Peptide hydrolase activity of mitochondrial-lysosomal fraction of tissue of whole 6-day-old embryos 50 hours post irradiation is higher than the activity of non-irradiated embryos. Later the activity of this fraction diminishes and on the 168 hr post irradiation it drops below the normal. Microsomal fraction and cell hyaloplasm activity likewise show deviation from the norm. Peptide hydrolase activity of mitochondrial-lysosomal fraction of liver, muscle and brain tissue of 14 and 18-day-old embryos is higher than the control 50 hours post irradiation and then declines. The activity of mitochondrial-lysosomal fraction of embryo brain tissue changes most strikingly on irradiation, while other brain cell fractions change less compared with liver and muscle fractions

  16. Anti-Inflammatory Activity of Methyl Salicylate Glycosides Isolated from Gaultheria yunnanensis (Franch. Rehder

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    Guan-Hua Du

    2011-05-01

    Full Text Available Gaultheria yunnanensis (Franch. Rehder is a kind of traditional Chinese herbal medicine used for the treatments of rheumatoid arthritis, swelling and pain. Two methyl salicylate glycosides, namely methyl benzoate-2-O-b-D-xylopyranosyl(1-6-O-b-D-gluco-pyranoside (J12122 and methyl benzoate-2-O-β-D-xylopyranosyl(1-2[O-β-D-xylopyranosyl(1-6]-O-β-D-glucopyranoside (J12123, are natural salicylic derivatives isolated from Gaultheria yunnanensis. In this study, we investigated the anti-inflammatory activity of J12122 and J12123 on LPS-induced RAW264.7 macrophage cells by measuring the production of pro-inflammatory cytokines, accumulation of nitric oxide (NO, and level of reactive oxygen species (ROS. The results showed that both methyl salicylate glycosides dose-dependently inhibited the production of tumor necrosis factor-α (TNF-α, interleukin-1β (IL-1β, and IL-6, respectively. Consistent with these observations, J12122 and J12123 significantly suppressed the accumulation of NO, with an inhibitory rate of 56.20% and 51.72% at 3.0 μg/mL concentration, respectively. Furthermore, the two methyl salicylate glycosides reduced the level of ROS induced by LPS. These results showed that the isolated compounds possess anti-inflammatory properties through inhibition the production pro-inflammatory cytokines, NO, and ROS.

  17. Anti-inflammatory activity of methyl salicylate glycosides isolated from Gaultheria yunnanensis (Franch.) Rehder.

    Science.gov (United States)

    Zhang, Dan; Liu, Rui; Sun, Lan; Huang, Chao; Wang, Chao; Zhang, Dong-Ming; Zhang, Tian-Tai; Du, Guan-Hua

    2011-05-09

    Gaultheria yunnanensis (Franch.) Rehder is a kind of traditional Chinese herbal medicine used for the treatments of rheumatoid arthritis, swelling and pain. Two methyl salicylate glycosides, namely methyl benzoate-2-O-β-D-xylopyranosyl(1-6)-O-β-D-gluco-pyranoside (J12122) and methyl benzoate-2-O-β-D-xylopyranosyl(1-2)[O-β-D-xylopyranosyl(1-6)]-O-β-D-glucopyranoside (J12123), are natural salicylic derivatives isolated from Gaultheria yunnanensis. In this study, we investigated the anti-inflammatory activity of J12122 and J12123 on LPS-induced RAW264.7 macrophage cells by measuring the production of pro-inflammatory cytokines, accumulation of nitric oxide (NO), and level of reactive oxygen species (ROS). The results showed that both methyl salicylate glycosides dose-dependently inhibited the production of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, respectively. Consistent with these observations, J12122 and J12123 significantly suppressed the accumulation of NO, with an inhibitory rate of 56.20% and 51.72% at 3.0 μg/mL concentration, respectively. Furthermore, the two methyl salicylate glycosides reduced the level of ROS induced by LPS. These results showed that the isolated compounds possess anti-inflammatory properties through inhibition the production pro-inflammatory cytokines, NO, and ROS.

  18. Acylated Flavone Glycosides from the Roots of Saussurea lappa and Their Antifungal Activity

    Directory of Open Access Journals (Sweden)

    Yemireddy Venkata Ramnareddy

    2007-03-01

    Full Text Available The isolation of four novel acylated flavonoid glycosides from the roots of Saussurea lappa and their identification using a combination of 1D and 2D NMR and mass spectrometry is described. The in vitro antifungal and antibacterial activities of the isolated compounds and their mixture were tested on nine fungal and four bacterial strains, using the microdilution method. The compounds and mixture showed moderate to high antifungal activity against most of the fungi tested, compared to a miconazole standard, while only one compound and the mixture showed antibacterial activity against all strains tested.

  19. New Flavonoid Glycoside and Pharmacological Activities of Pteranthus dichotomus Forssk.

    Directory of Open Access Journals (Sweden)

    Emad, M. Atta

    2013-03-01

    Full Text Available Luteolin 6-rhamnosyl (1``` 4`` rhamnoside was isolated from Pteranthus dichotomus for the first time as a natural product along with eight compounds; kaempferol, quercetin, quercetin-7-glucoside, isoorientin, orientin-7-methoxide, luteolin, kaempferol-3-rhamnoside-7-glucouronic acid and myricetin-3-glucoside. The plant contained phenol, protocatechuic, p-hydroxy benzoic, gallic, p-coumaric and o-coumaric acids; they were identified by HPLC, the flavonoid compounds were purified by chromatographic methods, identified by chemical and physical methods including UV, 1H, 13C and 2D- NMR. The tested extract was highly safe as LD 50 (4 g/kg b.wt., it has anti-inflammatory, moderate analgesic effect and caused increase in urine volume, it also had no effect on liver functions of animals. Kidney functions were impaired after large dose (100 mg/kg -1. It has Anti-tumor activity against Ehrlish Ascites Carcinoma. The new isolated compound showed antipyretic effect and increased the urine volume while the tested extract had moderate antipyretic activity in rats.

  20. Lignans and aromatic glycosides from Piper wallichii and their antithrombotic activities.

    Science.gov (United States)

    Shi, Yan-Ni; Shi, Yi-Ming; Yang, Lian; Li, Xing-Cong; Zhao, Jin-Hua; Qu, Yan; Zhu, Hong-Tao; Wang, Dong; Cheng, Rong-Rong; Yang, Chong-Ren; Xu, Min; Zhang, Ying-Jun

    2015-03-13

    Piper wallichii (Miq.) Hand.-Mazz. is a medicinal plant used widely for the treatment of rheumatoid arthritis, inflammatory diseases, cerebral infarction and angina in China. Previous study showed that lignans and neolignans from Piper spp. had potential inhibitory activities on platelet aggregation. In the present study, we investigated the chemical constituents of Piper wallichii and their antithrombotic activities, to support its traditional uses. The methanolic extract of the air-dried stems of Piper wallichii was separated and purified using various chromatographic methods, including semi-preparative HPLC. The chemical structures of the isolates were determined by detailed spectroscopic analysis, and acidic hydrolysis in case of the new glycoside 2. Determination of absolute configurations of the new compound 1 was facilitated by calculated electronic circular dichroism using time-dependent density-functional theory. All compounds were tested for their inhibitory effects on platelet aggregation induced by platelet activating factor (PAF) in rabbits׳ blood model, from which the active ones were further evaluated the in vivo antithrombotic activity in zebrafish model. A new neolignan, piperwalliol A (1), and four new aromatic glycosides, piperwalliosides A-D (2-5) were isolated from the stems of Piper wallichii, along with 25 known compounds, including 13 lignans, six aromatic glycosides, two phenylpropyl aldehydes, and four biphenyls. Five known compounds (6-10) showed in vitro antiplatelet aggregation activities. Among them, (-)-syringaresinol (6) was the most active compound with an IC50 value of 0.52 mM. It is noted that in zebrafish model, the known lignan 6 showed good in vivo antithrombotic effect with a value of 37% at a concentration of 30 μM, compared with the positive control aspirin with the inhibitory value of 74% at a concentration of 125μM. This study demonstrated that lignans, phenylpropanoid and biphenyl found in Piper wallichii may be

  1. Molecular Basis of Prodrug Activation by Human Valacyclovirase, an [alpha]-Amino Acid Ester Hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Longsheng; Xu, Zhaohui; Zhou, Jiahai; Lee, Kyung-Dall; Amidon, Gordon L. (Michigan)

    2008-07-08

    Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of {alpha}-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific {alpha}-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.

  2. Systems Approaches to Predict the Functions of Glycoside Hydrolases during the Life Cycle of Aspergillus niger Using Developmental Mutants ∆brlA and ∆flbA

    NARCIS (Netherlands)

    van Munster, Jolanda M.; Nitsche, Benjamin M.; Akeroyd, Michiel; Dijkhuizen, Lubbert; van der Maarel, Marc J.E.C.; Ram, Arthur F.J.

    2015-01-01

    BACKGROUND: The filamentous fungus Aspergillus niger encounters carbon starvation in nature as well as during industrial fermentations. In response, regulatory networks initiate and control autolysis and sporulation. Carbohydrate-active enzymes play an important role in these processes, for example

  3. Systems Approaches to Predict the Functions of Glycoside Hydrolases during the Life Cycle of Aspergillus niger Using Developmental Mutants ∆brlA and ∆flbA

    Science.gov (United States)

    van Munster, Jolanda M.; Nitsche, Benjamin M.; Akeroyd, Michiel; Dijkhuizen, Lubbert; van der Maarel, Marc J. E. C.; Ram, Arthur F. J.

    2015-01-01

    Background The filamentous fungus Aspergillus niger encounters carbon starvation in nature as well as during industrial fermentations. In response, regulatory networks initiate and control autolysis and sporulation. Carbohydrate-active enzymes play an important role in these processes, for example by modifying cell walls during spore cell wall biogenesis or in cell wall degradation connected to autolysis. Results In this study, we used developmental mutants (ΔflbA and ΔbrlA) which are characterized by an aconidial phenotype when grown on a plate, but also in bioreactor-controlled submerged cultivations during carbon starvation. By comparing the transcriptomes, proteomes, enzyme activities and the fungal cell wall compositions of a wild type A. niger strain and these developmental mutants during carbon starvation, a global overview of the function of carbohydrate-active enzymes is provided. Seven genes encoding carbohydrate-active enzymes, including cfcA, were expressed during starvation in all strains; they may encode enzymes involved in cell wall recycling. Genes expressed in the wild-type during starvation, but not in the developmental mutants are likely involved in conidiogenesis. Eighteen of such genes were identified, including characterized sporulation-specific chitinases and An15g02350, member of the recently identified carbohydrate-active enzyme family AA11. Eight of the eighteen genes were also expressed, independent of FlbA or BrlA, in vegetative mycelium, indicating that they also have a role during vegetative growth. The ΔflbA strain had a reduced specific growth rate, an increased chitin content of the cell wall and specific expression of genes that are induced in response to cell wall stress, indicating that integrity of the cell wall of strain ΔflbA is reduced. Conclusion The combination of the developmental mutants ΔflbA and ΔbrlA resulted in the identification of enzymes involved in cell wall recycling and sporulation-specific cell wall

  4. Systems approaches to predict the functions of glycoside hydrolases during the life cycle of Aspergillus niger using developmental mutants ∆brlA and ∆flbA.

    Directory of Open Access Journals (Sweden)

    Jolanda M van Munster

    Full Text Available The filamentous fungus Aspergillus niger encounters carbon starvation in nature as well as during industrial fermentations. In response, regulatory networks initiate and control autolysis and sporulation. Carbohydrate-active enzymes play an important role in these processes, for example by modifying cell walls during spore cell wall biogenesis or in cell wall degradation connected to autolysis.In this study, we used developmental mutants (ΔflbA and ΔbrlA which are characterized by an aconidial phenotype when grown on a plate, but also in bioreactor-controlled submerged cultivations during carbon starvation. By comparing the transcriptomes, proteomes, enzyme activities and the fungal cell wall compositions of a wild type A. niger strain and these developmental mutants during carbon starvation, a global overview of the function of carbohydrate-active enzymes is provided. Seven genes encoding carbohydrate-active enzymes, including cfcA, were expressed during starvation in all strains; they may encode enzymes involved in cell wall recycling. Genes expressed in the wild-type during starvation, but not in the developmental mutants are likely involved in conidiogenesis. Eighteen of such genes were identified, including characterized sporulation-specific chitinases and An15g02350, member of the recently identified carbohydrate-active enzyme family AA11. Eight of the eighteen genes were also expressed, independent of FlbA or BrlA, in vegetative mycelium, indicating that they also have a role during vegetative growth. The ΔflbA strain had a reduced specific growth rate, an increased chitin content of the cell wall and specific expression of genes that are induced in response to cell wall stress, indicating that integrity of the cell wall of strain ΔflbA is reduced.The combination of the developmental mutants ΔflbA and ΔbrlA resulted in the identification of enzymes involved in cell wall recycling and sporulation-specific cell wall modification

  5. A thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 shows a novel domain organization among glycoside hydrolase family 13 enzymes.

    Science.gov (United States)

    Saburi, Wataru; Morimoto, Naoki; Mukai, Atsushi; Kim, Dae Hoon; Takehana, Toshihiko; Koike, Seiji; Matsui, Hirokazu; Mori, Haruhide

    2013-01-01

    α-Amylases (EC 3.2.1.1) hydrolyze internal α-1,4-glucosidic linkages of starch and related glucans. Bacillus sp. AAH-31 produces an alkalophilic thermophilic α-amylase (AmyL) of higher molecular mass, 91 kDa, than typical bacterial α-amylases. In this study, the AmyL gene was cloned to determine its primary structure, and the recombinant enzyme, produced in Escherichia coli, was characterized. AmyL shows no hydrolytic activity towards pullulan, but the central region of AmyL (Gly395-Asp684) was similar to neopullulanase-like α-amylases. In contrast to known neopullulanase-like α-amylases, the N-terminal region (Gln29-Phe102) of AmyL was similar to carbohydrate-binding module family 20 (CBM20), which is involved in the binding of enzymes to starch granules. Recombinant AmyL showed more than 95% of its maximum activity in a pH range of 8.2-10.5, and was stable below 65 °C and from pH 6.4 to 11.9. The kcat values for soluble starch, γ-cyclodextrin, and maltotriose were 103 s(-1), 67.6 s(-1), and 5.33 s(-1), respectively, and the Km values were 0.100 mg/mL, 0.348 mM, and 2.06 mM, respectively. Recombinant AmyL did not bind to starch granules. But the substitution of Trp45 and Trp84, conserved in site 1 of CBM20, with Ala reduced affinity to soluble starch, while the mutations did not affect affinity for oligosaccharides. Substitution of Trp61, conserved in site 2 of CBM20, with Ala enhanced hydrolytic activity towards soluble starch, indicating that site 2 of AmyL does not contribute to binding to soluble long-chain substrates.

  6. Cardiac glycoside activities link Na(+)/K(+) ATPase ion-transport to breast cancer cell migration via correlative SAR.

    Science.gov (United States)

    Magpusao, Anniefer N; Omolloh, George; Johnson, Joshua; Gascón, José; Peczuh, Mark W; Fenteany, Gabriel

    2015-02-20

    The cardiac glycosides ouabain and digitoxin, established Na(+)/K(+) ATPase inhibitors, were found to inhibit MDA-MB-231 breast cancer cell migration through an unbiased chemical genetics screen for cell motility. The Na(+)/K(+) ATPase acts both as an ion-transporter and as a receptor for cardiac glycosides. To delineate which function is related to breast cancer cell migration, structure-activity relationship (SAR) profiles of cardiac glycosides were established at the cellular (cell migration inhibition), molecular (Na(+)/K(+) ATPase inhibition), and atomic (computational docking) levels. The SAR of cardiac glycosides and their analogs revealed a similar profile, a decrease in potency when the parent cardiac glycoside structure was modified, for each activity investigated. Since assays were done at the cellular, molecular, and atomic levels, correlation of SAR profiles across these multiple assays established links between cellular activity and specific protein-small molecule interactions. The observed antimigratory effects in breast cancer cells are directly related to the inhibition of Na(+)/K(+) transport. Specifically, the orientation of cardiac glycosides at the putative cation permeation path formed by transmembrane helices αM1-M6 correlates with the Na(+) pump activity and cell migration. Other Na(+)/K(+) ATPase inhibitors that are structurally distinct from cardiac glycosides also exhibit antimigratory activity, corroborating the conclusion that the antiport function of Na(+)/K(+) ATPase and not the receptor function is important for supporting the motility of MDA-MB-231 breast cancer cells. Correlative SAR can establish new relationships between specific biochemical functions and higher-level cellular processes, particularly for proteins with multiple functions and small molecules with unknown or various modes of action.

  7. Effects of Cu, Zn and Pb Combined Pollution on Soil Hydrolase Activities

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    FENG Dan

    2015-08-01

    Full Text Available To study the relations between soil enzyme activities and heavy metal pollution, the combined effects of Cu, Zn and Pb on the three hydrolase activities, including invertase(IN, urease(Uand alkaline phosphatase(ALPwere investigated via an orthogonal experiment. Results showed as the following: When the concentration of Cu was 400 mg·kg-1, the U and ALP activities were decreased 51% and 44%, separately; When Zn was at 500 mg·kg-1, IN and ALP activities were only decreased 3% and 9%, while U activity was increased; When Pb was at 500 mg·kg-1, IN and U activities were increased, while ALP activity was decreased 13%. As a whole, Cu was considered as the most remarkable influence factor for IN, U and ALP activity regardless of interactions among the heavy metals, Zn came second, and Pb mainly showed activation. Considering interactions, Cu×Zn could significantly influence U activity(P<0.05, effects of Cu×Pb and Cu×Zn on ALP activity were remarkable(95% confidence interval. The response of ALP activity was more sensitive than the other two enzymes. Soil ALP activity might be a sensitive tool for assessing the pollution degree of Cu.

  8. Phenylethanoid Glycoside Profiles and Antioxidant Activities of Osmanthus fragrans Lour. Flowers by UPLC/PDA/MS and Simulated Digestion Model.

    Science.gov (United States)

    Jiang, Yirong; Mao, Shuqin; Huang, Weisu; Lu, Baiyi; Cai, Zengxuan; Zhou, Fei; Li, Maiquan; Lou, Tiantian; Zhao, Yajing

    2016-03-30

    Variations of phenylethanoid glycoside profiles and antioxidant activities in Osmanthus fragrans flowers through the digestive tract were evaluated by a simulated digestion model and UPLC/PDA/MS. Major phenylethanoid glycosides and phenolic acids, namely, salidroside, acteoside, isoacteoside, chlorogenic acid, and caffeic acid, were identified in four cultivars of O. fragrans flowers, and the concentration of acteoside was the highest, being up to 71.79 mg/g dry weight. After simulated digestion, total phenylethanoid glycoside contents and antioxidant activities were significantly decreased. Acteoside was identified as decomposing into caffeic acid, whereas salidroside was found to be stable during simulated digestion. According to Pearson's correlation analysis, acteoside contents showed good correlations with antioxidant activities during simulated digestion (R(2) = 0.994, P < 0.01). In conclusion, acteoside was the major contributor to the antioxidant activity of O. fragrans flowers, and salidroside was considered as the major antioxidant compound of O. fragrans flowers in vivo.

  9. Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

    Science.gov (United States)

    Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

    2009-01-01

    Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

  10. Bifidobacterium glycoside hydrolases and (potential) prebiotics

    NARCIS (Netherlands)

    Broek, van den L.A.M.; Voragen, A.G.J.

    2008-01-01

    Carbohydrates occur in food as natural constituents or are added as ingredients. In the last decade a number of novel dietary carbohydrates have been introduced as ingredients for food applications, responding to the growing awareness among consumers of the link between health and diet. One

  11. A new versatile microarray-based method for high-throughput screening of carbohydrate-active enzymes

    DEFF Research Database (Denmark)

    Vidal Melgosa, Silvia; Pedersen, Henriette Lodberg; Schückel, Julia

    2015-01-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing, together with associated bioinformatic tools have identified vast numbers of putative carbohydrate degrading and modifying enzymes including glycoside hydrolases...... that the technique can be used to analyse both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified un-characterised enzymes...

  12. The Natural Product Acivicin as a Tool for ABPP and the Activity of Serine Hydrolases in Uterine Fibroids

    OpenAIRE

    Kreuzer, Johannes

    2015-01-01

    The target proteins of acivicin and structure derived probes in tumor cells were identified using activity-based protein profiling. The target proteins were further characterized and their relation to the antitumor activity of acivicin pointed out. In a further project, the activity of serine hydrolases in myoma and myometrium was examined from tissue samples. This revealed a different activity of mast cell proteases. Mittels Activity-based Protein Profiling wurde eine Identifikation der Z...

  13. Soluble Epoxide Hydrolase Inhibitory Activity of Selaginellin Derivatives from Selaginella tamariscina

    Directory of Open Access Journals (Sweden)

    Jang Hoon Kim

    2015-12-01

    Full Text Available Selaginellin derivatives 1–3 isolated from Selaginella tamariscina were evaluated for their inhibition of soluble epoxide hydrolase (sEH to demonstrate their potential for the treatment of cardiovascular disease. All selaginellin derivatives (1–3 inhibited sEH enzymatic activity and PHOME hydrolysis, in a dose-dependent manner, with IC50 values of 3.1 ± 0.1, 8.2 ± 2.2, and 4.2 ± 0.2 μM, respectively. We further determined that the derivatives function as non-competitive inhibitors. Moreover, the predicted that binding sites and interaction between 1–3 and sEH were solved by docking simulations. According to quantitative analysis, 1–3 were confirmed to have high content in the roots of S. tamariscina; among them, selaginellin 3 exhibited the highest content of 189.3 ± 0.0 μg/g.

  14. Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity.

    Science.gov (United States)

    Mofford, David M; Adams, Spencer T; Reddy, G S Kiran Kumar; Reddy, Gadarla Randheer; Miller, Stephen C

    2015-07-15

    Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

  15. Synthesis of nonionic surfactants with azole ring bearing N-glycosides and their antibacterial activity

    Directory of Open Access Journals (Sweden)

    Fawzia Taieb Brahimi

    2017-05-01

    Full Text Available Six azoles with n-pentyl side chain 6–9, 11 and 12 were synthesized from n-hexanoic acid. Three N-glycosides namely: 5-pentyl-2-(d-amino arabinoside-1,3,4-oxadiazole (13, 5-pentyl-2-(d-aminoglycoside-1,3,4-thiadiazole (14, and 3-pentyl-4-(d-amino xyloside-4H-1,2,4-triazole-5-thiol (15 were prepared from already synthesized n-pentyl azoles 6, 7 and 11, respectively. Surface activity properties of water soluble synthesized compounds 6, 7, and 11–15 were studied in terms of surface tension, cloud point and critical micelle concentration. The antibacterial activities were assessed using the paper disk diffusion and broth dilution methods against gram-positive and gram-negative bacteria. Some of the synthetic compounds showed promising activity against microorganisms under test in comparison to commercially available antibiotics polymixine and oxytetracycline.

  16. A new aurone glycoside with antifungal activity from marine-derived fungus Penicillium sp. FJ-1.

    Science.gov (United States)

    Song, Yan-xia; Ma, Qiang; Li, Jie

    2015-03-01

    Endophytic fungi which reside in the tissue of mangrove plants seem to play an important role in the discovery of new biologically active substances. During the course of screening for the antimicrobial metabolites from the endophytic fugus Penicillium sp. FJ-1 of mangrove plant Avicennia marina, a new aurone glycoside (1) was isolated by repeated column chromatography on silica gel and recrystallization methods. The structure of 1 was elucidated as (Z)-7,4'-dimethoxy-6-hydroxy-aurone-4-O-β-glucopyranoside, on the basis of spectroscopic analysis. Compound 1 exhibited antifungal activity against Candida sp., with the potency comparable to amphotericin B and much better than fluconazole. Compound 1 can also inhibit extracellular phospholipase secretion in a concentration-dependent manner.

  17. Reactive oxygen species scavenging activity of flavone glycosides from Melilotus neapolitana.

    Science.gov (United States)

    Fiorentino, Antonio; D'Abrosca, Brigida; Pacifico, Severina; Golino, Annunziata; Mastellone, Claudio; Oriano, Palma; Monaco, Pietro

    2007-02-28

    One new and six known flavone glycosides were isolated from the MeOH extract of Melilotus neapolitana Ten. The new compound, identified as 7-O-beta-D-glucopyranosyloxy-4',5-dihydroxy-3-[O-alpha-L-rhamnopyranosyl-(1-->6)-3-O-beta-D-glucopyranosyloxy]flavone (1) by 1D and 2D NMR techniques and mass spectra, was isolated along with kaempferol-3-O-rutinoside (2), kaempferol-3-O-glucoside (3), rutin (4), quercetin-3-O-glucoside (5), isorhamnetin-3-O-rutinoside (6), and isorhamnetin-3-O-glucoside (7). The antioxidant and radical scavenging activities of these compounds and the whole crude methanol extract were evaluated. The organic extract can inhibit MDA marker's synthesis by 57%. All the metabolites displayed good reducing power, with the kaempferol (2,3) and isorhamnetin derivatives (6,7) being less active than the corresponding quercetin derivatives 4,5.

  18. Isolation, characterization and antimicrobial activities of polyacetylene glycosides from Coreopsis tinctoria Nutt.

    Science.gov (United States)

    Guo, Jia; Wang, Ao; Yang, Ke; Ding, Hao; Hu, Yimin; Yang, Yumeng; Huang, Siqi; Xu, Jingguo; Liu, Tianxing; Yang, Haiyan; Xin, Zhihong

    2017-04-01

    Polyacetylene glycosides, (6Z, 12E)-tetradecadiene-8,10-diyne-1-ol-3(R)-O-β-D-glucopyranoside (trivially named coreoside E) and (6Z, 12E)-tetradecadiene-8,10-diyne-1-ol-3(R)-O-β-L-arabinopyranosyl-(1 → 2)-β-D-glucopyranoside (trivially named coreoside F), were isolated from buds of Coreopsis tinctoria Nutt., together with one known compound, coreoside B. Their chemical structures were elucidated by extensive spectroscopic analysis and on the basis of their chemical reactivities. Coreoside E exhibited high levels of antimicrobial activity against Staphylococcus aureus and Bacillus anthracis with minimum inhibitory concentrations of 27 ± 0.27 and 18 ± 0.40 μM, respectively, whereas coreoside F and coreoside B showed weak antimicrobial activity against S. aureus and B. anthracis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Soluble epoxide hydrolase activity and pharmacologic inhibition in horses with chronic severe laminitis.

    Science.gov (United States)

    Guedes, A; Galuppo, L; Hood, D; Hwang, S H; Morisseau, C; Hammock, B D

    2017-05-01

    The roles of soluble epoxide hydrolase and lipid mediators in inflammatory and neuropathic pain could be relevant in laminitis pain management. To determine soluble epoxide hydrolase (sEH) activity in the digital laminae, sEH inhibitor potency in vitro, and efficacy of a sEH inhibitor as an adjunct analgesic therapy in chronic laminitic horses. In vitro experiments and clinical case series. sEH activity was measured in digital laminae from euthanised healthy and laminitic horses (n = 5-6/group). Potency of 7 synthetic sEH inhibitors was determined in vitro using equine liver cytosol. One of them (t-TUCB; 0.1 mg/kg bwt i.v. every 24 h) was selected based on potency and stability, and used as adjunct therapy in 10 horses with severe chronic laminitis (Obel grades 2, one horse; 3-4, nine horses). Daily assessments of forelimb lifts, pain scores, physiologic and laboratory examinations were performed before (baseline) and during t-TUCB treatment. Data are presented as mean ± s.d. and 95% confidence intervals (CI). sEH activity in the digital laminae from laminitic horses (0.9±0.6 nmol/min/mg; 95% CI 0.16-1.55 nmol/min/mg) was significantly greater (P = 0.01) than in healthy horses (0.17±0.09 nmol/min/mg; CI 0.07-0.26 nmol/min/mg). t-TUCB as an adjunct analgesic up to 10 days (4.3±3 days) in laminitic horses was associated with significant reduction in forelimb lifts (36±22%; 95% CI 9-64%) and in pain scores (18±23%; 95% CI 2-35%) compared with baseline (P = 0.04). One horse developed gas colic and another corneal vascularisation in a blind eye during treatment. No other significant changes were observed. Absence of control group and evaluator blinding in case series. sEH activity is significantly higher in the digital laminae of actively laminitic compared with healthy horses, and use of a potent inhibitor of equine sEH as adjunct analgesic therapy appears to decrease signs of pathologic pain in laminitic horses. © 2016 EVJ Ltd.

  20. A new bianthron glycoside as inhibitor of Trypanosoma cruzi glyceraldehyde 3-phosphate dehydrogenase activity

    International Nuclear Information System (INIS)

    Macedo, Edangelo M.S. de; Silva, Maria G.V.; Wiggers, Helton J.; Montanari, Carlos A.; Braz-Filho, Raimundo; Andricopulo, Adriano D.

    2009-01-01

    A phytochemical investigation of the ethanolic extract of stalks of Senna martiana Benth. (Leguminoseae), native specie of northeast Brazil, resulted in the isolation and spectroscopic characterization of a new bianthrone glycoside, martianine 1 (10,10'-il-chrysophanol-10-oxi- 10,10'-bi-glucosyl). Its identification was established by HRMS, IR and 2D NMR experiments. The evaluation of martianine trypanocidal activity was carried out against gliceraldehyde 3-phosphate dehydrogenase enzyme from Trypanosoma cruzi. Its inhibitory constant (K i ) is in the low micromolar concentration and it was determined by isothermal titration calorimetry to be 27.3 +-2.47 μmol L -1 . The non-competitive mechanism is asserted to be putative of the mode of action martianine displays against T. cruzi GAPDH. Results show that martianine has a great potential to become new lead molecule by inhibiting this key enzyme and for the development of new drugs against Chagas disease. (author)

  1. New flavonol glycoside from Scabiosa prolifera L. aerial parts with in vitro antioxidant and cytotoxic activities.

    Science.gov (United States)

    Al-Qudah, Mahmoud A; Otoom, Noor K; Al-Jaber, Hala I; Saleh, Ayman M; Abu Zarga, Musa H; Afifi, Fatma U; Abu Orabi, Sultan T

    2017-12-01

    Phytochemical investigation of the chemical constituents of the aerial parts of Scabiosa prolifera L. led to the isolation of one new flavonol glycoside, kaempferol-3-O-(4″,6″-di-E-p-coumaroyl)-β-D-galactopyranoside (1), along with ten other known compounds including luteolin-7-O-(2″-O-ethyl-β-glucopyranoside), β-sitosterol, β-sitosterylglucoside, ursolic acid, corosolic acid, ursolic acid 3-O-β-D-arabinopyranoside, apigenin, methyl-α-D-glucopyranoside, luteolin-7-O-β-glucopyranoside and isoorientin. The structures of all isolated compounds were established using chemical methods and spectroscopic methods including IR, UV, NMR (1D and 2D) and HRESIMS. All compounds were isolated for the first time from the plant. The antioxidant and cytotoxic activities of compounds 1 and 2 were also investigated.

  2. A sensitive and specific radiochromatographic assay of fatty acid amide hydrolase activity.

    Science.gov (United States)

    Maccarrone, M; Bari, M; Agrò, A F

    1999-02-15

    A radiochromatographic method has been set up in order to determine fatty acid amide hydrolase (FAAH) activity, based on reversed-phase high-performance liquid chromatography and on-line scintillation counting. The reaction products were separated using a C18 column eluted with methanol-water-acetic acid and quantitated with an external standard. Baseline separation of the acid product from the substrate was completed in less than 4 min, with a detection limit of 2.5 fmol arachidonic acid at a signal to noise ratio of 4:1. The method enabled to determine the kinetic constants (i.e., apparent Km of 2.0 +/- 0.2 microM and Vmax of 800 +/- 75 pmol. min-1. mg protein-1 toward anandamide) and the substrate specificity of human brain FAAH, as well as the extent of enzyme inhibition by some anandamide congeners. The femtomole sensitivity and the accuracy of the method allow detection and characterization of the activity of FAAH in very minute tissue samples or in samples where the enzymatic activity is very low. Copyright 1999 Academic Press.

  3. Zinc mediated activation of terminal alkynes: stereoselective synthesis of alkynyl glycosides.

    Science.gov (United States)

    Tatina, Madhu Babu; Kusunuru, Anil Kumar; Yousuf, Syed Khalid; Mukherjee, Debaraj

    2014-10-28

    Zinc mediated alkynylation reaction was studied for the preparation of C-glycosides from unactivated alkynes. Different glycosyl donors such as glycals and anomeric acetates were tested towards an alkynyl zinc reagent obtained from alkynes using zinc dust and ethyl bromoacetate as an additive. The method provides simple, mild and stereoselective access to alkynyl glycosides both from aromatic and aliphatic acetylenes.

  4. Genome mining and motif truncation of glycoside hydrolase family 5 endo-β-1,4-mannanase encoded by Aspergillus oryzae RIB40 for potential konjac flour hydrolysis or feed additive.

    Science.gov (United States)

    Tang, Cun-Duo; Shi, Hong-Ling; Tang, Qing-Hai; Zhou, Jun-Shi; Yao, Lun-Guang; Jiao, Zhu-Jin; Kan, Yun-Chao

    2016-11-01

    Two novel glycosyl hydrolase family 5 (GH5) β-mannanases (AoMan5A and AoMan5B) were identified from Aspergillus oryzae RIB40 by genome mining. The AoMan5A contains a predicted family 1 carbohydrate binding module (CBM-1), located at its N-terminal. The AoMan5A, AoMan5B and truncated mutant AoMan5AΔCL (truncating the N-terminal CBM and linker of AoMan5A) were expressed retaining the N-terminus of the native protein in Pichia pastoris GS115 by pPIC9K M . The specific enzyme activity of the purified reAoMan5A, reAoMan5B and reAoMan5AΔCL towards locust bean gum at pH 3.6 and 40°C for 10min, was 8.3, 104.2 and 15.8U/mg, respectively. The temperature properties of the reAoMan5AΔCL were improved by truncating CBM. They can degrade the pretreated konjac flour and produce prebiotics. In addition, they had excellent stability under simulative gastric fluid and simulative prilling process. All these properties make these recombinant β-mannanases potential additives for use in the food and feed industries. Copyright © 2016. Published by Elsevier Inc.

  5. An appraisal of eighteen commonly consumed edible plants as functional food based on their antioxidant and starch hydrolase inhibitory activities.

    Science.gov (United States)

    Lee, Yian Hoon; Choo, Candy; Watawana, Mindani I; Jayawardena, Nilakshi; Waisundara, Viduranga Y

    2015-11-01

    Eighteen edible plants were assessed for their antioxidant potential based on oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, total phenolics, vitamin C content and various lipophilic antioxidants. The inhibitory activities of the plant extracts against the enzymatic activities of α-amylase and α-glucosidase were also evaluated. The antioxidant and starch hydrolase activities of the plants varied widely across a single batch of analysis. The ORAC and DPPH radical scavenging EC50 values varied between 298 and 1984 Trolox equivalents g(-1) fresh weight and between 91 and 533 mg kg(-1) fresh weight, respectively. The total phenolics and vitamin C contents varied between 32 and 125 mg gallic acid equivalents g(-1) fresh weight and between 96 and 285 µg g(-1) fresh weight, respectively. All the plants contained neoxanthin, violaxanthin, and α- and β-carotene in varying amounts. Coccinia grandis, Asparagus racemosus, Costus speciosus, Amaranthus viridis and Annona muricata displayed the highest inhibitory activities against starch hydrolases. They were the most efficient against the breakdown of seven starches exposed to the two enzymes as well. Overall, the edible plants were observed to display a high antioxidant potential with starch hydrolase inhibitory properties, which were beneficial in their being recognized as functional food. © 2014 Society of Chemical Industry.

  6. Evaluation of the organophosphorus hydrolase enzyme activity in creams and investigation of its stability

    Directory of Open Access Journals (Sweden)

    Mariye Rajaie

    2016-06-01

    Full Text Available The main purpose of this project is investigation of the organophosphorus hydrolase (OPH enzyme activity in water in oil (w/o and oil in water (o/w creams and investigation of the OPH enzyme stability in formulated creams. OPH enzyme was extracted and purified from strain flavobacterium. The w/o and o/w creams were prepared using different formulations. In order to achieve an emulsion with maximum stability, appropriate percentage of the cream components was selected by studying different formulations and the physical and chemical stability of the produced cream were considered. 5Uenzyme/90gcream enzyme was used for each formulation. To measure the enzyme activity in creams, extraction method was used and enzyme activity was determined based on parathion hydrolysis. The thermal stability of OPH in both types of w/o and o/w creams was studied at 4 and 30  °C for various time periods. The average enzyme activity was about 0.0065 U/gcream and 0.018 U/gcream for w/o and o/w creams respectivly. According to the results, the relative activity at 4 °C was reduced to 50% after 26 and 45 days in w/o and o/w creams, respectivly. The results showed that the OPH enzyme activity in o/w cream was 2.6 times more than that of w/o cream, because of the higher hydrophobicity of o/w cream compared to w/o. The OPH enzyme stability in o/w cream was greater in comparison to w/o cream. The OPH enzyme was active for nearly 2 months on o/w creams at 4 °C .

  7. Bile Salt Hydrolase Activities: A Novel Target to Screen Anti-Giardia Lactobacilli?

    Directory of Open Access Journals (Sweden)

    Thibault Allain

    2018-02-01

    Full Text Available Giardia duodenalis is a protozoan parasite responsible for giardiasis, a disease characterized by intestinal malabsorption, diarrhea and abdominal pain in a large number of mammal species. Giardiasis is one of the most common intestinal parasitic diseases in the world and thus a high veterinary, and public health concern. It is well-established that some probiotic bacteria may confer protection against this parasite in vitro and in vivo and we recently documented the implication of bile-salt hydrolase (BSH-like activities from strain La1 of Lactobacillus johnsonii as mediators of these effects in vitro. We showed that these activities were able to generate deconjugated bile salts that were toxic to the parasite. In the present study, a wide collection of lactobacilli strains from different ecological origins was screened to assay their anti-giardial effects. Our results revealed that the anti-parasitic effects of some of the strains tested were well-correlated with the expression of BSH-like activities. The two most active strains in vitro, La1 and Lactobacillus gasseri CNCM I-4884, were then tested for their capacity to influence G. duodenalis infection in a suckling mice model. Strikingly, only L. gasseri CNCM I-4884 strain was able to significantly antagonize parasite growth with a dramatic reduction of the trophozoites load in the small intestine. Moreover, this strain also significantly reduced the fecal excretion of Giardia cysts after 5 days of treatment, which could contribute to blocking the transmission of the parasite, in contrast of La1 where no effect was observed. This study represents a step toward the development of new prophylactic strategies to combat G. duodenalis infection in both humans and animals.

  8. Dipeptidyl peptidase-IV inhibitory activity of dimeric dihydrochalcone glycosides from flowers of Helichrysum arenarium.

    Science.gov (United States)

    Morikawa, Toshio; Ninomiya, Kiyofumi; Akaki, Junji; Kakihara, Namiko; Kuramoto, Hiroyuki; Matsumoto, Yurie; Hayakawa, Takao; Muraoka, Osamu; Wang, Li-Bo; Wu, Li-Jun; Nakamura, Seikou; Yoshikawa, Masayuki; Matsuda, Hisashi

    2015-10-01

    A methanol extract of everlasting flowers of Helichrysum arenarium L. Moench (Asteraceae) was found to inhibit the increase in blood glucose elevation in sucrose-loaded mice at 500 mg/kg p.o. The methanol extract also inhibited the enzymatic activity against dipeptidyl peptidase-IV (DPP-IV, IC50 = 41.2 μg/ml), but did not show intestinal α-glucosidase inhibitory activities. From the extract, three new dimeric dihydrochalcone glycosides, arenariumosides V-VII (2-4), were isolated, and the stereostructures were elucidated based on their spectroscopic properties and chemical evidence. Of the constituents, several flavonoid constituents, including 2-4, were isolated, and these isolated constituents were investigated for their DPP-IV inhibitory effects. Among them, chalconaringenin 2'-O-β-D-glucopyranoside (16, IC50 = 23.1 μM) and aureusidin 6-O-β-D-glucopyranoside (35, 24.3 μM) showed relatively strong inhibitory activities.

  9. Reactive Oxygen Species Scavenging Activity of Flavone Glycosides from Melilotus neapolitana

    Directory of Open Access Journals (Sweden)

    Pietro Monaco

    2007-02-01

    Full Text Available One new and six known flavone glycosides were isolated from the MeOH extract of Melilotus neapolitana Ten. The new compound, identified as 7-O-β-D-gluco-pyranosyloxy-4',5-dihydroxy-3-[O-α-L-rhamnopyranosyl-(1→6-3-O-β-D-glucopyrano-syloxy]flavone (1 by 1D and 2D NMR techniques and mass spectra, was isolated along with kaempferol-3-O-rutinoside (2, kaempferol-3-O-glucoside (3, rutin (4, quercetin-3-O-glucoside (5, isorhamnetin-3-O-rutinoside (6, and isorhamnetin-3-O-glucoside (7. The antioxidant and radical scavenging activities of these compounds and the whole crude methanol extract were evaluated. The organic extract can inhibit MDA marker’s synthesis by 57%. All the metabolites displayed good reducing power, with the kaempferol (2,3 and isorhamnetin derivatives (6,7 being less active than the corresponding quercetin derivatives 4,5.

  10. Phenylpropanoid Glycoside Analogues: Enzymatic Synthesis, Antioxidant Activity and Theoretical Study of Their Free Radical Scavenger Mechanism

    Science.gov (United States)

    López-Munguía, Agustín; Hernández-Romero, Yanet; Pedraza-Chaverri, José; Miranda-Molina, Alfonso; Regla, Ignacio; Martínez, Ana; Castillo, Edmundo

    2011-01-01

    Phenylpropanoid glycosides (PPGs) are natural compounds present in several medicinal plants that have high antioxidant power and diverse biological activities. Because of their low content in plants (less than 5% w/w), several chemical synthetic routes to produce PPGs have been developed, but their synthesis is a time consuming process and the achieved yields are often low. In this study, an alternative and efficient two-step biosynthetic route to obtain natural PPG analogues is reported for the first time. Two galactosides were initially synthesized from vanillyl alcohol and homovanillyl alcohol by a transgalactosylation reaction catalyzed by Kluyveromyces lactis β-galactosidase in saturated lactose solutions with a 30%–35% yield. To synthesize PPGs, the galactoconjugates were esterified with saturated and unsaturated hydroxycinnamic acid derivatives using Candida antarctica Lipase B (CaL-B) as a biocatalyst with 40%–60% yields. The scavenging ability of the phenolic raw materials, intermediates and PPGs was evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) method. It was found that the biosynthesized PPGs had higher scavenging abilities when compared to ascorbic acid, the reference compound, while their antioxidant activities were found similar to that of natural PPGs. Moreover, density functional theory (DFT) calculations were used to determine that the PPGs antioxidant mechanism proceeds through a sequential proton loss single electron transfer (SPLET). The enzymatic process reported in this study is an efficient and versatile route to obtain PPGs from different phenylpropanoid acids, sugars and phenolic alcohols. PMID:21674039

  11. Antimicrobial, anti-inflammatory activities and toxicology of phenylethanoid glycosides from Monochasma savatieri Franch. ex Maxim.

    Science.gov (United States)

    Liu, Yan-li; He, Wen-jun; Mo, Li; Shi, Meng-fan; Zhu, Ying-ying; Pan, Shu; Li, Xiao-ran; Xu, Qiong-ming; Yang, Shi-lin

    2013-09-16

    Monochasma savatieri Franch. ex Maxim is used for treating many diseases in Traditional Chinese Medicine. The present study was designed to evaluate antibacterial, anti-inflammatory activities and toxicity of the total phenylethanoid glycosides from Monochasma savatieri Franch. ex Maxim (TPG). The antibacterial activity of TPG was checked by MIC and MBC in vitro; survival of mice with Pseudomonas aeruginosa or Staphylococcus aureus infection-induced sepsis was investigated to evaluate antibacterial activity of TPG in vivo. Additionally, antibacterial activities of TPG were also investigated in a Pseudomonas aeruginosa infection-induced pneumonia in the mice model. Cotton pellet induced granuloma and xylene induced ear swelling in mice models were used to quantify the anti-inflammatory activity. TPG showed a significant possess bacteriostatic properties against five bacteria strains at a concentration between 0.0625 and 16 mg/ml. Moreover, TPG has bactericidal activity against Pseudomonas aeruginosa, Streptococcus pneumoniae or Escherichia coli. TPG (60, 120, and 180 mg/kg) prolonged survival rate of mice with Pseudomonas aeruginosa or Staphylococcus aureus infection-induced sepsis. In addition, TPG (180 mg/kg) could reduce the bacterial colony-forming units in lung tissue. Furthermore, TPG (60-180 mg/kg) had significantly reduced xylene-induced ear edema and granulomat formation induced by cotton pellet at a dose-dependent manner. In addition, administration of TPG (1.5 g/kg) for 15 days did not result in toxicities in liver, kidney, spleen and thymus tissue in rats. These results indicated that TPG might be useful for the development of a novel treatment for respiratory infections or pneumonia caused by Pseudomonas aeruginosa or Staphylococcus aureus. © 2013 Elsevier Ireland Ltd. All rights reserved.

  12. Lipoxin Generation Is Related to Soluble Epoxide Hydrolase Activity in Severe Asthma

    Science.gov (United States)

    Ono, Emiko; Dutile, Stefanie; Kazani, Shamsah; Wechsler, Michael E.; Yang, Jun; Hammock, Bruce D.; Douda, David Nobuhiro; Tabet, Yacine; Khaddaj-Mallat, Rayan; Sirois, Marco; Sirois, Chantal; Rizcallah, Edmond; Rousseau, Éric; Martin, Richard; Sutherland, E. Rand; Castro, Mario; N. Jarjour, Nizar; Israel, Elliot

    2014-01-01

    Rationale: Severe asthma is characterized by airway inflammatory responses associated with aberrant metabolism of arachidonic acid. Lipoxins (LX) are arachidonate-derived pro-resolving mediators that are decreased in severe asthma, yet mechanisms for defective LX biosynthesis and a means to increase LXs in severe asthma remain to be established. Objectives: To determine if oxidative stress and soluble epoxide hydrolase (sEH) activity are linked to decreased LX biosynthesis in severe asthma. Methods: Aliquots of blood, sputum, and bronchoalveolar lavage fluid were obtained from asthma subjects for mediator determination. Select samples were exposed to t-butyl-hydroperoxide or sEH inhibitor (sEHI) before activation. Peripheral blood leukocyte–platelet aggregates were monitored by flow cytometry, and bronchial contraction was determined with cytokine-treated human lung sections. Measurements and Main Results: 8-Isoprostane levels in sputum supernatants were inversely related to LXA4 in severe asthma (r = −0.55; P = 0.03) and t-butyl-hydroperoxide decreased LXA4 and 15-epi-LXA4 biosynthesis by peripheral blood leukocytes. LXA4 and 15-epi-LXA4 levels were inversely related to sEH activity in sputum supernatants and sEHIs significantly increased 14,15-epoxy-eicosatrienoic acid and 15-epi-LXA4 generation by severe asthma whole blood and bronchoalveolar lavage fluid cells. The abundance of peripheral blood leukocyte–platelet aggregates was related to asthma severity. In a concentration-dependent manner, LXs significantly inhibited platelet-activating factor–induced increases in leukocyte–platelet aggregates (70.8% inhibition [LXA4 100 nM], 78.3% inhibition [15-epi-LXA4 100 nM]) and 15-epi-LXA4 markedly inhibited tumor necrosis factor-α–induced increases in bronchial contraction. Conclusions: LX levels were decreased by oxidative stress and sEH activity. Inhibitors of sEH increased LXs that mediated antiphlogistic actions, suggesting a new therapeutic approach

  13. A new flavonol glycoside and activity of compounds from the flower of Nymphaea candida.

    Science.gov (United States)

    Liu, R-N; Wang, W; Ding, Y; Xie, W-D; Ma, C; Du, L-J

    2007-01-01

    A new compound, kaempferol 3-O-(2''-O-galloylrutinoside) (1), was isolated from the white flower of Nymphaea candida, together with nine known flavonol glycosides, kaempferol (2), kaempferol 3-O-beta-D-glucopyranoside (3), kaempferol 3-O-alpha-l-rhamnopyranoside (4), kaempferol 3-O-alpha-l-rhamnopyranosylglucopyranoside (5), kaempferol 7-O-beta-D-glucopyranoside 3-(O-alpha-l-rhamnopyranosylglucopyranoside) (6), quercetin (7), quercetin 3-O-beta-D-xylopyranoside (8), myricetin (9), myricetin 3'-O-beta-D-xylopyranoside (10). The structure of 1 was established on the basis of the analysis of its 1D and 2D NMR spectral data. Compounds 1-7 and 9 exhibited moderate to significant antioxidant activities, which were evaluated by measurement of low-density lipoprotein (LDL) and malondialdehyde (MDA) levels in vitro. Compounds 1, 3, 4, 6 and 9 exhibited promising neuroprotective effects on ischemic injury model of cultured rat cortical neurons treated with sodium dithionite in glucose-free medium. Furthermore, compounds 1, 5, and 9 had distinct cytotoxicity to adrenal gland pheochromocytoma, PC12 cells, being treated by the same way.

  14. Experimental and DFT studies on the antioxidant activity of a C-glycoside from Rhynchosia capitata

    Science.gov (United States)

    Praveena, R.; Sadasivam, K.; Kumaresan, R.; Deepha, V.; Sivakumar, Raman

    2013-02-01

    Rhynchosia capitata (=Glycine capitata) Heyne ex roth, was found to possess polyphenolics including flavonoids, which acts as potential antioxidant. The study of ethanolic extract of roots and leaves reveals that the leaves possess high polyphenolics including flavonoids than roots. This was also confirmed by DPPH radical scavenging activity. Leaf powder of the plant was extracted with different solvents by soxhlet apparatus in the order of increasing polarity. The DPPH scavenging activity of methanol fraction was found to be high compared to the crude extract and other fractions. Nitric oxide scavenging activity was dominant in chloroform fraction compared to methanol fraction. Presence of flavonoids especially vitexin, a C-glycoside in methanol and chloroform fractions were confirmed by high pressure thin layer chromatography (HPTLC) analysis. The structural and molecular characteristics of naturally occurring flavonoid, vitexin was investigated in gas phase using density functional theory (DFT) approach with B3LYP/6-311G(d,p) level of theory. Analysis of bond dissociation enthalpy (BDE) reveals that the OH site that requires minimum energy for dissociation is 4'-OH from B-ring. To explore the radical scavenging activity of vitexin, the adiabatic ionization potential, electron affinity, hardness, softness, electronegativity and electrophilic index properties were computed and interpreted. The nonvalidity of Koopman's theorem has been verified by the computation of Eo and Ev energy magnitudes. Interestingly, from BDE calculations it was observed that BDE for 4'-OH, 5-OH and 7-OH are comparatively low for vitexin than its aglycone apigenin and this may be due to the presence of C-8 glucoside in vitexin. To substantiate this, plot of frontier molecular orbital and spin density distribution analysis for neutral and the corresponding radical species for the compound vitexin have been presented.

  15. Antiallergic activity of unripe Citrus hassaku fruits extract and its flavanone glycosides on chemical substance-induced dermatitis in mice.

    Science.gov (United States)

    Itoh, Kimihisa; Masuda, Megumi; Naruto, Shunsuke; Murata, Kazuya; Matsuda, Hideaki

    2009-10-01

    Oral administration of a 50% ethanolic extract (CH-ext) obtained from unripe Citrus hassaku fruits collected in July exhibited a potent dose-dependent inhibition of IgE (immunoglobulin E)-mediated triphasic cutaneous reaction at 1 h [immediate phase response (IPR)], 24 h [late phase response (LPR)] and 8 days [very late phase response (vLPR)] after dinitrofluorobenzene challenge in mice. Naringin, a major flavanone glycoside component of CH-ext, showed a potent dose-dependent inhibition against IPR, LPR and vLPR. Neohesperidin, another major glycoside component of CH-ext, showed an inhibition against vLPR. The effect of CH-ext on type IV allergic reaction was examined by determining inhibitory activity against ear swelling in mice by using the picryl chloride-induced contact dermatitis (PC-CD) model. Oral administration (p.o.) of CH-ext and subcutaneous administration (s.c.) of prednisolone inhibited ear swelling during the induction phase of PC-CD. The inhibitory activities of combinations of CH-ext (p.o.) and prednisolone (s.c.) against PC-CD in mice were more potent than those of CH-ext alone and prednisolone alone, without enhancing the adverse effects. Other combinations of prednisolone (s.c.) and flavanone glycoside (p.o.) components of CH-ext, i.e. naringin and neohesperidin, exerted similar synergistic effects.

  16. Peptidoglycan Hydrolases of Escherichia coli

    Science.gov (United States)

    van Heijenoort, Jean

    2011-01-01

    Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

  17. Sweet antibiotics – the role of glycosidic residues in antibiotic and antitumor activity and their randomization

    Czech Academy of Sciences Publication Activity Database

    Křen, Vladimír; Řezanka, Tomáš

    2008-01-01

    Roč. 32, č. 5 (2008), s. 858-889 ISSN 0168-6445 R&D Projects: GA MŠk(CZ) LC06010; GA AV ČR IAA400200503 Institutional research plan: CEZ:AV0Z50200510 Keywords : glycosides * sweet antibiotics * aglycone Subject RIV: CE - Biochemistry Impact factor: 7.963, year: 2008

  18. Characterization of Carbohydrate Active Enzymes Involved in Arabinogalactan Protein Metabolism

    DEFF Research Database (Denmark)

    Knoch, Eva

    and tissues, their functions and synthesis are still poorly understood. The aim of the research presented in the thesis was to characterize carbohydrate active enzymes involved in AGP biosynthesis and modification to gain insights into the biosynthesis of the glycoproteins in plants. Candidate...... glycosyltransferases and glycoside hydrolases were selected based on co-expression profiles from a transcriptomics analysis. Reverse genetics approach on a novel glucuronosyltransferase involved in AGP biosynthesis has revealed that the enzyme activity is required for normal cell elongation in etiolated seedlings....... The enzymatic activity of a hydrolase from GH family 17 was investigated, without successful determination of the activity. Members of hydrolase family 43 appeared to be localized in the Golgi-apparatus, which is also the compartment for glycan biosynthesis. The localization of these glycoside hydrolases...

  19. Anthracycline glycosides

    International Nuclear Information System (INIS)

    Vicario, G.P.; Penco, S.; Arcamone, F.

    1980-01-01

    An invention is described which relates to anthracycline glycosides, and provides as new compounds the radiochemically labelled [14- 14 C] daunorubicin and [14- 14 C] doxorubicin and their hydrochlorides. These are important for the study of the distribution pharmaco-kinetics and metabolism of these compounds which are antitumour medicines. The stability and specificity of the 14 C-label makes these compounds useful for both experimental and medical purposes. (author)

  20. Some hydrolase activities from the tick Hyalomma lusitanicum Koch, 1844 (Ixodoidea: Ixodida

    Directory of Open Access Journals (Sweden)

    Giménez-Pardo C.

    2008-12-01

    Full Text Available In this work has been made a detection and preliminary characterization of some hydrolases in whole extracts from unfed adult males and females of Hyalomma lusitanicum, one of the vectors for Theileria annulata that causes Mediterranean theileriosis in cattle. We have elected as targets, proteases as enzymes implicated in the nutritional processes of ticks, esterases that are usually implicated in resistance to organophosphates and phosphatises often implicated in protein phosphorilation and control of ticks salivary gland. The biological role and physiological significance are discussed in terms of the possibility of use these enzymes as possible in future anti-tick vaccination or acaricide resistance.

  1. Antifungal (Gaeumannomyces graminis var. tritici) activity of various glycosides of medicagenic acid

    OpenAIRE

    Stefan Martyniuk; Marian Jurzysta

    2012-01-01

    Different concentrations of medicagenic acid and five glycosides of this acid isolated from alfalfa (Medicago sativa) were added to agar medium (corn meal agar, CMA) inoculated with cultures of Gaeumannomyces graminis var. tritici (Ggt). After 7 days of incubation at 25oC colony radius was measured and % of inhibition calculated in relation to the control medium (CMA enriched with the solvent of the tested compounds). Within the tested concentrations, only 3-O-β -D -glucopiranoside medicagena...

  2. A sulphated flavone glycoside from Livistona australis and its antioxidant and cytotoxic activity.

    Science.gov (United States)

    Kassem, Mona E S; Shoela, Soha; Marzouk, Mona M; Sleem, Amany A

    2012-01-01

    A new flavone glycoside tricin 7-O-β-glucopyranoside-2″-sulphate sodium salt along with 14 known flavonoid compounds were isolated and identified from the aqueous methanol extract of Livistona australis leaves. Their structures were established on the basis of extensive NMR (¹H, ¹³C, HSQC and H-H COSY) and ESIMS data. Antioxidant and cytotoxicity properties of the methanol extract of the leaves as well as the new compound were investigated.

  3. A GH57 4-alpha-glucanotransferase of hyperthermophilic origin with potential for alkyl glycoside production

    NARCIS (Netherlands)

    Paul, Catherine J.; Leemhuis, Hans; Dobruchowska, Justyna M.; Grey, Carl; Onnby, Linda; van Leeuwen, Sander S.; Dijkhuizen, Lubbert; Karlsson, Eva Nordberg

    4-alpha-Glucanotransferase (GTase) enzymes (EC 2.4.1.25) modulate the size of alpha-glucans by cleaving and reforming alpha-1,4 glycosidic bonds in alpha-glucans, an essential process in starch and glycogen metabolism in plants and microorganisms. The glycoside hydrolase family 57 enzyme (GTase57)

  4. Changes in Levels of Phenylethanoid Glycosides, Antioxidant Activity, and Other Quality Traits in Cistanche deserticola Slices by Steam Processing.

    Science.gov (United States)

    Peng, Fang; Chen, Jun; Wang, Xia; Xu, Changqing; Liu, Tongning; Xu, Rong

    2016-07-01

    We investigated the effect of steaming time on Cistanche deserticola Y. C. MA slices by analyzing levels of bioactive compounds, antioxidant activity, and weight loss compared with fresh, directly oven-dried, and blanched samples. Fresh samples had extremely low levels of phenylethanoid glycosides and antioxidant activity. Lower levels of weight loss and higher amounts of soluble sugars, polysaccharides, and dilute ethanol-soluble extracts were found when the slices were steamed rather than blanched. Slices steamed for 5 and 7 min contained significantly (pprocess. The concentration of polysaccharides fluctuated during the steaming process. The steaming time had a consistent effect on antioxidant properties evaluated by oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity (DPPH) and ferric reducing antioxidant property (FRAP), showing a significant increase and reaching 108.62, 23.08, and 11.68 micromoles Trolox per mass of fresh slice (μmol TE/g FW), respectively. The present results suggest that fresh-cut C. deserticola can be subjected to approximately 5-7 min of steaming to improve phenylethanoid glycoside levels and antioxidant activity, while still preserving the amounts of soluble sugars, polysaccharides, and dilute ethanol-soluble extracts. These results would help to improve the production process for fresh-cut Chinese medicines, and increase the understanding of their associated health benefits.

  5. Transient changes of enzyme activity of five acid hydrolases in the supernatants of homogenates of hearts of mice due to ultraviolet irradiation

    International Nuclear Information System (INIS)

    Droba, B.; Jagiellonian Univ., Krakow

    1977-01-01

    Enzymatic activity of five lysosomal hydrolases: acid p-nitrophenyl phosphatase (EC 3.1.3.2), acid β-glycerophosphatase (EC 3.1.3.2), arylsulphatase (EC 3.1.6.1), β-galactosidase (EC 3.2.1.23) and β-N-acetylhexoaminidase (EC 3.2.1.30) was studied in the supernatants of homogenates of hearts of unirradiated mice, serving as controls, and a group of UV-irradiated mice. In the control group, determinations made at 6-hr intervals showed rhythmic diurnal changes in activities of three acid hydrolases. These changes were statistically significant in the case of acid p-nitrophenyl phosphatase, acid β-glycerophosphatase, and β-N-acetylhexosaminidase. The effect of UV-irradiation was manifested mainly by depression of enzyme activities of the acid hydrolases during the first few hours after exposure. Depression of activities of arylsulphatase and β-N-acetylhexosaminidase by UV light was statistically significant. Presumably, the fall in enzyme activities of the acid hydrolases was due to chemical mediators formed in the skin under the influence of UV-radiation and adrenal corticoids secreted into the blood

  6. The Activation of Phytophthora Effector Avr3b by Plant Cyclophilin is Required for the Nudix Hydrolase Activity of Avr3b.

    Science.gov (United States)

    Kong, Guanghui; Zhao, Yao; Jing, Maofeng; Huang, Jie; Yang, Jin; Xia, Yeqiang; Kong, Liang; Ye, Wenwu; Xiong, Qin; Qiao, Yongli; Dong, Suomeng; Ma, Wenbo; Wang, Yuanchao

    2015-08-01

    Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a Phytophthora sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta; and this enzymatic activity is required for full virulence of P. sojae strain P6497 in soybean (Glycine max). Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase), cyclophilin, in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1, which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYP1; as a result, the mutant Avr3bP132A can no longer be activated by GmCYP1, and is also unable to promote Phytophthora infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3bP132A lost its ability to trigger HR. Furthermore, silencing of GmCYP1 rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of Nicotiana benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a "helper" that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b.

  7. HPLC-ESI-IT-MS/MS Analysis and Biological Activity of Triterpene Glycosides from the Colombian Marine Sponge Ectyoplasia ferox

    Directory of Open Access Journals (Sweden)

    Jhonny Colorado-Ríos

    2013-12-01

    Full Text Available The marine sponge Ectyoplasia ferox produces antipredatory and allelopathic triterpenoid glycosides as part of its chemical defense repertoire against predators, competitors, and fouling organisms. These molecules are responsible for the pharmacological potential found in the glycosides present in this species. In order to observe the glycochemical diversity present in E. ferox, a liquid chromatography coupled to a tandem mass spectrometry approach to analyse a complex polar fraction of this marine sponge was performed. This gave valuable information for about twenty-five compounds three of which have been previously reported and another three which were found to be composed of known aglycones. Furthermore, a group of four urabosides, sharing two uncommon substitutions with carboxyl groups at C-4 on the terpenoid core, were identified by a characteristic fragmentation pattern. The oxidized aglycones present in this group of saponins can promote instability, making the purification process difficult. Cytotoxicity, cell cycle modulation, a cell cloning efficiency assay, as well as its hemolytic activity were evaluated. The cytotoxic activity was about IC50 40 µg/mL on Jurkat and CHO-k1 cell lines without exhibiting hemolysis. Discussion on this bioactivity suggests the scanning of other biological models would be worthwhile.

  8. Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion associated peptidoglycan hydrolase: fusions, deletions and synergy with LysH5

    Science.gov (United States)

    Virion-associated peptidoglycan hydrolases have a potential as antimicrobial agents due to their ability to lyse Gram positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriopha...

  9. 4-alkyl-L-(Dehydro)proline biosynthesis in actinobacteria involves N-terminal nucleophile-hydrolase activity of γ-glutamyltranspeptidase homolog for C-C bond cleavage

    Science.gov (United States)

    Zhong, Guannan; Zhao, Qunfei; Zhang, Qinglin; Liu, Wen

    2017-07-01

    γ-Glutamyltranspeptidases (γ-GTs), ubiquitous in glutathione metabolism for γ-glutamyl transfer/hydrolysis, are N-terminal nucleophile (Ntn)-hydrolase fold proteins that share an autoproteolytic process for self-activation. γ-GT homologues are widely present in Gram-positive actinobacteria where their Ntn-hydrolase activities, however, are not involved in glutathione metabolism. Herein, we demonstrate that the formation of 4-Alkyl-L-(dehydro)proline (ALDP) residues, the non-proteinogenic α-amino acids that serve as vital components of many bioactive metabolites found in actinobacteria, involves unprecedented Ntn-hydrolase activity of γ-GT homologue for C-C bond cleavage. The related enzymes share a key Thr residue, which acts as an internal nucleophile for protein hydrolysis and then as a newly released N-terminal nucleophile for carboxylate side-chain processing likely through the generation of an oxalyl-Thr enzyme intermediate. These findings provide mechanistic insights into the biosynthesis of various ALDP residues/associated natural products, highlight the versatile functions of Ntn-hydrolase fold proteins, and particularly generate interest in thus far less-appreciated γ-GT homologues in actinobacteria.

  10. Quercetin and quercetin 3-O-glycosides from Bauhinia longifolia (Bong.) Steud. show anti-Mayaro virus activity.

    Science.gov (United States)

    dos Santos, Alda E; Kuster, Ricardo M; Yamamoto, Kristie A; Salles, Tiago S; Campos, Renata; de Meneses, Marcelo D F; Soares, Márcia R; Ferreira, Davis

    2014-03-28

    The arthropod-borne Mayaro virus (MAYV) causes 'Mayaro fever', a disease of medical significance, primarily affecting individuals in permanent contact with forested areas in tropical South America. Recently, MAYV has attracted attention due to its likely urbanization. Currently, there are no licensed drugs against most mosquito-transmitted viruses. Here, we investigated the in vitro anti-MAYV activity of the flavonoids quercetin and its derivatives from the Brazilian shrub Bauhinia longifolia (Bong.) Steud. Flavonoids were purified by chromatographic fractionation from leaf extracts of B. longifolia and chemically identified as quercetin and quercetin glycosides using spectroscopic techniques. Cytotoxicity of purified flavonoids and of EtOAc- and n-BuOH-containing flavonoid mixtures was measured by the dye-uptake assay while their antiviral activity was evaluated by a virus yield inhibition assay. The following flavonoids were purified from B. longifolia leaves: non-glycosylated quercetin and its glycosides guaijaverin, quercitrin, isoquercitrin, and hyperin. EtOAc and n-BuOH fractions containing these flavonoids demonstrated the highest antiviral activity of all tested substances, while quercetin had the highest antiviral activity amongst purified flavonoids. Quercetin, EtOAc, or n-BuOH fractions inhibited MAYV production by more than 90% at 25 μg/mL, displaying a stronger antiviral effect than the licensed antiviral ribavirin. A mixture of the isomers isoquercitrin and hyperin had a modest antiviral effect (IC90 = 104.9), while guaijaverin and quercitrin did not show significant antiviral activity. B. longifolia is a good source of flavonoids with anti-Mayaro virus activity. This is the first report of the activity of quercetin and its derivatives against an alphavirus.

  11. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

    OpenAIRE

    Oguro, Ami; Imaoka, Susumu

    2012-01-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hyd...

  12. Naturally occurring methyl salicylate glycosides.

    Science.gov (United States)

    Mao, Ping; Liu, Zizhen; Xie, Meng; Jiang, Rui; Liu, Weirui; Wang, Xiaohong; Meng, Shen; She, Gaimei

    2014-01-01

    As an important part of non steroids anti-inflammation drug (NSAIDs), salicylate has developed from natural substance salicylic acid to natrium salicylicum, to aspirin. Now, methyl salicylate glycoside, a new derivative of salicylic acid, is modified with a -COOH group integrated one methyl radical into formic ether, and a -OH linked with a monosaccharide, a disaccharide or a trisaccharide unit by glycosidic linkage. It has the similar pharmacological activities, anti-inflammatory, analgesic, antipyretic and antithrombotic as the previous salicylates' without resulting in serious side effects, particularly the gastrointestinal toxicity. Owing to the superiority of those significant bioactivities, methyl salicylate glycosides have became a hot research area in NSAIDs for several years. This paper compiles all 9 naturally occurring methyl salicylate glycosides, their distribution of the resource and pharmacological mechanism, which could contribute to the new drug discovery.

  13. Activity of xyloglucan endotransglucosylases/hydrolases suggests a role during host invasion by the parasitic plant Cuscuta reflexa.

    Science.gov (United States)

    Olsen, Stian; Krause, Kirsten

    2017-01-01

    The parasitic vines of the genus Cuscuta form haustoria that grow into other plants and connect with their vascular system, thus allowing the parasite to feed on its host. A major obstacle that meets the infection organ as it penetrates the host tissue is the rigid plant cell wall. In the present study, we examined the activity of xyloglucan endotransglucosylases/hydrolases (XTHs) during the host-invasive growth of the haustorium. The level of xyloglucan endotransglucosylation (XET) activity was found to peak at the penetrating stage of Cuscuta reflexa on its host Pelargonium zonale. In vivo colocalization of XET activity and donor substrate demonstrated XET activity at the border between host and parasite. A test for secretion of XET-active enzymes from haustoria of C. reflexa corroborated this and further indicated that the xyloglucan-modifying enzymes originated from the parasite. A known inhibitor of XET, Coomassie Brilliant Blue R250, was shown to reduce the level of XET in penetrating haustoria of C. reflexa. Moreover, the coating of P. zonale petioles with the inhibitor compound lowered the number of successful haustorial invasions of this otherwise compatible host plant. The presented data indicate that the activity of Cuscuta XTHs at the host-parasite interface is essential to penetration of host plant tissue.

  14. Antifungal (Gaeumannomyces graminis var. tritici activity of various glycosides of medicagenic acid

    Directory of Open Access Journals (Sweden)

    Stefan Martyniuk

    2012-12-01

    Full Text Available Different concentrations of medicagenic acid and five glycosides of this acid isolated from alfalfa (Medicago sativa were added to agar medium (corn meal agar, CMA inoculated with cultures of Gaeumannomyces graminis var. tritici (Ggt. After 7 days of incubation at 25oC colony radius was measured and % of inhibition calculated in relation to the control medium (CMA enriched with the solvent of the tested compounds. Within the tested concentrations, only 3-O-β -D -glucopiranoside medicagenate (monoglucoside significantly reduced the growth of Ggt on CMA medium. This compound at 0.05 mM concentration completely inhibited the development of the fungus and the effect was shown to be fungi-toxic.

  15. Kaempferol glycosides from the twigs of Cinnamomum osmophloeum and their nitric oxide production inhibitory activities.

    Science.gov (United States)

    Lin, Huan-You; Chang, Shang-Tzen

    2012-12-15

    In the present study, ethanolic extract of twigs from Cinnamomum osmophloeum led to isolate nine kaempferol glycosides including two new kaempferol triglycosides that were characterized as kaempferol 3-O-β-D-xylopyranosyl-(1→2)-α-L-arabinofuranosyl-7-O-α-L-rhamnopyranoside (1) and kaempferol 3-O-β-D-xylopyranosyl-(1→2)-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranoside (2). The structures of these compounds were assigned by the application of 1D and 2D NMR spectroscopy and other techniques. Among these nine compounds, kaempferol 7-O-α-L-rhamnopyranoside (9) revealed inhibitory effect against LPS-induced production of nitric oxide in RAW 264.7 macrophages with an IC(50) value of 41.2 μM. It also slightly reduced PGE(2) accumulation by 26% at the concentration of 50 μM. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Characterization of fatty acid amide hydrolase activity by a fluorescence-based assay.

    Science.gov (United States)

    Dato, Florian M; Maaßen, Andreas; Goldfuß, Bernd; Pietsch, Markus

    2018-04-01

    Fatty acid amide hydrolase (FAAH) is involved in many human diseases, particularly cancer, pain and inflammation as well as neurological, metabolic and cardiovascular disorders. Therefore, FAAH is an attractive target for the development of low-molecular-weight inhibitors as therapeutics, which requires robust assays that can be used for high-throughput screening (HTS) of compound libraries. Here, we report the development of a fluorometric assay based on FAAH's ability to effectively hydrolyze medium-chain fatty acid amides, introducing N-decanoyl-substituted 5-amino-2-methoxypyridine (D-MAP) as new amide substrate. D-MAP is cleaved by FAAH with an 8-fold larger specificity constant than the previously reported octanoyl-analog Oc-MAP (V max /K m of 1.09 and 0.134 mL min -1 mg -1 , respectively), with both MAP derivatives possessing superior substrate properties and much increased aqueous solubility compared to the respective p-nitroaniline compounds D-pNA and Oc-pNA. The new assay with D-MAP as substrate is highly sensitive using a lower enzyme concentration (1 μg mL -1 ) than literature-reported fluorimetric FAAH assays. In addition, D-MAP was validated in comparison to the substrate Oc-MAP for the characterization of FAAH inhibitors by means of the reference compounds URB597 and TC-F2 and was shown to be highly suitable for HTS in both kinetic and endpoint assays (Z' factors of 0.81 and 0.78, respectively). Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Ouabain, a cardiac glycoside, inhibits the Fanconi anemia/BRCA pathway activated by DNA interstrand cross-linking agents.

    Directory of Open Access Journals (Sweden)

    Dong Wha Jun

    Full Text Available Modulation of the DNA repair pathway is an emerging target for the development of anticancer drugs. DNA interstrand cross-links (ICLs, one of the most severe forms of DNA damage caused by anticancer drugs such as cisplatin and mitomycin C (MMC, activates the Fanconi anemia (FA/BRCA DNA repair pathway. Inhibition of the FA/BRCA pathway can enhance the cytotoxic effects of ICL-inducing anticancer drugs and can reduce anticancer drug resistance. To find FA/BRCA pathway inhibitory small molecules, we established a cell-based high-content screening method for quantitating the activation of the FA/BRCA pathway by measuring FANCD2 foci on DNA lesions and then applied our method to chemical screening. Using commercial LOPAC1280 chemical library screening, ouabain was identified as a competent FA/BRCA pathway inhibitory compound. Ouabain, a member of the cardiac glycoside family, binds to and inhibits Na(+/K(+-ATPase and has been used to treat heart disease for many years. We observed that ouabain, as well as other cardiac glycoside family members--digitoxin and digoxin--down-regulated FANCD2 and FANCI mRNA levels, reduced monoubiquitination of FANCD2, inhibited FANCD2 foci formation on DNA lesions, and abrogated cell cycle arrest induced by MMC treatment. These inhibitory activities of ouabain required p38 MAPK and were independent of cellular Ca(2+ ion increase or the drug uptake-inhibition effect of ouabain. Furthermore, we found that ouabain potentiated the cytotoxic effects of MMC in tumor cells. Taken together, we identified an additional effect of ouabain as a FA/BRCA pathway-inhibiting chemosensitization compound. The results of this study suggest that ouabain may serve as a chemosensitizer to ICL-inducing anticancer drugs.

  18. Four New Acylated Iridoid Glycosides from the Aerial Part of Veronicastrum sibiricum and Their Antioxidant Response Element-Inducing Activity.

    Science.gov (United States)

    Kim, Myeong Il; Kim, Chul Young

    2018-01-01

    Four new (1 - 4) and one known (5) acylated iridoid glycosides were isolated from the aerial parts of Veronicastrum sibiricum (L.) Pennell. The chemical structures of the isolated compounds were determined to be 3″,4″-dicinnamoyl-6-O-rhamnopyranosyl-10-O-bergaptol-5,7-bisdeoxycynanchoside (1), 3″,4″-dicinnamoyl-6-O-rhamnopyranosylpaulownioside (2), 2″,4″-dicinnamoyl-6-O-rhamnopyranosylcatalpol (3), 3″,4″-dicinnamoyl-6-O-rhamnopyranosylaucubin (4), and 3″,4″-dicinnamoyl-6-O-rhamnopyranosylcatalpol (5) using spectroscopic techniques. Among these compounds, compound 5 increased antioxidant response element (ARE) luciferase activity. © 2018 Wiley-VHCA AG, Zurich, Switzerland.

  19. Evaluation of the Stability of the Total Antioxidant Capacity, Polyphenol Contents, and Starch Hydrolase Inhibitory Activities of Kombucha Teas Using an In Vitro Model of Digestion

    Directory of Open Access Journals (Sweden)

    Mindani I. Watawana

    2015-01-01

    Full Text Available The objective of this study was to evaluate and compare antioxidant and starch hydrolase inhibitory activity of three different types of Kombucha beverages prepared by three pellicles with different microbial compositions. The fermentation process was carried out for 7 days and the assessments of antioxidant and starch hydrolase inhibitory activities as well as tea phenolic compounds were carried out. These parameters were also evaluated after subjecting the final fermented samples to gastric and duodenal digestion in an in vitro digestion model. The pH had a statistically significant decrease during the period of fermentation. The total phenolics content and antioxidant activities had increased during the fermentation process as well as when subjected to digestion. The starch hydrolase inhibitory activities also increased in a similar manner during the different phases. The α-amylase and α-glucosidase inhibitory activities showed statistically significant increases (P<0.05 as the fermentation progressed, while an increase was observed after being subjected to pancreatic and duodenal digestion as well. All three types of tea showed a higher α-amylase inhibitory activity than α-glucosidase inhibitory activity.

  20. Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG.

    Directory of Open Access Journals (Sweden)

    Ingmar J J Claes

    Full Text Available Lactobacillus rhamnosus GG (LGG produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75 and Msp2 (LGG_00031 or p40, which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with cell wall hydrolases, a physiological function that correlates with such an enzymatic activity remained to be substantiated in LGG. To investigate the bacterial function, we constructed knock-out mutants in the corresponding genes aiming to establish a genotype to phenotype relation. Microscopic examination of the msp1 mutant showed the presence of rather long and overly extended cell chains, which suggests that normal daughter cell separation is hampered. Subsequent observation of the LGG wild-type cells by immunofluorescence microscopy revealed that the Msp1 protein accumulates at the septum of exponential-phase cells. The cell wall hydrolyzing activity of the Msp1 protein was confirmed by zymogram analysis. Subsequent analysis by RP-HPLC and mass spectrometry of the digestion products of LGG peptidoglycan (PG by Msp1 indicated that the Msp1 protein has D-glutamyl-L-lysyl endopeptidase activity. Immunofluorescence microscopy and the failure to construct a knock-out mutant suggest an indispensable role for Msp2 in priming septum formation in LGG.

  1. Ubiquitin carboxyl terminal hydrolase L1 negatively regulates TNFα-mediated vascular smooth muscle cell proliferation via suppressing ERK activation

    International Nuclear Information System (INIS)

    Ichikawa, Tomonaga; Li, Jinqing; Dong, Xiaoyu; Potts, Jay D.; Tang, Dong-Qi; Li, Dong-Sheng; Cui, Taixing

    2010-01-01

    Deubiquitinating enzymes (DUBs) appear to be critical regulators of a multitude of processes such as proliferation, apoptosis, differentiation, and inflammation. We have recently demonstrated that a DUB of ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) inhibits vascular lesion formation via suppressing inflammatory responses in vasculature. However, the precise underlying mechanism remains to be defined. Herein, we report that a posttranscriptional up-regulation of UCH-L1 provides a negative feedback to tumor necrosis factor alpha (TNFα)-mediated activation of extracellular signal-regulated kinases (ERK) and proliferation in vascular smooth muscle cells (VSMCs). In rat adult VSMCs, adenoviral over-expression of UCH-L1 inhibited TNFα-induced activation of ERK and DNA synthesis. In contrast, over-expression of UCH-L1 did not affect platelet derived growth factor (PDGF)-induced VSMC proliferation and activation of growth stimulating cascades including ERK. TNFα hardly altered UCH-L1 mRNA expression and stability; however, up-regulated UCH-L1 protein expression via increasing UCH-L1 translation. These results uncover a novel mechanism by which UCH-L1 suppresses vascular inflammation.

  2. Contribution of the Staphylococcus aureus Atl AM and GL murein hydrolase activities in cell division, autolysis, and biofilm formation.

    Directory of Open Access Journals (Sweden)

    Jeffrey L Bose

    Full Text Available The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM and a glucosaminidase (GL. Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development.

  3. Contribution of the Staphylococcus aureus Atl AM and GL murein hydrolase activities in cell division, autolysis, and biofilm formation.

    Science.gov (United States)

    Bose, Jeffrey L; Lehman, McKenzie K; Fey, Paul D; Bayles, Kenneth W

    2012-01-01

    The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM) and a glucosaminidase (GL). Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development.

  4. Isolation, Identification and Partial Characterization of a Lactobacillus casei Strain with Bile Salt Hydrolase Activity from Pulque.

    Science.gov (United States)

    González-Vázquez, R; Azaola-Espinosa, A; Mayorga-Reyes, L; Reyes-Nava, L A; Shah, N P; Rivera-Espinoza, Y

    2015-12-01

    The aim of this study was to isolate, from pulque, Lactobacillus spp. capable of survival in simulated gastrointestinal stress conditions. Nine Gram-positive rods were isolated; however, only one strain (J57) shared identity with Lactobacillus and was registered as Lactobacillus casei J57 (GenBank accession: JN182264). The other strains were identified as Bacillus spp. The most significant observation during the test of tolerance to simulated gastrointestinal conditions (acidity, gastric juice and bile salts) was that L. casei J57 showed a rapid decrease (p ≤ 0.05) in the viable population at 0 h. Bile salts were the stress condition that most affected its survival, from which deoxycholic acid and the mix of bile salts (oxgall) were the most toxic. L. casei J57 showed bile salt hydrolase activity over primary and secondary bile salts as follows: 44.91, 671.72, 45.27 and 61.57 U/mg to glycocholate, taurocholate, glycodeoxycholate and taurodeoxycholate. In contrast, the control strain (L. casei Shirota) only showed activity over tauroconjugates. These results suggest that L. casei J57 shows potential for probiotic applications.

  5. Evaluation of the precision-cut liver and lung slice systems for the study of induction of CYP1, epoxide hydrolase and glutathione S-transferase activities.

    Science.gov (United States)

    Pushparajah, Daphnee S; Umachandran, Meera; Plant, Kathryn E; Plant, Nick; Ioannides, Costas

    2007-02-28

    The principal objective was to ascertain whether precision-cut tissue slices can be used to evaluate the potential of chemicals to induce CYP1, epoxide hydrolase and glutathione S-transferase activities, all being important enzymes involved in the metabolism of polycyclic aromatic hydrocarbons. Precision-cut rat liver and lung slices were incubated with a range of benzo[a]pyrene concentrations for various time periods. A rise in the O-deethylation of ethoxyresorufin was seen in both liver and lung slices exposed to benzo[a]pyrene, which was accompanied by increased CYP1A apoprotein levels. Pulmonary CYP1B1 apoprotein levels and hepatic mRNA levels were similarly enhanced. Elevated epoxide hydrolase and glutathione S-transferase activities were also observed in liver slices following incubation for 24h; similarly, a rise in apoprotein levels of both enzymes was evident, peak levels occurring at the same time point. When mRNA levels were monitored, a rise in the levels of both enzymes was seen as early as 4h after incubation, but maximum levels were attained at 24 h. In lung slices, induction of epoxide hydrolase by benzo[a]pyrene was observed after a 24-h incubation, and at a concentration of 1 microM; a rise in apoprotein levels was seen at this time point. Glutathione S-transferase activity was not inducible in lung slices by benzo[a]pyrene but a modest increase was observed in hepatic slices. Collectively, these studies confirmed CYP1A induction in rat liver slices and established that CYP1B1 expression, and epoxide hydrolase and glutathione S-transferase activities are inducible in precision-cut tissue slices.

  6. Melanogenesis-inhibitory activity of aromatic glycosides from the stem bark of Acer buergerianum.

    Science.gov (United States)

    Akihisa, Toshihiro; Orido, Masashi; Akazawa, Hiroyuki; Takahashi, Akitomo; Yamamoto, Ayako; Ogihara, Eri; Fukatsu, Makoto

    2013-02-01

    A new benzyl glucoside, 3-O-demethylnikoenoside (1), along with eleven known compounds, including seven aromatic glycosides, 2-8, three lignans, 9-11, and one cyclitol, 12, were isolated from the BuOH-soluble fraction of a MeOH extract of Acer buergerianum stem bark. The structures of the new compound were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. Upon evaluation of compounds 1-12 on melanogenesis in B16 melanoma cells induced with α-melanocyte-stimulating hormone (α-MSH), three compounds, i.e., hovetrichoside B (8), pinoresinol 4-O-β-D-glucopyranoside (9), and pinoresinol 4-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside (10), have been found to exhibit inhibitory effects with 41-49% melanin content compared to the control at 100 μM and low cytotoxicity to the cells (81-92% cell viability at 100 μM). Western blot analysis showed that compound 8 reduced the protein levels of MITF (=microphtalmia-associated transcription factor) and tyrosinase, in a concentration-dependent manner, suggesting that 8 inhibits melanogenesis in α-MSH-stimulated B16 melanoma cells by, at least in part, inhibiting the expression of MITF, followed by decreasing the expression of tyrosinase. On the other hand, upon Western blotting, compound 9 was found to reduce the protein levels of tyrosinase and TRP-2, while it increased MITF and TRP-1 (=tyrosine-related protein 1), in a concentration-dependent manner, indicating that 9 inhibits melanogenesis in α-MSH-stimulated B16 melanoma cells by, at least in part, inhibiting the expression of tyrosinase and TRP-2. Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.

  7. Anaerobic accumulation of short-chain fatty acids from algae enhanced by damaging cell structure and promoting hydrolase activity.

    Science.gov (United States)

    Feng, Leiyu; Chen, Yunzhi; Chen, Xutao; Duan, Xu; Xie, Jing; Chen, Yinguang

    2018-02-01

    Short-chain fatty acid (SCFAs) produced from harvested algae by anaerobic fermentation with uncontrolled pH was limited due to the solid cell structure of algae. This study, therefore, was undertaken to enhance the generation of SCFAs from algae by controlling the fermentation pH. pH influenced not only the total SCFAs production, but the percentage of individual SCFA. The maximal yield of SCFAs occurred at pH 10.0 and fermentation time of 6 d (3161 mg COD/L), which mainly contained acetic and iso-valeric acids and was nearly eight times that at uncontrolled pH (392 mg COD/L). Mechanism exploration revealed at alkaline pH, especially at pH 10.0, not only the cell structure of algae was damaged effectively, but also activities and relative quantification of hydrolases as well as the abundance of microorganisms responsible for organics hydrolysis and SCFAs production were improved. Also, the released microcystins from algae were removed efficiently during alkaline anaerobic fermentation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Hydrolase and fructosyltransferase activities implicated in the accumulation of different chain size fructans in three Asteraceae species.

    Science.gov (United States)

    Itaya, Nair M; Asega, Amanda F; Carvalho, Maria Angela M; Figueiredo-Ribeiro, Rita de Cássia L

    2007-09-01

    Fructans are widely distributed in Asteraceae from floras with seasonal growth and are thought to be involved in drought and freezing tolerance, in addition to storage function. Reserve organs of Vernonia herbacea and Viguiera discolor, from the cerrado, and of the perennial herb Smallanthus sonchifolius, endemic to Andean region, store over 80% inulin, with different DP (35, 150, and 15, respectively). The fructan pattern in Asteraceae species could be explained by characteristics of their respective 1-FFTs. Hydrolases and fructosyltransferases from S. sonchifolius, V. herbacea and V. discolor were analyzed in plants at the same environmental conditions. The higher 1-FEH activities found in the species with lower DP, S. sonchifolius and V. herbacea reinforce the hypothesis of the involvement of 1-FEH in fructan profile and suggest that the high DP fructan of V. discolor is a consequence of the low affinity of its 1-FEH to the native long chain inulin. Long term incubation with sucrose suggested that the affinity of 1-FFT of V. discolor for 1-kestose is low when compared to that of V. herbacea. Indeed 1-FFT from V. discolor was shown to be an hDP 1-FFT, preferring longer inulins as acceptors. Conversely, 1-FFT from V. herbacea seems to have a higher affinity for short fructo-oligosaccharides, including 1-kestose, as acceptor substrates. Differences in fructan enzymes of the three Asteraceae provide new information towards the understanding of fructan metabolism and control of carbon flow between low and high DP fructans.

  9. Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis.

    Science.gov (United States)

    Lentz, Christian S; Ordonez, Alvaro A; Kasperkiewicz, Paulina; La Greca, Florencia; O'Donoghue, Anthony J; Schulze, Christopher J; Powers, James C; Craik, Charles S; Drag, Marcin; Jain, Sanjay K; Bogyo, Matthew

    2016-11-11

    Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb "Hydrolase important for pathogenesis 1" (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections.

  10. Structure-activity relationships of amide-phosphonate derivatives as inhibitors of the human soluble epoxide hydrolase.

    Science.gov (United States)

    Kim, In-Hae; Park, Yong-Kyu; Nishiwaki, Hisashi; Hammock, Bruce D; Nishi, Kosuke

    2015-11-15

    Structure-activity relationships of amide-phosphonate derivatives as inhibitors of the human soluble epoxide hydrolase (sEH) were investigated. First, a series of alkyl or aryl groups were substituted on the carbon alpha to the phosphonate function in amide compounds to see whether substituted phosphonates can act as a secondary pharmacophore. A tert-butyl group (16) on the alpha carbon was found to yield most potent inhibition on the target enzyme. A 4-50-fold drop in inhibition was induced by other substituents such as aryls, substituted aryls, cycloalkyls, and alkyls. Then, the modification of the O-substituents on the phosphonate function revealed that diethyl groups (16 and 23) were preferable for inhibition to other longer alkyls or substituted alkyls. In amide compounds with the optimized diethylphosphonate moiety and an alkyl substitution such as adamantane (16), tetrahydronaphthalene (31), or adamantanemethane (36), highly potent inhibitions were gained. In addition, the resulting potent amide-phosphonate compounds had reasonable water solubility, suggesting that substituted phosphonates in amide inhibitors are effective for both inhibition potency on the human sEH and water solubility as a secondary pharmacophore. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Impact of the Staphylococcus epidermidis LytSR two-component regulatory system on murein hydrolase activity, pyruvate utilization and global transcriptional profile

    Directory of Open Access Journals (Sweden)

    Yu Fangyou

    2010-11-01

    Full Text Available Abstract Background Staphylococcus epidermidis has emerged as one of the most important nosocomial pathogens, mainly because of its ability to colonize implanted biomaterials by forming a biofilm. Extensive studies are focused on the molecular mechanisms involved in biofilm formation. The LytSR two-component regulatory system regulates autolysis and biofilm formation in Staphylococcus aureus. However, the role of LytSR played in S. epidermidis remained unknown. Results In the present study, we demonstrated that lytSR knock-out in S. epidermidis did not alter susceptibility to Triton X-100 induced autolysis. Quantitative murein hydrolase assay indicated that disruption of lytSR in S. epidermidis resulted in decreased activities of extracellular murein hydrolases, although zymogram showed no apparent differences in murein hydrolase patterns between S. epidermidis strain 1457 and its lytSR mutant. Compared to the wild-type counterpart, 1457ΔlytSR produced slightly more biofilm, with significantly decreased dead cells inside. Microarray analysis showed that lytSR mutation affected the transcription of 164 genes (123 genes were upregulated and 41 genes were downregulated. Specifically, genes encoding proteins responsible for protein synthesis, energy metabolism were downregulated, while genes involved in amino acid and nucleotide biosynthesis, amino acid transporters were upregulated. Impaired ability to utilize pyruvate and reduced activity of arginine deiminase was observed in 1457ΔlytSR, which is consistent with the microarray data. Conclusions The preliminary results suggest that in S. epidermidis LytSR two-component system regulates extracellular murein hydrolase activity, bacterial cell death and pyruvate utilization. Based on the microarray data, it appears that lytSR inactivation induces a stringent response. In addition, LytSR may indirectly enhance biofilm formation by altering the metabolic status of the bacteria.

  12. The Antioxidant and Starch Hydrolase Inhibitory Activity of Ten Spices in an In Vitro Model of Digestion: Bioaccessibility of Anthocyanins and Carotenoids

    Directory of Open Access Journals (Sweden)

    Nilakshi Jayawardena

    2015-01-01

    Full Text Available The antioxidant and starch hydrolase inhibitory activities of cardamom, cloves, coriander, cumin seeds, curry leaves, fenugreek, mustard seeds, nutmeg, sweet cumin, and star anise extracts were investigated in an in vitro model of digestion mimicking the gastric and duodenal conditions. The total phenolic contents in all spice extracts had statistically significantly (P<0.05 increased following both gastric and duodenal digestion. This was also in correlation with the antioxidant assays quantifying the water-soluble antioxidant capacity of the extracts. The lipophilic Oxygen Radical Absorbance Capacity assay did not indicate a statistically significant change in the values during any of the digestion phases. Statistically significant (P<0.05 reductions in the anthocyanin contents were observed during the digestion phases in contrast to the carotenoid contents. With the exception of the cumin seed extract, none of the spice extracts showed statistically significant changes in the initial starch hydrolase enzyme inhibitory values prior to gastric and duodenal digestion. In conclusion, this study was able to prove that the 10 spices were a significant source of total phenolics, antioxidant, and starch hydrolase inhibitory activities.

  13. Two New Monoterpene Glycosides from Qing Shan Lu Shui Tea with Inhibitory Effects on Leukocyte-Type 12-Lipoxygenase Activity

    Directory of Open Access Journals (Sweden)

    Ding Zhi Fang

    2013-04-01

    Full Text Available We evaluated the inhibitory effect of 12 Chinese teas on leukocyte-type 12-lipoxygenase (LOX activity. Tea catechins such as epigallocatechin gallate have been known to exhibit leukocyte-type 12-LOX inhibition. Qing Shan Lu Shui, which contains lower catechin levels than the other tested teas, suppressed leukocyte-type 12-LOX activity. To characterize the bioactive components of Qing Shan Lu Shui, leukocyte-type 12-LOX inhibitory activity–guided fractionation of the aqueous ethanol extract of the tea was performed, resulting in the isolation of two new monoterpene glycosides: liguroside A (1 and B (2. The structures of compounds 1 and 2 were characterized as (2E,5E-7-hydroperoxy-3,7-dimethyl-2,5-octadienyl-O-(α-L-rhamnopyranosyl-(1″→3′-(4′″-O-trans-p-coumaroyl-β-D-glucopyranoside and (2E,5E-7-hydroperoxy-3,7-dimethyl-2,5-octa-dienyl- O-(α-L-rhamnopyranosyl-(1″→3′-(4′″-O-cis-p-coumaroyl-β-D-glucopyranoside, respectively, based on spectral and chemical evidence. Ligurosides A (1 and B (2 showed inhibitory effects on leukocyte-type 12-LOX activity, with IC50 values of 1.7 and 0.7 μM, respectively.

  14. Lipopolysaccharide-induced pulmonary inflammation is not accompanied by a release of anandamide into the lavage fluid or a down-regulation of the activity of fatty acid amide hydrolase

    DEFF Research Database (Denmark)

    Holt, S.; J. Fowler, C.; Rocksén, D.

    2004-01-01

    The effect of lipopolysaccharide inhalation upon lung anandamide levels, anandamide synthetic enzymes and fatty acid amide hydrolase has been investigated. Lipopolysaccharide exposure produced a dramatic extravasation of neutrophils and release of tumour necrosis factor a into the bronchoalveolar......-acyltransferase and N-acylphosphatidylethanolamine phospholipase D and the activity of fatty acid amide hydrolase in lung membrane fractions did not change significantly following the exposure to lipopolysaccharide. The non-selective fatty acid amide hydrolase inhibitor phenylmethylsulfonyl fluoride was a less potent...... inhibitor of lung fatty acid amide hydrolase than expected from the literature, and a dose of 30 mg/kg i.p. of this compound, which produced a complete inhibition of brain anandamide metabolism, only partially inhibited the lung metabolic activity....

  15. Modification of soil microbial activity and several hydrolases in a forest soil artificially contaminated with copper

    Science.gov (United States)

    Bellas, Rosa; Leirós, Mā Carmen; Gil-Sotres, Fernando; Trasar-Cepeda, Carmen

    2010-05-01

    Soils have long been exposed to the adverse effects of human activities, which negatively affect soil biological activity. As a result of their functions and ubiquitous presence microorganisms can serve as environmental indicators of soil pollution. Some features of soil microorganisms, such as the microbial biomass size, respiration rate, and enzyme activity are often used as bioindicators of the ecotoxicity of heavy metals. Although copper is essential for microorganisms, excessive concentrations have a negative influence on processes mediated by microorganisms. In this study we measured the response of some microbial indicators to Cu pollution in a forest soil, with the aim of evaluating their potential for predicting Cu contamination. Samples of an Ah horizon from a forest soil under oakwood vegetation (Quercus robur L.) were contaminated in the laboratory with copper added at different doses (0, 120, 360, 1080 and 3240 mg kg-1) as CuCl2×2H2O. The soil samples were kept for 7 days at 25 °C and at a moisture content corresponding to the water holding capacity, and thereafter were analysed for carbon and nitrogen mineralization capacity, microbial biomass C, seed germination and root elongation tests, and for urease, phosphomonoesterase, catalase and ß-glucosidase activities. In addition, carbon mineralization kinetics were studied, by plotting the log of residual C against incubation time, and the metabolic coefficient, qCO2, was estimated. Both organic carbon and nitrogen mineralization were lower in polluted samples, with the greatest decrease observed in the sample contaminated with 1080 mg kg-1. In all samples carbon mineralization followed first order kinetics; the C mineralization constant was lower in contaminated than in uncontaminated samples and, in general, decreased with increasing doses of copper. Moreover, it appears that copper contamination not only reduced the N mineralization capacity, but also modified the N mineralization process, since in

  16. Characterization of the Paenibacillus beijingensis DSM 24997 GtfD and its glucan polymer products representing a new glycoside hydrolase 70 subfamily of 4,6-α-glucanotransferase enzymes.

    Directory of Open Access Journals (Sweden)

    Joana Gangoiti

    Full Text Available Previously we have reported that the Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 uses the 4,6-α-glucanotransferase GtfD to convert maltodextrins and starch into a reuteran-like polymer consisting of (α1→4 glucan chains connected by alternating (α1→4/(α1→6 linkages and (α1→4,6 branching points. This enzyme constituted the single evidence for this reaction and product specificity in the GH70 family, mostly containing glucansucrases encoded by lactic acid bacteria (http://www.CAZy.org. In this work, 4 additional GtfD-like proteins were identified in taxonomically diverse plant-associated bacteria forming a new GH70 subfamily with intermediate characteristics between the evolutionary related GH13 and GH70 families. The GtfD enzyme encoded by Paenibacillus beijingensis DSM 24997 was characterized providing the first example of a reuteran-like polymer synthesizing 4,6-α-glucanotransferase in a Gram-positive bacterium. Whereas the A. chroococcum GtfD activity on amylose resulted in the synthesis of a high molecular polymer, in addition to maltose and other small oligosaccharides, two reuteran-like polymer distributions are produced by P. beijingensis GtfD: a high-molecular mass polymer and a low-molecular mass polymer with an average Mw of 27 MDa and 19 kDa, respectively. Compared to the A. chroooccum GtfD product, both P. beijingensis GtfD polymers contain longer linear (α1→4 sequences in their structure reflecting a preference for transfer of even longer glucan chains by this enzyme. Overall, this study provides new insights into the evolutionary history of GH70 enzymes, and enlarges the diversity of natural enzymes that can be applied for modification of the starch present in food into less and/or more slowly digestible carbohydrate structures.

  17. Development and Application of a New Microarray- Based Method for High-Throughput Screening of Carbohydrate Active Enzymes

    DEFF Research Database (Denmark)

    Vidal Melgosa, Silvia

    single defined polysaccharides, mixtures of defined polysaccharides and complex biomass extracts. Furthermore, the capacity of the technique to analyse endo- and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases and lytic polysaccharide monooxygenases has been confirmed...... will contribute to both the discovery of CAZymes and the empirical characterisation of their activities, thus aiding their industrial utilisation and biological understanding...

  18. The dead, hardened floral bracts of dispersal units of wild wheat function as storage for active hydrolases and in enhancing seedling vigor.

    Directory of Open Access Journals (Sweden)

    Buzi Raviv

    Full Text Available It is commonly assumed that the dead, hardened floral bracts of the dispersal unit of grasses have been evolved to protect seeds from predation and / or assist in fruit/caryopsis dispersal. While these structures have important agronomical and economical implications, their adaptive value has not been fully explored. We investigated the hypothesis that the maternally derived hardened floral bracts have been evolved not just as a means for caryopsis protection and dispersal, but also as storage for substances that might affect seed germination and seedling vigor. Dead glumes as well as lemmas and paleas of wild emmer wheat (Triticum turgidum var dicoccoides were found to store and release upon hydration active hydrolases including nucleases and chitinases. High nuclease activity was released upon hydration from glumes derived from wild strains of wheat including Triticum urartu and wild emmer wheat, while very low nuclease activity was detected in glumes derived from domesticated, free-threshing wheat cultivars (e.g., durum wheat. Germination from the intact dispersal unit of wild emmer wheat was delayed, but post germination growth was better than those of separated caryopses. Most notable was a significant increase in lateral root production on seedlings germinated from the intact dispersal unit. Proteome analysis of wild emmer wheat glumes revealed many proteins stored and released upon hydration including S1-type nucleases, peptidases, antifungal hydrolases such as chitinases and β-1,3-glucanase as well as pectin acetylesterase, a protein involved in cell wall degradation and remodeling. Also, reactive oxygen species (ROS-detoxifying enzymes such as superoxide dismutase and ascorbate peroxidase were overrepresented in dead glumes of wild emmer wheat. Thus our study highlighted previously unknown features of the dispersal unit in wild wheat in which the dead, hardened floral bracts enclosing the caryopsis store active hydrolases and

  19. Amarogentin, a secoiridoid glycoside, abrogates platelet activation through PLC γ 2-PKC and MAPK pathways.

    Science.gov (United States)

    Yen, Ting-Lin; Lu, Wan-Jung; Lien, Li-Ming; Thomas, Philip Aloysius; Lee, Tzu-Yin; Chiu, Hou-Chang; Sheu, Joen-Rong; Lin, Kuan-Hung

    2014-01-01

    Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60  μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC) γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLC γ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

  20. Amarogentin, a Secoiridoid Glycoside, Abrogates Platelet Activation through PLCγ2-PKC and MAPK Pathways

    Directory of Open Access Journals (Sweden)

    Ting-Lin Yen

    2014-01-01

    Full Text Available Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60 μM inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (MAPKs. It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLCγ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

  1. A New Kaempferol Glycoside with Antioxidant Activity from Chenopodium ambrosioides Growing in Egypt

    OpenAIRE

    Mosad Ahmed Ghareeb; Amal Mohamed Saad; Allia Mahmoud Abdou; Laila Abdel-Ghany Refahy; Wafaa Sabry Ahmed; Wafaa Sabry Ahmed

    2016-01-01

    The current study aimed to identify the chemical constituents of Chenopodium ambrosioides (Linn.), and the assessment of the in vitro antioxidant activity of the different extracts and pure isolates. Methods: The antioxidant activity was estimated via free radical scavenging and Phosphomolybdenum assays. Structure elucidation of pure compounds was achieved via UV, IR, 1H & 13C-NMR, 1H-1H COSY, HMQC, and HMBC, spectroscopy. Bioassay-guided fractionation and isolation of the n-butanol fraction ...

  2. Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature.

    Directory of Open Access Journals (Sweden)

    Peter K Busk

    Full Text Available The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls.

  3. Eremophila maculata-Isolation of a rare naturally-occurring lignan glycoside and the hepatoprotective activity of the leaf extract.

    Science.gov (United States)

    Youssef, Fadia S; Ashour, Mohamed L; Sobeh, Mansour; El-Beshbishy, Hesham A; Singab, Abdel Nasser; Wink, Michael

    2016-11-15

    The Australian plant Eremophila maculata F. Muell (Scrophulariaceae) is cultivated worldwide as an ornamental plant. This study was designed to assess the antioxidant and hepatoprotective activities of a methanol extract from E. maculata leaves (EMM) both in vitro and in vivo (rats) experiments. Detailed phytochemical study was done on the extract followed by molecular docking experiments on TNF-α ascertain the efficacy of the isolated compounds. The antiproliferative activity was evaluated in the human cancer cell lines A-495, PC3 and HepG2 cells using the SRB method. The antioxidant activitywas evaluated in vitro using the DPPH• assay while the hepatoprotective properties were investigated by determining the amelioration of CCl 4 -induced cytotoxicity and oxidative stress in HepG2 cells. The activity was confirmed in vivo by studying tamoxifen-induced hepatotoxicity in rats. An in-depth phytochemical investigation of a methanol extract was performed using 1D and 2D NMR experiments. In silico molecular modeling studies of the isolated compounds on TNF-α (PDB ID 2AZ5) were carried out using Discovery Studio 2.5 software applying C-Docker protocol. The IC 50 values of EMM were >500µg/ml for both PC3 and HepG2 cells indicating its safety. Similar to the standard drug silymarin, EMM could restore AST, ALT values; replenish GSH level, SOD activity and TAC in vitro. The hepatoprotective activity was confirmed in vivo in which the extract (20mg/kg body weight) decreased ALT and AST levels by 45.23 and 45.79%, respectively as compared to the tamoxifen treated groups. Oxidative stress was reduced by lowering of thiobarbituric acid reactive substances by 28.57%. Additionally, hepatocyte inflammation was improved by reducing the pro-inflammatory mediator TNF-α by 54.29%. Phytochemical investigation resulted in the isolation of a rare naturally-occurring lignan glycoside, namely pinoresinol-4-O-[6″-O-(E)-feruloyl]-β-D-glucopyranoside for the first time from the

  4. Microbial hydroxylation and glycosidation of oleanolic acid by Circinella muscae and their anti-inflammatory activities.

    Science.gov (United States)

    Yan, Sensen; Lin, Haijun; Huang, Huilian; Yang, Min; Xu, Bohui; Chen, Guangtong

    2018-05-29

    Biotransformation of oleanolic acid (OA) by Circinella muscae AS 3.2695 was investigated. Nine hydroxylated and glycosylated metabolites (1-9) were obtained. Their structures were elucidated as 3β,7β-dihydroxyolean-12-en-28-oic acid (1), 3β,7β,21β-trihydroxyolean-12-en-28-oic acid (2), 3β,7α,21β-trihydroxyolean-12-en- 28-oic acid (3), 3β,7β,15α-trihydroxyolean-12-en-28-oic acid (4), 7β,15α-dihydroxy- 3-oxo-olean-12-en-28-oic acid (5), 7β-hydroxy-3-oxo-olean-12-en-28-oic acid (6), oleanolic acid-28-O-β-D-glucopyranosyl ester (7), 3β,21β-dihydroxyolean-12-en-28- oic acid-28-O-β-D-glucopyranosyl ester (8), and 3β,7β,15α-trihydroxyolean-12-en- 28-oic acid-28-O-β-D-glucopyranosyl ester (9) by spectroscopic analysis. Among them, compounds 4 and 9 were new compounds. In addition, anti-inflammatory activities were assayed and evaluated for the isolated metabolites. Most of the metabolites exhibited significant inhibitory activities on lipopolysaccharides-induced NO production in RAW 264.7 cells.

  5. Antioxidant Activities of Hydrolysable Tannins and Flavonoid Glycosides Isolated from Eugenia uniflora L.

    Directory of Open Access Journals (Sweden)

    Gilmara A. C. Fortes

    2015-01-01

    Full Text Available Aphytochemical investigation of Eugenia uniflora ’s leaf extract resulted in the isolation of eleven phenolic compounds: 2,3-di-O-galloyl- b - D -glucose (1, 1,2,3,4,6-penta-O-galloyl- b - D -glucose (2, gemin D (3, hippomanin A (4, oenothein B (5, eugeniflorin D 2 (6, camptothin A (7, afzelin (8, quercitrin (9, myricitrin, (10 and desmanthin-1 (11. These compounds were identified by spectroscopic methods including 1D- and 2D-NMR, UV, IR, and TOF/MS. Compounds 1, 2, 3, 4, 7, 8, 9, and 11 were isolated from this species for the first time. Ten isolates were evaluated for antioxidant activity by DPPH free radical and Oxygen Radical Absorbance Capacity (ORAC-Fluorecein assay . Dimeric tannins, oenothein B (5, eugeniflorin D 2 (6, and camptothin A (7 showed a remarkable radical scavenging capacity.

  6. Activity-based protein profiling of glucosidases, fucosidases and glucuronidases

    NARCIS (Netherlands)

    Jiang, J.

    2016-01-01

    Glycoside hydrolases (GHs), enzymes that catalyze the hydrolytic cleavage of glycosidic bonds, receive continuing interest both in fundamental and applied biology and biomedicine. Lysosomal storage disorders (LSDs) are caused by inborn metabolic errors due to deficiency in specific lysosomal

  7. Identification of the Gene Encoding Isoprimeverose-producing Oligoxyloglucan Hydrolase in Aspergillus oryzae*

    Science.gov (United States)

    Matsuzawa, Tomohiko; Mitsuishi, Yasushi; Kameyama, Akihiko

    2016-01-01

    Aspergillus oryzae produces a unique β-glucosidase, isoprimeverose-producing oligoxyloglucan hydrolase (IPase), that recognizes and releases isoprimeverose (α-d-xylopyranose-(1→6)-d-glucopyranose) units from the non-reducing ends of oligoxyloglucans. A gene encoding A. oryzae IPase, termed ipeA, was identified and expressed in Pichia pastoris. With the exception of cellobiose, IpeA hydrolyzes a variety of oligoxyloglucans and is a member of the glycoside hydrolase family 3. Xylopyranosyl branching at the non-reducing ends was vital for IPase activity, and galactosylation at a α-1,6-linked xylopyranosyl side chain completely abolished IpeA activity. Hepta-oligoxyloglucan saccharide (Xyl3Glc4) substrate was preferred over tri- (Xyl1Glc2) and tetra- (Xyl2Glc2) oligoxyloglucan saccharides substrates. IpeA transferred isoprimeverose units to other saccharides, indicating transglycosylation activity. The ipeA gene was expressed in xylose and xyloglucan media and was strongly induced in the presence of xyloglucan endo-xyloglucanase-hydrolyzed products. This is the first study to report the identification of a gene encoding IPase in eukaryotes. PMID:26755723

  8. Phytosterol glycosides reduce cholesterol absorption in humans

    OpenAIRE

    Lin, Xiaobo; Ma, Lina; Racette, Susan B.; Anderson Spearie, Catherine L.; Ostlund, Richard E.

    2009-01-01

    Dietary phytosterols inhibit intestinal cholesterol absorption and regulate whole body cholesterol excretion and balance. However, they are biochemically heterogeneous and a portion is glycosylated in some foods with unknown effects on biological activity. We tested the hypothesis that phytosterol glycosides reduce cholesterol absorption in humans. Phytosterol glycosides were extracted and purified from soy lecithin in a novel two-step process. Cholesterol absorption was measured in a series ...

  9. Identification and characterization of epoxide hydrolase activity of polycyclic aromatic hydrocarbon-degrading bacteria for biocatalytic resolution of racemic styrene oxide and styrene oxide derivatives.

    Science.gov (United States)

    Woo, Jung-Hee; Kwon, Tae-Hyung; Kim, Jun-Tae; Kim, Choong-Gon; Lee, Eun Yeol

    2013-04-01

    A novel epoxide hydrolase (EHase) from polycyclic aromatic hydrocarbon (PAH)-degrading bacteria was identified and characterized. EHase activity was identified in four strains of PAH-degrading bacteria isolated from commercial gasoline and oil-contaminated sediment based on their growth on styrene oxide and its derivatives, such as 2,3- and 4-chlorostyrene oxides, as a sole carbon source. Gordonia sp. H37 exhibited high enantioselective hydrolysis activity for 4-chlorostyrene oxide with an enantiomeric ratio of 27. Gordonia sp. H37 preferentially hydrolyzed the (R)-enantiomer of styrene oxide derivatives resulting in the preparation of a (S)-enantiomer with enantiomeric excess greater than 99.9 %. The enantioselective EHase activity was identified and characterized in various PAH-degrading bacteria, and whole cell Gordonia sp. H37 was employed as a biocatalyst for preparing enantiopure (S)-styrene oxide derivatives.

  10. Synthesis and evaluation of a series of 6-chloro-4-methylumbelliferyl glycosides as fluorogenic reagents for screening metagenomic libraries for glycosidase activity.

    Science.gov (United States)

    Chen, Hong-Ming; Armstrong, Zachary; Hallam, Steven J; Withers, Stephen G

    2016-02-08

    Screening of large enzyme libraries such as those derived from metagenomic sources requires sensitive substrates. Fluorogenic glycosides typically offer the best sensitivity but typically must be used in a stopped format to generate good signal. Use of fluorescent phenols of pKa libraries yielded a "hit rate" of 1 in 60. Hits were then readily deconvoluted with the individual substrates in a single plate to identify specific activities within each clone. The use of such a collection of substrates greatly accelerates the screening process. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Deconjugated bile salts produced by extracellular bile-salt hydrolase-like activities from the probiotic Lactobacillus johnsonii La1 inhibit Giardia duodenalis in vitro growth

    Directory of Open Access Journals (Sweden)

    Marie-Agnès Travers

    2016-09-01

    Full Text Available Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting or releasing BSH-like activity(ies in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present.

  12. Suite of Activity-Based Probes for Cellulose-Degrading Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Chauvigne-Hines, Lacie M.; Anderson, Lindsey N.; Weaver, Holly M.; Brown, Joseph N.; Koech, Phillip K.; Nicora, Carrie D.; Hofstad, Beth A.; Smith, Richard D.; Wilkins, Michael J.; Callister, Stephen J.; Wright, Aaron T.

    2012-12-19

    Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degradation of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome producing bacterium that couples rapid reproduction rates to highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chemistry, and to increase enzyme active site inclusion for LC-MS analysis. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes in addition to a large collection of integral cellulosome-containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic cellulose degrading systems, and facilitates a greater understanding of the organismal role associated within biofuel development.

  13. Cyanohydrin glycosides of Passiflora

    DEFF Research Database (Denmark)

    Jaroszewski, Jerzy W; Olafsdottir, Elin S; Wellendorph, Petrine

    2002-01-01

    this glycoside co-occurs with simple beta-D-glucopyranosides: tetraphyllin A, deidaclin, tetraphyllin B, volkenin, epivolkenin and taraktophyllin. P. citrina contains passicapsin, a rare glycoside with the 2,6-dideoxy-beta-D-xylo-hexopyranosyl moiety, while P. herbertiana contains tetraphyllin A, deidaclin...... Passiflora species. These alpha-hydroxyamides, presumably formed during processing of the plant material, behave as cyanogenic compounds when treated with commercial Helix pomatia crude enzyme preparation. Thus, the enzyme preparation appears to contain an amide dehydratase, which converts alpha......-hydroxyamides to cyanohydrins that liberate cyanide; this finding is of interest in connection with analysis of plant tissues and extracts using Helix pomatia enzymes....

  14. Antifungal activity and fungal metabolism of steroidal glycosides of Easter lily (Lilium longiflorum Thunb.) by the plant pathogenic fungus, Botrytis cinerea.

    Science.gov (United States)

    Munafo, John P; Gianfagna, Thomas J

    2011-06-08

    Botrytis cinerea Pers. Fr. is a plant pathogenic fungus and the causal organism of blossom blight of Easter lily (Lilium longiflorum Thunb.). Easter lily is a rich source of steroidal glycosides, compounds which may play a role in the plant-pathogen interaction of Easter lily. Five steroidal glycosides, including two steroidal glycoalkaloids and three furostanol saponins, were isolated from L. longiflorum and evaluated for fungal growth inhibition activity against B. cinerea, using an in vitro plate assay. All of the compounds showed fungal growth inhibition activity; however, the natural acetylation of C-6''' of the terminal glucose in the steroidal glycoalkaloid, (22R,25R)-spirosol-5-en-3β-yl O-α-L-rhamnopyranosyl-(1→2)-[6-O-acetyl-β-D-glucopyranosyl-(1→4)]-β-D-glucopyranoside (2), increased antifungal activity by inhibiting the rate of metabolism of the compound by B. cinerea. Acetylation of the glycoalkaloid may be a plant defense response to the evolution of detoxifying mechanisms by the pathogen. The biotransformation of the steroidal glycoalkaloids by B. cinerea led to the isolation and characterization of several fungal metabolites. The fungal metabolites that were generated in the model system were also identified in Easter lily tissues infected with the fungus by LC-MS. In addition, a steroidal glycoalkaloid, (22R,25R)-spirosol-5-en-3β-yl O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (6), was identified as both a fungal metabolite of the steroidal glycoalkaloids and as a natural product in L. longiflorum for the first time.

  15. Phytosterol glycosides reduce cholesterol absorption in humans.

    Science.gov (United States)

    Lin, Xiaobo; Ma, Lina; Racette, Susan B; Anderson Spearie, Catherine L; Ostlund, Richard E

    2009-04-01

    Dietary phytosterols inhibit intestinal cholesterol absorption and regulate whole body cholesterol excretion and balance. However, they are biochemically heterogeneous and a portion is glycosylated in some foods with unknown effects on biological activity. We tested the hypothesis that phytosterol glycosides reduce cholesterol absorption in humans. Phytosterol glycosides were extracted and purified from soy lecithin in a novel two-step process. Cholesterol absorption was measured in a series of three single-meal tests given at intervals of 2 wk to each of 11 healthy subjects. In a randomized crossover design, participants received approximately 300 mg of added phytosterols in the form of phytosterol glycosides or phytosterol esters, or placebo in a test breakfast also containing 30 mg cholesterol-d7. Cholesterol absorption was estimated by mass spectrometry of plasma cholesterol-d7 enrichment 4-5 days after each test. Compared with the placebo test, phytosterol glycosides reduced cholesterol absorption by 37.6+/-4.8% (Pphytosterol esters 30.6+/-3.9% (P=0.0001). These results suggest that natural phytosterol glycosides purified from lecithin are bioactive in humans and should be included in methods of phytosterol analysis and tables of food phytosterol content.

  16. Cholesterol-Lowering Potentials of Lactic Acid Bacteria Based on Bile-Salt Hydrolase Activity and Effect of Potent Strains on Cholesterol Metabolism In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Cheng-Chih Tsai

    2014-01-01

    Full Text Available This study collected different probiotic isolates from animal and plant sources to evaluate the bile-salt hydrolase activity of probiotics in vitro. The deconjugation potential of bile acid was determined using high-performance liquid chromatography. HepG2 cells were cultured with probiotic strains with high BSH activity. The triglyceride (TG and apolipoprotein B (apo B secretion by HepG2 cells were evaluated. Our results show that the BSH activity and bile-acid deconjugation abilities of Pediococcus acidilactici NBHK002, Bifidobacterium adolescentis NBHK006, Lactobacillus rhamnosus NBHK007, and Lactobacillus acidophilus NBHK008 were higher than those of the other probiotic strains. The cholesterol concentration in cholesterol micelles was reduced within 24 h. NBHK007 reduced the TG secretion by 100% after 48 h of incubation. NBHK002, NBHK006, and NBHK007 could reduce apo B secretion by 33%, 38%, and 39%, respectively, after 24 h of incubation. The product PROBIO S-23 produced a greater decrease in the total concentration of cholesterol, low-density lipoprotein, TG, and thiobarbituric acid reactive substance in the serum or livers of hamsters with hypercholesterolemia compared with that of hamsters fed with a high-fat and high-cholesterol diet. These results show that the three probiotic strains of lactic acid bacteria are better candidates for reducing the risk of cardiovascular disease.

  17. The activity of the endocannabinoid metabolising enzyme fatty acid amide hydrolase in subcutaneous adipocytes correlates with BMI in metabolically healthy humans

    Directory of Open Access Journals (Sweden)

    Alexander Stephen PH

    2011-08-01

    Full Text Available Abstract Background The endocannabinoid system (ECS is a ubiquitously expressed signalling system, with involvement in lipid metabolism and obesity. There are reported changes in obesity of blood concentrations of the endocannabinoids anandamide (AEA and 2-arachidonoylglcyerol (2-AG, and of adipose tissue expression levels of the two key catabolic enzymes of the ECS, fatty acid amide hydrolase (FAAH and monoacylglycerol lipase (MGL. Surprisingly, however, the activities of these enzymes have not been assayed in conditions of increasing adiposity. The aim of the current study was to investigate whether FAAH and MGL activities in human subcutaneous adipocytes are affected by body mass index (BMI, or other markers of adiposity and metabolism. Methods Subcutaneous abdominal mature adipocytes, fasting blood samples and anthropometric measurements were obtained from 28 metabolically healthy subjects representing a range of BMIs. FAAH and MGL activities were assayed in mature adipocytes using radiolabelled substrates. Serum glucose, insulin and adipokines were determined using ELISAs. Results MGL activity showed no relationship with BMI or other adiposity indices, metabolic markers (fasting serum insulin or glucose or serum adipokine levels (adiponectin, leptin or resistin. In contrast, FAAH activity in subcutaneous adipocytes correlated positively with BMI and waist circumference, but not with skinfold thickness, metabolic markers or serum adipokine levels. Conclusions In this study, novel evidence is provided that FAAH activity in subcutaneous mature adipocytes increases with BMI, whereas MGL activity does not. These findings support the hypothesis that some components of the ECS are upregulated with increasing adiposity in humans, and that AEA and 2-AG may be regulated differently.

  18. Bile-Salt-Hydrolases from the Probiotic Strain Lactobacillus johnsonii La1 Mediate Anti-giardial Activity in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Thibault Allain

    2018-01-01

    Full Text Available Giardia duodenalis (syn. G. lamblia, G. intestinalis is the protozoan parasite responsible for giardiasis, the most common and widely spread intestinal parasitic disease worldwide, affecting both humans and animals. After cysts ingestion (through either contaminated food or water, Giardia excysts in the upper intestinal tract to release replicating trophozoites that are responsible for the production of symptoms. In the gut, Giardia cohabits with the host's microbiota, and several studies have revealed the importance of this gut ecosystem and/or some probiotic bacteria in providing protection against G. duodenalis infection through mechanisms that remain incompletely understood. Recent findings suggest that Bile-Salt-Hydrolase (BSH-like activities from the probiotic strain of Lactobacillus johnsonii La1 may contribute to the anti-giardial activity displayed by this strain. Here, we cloned and expressed each of the three bsh genes present in the L. johnsonii La1 genome to study their enzymatic and biological properties. While BSH47 and BSH56 were expressed as recombinant active enzymes, no significant enzymatic activity was detected with BSH12. In vitro assays allowed determining the substrate specificities of both BSH47 and BSH56, which were different. Modeling of these BSHs indicated a strong conservation of their 3-D structures despite low conservation of their primary structures. Both recombinant enzymes were able to mediate anti-giardial biological activity against Giardia trophozoites in vitro. Moreover, BSH47 exerted significant anti-giardial effects when tested in a murine model of giardiasis. These results shed new light on the mechanism, whereby active BSH derived from the probiotic strain Lactobacillus johnsonii La1 may yield anti-giardial effects in vitro and in vivo. These findings pave the way toward novel approaches for the treatment of this widely spread but neglected infectious disease, both in human and in veterinary medicine.

  19. Pregnane glycosides from Sansevieria trifasciata.

    Science.gov (United States)

    Mimaki, Y; Inoue, T; Kuroda, M; Sashida, Y

    1997-01-01

    Phytochemical analysis of the whole plant of Sansevieria trifasciata, one of the most common Agavaceae plants, has resulted in the isolation of four new pregnane glycosides. Their structures have been determined by spectroscopic analysis and acid- and alkaline-catalysed hydrolysis to be 1 beta,3 beta-dihydroxypregna-5,16-dien-20-one glycosides. This is believed to be the first report of the isolation of the pregnane glycosides from a plant of the family Agavaceae.

  20. Inhibition of Nuclear Transcription Factor-κB and Activation of Peroxisome Proliferator-Activated Receptors in HepG2 Cells by Cucurbitane-Type Triterpene Glycosides from Momordica charantia

    Science.gov (United States)

    Nhiem, Nguyen Xuan; Yen, Pham Hai; Ngan, Nguyen Thi Thanh; Quang, Tran Hong; Kiem, Phan Van; Minh, Chau Van; Tai, Bui Huu; Cuong, Nguyen Xuan; Song, Seok Bean

    2012-01-01

    Abstract Momordica charantia: is used to treat various diseases, including inflammatory conditions. Previous reports indicated that the extract of this plant inhibits activation of nuclear transcription factor-κB (NF-κB) but activates peroxisome proliferator-activated receptor (PPAR). Additionally, cucurbitane-type triterpene glycosides are the main bioactive components of the fruit of M. charantia. Therefore, we investigated the anti-inflammatory activity of 17 cucurbitane-type triterpene glycosides (1–17) isolated from this plant. Their inhibition of NF-κB and activation of PPAR activities in HepG2 cells were measured using luciferase reporter and PPAR subtype transactivation assays. Compounds 6 and 8 were found to inhibit NF-κB activation stimulated by tumor necrosis factor-α (TNFα) in a dose-dependent manner. With 50% inhibition concentration (IC50) values of 0.4 μM, compounds 6 and 8 were more potent inhibitors than the positive control, sulfasalazine (IC50=0.9 μM). Compounds 4, 6, and 8 also inhibited TNFα-induced expressions of inducible nitric oxide synthase and cyclooxygenase-2 mRNA. However, only compound 13 significantly increased PPARγ transactivation. PMID:22248180

  1. Lauroside B, a megastigmane glycoside from Laurus nobilis (bay laurel) leaves, induces apoptosis in human melanoma cell lines by inhibiting NF-κB activation.

    Science.gov (United States)

    Panza, Elisabetta; Tersigni, Mariaroberta; Iorizzi, Maria; Zollo, Franco; De Marino, Simona; Festa, Carmen; Napolitano, Maria; Castello, Giuseppe; Ialenti, Armando; Ianaro, Angela

    2011-02-25

    Malignant melanoma is a highly aggressive tumor that frequently resists chemotherapy, so the search for new agents for its treatment is of great importance. In the present study, the antiproliferative propensity against human melanoma cell lines of lauroside B (1), a megastigmane glycoside isolated from Laurus nobilis (bay laurel) leaves, was investigated. This compound suppressed the proliferation of three human melanoma cell lines, namely, A375, WM115, and SK-Mel-28. The 1-induced inhibition of human melanoma cell proliferation was due to the induction of apoptosis, as demonstrated by FACS analysis with annexin V/PI staining and confirmed by activation of caspase-3 and by the cleavage of poly(ADP-ribose) polymerase (PARP). Growing evidence implicates NF-κB as an important contributor to metastasis and increased chemoresistance of melanoma. Thus, it was hypothesized that 1-induced apoptosis could be associated with suppression of NF-κB activation. The results showed that exposure of human melanoma cells to 1 inhibited IκB-α degradation and constitutive NF-κB DNA-binding activity as well as the expression, regulated by NF-κB, of two antiapoptotic genes, XIAP and c-FLIP. Induction of apoptosis by 1 in human aggressive melanoma cell lines has a potential high biological value.

  2. Cytosolic cholesterol ester hydrolase in adrenal cortex

    OpenAIRE

    Tocher, Douglas R.

    1983-01-01

    Cholesterol ester hydrolase (CEH) in adrenocortical cytosol was known to be phosphorylated and activated, in response to ACTH in a cAMPdependent protein kinase mediated process. The purification of CEH from bovine adrenocortical cytosol was attempted. The use of detergents to solubilise the enzyme from lipid-rich aggregates was investigated and sodium cholate was found to be effective. A purification procedure using cholate solubilised enzyme was developed. The detergent int...

  3. Hepatitis B spliced protein (HBSP) promotes the carcinogenic effects of benzo [alpha] pyrene by interacting with microsomal epoxide hydrolase and enhancing its hydrolysis activity

    International Nuclear Information System (INIS)

    Chen, Jin-Yan; Chen, Wan-Nan; Jiao, Bo-Yan; Lin, Wan-Song; Wu, Yun-Li; Liu, Ling-Ling; Lin, Xu

    2014-01-01

    The risk of hepatocellular carcinoma (HCC) increases in chronic hepatitis B surface antigen (HBsAg) carriers who often have concomitant increase in the levels of benzo[alpha]pyrene-7,8-diol-9,10-epoxide(±) (BPDE)-DNA adduct in liver tissues, suggesting a possible co-carcinogenesis of Hepatitis B virus (HBV) and benzo[alpha]pyrene in HCC; however the exact mechanisms involved are unclear. The interaction between hepatitis B spliced protein (HBSP) and microsomal epoxide hydrolase (mEH) was confirmed using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assay; the effects of HBSP on mEH-mediated B[alpha]P metabolism was examined by high performance liquid chromatography (HPLC); and the influences of HBSP on B[alpha]P carcinogenicity were evaluated by bromodeoxyuridine cell proliferation, anchorage-independent growth and tumor xenograft. HBSP could interact with mEH in vitro and in vivo, and this interaction was mediated by the N terminal 47 amino acid residues of HBSP. HBSP could greatly enhance the hydrolysis activity of mEH in cell-free mouse liver microsomes, thus accelerating the metabolism of benzo[alpha]pyrene to produce more ultimate carcinnogen, BPDE, and this effect of HBSP requires the intact HBSP molecule. Expression of HBSP significantly increased the formation of BPDE-DNA adduct in benzo[alpha]pyrene-treated Huh-7 hepatoma cells, and this enhancement was blocked by knockdown of mEH. HBSP could enhance the cell proliferation, accelerate the G1/S transition, and promote cell transformation and tumorigenesis of B[alpha]P-treated Huh-7 hepatoma cells. Our results demonstrated that HBSP could promote carcinogenic effects of B[alpha]P by interacting with mEH and enhancing its hydrolysis activity

  4. [Response of soil hydrolase and oxidoreductase activities to free-air carbon dioxide enrichment (FACE) under rice-wheat rotation].

    Science.gov (United States)

    Zhang, Yulan; Zhang, Lili; Chen, Lijun; Wu, Zhijie

    2004-06-01

    This paper studied the response of soil urease, phosphatase, arylsulphatase and dehydrogenase to 200 micromol x mol(-1) CO2 elevation under rice-wheat rotation. The results showed that under CO2 elevation, the urease activity in 0-10 cm soil layer was decreased at the early growth stages of wheat but increased at its booting stage; the activity increased at the early growth stages of rice but decreased at its ripening stage. Phosphatase activity was increased during the whole growth period of wheat; the activity increased at the tillering stage of rice but decreased at its later growth stages. Arylsulphatase activity was decreased at the over-wintering and booting stages of wheat but increased at its tillering and ripening stages. Dehydrogenase activity was decreased at the early growth stages of wheat and rice, but increased at their late growth stages.

  5. Photochemistry and pharmacology of 9, 19-cyclolanostane glycosides isolated from genus Cimicifuga.

    Science.gov (United States)

    Su, Yang; Chi, Wen-Cheng; Wu, Lun; Wang, Qiu-Hong; Kuang, Hai-Xue

    2016-10-01

    The constituents of Cimicifuga plants have been extensively investigated, and the principal metabolites are 9, 19-cyclolanostane triterpenoid glycosides, which often exhibit extensive pharmacological activities. 9, 19-Cyclolanostane triterpenoid glycosides are distributed widely in genus Cimicifuga rather than in other members of the Ranunculaceae family. So far, more than 140 cycloartane triterpene glycosides have been isolated from Cimicifuga spp.. The aim of this review was to summarize all 9, 19-cyclolanostane triterpenoid glycosides based on the available relevant scientific literatures from 2000 to 2014. Biological studies of cycloartane triterpene glycosides from Cimicifuga spp. are also discussed. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  6. Phytosterol glycosides reduce cholesterol absorption in humans

    Science.gov (United States)

    Lin, Xiaobo; Ma, Lina; Racette, Susan B.; Anderson Spearie, Catherine L.; Ostlund, Richard E.

    2009-01-01

    Dietary phytosterols inhibit intestinal cholesterol absorption and regulate whole body cholesterol excretion and balance. However, they are biochemically heterogeneous and a portion is glycosylated in some foods with unknown effects on biological activity. We tested the hypothesis that phytosterol glycosides reduce cholesterol absorption in humans. Phytosterol glycosides were extracted and purified from soy lecithin in a novel two-step process. Cholesterol absorption was measured in a series of three single-meal tests given at intervals of 2 wk to each of 11 healthy subjects. In a randomized crossover design, participants received ∼300 mg of added phytosterols in the form of phytosterol glycosides or phytosterol esters, or placebo in a test breakfast also containing 30 mg cholesterol-d7. Cholesterol absorption was estimated by mass spectrometry of plasma cholesterol-d7 enrichment 4–5 days after each test. Compared with the placebo test, phytosterol glycosides reduced cholesterol absorption by 37.6 ± 4.8% (P lecithin are bioactive in humans and should be included in methods of phytosterol analysis and tables of food phytosterol content. PMID:19246636

  7. Changes of oxidase and hydrolase activities in pecan leaves elicited by black pecan aphid (Hemiptera: Aphididae) feeding.

    Science.gov (United States)

    Chen, Yigen; Ni, Xinzhi; Cottrell, Ted E; Wood, Bruce W; Buntin, G David

    2009-06-01

    The black pecan aphid, Melanocallis caryaefoliae (Davis) (Hemiptera: Aphididae), is a foliar feeder of pecan, Carya illinoinensis (Wangenh.) K. Koch (Juglandaceae). The pest causes chlorosis of leaflet lamina, physiological damage to foliage and trees, and commonly limits the profitability of commercial pecan orchard enterprises. However, key aspects of this host-pest interaction are poorly understood. We report here the effects of M. caryaefoliae feeding on the foliar activity of oxidative (i.e., catalase, lipoxygenase [LOX]-1 and 3, and peroxidase) and hydrolytic (i.e., esterase) enzymes in relation to the degree of aphid resistance among pecan varieties. The 2-yr study showed that M. caryaefoliae-infested foliage exhibited elevated peroxidase activity only in susceptible ('Desirable', 'Sumner', and 'Schley'), but not in resistant ('Cape Fear', 'Gloria Grande', and 'Money Maker') genotypes. Susceptible genotypes also exhibited more severe leaf chlorosis in response to M. caryaefoliae feeding than the resistant genotypes; however, the aphid feeding did not influence catalase or esterase activity in all varieties, except the increase of esterase activity in Desirable and Gloria Grande. Melanocallis caryaefoliae feeding also influences activity of two lipoxygenase isozymes, with LOX3 being more frequently induced than LOX1. Foliar LOX3 activity was more frequently induced by M. caryaefoliae feeding in the moderately resistant 'Oconee' and highly resistant Money Maker and Cape Fear than in the susceptible genotypes. Therefore, the elevation of peroxidase is likely to be associated with aphid susceptibility and contributed to the severe leaf chlorosis, whereas the increase of LOX3 activity might be associated with aphid resistance in pecan. These findings contribute to our understanding of the etiology of M. caryaefoliae-elicited leaf chlorosis on pecan foliage. Such information may also be used to develop enzyme markers for identifying black pecan aphid resistance

  8. Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A*

    OpenAIRE

    Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit

    2016-01-01

    Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and ...

  9. Disruption of Trichoderma reesei cre2, encoding an ubiquitin C-terminal hydrolase, results in increased cellulase activity

    Science.gov (United States)

    2011-01-01

    Background The filamentous fungus Trichoderma reesei (Hypocrea jecorina) is an important source of cellulases for use in the textile and alternative fuel industries. To fully understand the regulation of cellulase production in T. reesei, the role of a gene known to be involved in carbon regulation in Aspergillus nidulans, but unstudied in T. reesei, was investigated. Results The T. reesei orthologue of the A. nidulans creB gene, designated cre2, was identified and shown to be functional through heterologous complementation of a creB mutation in A. nidulans. A T. reesei strain was constructed using gene disruption techniques that contained a disrupted cre2 gene. This strain, JKTR2-6, exhibited phenotypes similar to the A. nidulans creB mutant strain both in carbon catabolite repressing, and in carbon catabolite derepressing conditions. Importantly, the disruption also led to elevated cellulase levels. Conclusions These results demonstrate that cre2 is involved in cellulase expression. Since the disruption of cre2 increases the amount of cellulase activity, without severe morphological affects, targeting creB orthologues for disruption in other industrially useful filamentous fungi, such as Aspergillus oryzae, Trichoderma harzianum or Aspergillus niger may also lead to elevated hydrolytic enzyme activity in these species. PMID:22070776

  10. Disruption of Trichoderma reesei cre2, encoding an ubiquitin C-terminal hydrolase, results in increased cellulase activity

    Directory of Open Access Journals (Sweden)

    Denton Jai A

    2011-11-01

    Full Text Available Abstract Background The filamentous fungus Trichoderma reesei (Hypocrea jecorina is an important source of cellulases for use in the textile and alternative fuel industries. To fully understand the regulation of cellulase production in T. reesei, the role of a gene known to be involved in carbon regulation in Aspergillus nidulans, but unstudied in T. reesei, was investigated. Results The T. reesei orthologue of the A. nidulans creB gene, designated cre2, was identified and shown to be functional through heterologous complementation of a creB mutation in A. nidulans. A T. reesei strain was constructed using gene disruption techniques that contained a disrupted cre2 gene. This strain, JKTR2-6, exhibited phenotypes similar to the A. nidulans creB mutant strain both in carbon catabolite repressing, and in carbon catabolite derepressing conditions. Importantly, the disruption also led to elevated cellulase levels. Conclusions These results demonstrate that cre2 is involved in cellulase expression. Since the disruption of cre2 increases the amount of cellulase activity, without severe morphological affects, targeting creB orthologues for disruption in other industrially useful filamentous fungi, such as Aspergillus oryzae, Trichoderma harzianum or Aspergillus niger may also lead to elevated hydrolytic enzyme activity in these species.

  11. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

    Science.gov (United States)

    Oguro, Ami; Imaoka, Susumu

    2012-01-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3–7 μM; Vmax, 150–193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism. PMID:22217705

  12. Ubiquitin C-Terminal Hydrolase-Activity Is Involved in Sperm Acrosomal Function and Anti-polyspermy Defense During Porcine Fertilization

    Czech Academy of Sciences Publication Activity Database

    Yi, Y. J.; Manandhar, G.; Sutovsky, M.; Rongfeng, L.; Jonáková, Věra; Oko, R.; Park, C. S.; Prather, R.S.; Sutovsky, P.

    2007-01-01

    Roč. 77, č. 5 (2007), s. 780-793 ISSN 0006-3363 R&D Projects: GA ČR GA303/06/0895; GA MŠk 1M06011 Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z50520701 Keywords : Ubiquitin * proteasome * hydrolase * spermadhesin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.670, year: 2007

  13. Pharmacological treatment of cardiac glycoside poisoning

    OpenAIRE

    Roberts, Darren M.; Gallapatthy, Gamini; Dunuwille, Asunga; Chan, Betty S.

    2015-01-01

    Cardiac glycosides are an important cause of poisoning, reflecting their widespread clinical usage and presence in natural sources. Poisoning can manifest as varying degrees of toxicity. Predominant clinical features include gastrointestinal signs, bradycardia and heart block. Death occurs from ventricular fibrillation or tachycardia. A wide range of treatments have been used, the more common including activated charcoal, atropine, ??adrenoceptor agonists, temporary pacing, anti?digoxin Fab a...

  14. Cell wall degrading enzymes in Trichoderma asperellum grown on wheat bran

    DEFF Research Database (Denmark)

    Bech, Lasse; Busk, Peter Kamp; Lange, Lene

    2015-01-01

    . asperellum was grown on wheat bran, the greatest range of enzymes activity was detected and a total of 175 glycoside hydrolases from 48 glycoside hydrolase families were identified in the transcriptome. The glycoside hydrolases were identified on a functional level using the bioinformatical tool Peptide...... the theory that the glycoside hydrolases have evolved from a common ancestor, followed by a specialization in which saprotrophic fungi such as T. reesei and T. longibrachiatum lost a significant number of genes including several glycoside hydrolases....

  15. Inhibition of dipeptidyl peptidase activity by flavonol glycosides of guava (Psidium guajava L.): a key to the beneficial effects of guava in type II diabetes mellitus.

    Science.gov (United States)

    Eidenberger, Thomas; Selg, Manuel; Krennhuber, Klaus

    2013-09-01

    Based on the traditional use in popular medicine, the effect of extracts from Psidium guajava L. leaves and of the main flavonol-glycoside components on dipeptidyl-peptidase IV (DP-IV), a key enzyme of blood glucose homoeostasis, has been investigated in-vitro. An ethanolic extract was prepared from dried, powdered leaves of guava and was found to contain seven main flavonol-glycosides, which were isolated by semipreparative HPLC and tested individually. The ethanolic guava leave extract was shown to exert a dose-dependent inhibition of DP-IV, with an IC50 of 380 μg/ml test assay solution. Also the individual flavonol-glycosides inhibited DP-IV dose-dependently, with variations of the effects by a factor of 10, and an overall effect accounting for 100% of that observed for the total guava extract. The recovery of individual flavonol-glycosides in CaCo-2 epithelial cells, a model of gastrointestinal tract absorption, amounted to 2.3-5.3% of the amount available for absorption over 60 min at 37°C. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. New benzophenone and quercetin galloyl glycosides from Psidium guajava L.

    Science.gov (United States)

    Matsuzaki, Keiichi; Ishii, Rie; Kobiyama, Kaori

    2010-01-01

    New benzophenone and flavonol galloyl glycosides were isolated from an 80% MeOH extract of Psidium guajava L. (Myrtaceae) together with five known quercetin glycosides. The structures of the novel glycosides were elucidated to be 2,4,6-trihydroxybenzophenone 4-O-(6″-O-galloyl)-β-d-glucopyranoside (1, guavinoside A), 2,4,6-trihydroxy-3,5-dimethylbenzophenone 4-O-(6″-O-galloyl)-β-d-glucopyranoside (2, guavinoside B), and quercetin 3-O-(5″-O-galloyl)-α-l-arabinofuranoside (3, guavinoside C) by NMR, MS, UV, and IR spectroscopies. Isolated phenolic glycosides showed significant inhibitory activities against histamine release from rat peritoneal mast cells, and nitric oxide production from a murine macrophage-like cell line, RAW 264.7. PMID:20354804

  17. Low and moderate photosynthetically active radiation affects the flavonol glycosides and hydroxycinnamic acid derivatives in kale (Brassica oleracea var. sabellica) dependent on two low temperatures.

    Science.gov (United States)

    Neugart, Susanne; Fiol, Michaela; Schreiner, Monika; Rohn, Sascha; Zrenner, Rita; Kroh, Lothar W; Krumbein, Angelika

    2013-11-01

    Kale (Brassica oleracea var. sabellica) contains a large number of naturally occurring structurally different non-acylated and acylated flavonol glycosides as well as hydroxycinnamic acid derivatives. The objective of this study was to determine the effect of low and moderate photosynthetic active radiation (PAR) and how these levels interact with low temperature in these phenolic compounds. Juvenile kale plants were treated with PAR levels from 200 to 800 μmol m(-2) s(-1) at 5 and 10 °C under defined conditions in climate chambers. Of the investigated 20 compounds, 11 and 17 compounds were influenced by PAR and temperature, respectively. In addition, an interaction between PAR and temperature was found for eight compounds. The response of the phenolic compounds to PAR was structure-dependent. While quercetin triglycosides increased with higher PAR at 5 and 10 °C, the kaempferol triglycosides exhibited the highest concentrations at 400 μmol m(-2) s(-1). In contrast, kaempferol diglycosides exhibited the highest concentrations at increased PAR levels of 600 and 800 μmol m(-2) s(-1) at 10 °C. However, key genes of flavonol biosynthesis were influenced by temperature but remained unaffected by PAR. Furthermore, there was no interaction between the PAR level and the low temperature in the response of hydroxycinnamic acid derivatives in kale with the exception of caffeoylquinic acid, which decreased with higher PAR levels of 600 and 800 μmol m(-2) s(-1) and at a lower temperature. In conclusion, PAR and its interaction with temperature could be a suitable tool for modifying the profile of phenolic compounds. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  18. Iridoid glycosides from Thunbergia grandiflora.

    Science.gov (United States)

    Ismail, L D; el-Azizi, M M; Khalifa, T I; Stermitz, F R

    1996-07-01

    The novel iridoid glycosides, isounedoside and grandifloric acid, were isolated from Thunbergia grandiflora. Grandifloric acid contains C-10 as a carboxylic acid group, the presence of which was predicted by recent iridoid biosynthesis studies carried out within T. alata. Isounedoside contains a rare 6,7-epoxide functional group. A revision in some of the NMR spectral assignments for the known iridoid glycoside alatoside was also made.

  19. New Apigenin Glycoside, Polyphenolic Constituents, Anti-inflammatory and Hepatoprotective Activities of Gaillardia grandiflora and Gaillardia pulchella Aerial Parts.

    Science.gov (United States)

    Moharram, Fatma A; El Dib, Rabab Abd El Moneim; Marzouk, Mohamed S; El-Shenawy, Siham M; Ibrahim, Haitham A

    2017-07-01

    Gaillardia grandiflora Hort. ex Van Houte and Gaillardia pulchella Foug are flowering plants widely cultivated in Egypt for their ornamental value. Previous reports demonstrated that sesquiterpene derivatives represent the major compounds in both species. Moreover, only few flavones were identified from genus Gaillardia and few studies on the cytotoxicity of G. pulchella were found. Investigation of the phenolic constituents of the aerial parts of both species and evaluation of their anti-inflammatory and hepatoprotective activities. The 80% aqueous methanol extracts (AME) were prepared for both plants and evaluated for their biological activities. Phytochemical investigation of both extracts resulted in isolation of twelve compounds, which have been identified on the basis of ultraviolet, 1D and 2D nuclear magnetic resonance spectroscopy and negative ESI-MS. The new 8-hydroxyapigenin 6- O -β-D-apiofuranosyl-(1'''→6'')- C -β-D- 4 C 1 -glucopyranoside was isolated from G. grandiflora for the first time in nature, along with schaftoside, luteolin 6-C-β-D- 4 C 1 -glucopyranoside 8-methyl ether, apigenin 6- C -β-D- 4 C 1 -glucopyranoside 8-methyl ether, isoorientin, isovitexin, 6-methoxyluteolin and hispidulin, as well as vicenin-2, vitexin, luteolin and apigenin, which were isolated from G. pulchella together with 6-methoxyluteolin. Furthermore, the AME of both species were found to be nontoxic to mice and exhibited significant anti-inflammatory and hepatoprotective activities in dose dependent manner. Current results shed light on the phenolic constituents of G. grandiflora and G. pulchella aerial parts and the safety of the AME of both species, in addition to their significant anti-inflammatory and hepatoprotective activities. Both plant species may be promising candidates for natural anti-inflammatory and hepatoprotective drugs. Phytochemical investigation of Gaillardia grandiflora and Gaillardia pulchella 80% aqueous methanol extracts of the aerial parts led

  20. Diversity of Microbial Carbohydrate-Active enZYmes (CAZYmes) Associated with Freshwater and Soil Samples from Caatinga Biome.

    Science.gov (United States)

    Andrade, Ana Camila; Fróes, Adriana; Lopes, Fabyano Álvares Cardoso; Thompson, Fabiano L; Krüger, Ricardo Henrique; Dinsdale, Elizabeth; Bruce, Thiago

    2017-07-01

    Semi-arid and arid areas occupy about 33% of terrestrial ecosystems. However, little information is available about microbial diversity in the semi-arid Caatinga, which represents a unique biome that extends to about 11% of the Brazilian territory and is home to extraordinary diversity and high endemism level of species. In this study, we characterized the diversity of microbial genes associated with biomass conversion (carbohydrate-active enzymes, or so-called CAZYmes) in soil and freshwater of the Caatinga. Our results showed distinct CAZYme profiles in the soil and freshwater samples. Glycoside hydrolases and glycosyltransferases were the most abundant CAZYme families, with glycoside hydrolases more dominant in soil (∼44%) and glycosyltransferases more abundant in freshwater (∼50%). The abundances of individual glycoside hydrolase, glycosyltransferase, and carbohydrate-binding module subfamilies varied widely between soil and water samples. A predominance of glycoside hydrolases was observed in soil, and a higher contribution of enzymes involved in carbohydrate biosynthesis was observed in freshwater. The main taxa associated with the CAZYme sequences were Planctomycetia (relative abundance in soil, 29%) and Alphaproteobacteria (relative abundance in freshwater, 27%). Approximately 5-7% of CAZYme sequences showed low similarity with sequences deposited in non-redundant databases, suggesting putative homologues. Our findings represent a first attempt to describe specific microbial CAZYme profiles for environmental samples. Characterizing these enzyme groups associated with the conversion of carbohydrates in nature will improve our understanding of the significant roles of enzymes in the carbon cycle. We identified a CAZYme signature that can be used to discriminate between soil and freshwater samples, and this signature may be related to the microbial species adapted to the habitat. The data show the potential ecological roles of the CAZYme repertoire and

  1. Cardenolide glycosides from Elaeodendron australe var. integrifolium.

    Science.gov (United States)

    Butler, Mark S; Towerzey, Leanne; Pham, Ngoc B; Hyde, Edward; Wadi, Sao Khemar; Guymer, Gordon P; Quinn, Ronald J

    2014-02-01

    Extracts from dried leaf and stems of Elaeodendron australe var. integrifolium (Celastraceae) collected in South East Queensland, Australia, were active in an assay that measured Ca(2+) driven expression of IL-2/luciferase designed to identify inhibitors of the ICRAC channel. Bioassay-guided isolation using C18 and polyamide column chromatography, HPLC (Phenyl and C18) and centrifugal partition chromatography (CPC) led to the isolation of digitoxigenin (1) and three cardenolide glycosides, glucoside 2, quinovoside 3 and the new natural product xyloside 4, as the active components with low nM activity in the reporter assay. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Autolysis of dairy leuconostocs and detection of peptidoglycan hydrolases by renaturing SDS-PAGE.

    Science.gov (United States)

    Cibik, R; Chapot-Chartier, M P

    2000-11-01

    The autolysis of lactic acid bacteria plays a major role during cheese ripening. The aim of this study was to evaluate the autolytic properties and peptidoglycan hydrolase content of dairy leuconostocs. Autolysis of 59 strains of dairy Leuconostoc was examined under starvation conditions in potassium phosphate buffer. The ability of dairy leuconostocs to lyse is strain dependant and not related to the species. The peptidoglycan hydrolase profile of Leuc. mesenteroides subsp. mesenteroides 10L was analysed by renaturing gel electrophoresis. Two major activity bands migrating at 41 and 52 kDa were observed. According to the specificity analysis, strain 10L seems to contain a glycosidase and an N-acetyl-muramyl-L-alanine amidase, or an endopeptidase. The peptidoglycan hydrolase profiles of various Leuconostoc species were also compared. Several peptidoglycan hydrolase activities could be detected in the different Leuconostoc species. Further characterization of the peptidoglycan hydrolases will help to control autolysis of leuconostocs in cheese.

  3. Acylated flavone glycosides from Veronica

    DEFF Research Database (Denmark)

    Albach, Dirk C.; Grayer, Renée J.; Jensen, Søren Rosendal

    2003-01-01

    A survey of the flavonoid glycosides of selected taxa in the genus Veronica yielded two new acylated 5,6,7,3',4'-pentahydroxyflavone (6-hydroxyluteolin) glycosides and two rare allose-containing acylated 5,7,8,4'-tetrahydroxyflavone (isoscutellarein) glycosides. The new compounds were isolated from...

  4. H-1-NMR Fingerprinting of Vaccinium vitis-idaea Flavonol Glycosides

    NARCIS (Netherlands)

    Riihinen, K.R.; Mihaleva, V.V.; Gödecke, T.; Soininen, P.; Laatikainen, R.; Vervoort, J.; Lankin, D.C.; Pauli, G.F.

    2013-01-01

    Introduction - The fruits of Vaccinium vitis-idaea L. are a valuable source of biologically active flavonoid derivatives. For studies focused on the purification of its quercetin glycosides (QGs) and related glycosides from plants and for the purpose of biological studies, the availability of

  5. Quercitrin: an antileishmanial flavonoid glycoside from Kalanchoe pinnata.

    Science.gov (United States)

    Muzitano, Michelle F; Cruz, Elaine A; de Almeida, Ana Paula; Da Silva, Silvia A G; Kaiser, Carlos R; Guette, Catherine; Rossi-Bergmann, Bartira; Costa, Sônia S

    2006-01-01

    Quercitrin (quercetin 3- O-alpha- L-rhamnopyranoside), one of the constituents of the biologically active aqueous extract obtained from Kalanchoe pinnata, is demonstrated to be a potent antileishmanial compound (IC50 approximately 1 microg/mL) with a low toxicity profile. This is the first time that antileishmanial activity is demonstrated for a flavonoid glycoside.

  6. Glycosidic Bond Cleavage is Not Required for Phytosteryl Glycoside-Induced Reduction of Cholesterol Absorption in Mice

    Science.gov (United States)

    Lin, Xiaobo; Ma, Lina; Moreau, Robert A.

    2012-01-01

    Phytosteryl glycosides occur in natural foods but little is known about their metabolism and bioactivity. Purified acylated steryl glycosides (ASG) were compared with phytosteryl esters (PSE) in mice. Animals on a phytosterol-free diet received ASG or PSE by gavage in purified soybean oil along with tracers cholesterol-d7 and sitostanol-d4. In a three-day fecal recovery study, ASG reduced cholesterol absorption efficiency by 45 ± 6% compared with 40 ± 6% observed with PSE. Four hours after gavage, plasma and liver cholesterol-d7 levels were reduced 86% or more when ASG was present. Liver total phytosterols were unchanged after ASG administration but were significantly increased after PSE. After ASG treatment both ASG and deacylated steryl glycosides (SG) were found in the gut mucosa and lumen. ASG was quantitatively recovered from stool samples as SG. These results demonstrate that ASG reduces cholesterol absorption in mice as efficiently as PSE while having little systemic absorption itself. Cleavage of the glycosidic linkage is not required for biological activity of ASG. Phytosteryl glycosides should be included in measurements of bioactive phytosterols. PMID:21538209

  7. Complementary action of jasmonic acid on salicylic acid in mediating fungal elicitor-induced flavonol glycoside accumulation of Ginkgo biloba cells.

    Science.gov (United States)

    Xu, Maojun; Dong, Jufang; Wang, Huizhong; Huang, Luqi

    2009-08-01

    The antagonistic action between jasmonic acid (JA) and salicylic acid (SA) in plant defence responses has been well documented. However, their relationship in secondary metabolite production is largely unknown. Here, we report that PB90, a protein elicitor from Phytophthora boehmeriae, triggers JA generation, SA accumulation and flavonol glycoside production of Ginkgo biloba cells. JA inhibitors suppress not only PB90-triggered JA generation, but also the elicitor-induced flavonol glycoside production. However, the elicitor can still enhance flavonol glycoside production even though the JA generation is totally inhibited. Over-expression of SA hydrolase gene NahG not only abolishes SA accumulation, but also suppresses the elicitor-induced flavonol glycoside production when JA signalling is inhibited. Interestingly, expression of NahG does not inhibit the elicitor-induced flavonol glycoside accumulation in the absence of JA inhibitors. Moreover, JA levels are significantly enhanced when SA accumulation is impaired in the transgenic cells. Together, the data suggest that both JA and SA are involved in PB90-induced flavonol glycoside production. Furthermore, we demonstrate that JA signalling might be enhanced to substitute for SA to mediate the elicitor-induced flavonol glycoside accumulation when SA signalling is impaired, which reveals an unusual complementary relationship between JA and SA in mediating plant secondary metabolite production.

  8. Antioxidant phenylpropanoid glycosides from Buddleja davidii.

    Science.gov (United States)

    Ahmad, Ijaz; Ahmad, Nisar; Wang, Fanghai

    2009-08-01

    Phytochemical investigations on the n-BuOH-soluble fraction of the whole plant of Buddleja davidii led to the isolation of the phenylpropanoid glycosides 1-10. Their structures were determined by 1D and 2D NMR spectroscopic techniques. All the compounds showed potent antioxidative activity in three different tests, with IC(50) values in the range 4.15-9.47 microM in the hydroxyl radical ( OH) inhibitory activity test, 40.32-81.15 microM in the total ROS (reactive oxygen species) inhibitory activity test, and 2.26-7.79 microM in the peroxynitrite (ONOO(-)) scavenging activity test. Calceolarioside A (1) displayed the strongest scavenging potential with IC(50) values of (4.15 +/- 0.07, 40.32 +/- 0.09, 2.26 +/- 0.03 microM) for OH, total ROS and scavenging of ONOO(-), respectively.

  9. Benzofuran Glycosides from Styrax Benzoin

    International Nuclear Information System (INIS)

    Hasliza Yusof; Laily Din; Wan Ahmad Yaacob

    2014-01-01

    Separation of methanol extracts of the fruits and stem bark of Styrax benzoin using various chromatography (vacuum liquid chromatography, column chromatography and preparative thin layer chromatography) gave four benzofuran glycosides namely egonol gentiobioside (1), egonol gentiotrioside (2), egonol glucoside (3) and masutakeside (4). The compounds were identified by spectroscopic analysis (NMR, mass and infra-red spectral data) and by comparison of the data with that of the literature. Isolation of compounds from this plant has never been reported before. (author)

  10. Soulieoside R : A New Cycloartane Triterpenoid Glycoside from Souliea vaginata

    Directory of Open Access Journals (Sweden)

    Qiongyu Zou

    2018-01-01

    Full Text Available A new cycloartane triterpenoid glycoside, named soulieoside R, was isolated from the rhizomes of Souliea vaginata. Its structure was characterized by comprehensive analyses of 1H, 13C NMR, COSY, HSQC, HMBC, NOESY spectroscopic, and HRESIMS mass spectrometric data, as well as chemical methods. The new compound showed weak inhibitory activity against three human cancer cell lines.

  11. A new phenolic glycoside from the stem of Dendrobium nobile.

    Science.gov (United States)

    Zhou, Xue-Ming; Zheng, Cai-Juan; Wu, Jia-Ting; Chen, Guang-Ying; Zhang, Bin; Sun, Chong-Ge

    2017-05-01

    A new phenolic glycoside dendroside (1), together with seven known compounds (2-8) were isolated from the stems of Dendrobium nobile. The structures of these compounds were elucidated using comprehensive spectroscopic methods. The inhibitory activities of all compounds against three cancer cell lines HeLa, MCF-7 and A549 were evaluated.

  12. Inhibition of Xenobiotic-Degrading Hydrolases by Organophosphinates

    Science.gov (United States)

    1986-07-01

    M 4 Q r 000 44 Table 11. Purification of arylester hydrolase Specific Total Total Activity Volume Activity Proteina (Umoles/ Purifi- Fraction (mL...did get re-adjusted after the sample was applied. After the sample was applied the column was washed with the above MES buffer an.+eluted with 100 ml...Lieske (94) and compared them to the reversed phase HPLC retention times we have previously reported (16). We get an excellent linear correlation

  13. Method Development for Extraction and Quantification of Glycosides in Leaves of Stevia Rebaudiana

    International Nuclear Information System (INIS)

    Salmah Moosa; Hazlina Ahmad Hassali; Norazlina Noordin

    2015-01-01

    A solid-liquid extraction and an UHPLC method for determination of glycosides from the leave parts of Stevia rebaudiana were developed. Steviol glycosides found in the leaves of Stevia are natural sweetener and commercially sold as sugar substitutes. Extraction of the glycosides consisted of solvent extraction of leaf powder using various solvents followed by its concentration using rotary evaporator and analysis using Ultra High Performance Liquid Chromatography (UHPLC). Existing analytical methods are mainly focused on the quantification of either rebaudioside A or stevioside, whereas other glycosides, such as rebaudioside B and rebaudioside D present in the leaves also contribute to sweetness or its biological activity. Therefore, we developed an improved method by changing the UHPLC conditions to enable a rapid and reliable determination of four steviol glycosides rather than just two using an isocratic UHPLC method. (author)

  14. Bioaccessibility, Intestinal Permeability and Plasma Stability of Isorhamnetin Glycosides from Opuntia ficus-indica (L.).

    Science.gov (United States)

    Antunes-Ricardo, Marilena; Rodríguez-Rodríguez, César; Gutiérrez-Uribe, Janet A; Cepeda-Cañedo, Eduardo; Serna-Saldívar, Sergio O

    2017-08-22

    Isorhamnetin glycosides are representative compounds of Opuntia ficus-indica that possess different biological activities. There is slight information about the changes in bioaccessibility induced by the glycosylation pattern of flavonoids, particularly for isorhamnetin. In this study, the bioaccessibility and permeability of isorhamnetin glycosides extracted from O. ficus-indica were contrasted with an isorhamnetin standard. Also, the plasma stability of these isorhamnetin glycosides after intravenous administration in rats was evaluated. Recoveries of isorhamnetin after oral and gastric digestion were lower than that observed for its glycosides. After intestinal digestion, isorhamnetin glycosides recoveries were reduced to less than 81.0%. The apparent permeability coefficient from apical (AP) to basolateral (BL) direction (Papp (AP-BL) ) of isorhamnetin was 2.6 to 4.6-fold higher than those obtained for its glycosides. Isorhamnetin diglycosides showed higher Papp (AP-BL) values than triglycosides. Sugar substituents affected the Papp (AP-BL) of the triglycosides. Isorhamnetin glycosides were better retained in the circulatory system than the aglycone. After intravenous dose of the isorhamnetin standard, the elimination half-life was 0.64 h but increased to 1.08 h when the O. ficus-indica extract was administered. These results suggest that isorhamnetin glycosides naturally found in O. ficus-indica could be a controlled delivery system to maintain a constant plasmatic concentration of this important flavonoid to exert its biological effects in vivo.

  15. Cardiac Glycoside Plants Self-Poisoning

    Directory of Open Access Journals (Sweden)

    Radenkova-Saeva J.

    2014-06-01

    Full Text Available Cardiac glycosides are found in a diverse group of plants including Digitalis purpurea and Digitalis lanata (foxgloves, Nerium oleander, Convallaria majalis (lily of the valley, Strophanthus gratus, etc. Nerium Oleander is an indoor and ornamental plant of an evergreen shrub. It’s widespread in countries with a Mediterranean climate. Oleander is one of the most poisonous plants known to humans. All parts of the nerium oleander are poisonous, primarily due to the contained cardiac glycosides - oleandrin, nerin, digitoxigenin, and olinerin of which oleandrin is the principal toxin. The bark contains the toxic substances of rosagenin which causes strychnine-like effects. Signs of poisoning appear a few hours after the adoption of the parts of the plant. Two cases of Nerium Oleander poisoning were presented. Clinical picture included gastrointestinal, cardiovascular and central nervous system effects. The clinical symptoms were characterized by nausea, vomiting, salivation, colic, diarrhoea, ventricular tachycardia, dysrhythmia, heart block, ataxia, drowsiness, muscular tremor. Treatment included administration of activated charcoal, symptomatic and supportive care.

  16. Sea Cucumber Glycosides: Chemical Structures, Producing Species and Important Biological Properties.

    Science.gov (United States)

    Mondol, Muhammad Abdul Mojid; Shin, Hee Jae; Rahman, M Aminur; Islam, Mohamad Tofazzal

    2017-10-17

    Sea cucumbers belonging to echinoderm are traditionally used as tonic food in China and other Asian countries. They produce abundant biologically active triterpene glycosides. More than 300 triterpene glycosides have been isolated and characterized from various species of sea cucumbers, which are classified as holostane and nonholostane depending on the presence or absence of a specific structural unit γ(18,20)-lactone in the aglycone. Triterpene glycosides contain a carbohydrate chain up to six monosaccharide units mainly consisting of d-xylose, 3-O-methy-d-xylose, d-glucose, 3-O-methyl-d-glucose, and d-quinovose. Cytotoxicity is the common biological property of triterpene glycosides isolated from sea cucumbers. Besides cytotoxicity, triterpene glycosides also exhibit antifungal, antiviral and hemolytic activities. This review updates and summarizes our understanding on diverse chemical structures of triterpene glycosides from various species of sea cucumbers and their important biological activities. Mechanisms of action and structural-activity relationships (SARs) of sea cucumber glycosides are also discussed briefly.

  17. HYDROLASING OF CONTAMINATED UNDERWATER BASIN SURFACES AT THE HANFORD K AREA

    International Nuclear Information System (INIS)

    CHRONISTER, G.B.

    2005-01-01

    This paper discusses selecting and implementing hydrolasing technology to reduce radioactive contamination in preparing to dispose of the K Basins; two highly contaminated concrete basins at the Hanford Site. A large collection of spent nuclear fuel stored for many years underwater at the K Basins has been removed to stable, dry, safe storage. Remediation activities have begun for the remaining highly contaminated water. sludge, and concrete basin structures. Hydrolasing will be used to decontaminate and prepare the basin structures for disposal

  18. Clostridium beijerinckii cells expressing Neocallimastix patriciarum glycoside hydrolases show enhanced Lichenan utilization and solvent production

    NARCIS (Netherlands)

    Lopez-Contreras, A.; Oost, van der J.; Claassen, P.; Mooibroek, H.; Vos, de W.M.

    2001-01-01

    Growth and the production of acetone, butanol, and ethanol by Clostridium beijerinckii NCIMB 8052 on several polysaccharides and sugars were analyzed. On crystalline cellulose, growth and solvent production were observed only when a mixture of fungal cellulases was added to the medium. On lichenan

  19. Structural insight into catalytic mechanism of PET hydrolase

    OpenAIRE

    Han, Xu; Liu, Weidong; Huang, Jian-Wen; Ma, Jiantao; Zheng, Yingying; Ko, Tzu-Ping; Xu, Limin; Cheng, Ya-Shan; Chen, Chun-Chi; Guo, Rey-Ting

    2017-01-01

    PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.

  20. Structural insight into catalytic mechanism of PET hydrolase.

    Science.gov (United States)

    Han, Xu; Liu, Weidong; Huang, Jian-Wen; Ma, Jiantao; Zheng, Yingying; Ko, Tzu-Ping; Xu, Limin; Cheng, Ya-Shan; Chen, Chun-Chi; Guo, Rey-Ting

    2017-12-13

    PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.

  1. Identification of a flavonoid C-glycoside as potent antioxidant.

    Science.gov (United States)

    Wen, Lingrong; Zhao, Yupeng; Jiang, Yueming; Yu, Limei; Zeng, Xiaofang; Yang, Jiali; Tian, Miaomiao; Liu, Huiling; Yang, Bao

    2017-09-01

    Flavonoids have been documented to have good antioxidant activities in vitro. However, reports on the cellular antioxidant activities of flavonoid C-glycosides are very limited. In this work, an apigenin C-glycoside was purified from Artocarpus heterophyllus by column chromatography and was identified to be 2″-O-β-D-xylosylvitexin by nuclear magnetic resonance spectroscopy. The cellular antioxidant activity and anticancer activity of 2″-O-β-D-xylosylvitexin were evaluated for the first time. The quantitative structure-activity relationship was analysed by molecular modeling. Apigenin presented an unexpected cellular antioxidation behaviour. It had an antioxidant activity at low concentration and a prooxidant activity at high concentration, whereas 2″-O-β-D-xylosylvitexin showed a dose-dependent cellular antioxidant activity. It indicated that C-glycosidation improved the cellular antioxidation performance of apigenin and eliminated the prooxidant effect. The ortho-dihydroxyl at C-3'/C-4' and C-3 hydroxyl in the flavonoid skeleton play important roles in the antioxidation behaviour. The cell proliferation assay revealed a low cytotoxicity of 2″-O-β-D-xylosylvitexin. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Unliganded and substrate bound structures of the cellooligosaccharide active lytic polysaccharide monooxygenase LsAA9A at low pH

    DEFF Research Database (Denmark)

    Frandsen, Kristian Erik Høpfner; Poulsen, Jens-Christian Navarro; Tandrup, Tobias

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) have been found to be key components in microbial (bacterial and fungal) degradation of biomass. They are copper metalloenzymes that degrade polysaccharides oxidatively and act in synergy with glycoside hydrolases. Recently crystallographic studies...

  3. Evaluation of the content variation of anthraquinone glycosides in rhubarb by UPLC-PDA

    Science.gov (United States)

    2013-01-01

    Background Rhubarb is an important Chinese medicinal herb with a long history of over 2000 years and has been commonly used as a laxative. It is the radix and rhizome of Rheum officinale Baill., R. palmatum L. and R. tanguticum Maxim, all of which are mainly distributed in a broad region in the Tibetan plateau. Anthraquinone glycosides are a series of major active ingredients found in all three species. They are key intermediates in the anthraquinone secondary metabolism and the sennnoside biosynthesis. The variation of the anthraquinone glycoside content in rhubarb in response to specific factors remains an attractive topic. Results A simple and sensitive Ultra Performance Liquid Chromatography with Photo-Diode Array (UPLC-PDA) detector was developed for the simultaneous determination of six anthraquinone glycosides in rhubarb, i.e., aloeemodin-8-O-glucoside, rhein-8-O-glucoside, chrysophanol-1-O-glucoside, emodin-1-O-glucoside, chrysophanol-8-O-glucoside, emodin-8-O-glucoside. Twenty-seven batches from three species were submitted to the multi-component analysis. The results showed that the anthraquinone glycoside content varied significantly even within the same species. The results showed that the anthraquinone glycoside content varied significantly within the same species but not between different species. The PCA and content analysis results confirmed that the plant species has no obvious effect on the content variation. Neither was any significant correlation observed between the anthraquinone glycoside content and the geographic distribution of the rhubarb. Through correlational analysis, altitude was found to be the main factor that affects the anthraquinone glycoside content in rhubarb. Rhubarb grown at higher altitude has higher anthraquinone glycoside content. Conclusions This work provides a rapid, sensitive and accurate UPLC-PDA method for the simultaneous determination of six anthraquinone glycosides in rhubarb. The anthraquinone glycoside content

  4. A New Furostanol Glycoside from Tribulus terrestris

    Directory of Open Access Journals (Sweden)

    Tonghua Liu

    2010-01-01

    Full Text Available Besides two known glycosides, a new furostanol glycoside was isolated from the Fruits of Tribulus terrestris L. The structure of the new furostanol glycoside was established as 26-O-β-D-glucopyranosyl-(25S-5α-furostane-20(22-en-12-one-3β, 26-diol-3-O-α-L-rhamnopyranosyl-(1→2-[β-D-glucopyranosyl-(1→4]-β-D-galactopyranoside (1 on the basis of 1D and 2D-NMR techniques, including COSY, HMBC, and HMQC correlations.

  5. A new furostanol glycoside from Tribulus terrestris.

    Science.gov (United States)

    Xu, Yajuan; Liu, Yonghong; Xu, Tunhai; Xie, Shengxu; Si, Yunshan; Liu, Yue; Zhou, Haiou; Liu, Tonghua; Xu, Dongming

    2010-01-27

    Besides two known glycosides, a new furostanol glycoside was isolated from the Fruits of Tribulus terrestris L. The structure of the new furostanol glycoside was established as 26-O-beta-D-glucopyranosyl-(25S)-5alpha-furostane-20(22)-en-12-one-3beta, 26-diol-3-O-alpha-L-rhamnopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->4)]-beta-D-galactopyranoside (1) on the basis of 1D and 2D-NMR techniques, including COSY, HMBC, and HMQC correlations.

  6. Growth, hydrolases and ultrastructure of Fusarium oxysporum as affected by phenolic rich extracts from several xerophytic plants.

    Science.gov (United States)

    Mohamed, Mahmoud S M; Saleh, Ahmed M; Abdel-Farid, Ibrahim B; El-Naggar, Sabry A

    2017-09-01

    Fusarium oxysporum, the causal agent of rot and wilt diseases, is one of the most detrimental phytopathogens for the productivity of many economic crops. The present study was conducted to evaluate the potentiality of some xerophytic plants as eco-friendly approach for management of F. oxysporum. Phenolic rich extracts from five plants namely: Horwoodia dicksoniae, Citrullus colocynthis, Gypsophila capillaris, Pulicaria incisa and Rhanterium epapposum were examined in vitro. The different extracts showed high variability in their phenolic and flavonoid contents as well as total antioxidant capacity. A strong positive correlation existed between the antifungal activity of the tested extracts and their contents of both total phenolics and flavonoids (r values are 0.91 and 0.82, respectively). Extract of P. incisa was the most effective in reducing the mycelial growth (IC 50 =0.92mg/ml) and inhibiting the activities of CMCase, pectinase, amylase and protease by 36, 42, 58 and 55%, respectively. The high performance liquid chromatography analysis of P. incisa extract revealed the presence of eight phenolic acids along with five polyphenolic compounds. The flavonol, quercetin and its glycosides rutin and quercetrin were the most abundant followed by the phenolic acids, t-cinnamic, caffeic, ferulic and vanillic. P. incisa extract not only affects the growth and hydrolases of F. oxysporum but also induces ultrastructure changes in the mycelium, as revealed by transmission electron microscopy. To our knowledge, this is the first study to investigate the mechanisms underlying the antifungal activity of P. incisa. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Cameroonenoside A: A New Antialgal Phenolic Glycoside from Helichrysum cameroonense

    Directory of Open Access Journals (Sweden)

    Kakam Zanetsie Antoine

    2011-01-01

    Full Text Available Helichrysum cameroonense is known for its medicinal value . This paper deals with a phytochemical investigation of this species, from which cameroonenoside A (1, a new cinnamic acid glycoside ester has been isolated. Its structure was determined by comprehensive analyses of its 1H and 13C NMR, COSY, HMQC, and HMBC spectroscopic, and HREIMS mass spectrometric data. Preliminary studies showed that cameroonenoside A (1 showed algicidal activity against Chlorella fusca

  8. Soulieoside O, a new cyclolanostane triterpenoid glycoside from Souliea vaginata.

    Science.gov (United States)

    Wu, Hai-Feng; Li, Peng-Fei; Zhu, Yin-Di; Zhang, Xiao-Po; Ma, Guo-Xu; Xu, Xu-Dong; Liu, Yi-Lin; Luo, Zheng-Hong; Chen, Di-Zhao; Zou, Qiong-Yu; Zhao, Zi-Jian

    2017-12-01

    A new cyclolanostane triterpenoid glycoside, soulieoside O (1), together with 25-O-acetylcimigenol-3-O-β-d-xylopyranoside (2) and cimigenol-3-O-β-d-xylopyranoside (3), was isolated from the rhizomes of Souliea vaginata. Their structures were characterized by spectroscopic analysis and chemical methods. The new compound showed moderate inhibitory activity against three human cancer cell lines with IC 50 values of 9.3-22.5 μM.

  9. Ultrasonication-Assisted Solvent Extraction of Quercetin Glycosides from ‘Idared’ Apple Peels

    Directory of Open Access Journals (Sweden)

    Gwendolyn M. Huber

    2011-11-01

    Full Text Available Quercetin and quercetin glycosides are physiologically active flavonol molecules that have been attributed numerous health benefits. Recovery of such molecules from plant matrices depends on a variety of factors including polarity of the extraction solvent. Among the solvents of a wide range of dielectric constants, methanol recovered the most quercetin and its glycosides from dehydrated ‘Idared’ apple peels. When ultra-sonication was employed to facilitate the extraction, exposure of 15 min of ultrasound wavelengths of dehydrated apple peel powder in 80% to 100% (v/v methanol in 1:50 (w:v solid to solvent ratio provided the optimum extraction conditions for quercetin and its glycosides. Acidification of extraction solvent with 0.1% (v/v or higher concentrations of HCl led to hydrolysis of naturally occurring quercetin glycosides into the aglycone as an extraction artifact.

  10. How to find soluble proteins: a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli

    Directory of Open Access Journals (Sweden)

    Barth Sandra

    2005-04-01

    Full Text Available Abstract Background In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5:443–448. We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. Results The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. Conclusion A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used

  11. Xyloglucan endotransglucosylase/hydrolases (XTHs) are inactivated by binding to glass and cellulosic surfaces, and released in active form by a heat-stable polymer from cauliflower florets.

    Science.gov (United States)

    Sharples, Sandra C; Nguyen-Phan, Tu C; Fry, Stephen C

    2017-11-01

    Xyloglucan endotransglucosylase (XET) activity, which cuts and re-joins hemicellulose chains in the plant cell wall, contributing to wall assembly and growth regulation, is the major activity of XTH proteins. During purification, XTHs often lose XET activity which, however, is restored by treatment with certain cold-water-extractable, heat-stable polymers (CHPs), e.g. from cauliflower florets. It was not known whether the XTH-activating factor (XAF) present in CHPs works by promoting (e.g. allosterically) XET activity or by re-solubilising sequestered XTH proteins. We now show that XTHs in dilute solution bind to diverse surfaces (e.g. glass and cellulose), and that CHPs can re-solubilise the bound enzyme, re-activating it. Cell walls prepared from cauliflower florets, mung bean shoots and Arabidopsis cell-suspension cultures each contained endogenous, tightly bound, inactive XTHs, which were likewise rapidly solubilised (within 0.5h) and thus activated by cauliflower XAF. We present a convenient quantitative assay for XAF acting on the native sequestered XTHs of Arabidopsis cell walls; using this assay, we show that CHPs from all plants tested possess XAF activity. The XAF activity of diverse CHPs does not correlate with their conductivity, showing that this activity is not a simple ionic effect. The XAF action of cauliflower CHPs was augmented by NaCl, although NaCl alone was much less effective than a CHP solution of similar conductivity, confirming that the cauliflower polymers did not simply exert a salt effect. We suggest that XAF is an endogenous regulator of XET action, modulating cell-wall loosening and/or assembly in vivo. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

  12. High-throughput analysis of endogenous fruit glycosyl hydrolases using a novel chromogenic hydrogel substrate assay

    DEFF Research Database (Denmark)

    Schückel, Julia; Kracun, Stjepan Kresimir; Lausen, Thomas Frederik

    2017-01-01

    A broad range of enzyme activities can be found in a wide range of different fruits and fruiting bodies but there is a lack of methods where many samples can be handled in a high-throughput and efficient manner. In particular, plant polysaccharide degrading enzymes – glycosyl hydrolases (GHs) play...... led to a more profound understanding of the importance of GH activity and regulation, current methods for determining glycosyl hydrolase activity are lacking in throughput and fail to keep up with data output from transcriptome research. Here we present the use of a versatile, easy...

  13. Resin Glycosides from the Morning Glory Family

    Science.gov (United States)

    Pereda-Miranda, Rogelio; Rosas-Ramírez, Daniel; Castañeda-Gómez, Jhon

    Resin glycosides are part of a very extensive family of secondary metabolites known as glycolipids or lipo-oligosaccharides and are constituents of complex resins (glycoresins) (1) unique to the morning glory family, Convolvulaceae (2). These active principles are responsible for the drastic purgative action of all the important Convolvulaceous species used in traditional medicine throughout the world since ancient times. Several commercial purgative crude drugs can be prepared from the roots of different species of Mexican morning glories. Their incorporation as therapeutic agents in Europe is an outstanding example of the assimilation of botanical drugs from the Americas as substitutes for traditional Old World remedies (3). Even though phytochemical investigations on the constituents of these drugs were initiated during the second half of the nineteenth century, the structure of their active ingredients still remains poorly known for some examples of these purgative roots. During the last two decades, the higher resolution capabilities of modern analytical isolation techniques used in conjunction with powerful spectroscopic methods have facilitated the elucidation of the active principles of these relevant herbal products.

  14. Extraction, radiolabeling and in vivo biological evaluation of {sup 131}I labeled egonol glycosides extract

    Energy Technology Data Exchange (ETDEWEB)

    Akguel, Yurdanur; Pazar, Erdinc [Ege Univ., Izmir (Turkey). Chemistry Dept.; Yilmaz, Habibe; Sanlier, Senay Hamarat [Ege Univ., Izmir (Turkey). Biochemistry Dept.; Lambrecht, Fatma Yurt [Ege Univ., Izmir (Turkey). Dept. of Nuclear Applications; Yilmaz, Osman [Dokuz Eyluel Univ., Izmir (Turkey). Dept. of Lab. Animal Science

    2015-09-01

    Crude extract of S. officinalis L. was found to have suspending agent, hemolytic, antitumor, antioxidant and antimicrobial activities. Its major components benzofurans and benzofuran glycosides have antifungal, anticancer, antibacterial and anticomplement activities and display acetylcholinesterase-cyclooxygenase inhibitory and cytotoxic properties. Recently, it has been reported that egonolgentiobioside is a valuable target for structural modification and warrants further investigation for its potential as a novel pharmaceutical tool for the prevention of estrogen deficiency induced diseases. The aim of the current study is to perform in vivo biological evaluation of a glycosides extract, which was isolated from the fruits endocarp of Styrax officinalis L, identified as egonolgentiobioside and homoegonolgentiobioside and labeled with {sup 131}I. The radiolabeled glycosides extract was labeled with {sup 131}I with high yield. The labeled obtained radiolabeled compound was found to be quite stable and lipophilic. In order to determine its tissue distribution, an in vivo study was performed using healthy female Albino Wistar rats injected by {sup 131}I-glycosides. The biodistribution results showed that clearance of the radiolabeled compound is through the hepatobiliary pathway. The experimental study indicated that the radiolabeled glycosides extract accumulated in the large intestine. Therefore, the potential of {sup 131}I-glycosides might be evaluated in colon cancer cell lines and this might be a promising of tumor-imaging agent.

  15. N (6-substituted AMPs inhibit mammalian deoxynucleotide N-hydrolase DNPH1.

    Directory of Open Access Journals (Sweden)

    Claire Amiable

    Full Text Available The gene dnph1 (or rcl encodes a hydrolase that cleaves the 2'-deoxyribonucleoside 5'-monophosphate (dNMP N-glycosidic bond to yield a free nucleobase and 2-deoxyribose 5-phosphate. Recently, the crystal structure of rat DNPH1, a potential target for anti-cancer therapies, suggested that various analogs of AMP may inhibit this enzyme. From this result, we asked whether N (6-substituted AMPs, and among them, cytotoxic cytokinin riboside 5'-monophosphates, may inhibit DNPH1. Here, we characterized the structural and thermodynamic aspects of the interactions of these various analogs with DNPH1. Our results indicate that DNPH1 is inhibited by cytotoxic cytokinins at concentrations that inhibit cell growth.

  16. Discovery of the first dual inhibitor of the 5-lipoxygenase-activating protein and soluble epoxide hydrolase using pharmacophore-based virtual screening

    Czech Academy of Sciences Publication Activity Database

    Temml, V.; Garscha, U.; Romp, E.; Schubert, G.; Gerstmeier, J.; Kutil, Zsófia; Matuszczak, B.; Waltenberger, B.; Stuppner, H.; Werz, O.; Schuster, D.

    2017-01-01

    Roč. 7, FEB 20 (2017), č. článku 42751. ISSN 2045-2322 Institutional support: RVO:61389030 Keywords : activating protein * drug discovery * leukotriene biosynthesis * inflammatory diseases * conformer generation * arachidonic-acid * flap * challenges * asthma * risk Subject RIV: CE - Biochemistry OBOR OECD: Physiology (including cytology) Impact factor: 4.259, year: 2016

  17. A new insight into the physiological role of bile salt hydrolase among intestinal bacteria from the genus Bifidobacterium.

    Science.gov (United States)

    Jarocki, Piotr; Podleśny, Marcin; Glibowski, Paweł; Targoński, Zdzisław

    2014-01-01

    This study analyzes the occurrence of bile salt hydrolase in fourteen strains belonging to the genus Bifidobacterium. Deconjugation activity was detected using a plate test, two-step enzymatic reaction and activity staining on a native polyacrylamide gel. Subsequently, bile salt hydrolases from B. pseudocatenulatum and B. longum subsp. suis were purified using a two-step chromatographic procedure. Biochemical characterization of the bile salt hydrolases showed that the purified enzymes hydrolyzed all of the six major human bile salts under the pH and temperature conditions commonly found in the human gastrointestinal tract. Next, the dynamic rheometry was applied to monitor the gelation process of deoxycholic acid under different conditions. The results showed that bile acids displayed aqueous media gelating properties. Finally, gel-forming abilities of bifidobacteria exhibiting bile salt hydrolase activity were analyzed. Our investigations have demonstrated that the release of deconjugated bile acids led to the gelation phenomenon of the enzymatic reaction solution containing purified BSH. The presented results suggest that bile salt hydrolase activity commonly found among intestinal microbiota increases hydrogel-forming abilities of certain bile salts. To our knowledge, this is the first report showing that bile salt hydrolase activity among Bifidobacterium is directly connected with the gelation process of bile salts. In our opinion, if such a phenomenon occurs in physiological conditions of human gut, it may improve bacterial ability to colonize the gastrointestinal tract and their survival in this specific ecological niche.

  18. Peptidoglycan Hydrolases of Local Lactic Acid Bacteria from Kazakh Traditional Food

    Directory of Open Access Journals (Sweden)

    Serik Shaikhin

    2014-01-01

    Full Text Available Introduction: Peptidoglycan (PG is a major component of the cell wall of Gram-positive bacteria and is essential for maintaining the integrity of the bacterial cell and its shape. The bacteria synthesize PG hydrolases, which are capable of cleaving the covalent bonds of PG. They also play an important role in modeling PG, which is required for bacterial growth and division. In an era of increasing antibiotic-resistant pathogens, PG hydrolases that destroy these important structures of the cell wall act as a potential source of new antimicrobials. The aim of this study is to identify the main PG hydrolases of local lactic acid bacteria isolated from traditional foods that enhance probiotic activity of a biological preparation. Methods. Lactococcus lactis 17А and Lactococcus garvieae 19А were isolated from the traditional sausage-like meat product called kazy. They were isolated according to standards methods of microbiology. Genetic identification of the isolates were tested by determining the nucleotide sequences of 16S rDNA. The Republican collection of microorganisms took strains of Lactobacillus casei subsp. Rhamnosus 13-P, L. delbrueckii subsp. lactis CG-1 B-RKM 0044 from cheese, Lactobacillus casei subsp. casei B-RKM 0202 from homemade butter. They used the standard technique of renaturating polyacrylamide gel electrophoresis to detect PG hydrolases activity. Results. According to the profiles of PG hydrolase activity on zymograms, the enzymes of Lactococci 17A and 19A in kazy are similar in electrophoretic mobility to major autolysin AcmA, while the lactobacilli of industrial and home-made dairy products have enzymes similar to extracellular proteins p40 and p75, which have probiotic activity. Conclusions. Use of peptidoglycan hydrolases seems to be an interesting approach in the fight against multi-drug resistant strains of bacteria and could be a valuable tool for the treatment of diseases caused by these microorganisms in Kazakhstan.

  19. Fungal lytic polysaccharide monooxygenases bind starch and β-cyclodextrin similarly to amylolytic hydrolases

    DEFF Research Database (Denmark)

    Nekiunaite, Laura; Isaksen, Trine; Vaaje-Kolstad, Gustav

    2016-01-01

    , the clustering of CBM20s from starch-targeting LPMOs and hydrolases was in accord with taxonomy and did not correlate to appended catalytic activity. Altogether, these results demonstrate that the CBM20-binding scaffold is retained in the evolution of hydrolytic and oxidative starch-degrading activities....

  20. Pharmacological treatment of cardiac glycoside poisoning.

    Science.gov (United States)

    Roberts, Darren M; Gallapatthy, Gamini; Dunuwille, Asunga; Chan, Betty S

    2016-03-01

    Cardiac glycosides are an important cause of poisoning, reflecting their widespread clinical usage and presence in natural sources. Poisoning can manifest as varying degrees of toxicity. Predominant clinical features include gastrointestinal signs, bradycardia and heart block. Death occurs from ventricular fibrillation or tachycardia. A wide range of treatments have been used, the more common including activated charcoal, atropine, β-adrenoceptor agonists, temporary pacing, anti-digoxin Fab and magnesium, and more novel agents include fructose-1,6-diphosphate (clinical trial in progress) and anticalin. However, even in the case of those treatments that have been in use for decades, there is debate regarding their efficacy, the indications and dosage that optimizes outcomes. This contributes to variability in use across the world. Another factor influencing usage is access. Barriers to access include the requirement for transfer to a specialized centre (for example, to receive temporary pacing) or financial resources (for example, anti-digoxin Fab in resource poor countries). Recent data suggest that existing methods for calculating the dose of anti-digoxin Fab in digoxin poisoning overstate the dose required, and that its efficacy may be minimal in patients with chronic digoxin poisoning. Cheaper and effective medicines are required, in particular for the treatment of yellow oleander poisoning which is problematic in resource poor countries. © 2015 The British Pharmacological Society.

  1. Cardiac glycoside ouabain induces activation of ATF-1 and StAR expression by interacting with the α4 isoform of the sodium pump in Sertoli cells.

    Science.gov (United States)

    Dietze, Raimund; Konrad, Lutz; Shihan, Mazen; Kirch, Ulrike; Scheiner-Bobis, Georgios

    2013-03-01

    Sertoli cells express α1 and α4 isoforms of the catalytic subunit of Na(+),K(+)-ATPase (sodium pump). Our recent findings demonstrated that interactions of the α4 isoform with cardiotonic steroids (CTS) like ouabain induce signaling cascades that resemble the so-called non-classical testosterone pathway characterized by activation of the c-Src/c-Raf/Erk1/2/CREB signaling cascade. Here we investigate a possible physiological significance of the activated cascade. The results obtained in the current investigation show that the ouabain-induced signaling cascade also leads to the activation of the CREB-related activating transcription factor 1 (ATF-1) in the Sertoli cell line 93RS2 in a concentration- and time-dependent manner, as demonstrated by detection of ATF-1 phosphorylated on Ser63 in western blots. The ouabain-activated ATF-1 protein was found to localize to the cell nuclei. The sodium pump α4 isoform mediates this activation, as it is ablated when cells are incubated with siRNA to the α4 isoform. Ouabain also leads to increased expression of steroidogenic acute regulator (StAR) protein, which has been shown to be a downstream consequence of CREB/ATF-1 activation. Taking into consideration that CTS are most likely produced endogenously, the demonstrated induction of StAR expression by ouabain establishes a link between CTS, the α4 isoform of the sodium pump, and steroidogenesis crucial for male fertility and reproduction. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. The platelet activating factor acetyl hydrolase, oxidized low-density lipoprotein, paraoxonase 1 and arylesterase levels in treated and untreated patients with polycystic ovary syndrome.

    Science.gov (United States)

    Carlioglu, Ayse; Kaygusuz, Ikbal; Karakurt, Feridun; Gumus, Ilknur Inegol; Uysal, Aysel; Kasapoglu, Benan; Armutcu, Ferah; Uysal, Sema; Keskin, Esra Aktepe; Koca, Cemile

    2014-11-01

    To evaluate the platelet activating factor acetyl hydrolyze (PAF-AH), oxidized low-density lipoprotein (ox-LDL), paraoxonase 1 (PON1), arylesterase (ARE) levels and the effects of metformin and Diane-35 (ethinyl oestradiol + cyproterone acetate) therapies on these parameters and to determine the PON1 polymorphisms among PCOS patients. Ninety patients with PCOS, age 30, and body mass index-matched healthy controls were included in the study. Patients were divided into three groups: metformin treatment, Diane-35 treatment and no medication groups. The treatment with metformin or Diane-35 was continued for 6 months and all subjects were evaluated with clinical and biochemical parameters 6 months later. One-way Anova test, t test and non-parametric Mann-Whitney U tests were used for statistical analysis. PAF-AH and ox-LDL levels were statistically significantly higher in untreated PCOS patients than controls, and they were statistically significantly lower in patients treated with metformin or Diane-35 than untreated PCOS patients. In contrast, there were lower PON1 (not statistically significant) and ARE (statistically significant) levels in untreated PCOS patients than the control group and they significantly increased after metformin and Diane-35 treatments. In PCOS patients serum PON1 levels for QQ, QR and RR phenotypes were statistically significantly lower than the control group. In patients with PCOS, proatherogenic markers increase. The treatment of PCOS with metformin or Diane-35 had positive effects on lipid profile, increased PON1 level, which is a protector from atherosclerosis and decreased the proatherogenic PAF-AH and ox-LDL levels.

  3. Role of lecithin-cholesterol acyltransferase in the metabolism of oxidized phospholipids in plasma: studies with platelet-activating factor-acetyl hydrolase-deficient plasma.

    Science.gov (United States)

    Subramanian, V S; Goyal, J; Miwa, M; Sugatami, J; Akiyama, M; Liu, M; Subbaiah, P V

    1999-07-09

    To determine the relative importance of platelet-activating factor-acetylhydrolase (PAF-AH) and lecithin-cholesterol acyltransferase (LCAT) in the hydrolysis of oxidized phosphatidylcholines (OXPCs) to lyso-phosphatidylcholine (lyso-PC), we studied the formation and metabolism of OXPCs in the plasma of normal and PAF-AH-deficient subjects. Whereas the loss of PC following oxidation was similar in the deficient and normal plasmas, the formation of lyso-PC was significantly lower, and the accumulation of OXPC was higher in the deficient plasma. Isolated LDL from the PAF-AH-deficient subjects was more susceptible to oxidation, and stimulated adhesion molecule synthesis in endothelial cells, more than the normal LDL. Oxidation of 16:0-[1-14C]-18:2 PC, equilibrated with plasma PC, resulted in the accumulation of labeled short- and long-chain OXPCs, in addition to the labeled aqueous products. The formation of the aqueous products decreased by 80%, and the accumulation of short-chain OXPC increased by 110% in the deficient plasma, compared to the normal plasma, showing that PAF-AH is predominantly involved in the hydrolysis of the truncated OXPCs. Labeled sn-2-acyl group from the long-chain OXPC was not only hydrolyzed to free fatty acid, but was preferentially transferred to diacylglycerol, in both the normal and deficient plasmas. In contrast, the acyl group from unoxidized PC was transferred only to cholesterol, showing that the specificity of LCAT is altered by OXPC. It is concluded that, while PAF-AH carries out the hydrolysis of mainly truncated OXPCs, LCAT hydrolyzes and transesterifies the long-chain OXPCs.

  4. Accumulation of Flavonoid Glycosides and UFGT Gene Expression in Mulberry Leaves (Morus alba L.) before and after Frost.

    Science.gov (United States)

    Yu, Xiaofeng; Zhu, Yiling; Fan, Jingyi; Wang, Dujun; Gong, Xiaohui; Ouyang, Zhen

    2017-08-01

    In order to determine the molecular mechanism underlying the influence of frost on chemical changes in mulberry leaves, the UFGT activity, expression level, and accumulation of flavonoid glycosides in mulberry leaves (Morus alba L.) were studied. The expression of UFGT gene was investigated by quantitative real-time PCR (qRT-PCR) and the UFGT activity, accumulation of flavonoid glycosides was studied by high performance liquid chromatography. Then, the correlation between the expression level of UFGT, the UFGT activity, and the flavonoid glycosides accumulation with temperature was explored. The accumulation of isoquercitrin and astragalin is significantly positively correlated with UFGT gene expression and UFGT activity. On the contrary, the average temperature was significantly negatively correlated with the level of UFGT gene expression and UFGT activity. The results show that after frost, low temperature can induce the expression of UFGT gene in mulberry leaves, resulting in the accumulation of flavonoid glycosides. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

  5. Cellulase variants

    Science.gov (United States)

    Blazej, Robert; Toriello, Nicholas; Emrich, Charles; Cohen, Richard N.; Koppel, Nitzan

    2015-07-14

    This invention provides novel variant cellulolytic enzymes having improved activity and/or stability. In certain embodiments the variant cellulotyic enzymes comprise a glycoside hydrolase with or comprising a substitution at one or more positions corresponding to one or more of residues F64, A226, and/or E246 in Thermobifida fusca Cel9A enzyme. In certain embodiments the glycoside hydrolase is a variant of a family 9 glycoside hydrolase. In certain embodiments the glycoside hydrolase is a variant of a theme B family 9 glycoside hydrolase.

  6. Caffeoyl phenylethanoid glycosides in Sanango racemosum and in the gesneriaceae

    DEFF Research Database (Denmark)

    Jensen, Søren Rosendal

    1996-01-01

    An investigation of Samango racemosum for systematically useful glycosides has been performed. No iridoids could be detected, but reverse phase chromatography provided the caffeoyl phenylethanoid glycosides (CPGs) calceolarioside C and conandroside together with the new 2-(3,4-dihydroxyphenyl...

  7. Triisobutylaluminium (TIBAL Promoted Rearrangement of C-glycosides

    Directory of Open Access Journals (Sweden)

    P. Sinay

    2005-08-01

    Full Text Available Triisobutylaluminium-promoted rearrangement of unsaturated glycosides containing electron-donating aglycons, such as C-aryl glycosides, provides direct access to highly functionalised cyclohexane derivatives.

  8. Cardiac glycosides induce cell death in human cells by inhibiting general protein synthesis.

    Directory of Open Access Journals (Sweden)

    Andrea Perne

    2009-12-01

    Full Text Available Cardiac glycosides are Na(+/K(+-pump inhibitors widely used to treat heart failure. They are also highly cytotoxic, and studies have suggested specific anti-tumor activity leading to current clinical trials in cancer patients. However, a definitive demonstration of this putative anti-cancer activity and the underlying molecular mechanism has remained elusive.Using an unbiased transcriptomics approach, we found that cardiac glycosides inhibit general protein synthesis. Protein synthesis inhibition and cytotoxicity were not specific for cancer cells as they were observed in both primary and cancer cell lines. These effects were dependent on the Na(+/K(+-pump as they were rescued by expression of a cardiac glycoside-resistant Na(+/K(+-pump. Unlike human cells, rodent cells are largely resistant to cardiac glycosides in vitro and mice were found to tolerate extremely high levels.The physiological difference between human and mouse explains the previously observed sensitivity of human cancer cells in mouse xenograft experiments. Thus, published mouse xenograft models used to support anti-tumor activity for these drugs require reevaluation. Our finding that cardiac glycosides inhibit protein synthesis provides a mechanism for the cytotoxicity of CGs and raises concerns about ongoing clinical trials to test CGs as anti-cancer agents in humans.

  9. Perspectives for the Industrial Enzymatic Production of Glycosides

    NARCIS (Netherlands)

    Roode, de B.M.; Franssen, M.C.R.; Padt, van der A.; Boom, R.M.

    2003-01-01

    Glycosides are of commercial interest for industry in general and specifically for the pharmaceutical and food industry. Currently chemical preparation of glycosides will not meet EC food regulations, and therefore chemical preparation of glycosides is not applicable in the food industry. Thus,

  10. Glycosides from Medicinal Plants as Potential Anticancer Agents: Emerging Trends towards Future Drugs.

    Science.gov (United States)

    Khan, Haroon; Saeedi, Mina; Nabavi, Seyed Mohammad; Mubarak, Mohammad S; Bishayee, Anupam

    2018-04-03

    Cancer continues to be a global burden, despite the advancement of various technological and pharmaceutical improvements over the past two decades. Methods for treating cancer include surgery, radiotherapy and chemotherapy in addition to other specialized techniques. On the other hand, medicinal plants have been traditionally employed either as the complementary medicine or dietary agents in the treatment and management of cancer. Medicinal plants are a rich source of secondary metabolites with interesting biological and pharmacological activities. Among these metabolites, glycosides are naturally occurring substances and have outstanding therapeutic potential and clinical utility. Different medical research engines such GoogleScholar, PubMed, SpringerLink, ScienceDirect were used to collect related literature on the subject matter. In this regard, only peer reviewed journals were considered. Emerging results showed that numerous glycosides isolated from various plants possessed marked anticancer activity against a variety of cancer cell lines. Accordingly, the aim of the present review is to shed light on the anticancer effects of glycosides, analyze possible mechanisms of action, and highlight the role of these natural agents as complementary and alternative medicine in combating and managing cancer. The glycosides isolated from different plants demonstrated potent cytotoxic effects against various cancer cell lines in initial preclinical studies. The anticancer effect was mediated through multiple mechanisms; however further detail studies are needed to understand the full potential of glycosides for clinical utility. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  11. Kaempferol glycosides and cardenolide glycosides, cytotoxic constituents from the seeds of Draba nemorosa (Brassicaceae).

    Science.gov (United States)

    Moon, Surk-Sik; Rahman, Md Aziz Abdur; Manir, Md Maniruzzaman; Jamal Ahamed, V S

    2010-08-01

    Bioassay-directed fractionation of a methanolic extract from the seeds of Draba nemorosa (Brassicaceae) led to isolation of a new flavonol glycoside, drabanemoroside (5, kaempferol 3-O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranose) along with four known flavonoid derivatives (1-4), four cardenolide glycosides (6-9). Kaempferol glycosides 2 and 5 showed strong cytotoxicity against human small lung cancer cell line A549 and melanoma SK-Mel-2 with an IC(50) of 0.5 microg/mL and 1.9 microg/mL, respectively. Cardenolide glycosides 6-9 showed potent cytotoxicity (A549) in the range of 0.01-0.032 microg/mL. Their structures were characterized based on spectroscopic data (2D NMR, HRTOFMS, IR, and UV) and comparison of literature values. The carbohydrate units were also confirmed by comparing the hydrolysate of 5 with authentic monosaccharides.

  12. [Determination of phenylethanoid glycosides in Orobanche coerulescens].

    Science.gov (United States)

    Han, Guo-qing; Li, Cai-feng; Wang, Xiao-qin; Li, Min-hui; Li, Jing

    2015-11-01

    Orobanche caerulescens is an important medicinal resource in Orobanchaceae. The present study aims to establish methods for determination of acteoside, crenatoside, and total phenylethanoid glycosides in O. caerulescens, and determine the content in 15 samples to evaluate the resource utilization of this medicinal plant. The content of acteoside and crenatoside were quantitatively determined by HPLC, while total phenylpropanoid glycosides was estimated by UV-VIS spectrophotometry. According to the results, the content of acteoside was the highest in O. caerulescens, followed by crenatoside. The contents of acteoside, crenatoside, and total phenylethanoid glycosides were between 1.15% - 15.60%, 0.83% - 4.47%, and 6.78% - 27.43%, respectively, which had significant differences. The acquisition time has great influence on the content of main components of O. caerulescens. The content of phenylethanoid glycosides is higher in the samples which were collected at the flowering stage. The two determination methods were proved to be simple, accurate and reliable, and can be used to evaluate the quality and resource utilization of O. caerulescens.

  13. FURTHER FLAVONOL GLYCOSIDES OF EMBELIA SCHIMPERI ...

    African Journals Online (AJOL)

    a

    ABSTRACT. Fractionation of the methanolic extract of Embelia schimperi leaves has led to the isolation of two novel flavonol glycosides. The compounds were characterized as isorhamnetin 3-O- β-galactoysyl (1→ 4)-β-galactoside and quercetin 3-O-[α-rhamnosyl (1→2)] [α-rhamnosyl (1→ 4)]-α- rhamnoside. Also reported ...

  14. Veronica: Acylated flavone glycosides as chemosystematic markers

    DEFF Research Database (Denmark)

    Albach, Dirk C.; Grayer, Renée J.; Kite, Geoffrey C.

    2005-01-01

    HPLC/DAD and LCeMS of an extract of Veronica spicata subgenus Pseudolysimachium, Plantaginaceae) revealed the presence of six 6-hydroxyluteolin glycosides acylated with phenolic acids, three of which are new compounds and which we called spicosides. A flavonoid survey of seven more species...

  15. A new withanolide glycoside from physalis peruviana

    Science.gov (United States)

    Ahmad; Malik; Afza; Yasmin

    1999-03-01

    A new withanolide glycoside, 17beta-hydroxy-14, 20-epoxy-1-oxo-[22R]-3beta-[O-beta-D-glucopyranosyl]-witha-5, 24-dienolide (1), has been isolated from the whole plant of Physalis peruviana. Its identity was determined using a combination of spectroscopic data including 2D NMR techniques and chemical transformations.

  16. Two New Flavone Glycosides from Chenopodiumambrosioides Growing Wildly in Egypt

    Directory of Open Access Journals (Sweden)

    Hala M. Hammoda

    2015-06-01

    Full Text Available Chenopodiumambrosioides (Chenopodiaceae growing wildly in Egypt was subjected to antioxidant –guided phytochemical investigation and the EtOAc fraction afforded the two new flavone glycosides; scutellarein-7-O-α-rhamnopyranosyl-(1→2-α-rhamnopyranosyl-(1→2-α-rhamnopyranoside (1 and scutella-rein-7-O-α-rhamnopyranosyl-(1→2-α-rhamnopyranoside (2. In addition, the invitro antioxidant activities of the plant alcohol extract, CHCl 3 fraction, EtOAc fraction and isolates were studied.

  17. Two new monoterpenoid glycosides from the fresh rhizome of Tongling White Ginger (Zingiber officinale).

    Science.gov (United States)

    Guo, Tao; Tan, Su-Bei; Wang, Ya; Chang, Jun

    2018-01-01

    Two new monoterpenoid glycosides, trans-1,8-cineole-3,6-dihydroxy-3-O-β-D-glucopyranoside (1), and 5,9-dihydroxy borneol 2-O-β-D-glucopyranoside (2), together with four known monoterpenoid glycosides (3-6), were isolated from the water-soluble constituents of the fresh rhizome of Tongling White Ginger (Zingiber officinale). Their structures were decisively elucidated by spectroscopic analysis. In vitro tests for antimicrobial activity showed that compounds 1 and 3 possess significant activity against two Gram-positive organisms, Staphylococcus aureus and Staphylococcus epidermidis.

  18. Total peroxynitrite scavenging capacity of phenylethanoid and flavonoid glycosides from the flowers of Buddleja officinalis.

    Science.gov (United States)

    Tai, Bui Huu; Jung, Bong Yong; Cuong, Nguyen Manh; Linh, Pham Thuy; Tung, Nguyen Huu; Nhiem, Nguyen Xuan; Huong, Tran Thu; Anh, Ngo Thi; Kim, Jeong Ah; Kim, Sang Kyum; Kim, Young Ho

    2009-12-01

    Nine compounds, including six phenylethanoid glycosides: acteoside (1); bioside (2); echinacoside (3); poliumoside (4); phenylethyl glycoside (5); salidroside (6) and three flavonoids; linarin (7); apigenin (8); isorhoifolin (9), were isolated from the flowers of Buddleja officinalis MAXIM. (Buddlejaceae). Chemical structures were confirmed by (1)H-, and (13)C-NMR, and MS spectral methods and compared with those reported in the literature. Antioxidant activities of the methanol and water extracts, and all isolated compounds were evaluated using the total oxidant scavenging capacity (TOSC) assay against peroxynitrite. Results of the assay showed that the phenylethanoid glycosides, a major class of compounds of the flowers of B. officinalis, possess strong antioxidant activity. Of these, acteoside, echinacoside and poliumoside have 9.9-, 9.8- and 9.5-fold TOSC value, respectively, compared with the positive control, Trolox.

  19. Methods for the enzymatic modification of steviol glycosides, modified steviol glycosides obtainable thereby, and the use thereof as sweeteners

    NARCIS (Netherlands)

    te Poele, Evelien; Dijkhuizen, Lubbert; Gerwig, Gerrit; Kamerling, Johannis

    2016-01-01

    The present invention relates generally to the production of steviol glycosides. Provided is a method for enzymatically providing a modified steviol glycoside, comprising incubating a steviol glycoside substrate in the presence of sucrose and the glucansucrase GTF180 of Lactobacillus reuteri strain

  20. A screening method for β-glucan hydrolase employing Trypan Blue-coupled β-glucan agar plate and β-glucan zymography.

    Science.gov (United States)

    Park, Chang-Su; Yang, Hee-Jong; Kim, Dong-Ho; Kang, Dae-Ook; Kim, Min-Soo; Choi, Nack-Shick

    2012-06-01

    A new screening method for β-(1,3-1,6) glucan hydrolase was developed using a pure β-glucan from Aureobaisidum pullulans by zymography and an LB-agar plate. Paenibacillus sp. was screened as a producer a β-glucan hydrolase on the Trypan Blue-coupled β-glucan LB-agar plate and the activity of the enzyme was analyzed by SDS-β-glucan zymography. The β-glucan was not hydrolyzed by Bacillus spp. strains, which exhibit cellulolytic activity on CMC zymography. The gene, obtaining by shotgun cloning and encoding the β-glucan hydrolase of Paenibacillus sp. was sequenced.

  1. Antidepressant-like effect of peony glycosides in mice.

    Science.gov (United States)

    Mao, Qing-Qiu; Ip, Siu-Po; Tsai, Sam-Hip; Che, Chun-Tao

    2008-09-26

    The root part of Paeonia lactiflora Pall. (Ranunculaceae), known as peony, is often used in Chinese herbal formulae for the treatment of depression-like disorders. Previous studies in our laboratory have shown that an ethanol extract of peony produced antidepressive effects in mouse models of depression. It is well known that peony contains glycosides such as paeoniflorin and albiflorin, yet it remains unclear whether the total glycosides of peony (TGP) are effective. The present study aims to evaluate the antidepressant-like effects of TGP. The antidepressant-like effects of TGP was determined by using animal models of depression including forced swim and tail suspension tests. The acting mechanism was explored by determining the effect of TGP on the activities of monoamine oxidases. Intragastric administration of TGP at 80 and 160 mg/kg for seven days caused a significant reduction of immobility time in both forced swim and tail suspension tests, yet TGP did not stimulate locomotor activity in the open-field test. In addition, TGP treatment antagonized reserpine-induced ptosis and inhibited the activities of monoamine oxidases in mouse cerebrum. These results suggest that the antidepressive effects of TGP are mediated, at least in part, by the inhibition of monoamine oxidases.

  2. Development of glycoside-bound radiopharmaceuticals; Novel radioiodination method for digoxin

    Energy Technology Data Exchange (ETDEWEB)

    Takemura, Yasutaka; Dote, Nobuhito; Taniuchi, Hideyuki; Iijima, Naoko; Yokoyama, Akira (Kyoto Univ. (Japan). Faculty of Pharmaceutical Science); Fujibayashi, Yasuhisa; Konishi, Junji

    1994-01-01

    We combined 2-hydroxy-3-methylbenzoylhydrazide (HMBH) with glycosides as a novel method for the radioiodination of physiologically active glycosides. This method was tested using digoxin, which is one of the cardiac glycosides. A digoxin-HMBH conjugate was synthesized by periodate cleavage of the third sugar ring, and was readily radiolabelled with Na[[sup 125]I] by the chloramine-T method. [sup 125]I labelled digoxin-HMBH conjugate retained Na[sup +], K[sup +]-ATPase binding in vivo and in vitro, and also retained immunoreactivity to an anti-digoxin antibody. Thus, this [sup 125]I labelled digoxin-HMBH conjugate represents a potential radiopharmaceutical for Na[sup +], K[sup +]-ATPase imaging, as well as for the radioimmunoassay of digoxin. (author).

  3. Synthesis of O- and C-glycosides derived from β-(1,3)-D-glucans.

    Science.gov (United States)

    Marca, Eduardo; Valero-Gonzalez, Jessika; Delso, Ignacio; Tejero, Tomás; Hurtado-Guerrero, Ramon; Merino, Pedro

    2013-12-15

    A series of β-(1,3)-d-glucans have been synthesized incorporating structural variations specifically on the reducing end of the oligomers. Both O- and C-glucosides derived from di- and trisaccharides have been obtained in good overall yields and with complete selectivity. Whereas the O-glycosides were obtained via a classical Koenigs-Knorr glycosylation, the corresponding C-glycosides were obtained through allylation of the anomeric carbon and further cross-metathesis reaction. Finally, the compounds were evaluated against two glycosidases and two endo-glucanases and no inhibitory activity was observed. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Synthesis and evaluation of cardiac glycoside mimics as potential anticancer drugs

    DEFF Research Database (Denmark)

    Jensen, Marie; Schmidt, Steffen; Fedosova, Natalya

    2011-01-01

    recent years cardiac glycosides have furthermore been suggested to possess valuable anticancer activity. To mimic the labile trisaccharide of digitoxin with a stabile carbohydrate surrogate, we have used sulfur linked ethylene glycol moieties of varying length (mono-, di-, tri- or tetra-ethylene glycol...... the shortest mimics were found to have highest efficacy, with the best ligand having a monoethylene glycol unit (IC(50) 0.24 μM), which was slightly better than digitoxigenin (IC(50) 0.64 μM), while none of the novel cardiac glycoside mimics display an in vitro effect as high as digitoxin (IC(50) 0.02 μM)....

  5. Key role of chemical hardness to compare 2,2-diphenyl-1-picrylhydrazyl radical scavenging power of flavone and flavonol O-glycoside and C-glycoside derivatives.

    Science.gov (United States)

    Waki, Tsukasa; Nakanishi, Ikuo; Matsumoto, Ken-ichiro; Kitajima, Junichi; Chikuma, Toshiyuki; Kobayashi, Shigeki

    2012-01-01

    The antioxidant activities of flavonoids and their glycosides were measured with the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH radical, DPPH(·)) scavenging method. The results show that free hydroxyl flavonoids are not necessarily more active than O-glycoside. Quercetin and kaempferol showed higher activity than apigenin. The C- and O-glycosides of flavonoids generally showed higher radical scavenging activity than aglycones; however, kaempferol C3-O-glycoside (astragalin) showed higher activity than kaempferol. In the radical scavenging activity of flavonoids, it was expected that OH substitutions at C3 and C5 and catechol substitution at C2 of B ring and intramolecular hydrogen bonding between OH at C5 and ketone at C3 would increase the activity; however, the reasons have yet to be clarified. We here show that the radical scavenging activities of flavonoids are controlled by their absolute hardness (η) and absolute electronegativity (χ) as a electronic state. Kaempferol and quercetin provide high radical scavenging activity since (i) OH substitutions at C3 and C5 strikingly decrease η of flavones, (ii) OH substitutions at C3 and C7 decrease χ and η of flavones, and (iii) phenol or o-catechol substitution at C2 of B ring decrease χ of flavones. The coordinate r(χ, η) as the electron state must be small to increase the radical scavenging activity of flavonoids. The results show that chemically soft kaempferol and quercetin have higher DPPH radical scavenging activity than chemically hard genistein and daidzein.

  6. Composition and expression of genes encoding carbohydrate-active enzymes in the straw-degrading mushroom Volvariella volvacea.

    Directory of Open Access Journals (Sweden)

    Bingzhi Chen

    Full Text Available Volvariella volvacea is one of a few commercial cultivated mushrooms mainly using straw as carbon source. In this study, the genome of V. volcacea was sequenced and assembled. A total of 285 genes encoding carbohydrate-active enzymes (CAZymes in V. volvacea were identified and annotated. Among 15 fungi with sequenced genomes, V. volvacea ranks seventh in the number of genes encoding CAZymes. In addition, the composition of glycoside hydrolases in V. volcacea is dramatically different from other basidiomycetes: it is particularly rich in members of the glycoside hydrolase families GH10 (hemicellulose degradation and GH43 (hemicellulose and pectin degradation, and the lyase families PL1, PL3 and PL4 (pectin degradation but lacks families GH5b, GH11, GH26, GH62, GH93, GH115, GH105, GH9, GH53, GH32, GH74 and CE12. Analysis of genome-wide gene expression profiles of 3 strains using 3'-tag digital gene expression (DGE reveals that 239 CAZyme genes were expressed even in potato destrose broth medium. Our data also showed that the formation of a heterokaryotic strain could dramatically increase the expression of a number of genes which were poorly expressed in its parental homokaryotic strains.

  7. Oleic and linoleic acids are active principles in Nigella sativa and stabilize an E(2)P conformation of the Na,K-ATPase. Fatty acids differentially regulate cardiac glycoside interaction with the pump.

    Science.gov (United States)

    Mahmmoud, Yasser A; Christensen, S Brøgger

    2011-10-01

    Nigella sativa seed oil was found to contain a modulator of Na,K-ATPase. Separation analyses combined with (1)H NMR and GCMS identified the inhibitory fraction as a mixture of oleic and linoleic acids. These two fatty acids are specifically concentrated in several medicinal plant oils, and have particularly been implicated in decreasing high blood pressure. The ouabain binding site on Na,K-ATPase has also been implicated in blood pressure regulation. Thus, we aimed to determine how these two molecules modify pig kidney Na,K-ATPase. Oleic and linoleic acids did not modify reactions involving the E(1) (Na(+)) conformations of the Na,K-ATPase. In contrast, K(+) dependent reactions were strongly modified after treatment. Oleic and linoleic acids were found to stabilize a pump conformation that binds ouabain with high affinity, i.e., an ion free E(2)P form. Time-resolved binding assays using anthroylouabain, a fluorescent ouabain analog, revealed that the increased ouabain affinity is unique to oleic and linoleic acids, as compared with γ-linolenic acid, which decreased pump-mediated ATP hydrolysis but did not equally increase ouabain interaction with the pump. Thus, the dynamic changes in plasma levels of oleic and linoleic acids are important in the modulation of the sensitivity of the sodium pump to cardiac glycosides. Given the possible involvement of the cardiac glycoside binding site on Na,K-ATPase in the regulation of hypertension, we suggest oleic acid to be a specific chaperon that modulates interaction of cardiac glycosides with the sodium pump. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

  8. Non-targeted glycosidic profiling of international wines using neutral loss-high resolution mass spectrometry.

    Science.gov (United States)

    Barnaba, C; Dellacassa, E; Nicolini, G; Nardin, T; Serra, M; Larcher, R

    2018-07-06

    Many metabolites naturally occur as glycosides, since sugar moieties can be crucial for their biological activity and increase their water solubility. In the plant kingdom they may occur as glycosides or sugar esters, depending on precursor chemical structure, and in wine they have traditionally attracted attention due to their organoleptic properties, such as astringency and bitterness, and because they affect the colour and aroma of wines. A new approach directed at detailed description of glycosides in a large selection of monovarietal wines (8 samples each of Pinot Blanc, Muller Thurgau, Riesling, Traminer, Merlot, Pinot Noir and Cabernet Sauvignon) was developed by combining high performance liquid chromatography with high resolution tandem mass spectrometry. Analytical separation was performed on an Accucore™ Polar Premium LC column, while mass analysis was performed in negative ion mode with an non-targeted screening approach, using a Full MS/AIF/NL dd-MS 2 experiment at a resolving power of 140,000 FWHM. Over 280 glycoside-like compounds were detected, of which 133 (including low-molecular weight phenols, flavonoids and monoterpenols) were tentatively identified in the form of pentose (6), deoxyhexose (17), hexose (73), hexose-pentose (16), hexose-deoxyhexose (7), dihexose (5) and hexose ester (9) derivatives. It was not possible to univocally define the corresponding chemical structure for the remaining 149 glycosides. Non-parametric statistical analysis showed it was possible to well characterise the glycosylated profile of all red and Traminer wines, while the identified glycosides were almost entirely lacking in Pinot Blanc, Riesling and Muller Thurgau wines. Also Tukey's Honestly Significant Difference test (p wines from each other according to their glycosylated profile. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Metabolite Profiling of Triterpene Glycosides of the Far Eastern Sea Cucumber Eupentacta fraudatrix and Their Distribution in Various Body Components Using LC-ESI QTOF-MS.

    Science.gov (United States)

    Popov, Roman S; Ivanchina, Natalia V; Silchenko, Alexandra S; Avilov, Sergey A; Kalinin, Vladimir I; Dolmatov, Igor Yu; Stonik, Valentin A; Dmitrenok, Pavel S

    2017-10-02

    The Far Eastern sea cucumber Eupentacta fraudatrix is an inhabitant of shallow waters of the south part of the Sea of Japan. This animal is an interesting and rich source of triterpene glycosides with unique chemical structures and various biological activities. The objective of this study was to investigate composition and distribution in various body components of triterpene glycosides of the sea cucumber E. fraudatrix . We applied LC-ESI MS (liquid chromatography-electrospray mass spectrometry) of whole body extract and extracts of various body components for metabolic profiling and structure elucidation of triterpene glycosides from the E. fraudatrix . Totally, 54 compounds, including 26 sulfated, 18 non-sulfated and 10 disulfated glycosides were detected and described. Triterpene glycosides from the body walls, gonads, aquapharyngeal bulbs, guts and respiratory trees were extracted separately and the distributions of the detected compounds in various body components were analyzed. Series of new glycosides with unusual structural features were described in E. fraudatrix , which allow clarifying the biosynthesis of these compounds. Comparison of the triterpene glycosides contents from the five different body components revealed that the profiles of triterpene glycosides were qualitatively similar, and only some quantitative variabilities for minor compounds were observed.

  10. Steviol Glycosides Modulate Glucose Transport in Different Cell Types

    Science.gov (United States)

    Rizzo, Benedetta; Zambonin, Laura; Leoncini, Emanuela; Vieceli Dalla Sega, Francesco; Prata, Cecilia; Fiorentini, Diana; Hrelia, Silvana

    2013-01-01

    Extracts from Stevia rebaudiana Bertoni, a plant native to Central and South America, have been used as a sweetener since ancient times. Currently, Stevia extracts are largely used as a noncaloric high-potency biosweetener alternative to sugar, due to the growing incidence of type 2 diabetes mellitus, obesity, and metabolic disorders worldwide. Despite the large number of studies on Stevia and steviol glycosides in vivo, little is reported concerning the cellular and molecular mechanisms underpinning the beneficial effects on human health. The effect of four commercial Stevia extracts on glucose transport activity was evaluated in HL-60 human leukaemia and in SH-SY5Y human neuroblastoma cells. The extracts were able to enhance glucose uptake in both cellular lines, as efficiently as insulin. Our data suggest that steviol glycosides could act by modulating GLUT translocation through the PI3K/Akt pathway since treatments with both insulin and Stevia extracts increased the phosphorylation of PI3K and Akt. Furthermore, Stevia extracts were able to revert the effect of the reduction of glucose uptake caused by methylglyoxal, an inhibitor of the insulin receptor/PI3K/Akt pathway. These results corroborate the hypothesis that Stevia extracts could mimic insulin effects modulating PI3K/Akt pathway. PMID:24327825

  11. A New Acylated Flavonol Glycoside from Chenopodium foliosum

    Directory of Open Access Journals (Sweden)

    Zlatina Kokanova-Nedialkova, , , , , and

    2014-07-01

    Full Text Available A new acylated flavonol glycoside, namely gomphrenol-3-O-( 5 '''-O-E-feruloyl-β-D-apiofuranosyl-(1→2[β-D-glucopyranosyl-(1→6]-β-D-glucopyranoside (1 was isolated from the aerial parts of Chenopodium foliosum Asch. The structure of 1 was determined by means of spectroscopic methods (1D and 2D NMR, UV, IR, and HRESIMS. Radical scavenging and antioxidant activities of 1 were established using DPPH and ABTS radicals, FRAP assay and inhibition of lipid peroxidation (LP in linoleic acid system by the ferric thiocyanate method. Compound 1 showed low activity (DPPH and ABTS or lack of activity (FRAP and LP. In combination with CCl 4, 1 reduced the damage caused by the hepatotoxic agent and preserved cell viability and GSH level, decreased LDH leakage and reduced lipid damage. Effects were concentration dependent, most visible at the highest concentration (100 µg/m L , and similar to those of silymarin .

  12. A new antibacterial benzophenone glycoside from Psidium guajava (Linn.) leaves.

    Science.gov (United States)

    Ukwueze, Stanley E; Osadebe, Patience O; Okoye, Festus B C

    2015-01-01

    Bioactivity-guided fractionation of methanol extract from the leaves of Psidium guajava L. (Myrtaceae) yielded a new benzophenone glycoside, Guajaphenone A (2) together with two known compounds, Garcimangosone D (1) and Guaijaverin (3). Their structures were elucidated by analysis of spectroscopic data including 1D and 2D NMR and electrospray ionisation mass spectrometry (ESI-MS). The isolated compounds were screened against standard strains of Gram-positive and Gram-negative bacteria using broth dilution assay method, and the MIC values determined and compared with reference antibiotic ceftriaxone. They were found to have significant antibacterial activities against Escherichia coli and Staphylococcus aureus with all of them showing better activities against S. aureus, but displaying weaker activities, in comparison to ceftriaxone. However, despite reduced effect of these compounds against the organisms, this work opens the perspective to use these molecules as 'leads' for the design of novel and selective drug candidates for some tropical infectious diseases.

  13. Perspectives for the industrial enzymatic production of glycosides.

    Science.gov (United States)

    de Roode, B Mattheus; Franssen, Maurice C R; van der Padt, Albert; Boom, Remko M

    2003-01-01

    Glycosides are of commercial interest for industry in general and specifically for the pharmaceutical and food industry. Currently chemical preparation of glycosides will not meet EC food regulations, and therefore chemical preparation of glycosides is not applicable in the food industry. Thus, enzyme-catalyzed reactions are a good alternative. However, until now the low yields obtained by enzymatic methods prevent the production of glycosides on a commercial scale. Therefore, high yields should be established by a combination of optimum reaction conditions and continuous removal of the product. Unfortunately, a bioreactor for the commercial scale production of glycosides is not available. The aim of this article is to discuss the literature with respect to enzymatic production of glycosides and the design of an industrially viable bioreactor system.

  14. Induction of apoptosis in colon cancer cells treated with isorhamnetin glycosides from Opuntia ficus-indica pads.

    Science.gov (United States)

    Antunes-Ricardo, Marilena; Moreno-García, Beatriz E; Gutiérrez-Uribe, Janet A; Aráiz-Hernández, Diana; Alvarez, Mario M; Serna-Saldivar, Sergio O

    2014-12-01

    (OFI) contains health-promoting compounds like flavonoids, being the isorhamnetin glycosides the most abundant. We evaluated the effect of OFI extracts with different isorhamnetin glycosides against two different human colon cancer cells (HT-29 and Caco2). The extracts were obtained by alkaline hydrolysis with NaOH at 40 °C during 15, 30 or 60 min. Tri and diglycosides were the most abundant isorhamnetin glycosides, therefore these compounds were isolated to compare their cytotoxic effect with the obtained from the extracts. The OFI extracts and purified isorhamnetin glycosides were more cytotoxic against HT-29 cells than Caco2 cells. OFI-30 exhibited the lowest IC50 value against HT-29 (4.9 ± 0.5 μg/mL) and against Caco2 (8.2 ± 0.3 μg/mL). Isorhamnetin diglycosides IG5 and IG6 were more cytotoxic than pure isorhamnetin aglycone or triglycosides when they were tested in HT-29 cells. Bioluminescent analysis revealed increased activity of caspase 3/7 in OFI extracts-treated cells, particularly for the extract with the highest concentration of isorhamnetin triglycosides. Flow cytometry analysis confirmed that OFI extract and isorhamnetin glycosides induced a higher percentage of apoptosis in HT-29 than in Caco2, while isorhamnetin was more apoptotic in Caco2. This research demonstrated that glycosilation affected antiproliferative effect of pure isorhamnetin glycosides or when they are mixed with other phytochemicals in an extract obtained from OFI.

  15. Synthesis of Capsaicin Glycosides and 8-Nordihydrocapsaicin Glycosides as Potential Weight-Loss Formulations

    Directory of Open Access Journals (Sweden)

    Hisashi Katsuragi

    2010-03-01

    Full Text Available The enzymatic synthesis of capsaicin glycosides and 8-nordihydrocapsaicin glycosides was investigated using almond β-glucosidase and cyclodextrin glucanotransferase (CGTase. Capsaicin and 8-nordihydrocapsaicin were converted into their β-glucoside and β-maltooligosaccharide (amylose conjugate, i.e. β-maltoside and β-maltotrioside, by sequencial glycosylation with almond β-glucosidase and CGTase. The β-glucoside and β-maltoside of capsaicin and β-glucoside of 8-nordihydrocapsaicin showed inhibitory effects on high-fat-diet-induced elevations in body weight of mice.

  16. Synthesis of Capsaicin Glycosides and 8-Nordihydrocapsaicin Glycosides as Potential Weight-Loss Formulations

    Directory of Open Access Journals (Sweden)

    Hisashi Katsuragi

    2010-01-01

    Full Text Available The enzymatic synthesis of capsaicin glycosides and 8-nordihydrocapsaicin glycosides was investigated using almond β-glucosidase and cyclodextrin glucanotransferase (CGTase. Capsaicin and 8-nordihydrocapsaicin were converted into their β-glucoside and β-maltooligosaccharide (amylose conjugate, i.e. β-maltoside and β-maltotrioside, by sequencial glycosylation with almond β-glucosidase and CGTase. The β-glucoside and β-maltoside of capsaicin and β-glucoside of 8-nordihydrocapsaicin showed inhibitory effects on high-fat-diet-induced elevations in body weight of mice.

  17. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase.

    Science.gov (United States)

    Oguro, Ami; Imaoka, Susumu

    2012-03-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3-7 μM; Vmax, 150-193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism.

  18. Crystal structure of bile salt hydrolase from Lactobacillus salivarius.

    Science.gov (United States)

    Xu, Fuzhou; Guo, Fangfang; Hu, Xiao Jian; Lin, Jun

    2016-05-01

    Bile salt hydrolase (BSH) is a gut-bacterial enzyme that negatively influences host fat digestion and energy harvesting. The BSH enzyme activity functions as a gateway reaction in the small intestine by the deconjugation of glycine-conjugated or taurine-conjugated bile acids. Extensive gut-microbiota studies have suggested that BSH is a key mechanistic microbiome target for the development of novel non-antibiotic food additives to improve animal feed production and for the design of new measures to control obesity in humans. However, research on BSH is still in its infancy, particularly in terms of the structural basis of BSH function, which has hampered the development of BSH-based strategies for improving human and animal health. As an initial step towards the structure-function analysis of BSH, C-terminally His-tagged BSH from Lactobacillus salivarius NRRL B-30514 was crystallized in this study. The 1.90 Å resolution crystal structure of L. salivarius BSH was determined by molecular replacement using the structure of Clostridium perfringens BSH as a starting model. It revealed this BSH to be a member of the N-terminal nucleophile hydrolase superfamily. Crystals of apo BSH belonged to space group P21212, with unit-cell parameters a = 90.79, b = 87.35, c = 86.76 Å (PDB entry 5hke). Two BSH molecules packed perfectly as a dimer in one asymmetric unit. Comparative structural analysis of L. salivarius BSH also identified potential residues that contribute to catalysis and substrate specificity.

  19. New acylated flavone and cyanogenic glycosides from Linum grandiflorum

    DEFF Research Database (Denmark)

    Mohammed, Magdy M. D.; Christensen, Lars Porskjær; Ibrahim, Nabaweya A.

    2009-01-01

    The first investigation of Linum grandiflorum resulted in the isolation of one new acylated flavone O-diglycoside known as luteolin 7-O-a-D-(6000-E-feruloyl)glucopyranosyl (1!2)--D-glucopyranoside, and one new cyanogenic glycoside known as 2-[(30-isopropoxy-O--D-glucopyranosyl)oxy]-2......-methylbutanenitrile, together with four known flavonoid glycosides, three known cyanogenic glycosides and one alkyl glycoside. The new compounds were structurally elucidated via the extensive 1D, 2D NMR and DIFNOE together with ESI-TOFCID-MS/MS and HR-MALDI/MS....

  20. Hepatic cholesterol ester hydrolase in human liver disease.

    Science.gov (United States)

    Simon, J B; Poon, R W

    1978-09-01

    Human liver contains an acid cholesterol ester hydrolase (CEH) of presumed lysosomal origin, but its significance is unknown. We developed a modified CEH radioassay suitable for needle biopsy specimens and measured hepatic activity of this enzyme in 69 patients undergoing percutaneous liver biopsy. Histologically normal livers hydrolyzed 5.80 +/- 0.78 SEM mumoles of cholesterol ester per hr per g of liver protein (n, 10). Values were similar in alcoholic liver disease (n, 17), obstructive jaundice (n, 9), and miscellaneous hepatic disorders (n, 21). In contrast, mean hepatic CEH activity was more than 3-fold elevated in 12 patients with acute hepatitis, 21.05 +/- 2.45 SEM mumoles per hr per g of protein (P less than 0.01). In 2 patients studied serially, CEH returned to normal as hepatitis resolved. CEH activity in all patients paralleled SGOT levels (r, 0.84; P less than 0.01). There was no correlation with serum levels of free or esterified cholesterol nor with serum activity of lecithin-cholesterol acyltransferase, the enzyme responsible for cholesterol esterification in plasma. These studies confirm the presence of CEH activity in human liver and show markedly increased activity in acute hepatitis. The pathogenesis and clinical significance of altered hepatic CEH activity in liver disease require further study.

  1. Fractionation and Characterization of Tannin Acyl Hydrolase from Aspergillus niger

    Directory of Open Access Journals (Sweden)

    YUNITA ARIAN SANI ANWAR

    2009-09-01

    Full Text Available We previously produced tannin acyl hydrolase (tannase from Aspergillus niger isolated from cacao pod. In the present study the enzyme was subjected to fractionation by ammonium sulphate followed by dialysis process. The saturation level of ammonium sulphate used was 30-80% where the best enzyme activity was obtained at the saturation level of 60%. Compared to that of crude enzyme, specific activity of tannase after dialysis was four folds. Characterization results showed that optimum activity was at 35-50 oC and pH 6. Tannase was activated by K+ and Na+ at concentration of 0.01 and 0.05 M respectively. Mg2+ was found activate tannase only at 0.01 M. Addition of metal ions like Zn2+, Cu2+, Ca2+, Mn2+ and Fe2+ inhibited the enzyme activity. Kinetics analysis of various substrates tested showed that the Km value of tannic acid and gallotannin was 0.401 and 6.611 mM respectively. Vmax value of tannic acid was 10.804 U/ml and of gallotannin was 12.406 U/ml. Based on Michaelis-Menten constant (Km, the tannase obtained in the present study was more active in hydrolysing depside bonds rather than ester bonds.

  2. Fractionation and Characterization of Tannin Acyl Hydrolase from Aspergillus niger

    Directory of Open Access Journals (Sweden)

    YUNITA ARIAN SANI ANWAR

    2009-09-01

    Full Text Available We previously produced tannin acyl hydrolase (tannase from Aspergillus niger isolated from cacao pod. In the present study the enzyme was subjected to fractionation by ammonium sulphate followed by dialysis process. The saturation level of ammonium sulphate used was 30–80% where the best enzyme activity was obtained at the saturation level of 60%. Compared to that of crude enzyme, specific activity of tannase after dialysis was four folds. Characterization results showed that optimum activity was at 35–50 °C and pH 6. Tannase was activated by K+ and Na+ at concentration of 0.01 and 0.05 M respectively. Mg2+ was found activate tannase only at 0.01 M. Addition of metal ions like Zn2+, Cu2+, Ca2+, Mn2+ and Fe2+ inhibited the enzyme activity. Kinetics analysis of various substrates tested showed that the Km value of tannic acid and gallotannin was 0.401 and 6.611 mM respectively. Vmax value of tannic acid was 10.804 U/ml and of gallotannin was 12.406 U/ml. Based on Michaelis-Menten constant (Km, the tannase obtained in the present study was more active in hydrolysing depside bonds rather than ester bonds.

  3. Hepatoprotective glycosides from the rhizomes of Imperata cylindrical.

    Science.gov (United States)

    Ma, Jie; Sun, Hua; Liu, Hui; Shi, Gao-Na; Zang, Ying-Da; Li, Chuang-Jun; Yang, Jing-Zhi; Chen, Fang-You; Huang, Ji-Wu; Zhang, Dan; Zhang, Dong-Ming

    2018-05-01

    Three new C-methylated phenylpropanoid glycosides (1, 2), a new 8-4'-oxyneolignan (3), together with two known analogs (4, 5), were isolated from the rhizomes of Imperata cylindrical Beauv. var. major (Nees) C. E. Hubb. Their structures were determined by spectroscopic and chemical methods. Compounds 1, 2, and 5 (10 μM) exhibited pronounced hepatoprotective activity against N-acetyl-p-aminophenol (APAP)-induced HepG2 cell damage in vitro assays. Furthermore, their antioxidant activities against Fe 2+ -cysteine-induced rat liver microsomal lipid peroxidation and the effects on the secretion of TNF-α in murine peritoneal macrophages (RAW264.7) induced by lipopolysaccharides were evaluated.

  4. Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library.

    Science.gov (United States)

    Macdonald, Spencer S; Patel, Ankoor; Larmour, Veronica L C; Morgan-Lang, Connor; Hallam, Steven J; Mark, Brian L; Withers, Stephen G

    2018-03-02

    Glycoside phosphorylases have considerable potential as catalysts for the assembly of useful glycans for products ranging from functional foods and prebiotics to novel materials. However, the substrate diversity of currently identified phosphorylases is relatively small, limiting their practical applications. To address this limitation, we developed a high-throughput screening approach using the activated substrate 2,4-dinitrophenyl β-d-glucoside (DNPGlc) and inorganic phosphate for identifying glycoside phosphorylase activity and used it to screen a large insert metagenomic library. The initial screen, based on release of 2,4-dinitrophenyl from DNPGlc in the presence of phosphate, identified the gene bglP, encoding a retaining β-glycoside phosphorylase from the CAZy GH3 family. Kinetic and mechanistic analysis of the gene product, BglP, confirmed a double displacement ping-pong mechanism involving a covalent glycosyl-enzyme intermediate. X-ray crystallographic analysis provided insights into the phosphate-binding mode and identified a key glutamine residue in the active site important for substrate recognition. Substituting this glutamine for a serine swapped the substrate specificity from glucoside to N -acetylglucosaminide. In summary, we present a high-throughput screening approach for identifying β-glycoside phosphorylases, which was robust, simple to implement, and useful in identifying active clones within a metagenomics library. Implementation of this screen enabled discovery of a new glycoside phosphorylase class and has paved the way to devising simple ways in which enzyme specificity can be encoded and swapped, which has implications for biotechnological applications. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Functional analysis of the Escherichia coli genome for members of the alpha/beta hydrolase family.

    Science.gov (United States)

    Zhang, L; Godzik, A; Skolnick, J; Fetrow, J S

    1998-01-01

    Database-searching methods based on sequence similarity have become the most commonly used tools for characterizing newly sequenced proteins. Due to the often underestimated functional diversity in protein families and superfamilies, however, it is difficult to make the characterization specific and accurate. In this work, we have extended a method for active-site identification from predicted protein structures. The structural conservation and variation of the active sites of the alpha/beta hydrolases with known structures were studied. The similarities were incorporated into a three-dimensional motif that specifies essential requirements for the enzymatic functions. A threading algorithm was used to align 651 Escherichia coli open reading frames (ORFs) to one of the members of the alpha/beta hydrolase fold family. These ORFs were then screened according to our three-dimensional motif and with an extra requirement that demands conservation of the key active-site residues among the proteins that bear significant sequence similarity to the ORFs. 17 ORFs from E. coli were predicted to have hydrolase activity and their putative active-site residues were identified. Most were in agreement with the experiments and results of other database-searching methods. The study further suggests that YHET_ECOLI, a hypothetical protein classified as a member of the UPF0017 family (an uncharacterized protein family), bears all the hallmarks of the alpha/beta hydrolase family. The novel feature of our method is that it uses three-dimensional structural information for function prediction. The results demonstrate the importance and necessity of such a method to fill the gap between sequence alignment and function prediction; furthermore, the method provides a way to verify the structure predictions, which enables an expansion of the applicable scope of the threading algorithms.

  6. Aspergillus nidulans α-galactosidase of glycoside hydrolase family 36 catalyses the formation of α-galacto-oligosaccharides by transglycosylation

    DEFF Research Database (Denmark)

    Nakai, Hiroyuki; Baumann, Martin; Petersen, B. O.

    2010-01-01

    xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred α-galactosyl to 6-OH of the terminal residue in the α-linked melibiose, maltose, trehalose, sucrose and turanose in 6–46% yield and the β-linked lactose, lactulose and cellobiose in 28...

  7. A new group of exo-acting family 28 glycoside hydrolases of Aspergillus niger that are involved in pectin degradation

    NARCIS (Netherlands)

    Martens-Uzunova, E.S.; Zandleven, J.S.; Benen, J.A.E.; Awad, H.; Kools, H.J.; Beldman, G.; Voragen, A.G.J.; Berg, van den J.A.; Schaap, P.J.

    2006-01-01

    The fungus Aspergillus niger is an industrial producer of pectin degrading enzymes. The recent solving of the genomic sequence of A. niger allowed an inventory of the entire genome of the fungus for potential carbohydrate degrading enzymes. By applying bioinformatics tools 12 new genes putatively

  8. Distinct substrate specificities of three glycoside hydrolase family 42 β-galactosidases from Bifidobacterium longum subsp. infantis ATCC 15697

    DEFF Research Database (Denmark)

    Viborg, Alexander Holm; Katayama, Takane; Abou Hachem, Maher

    2014-01-01

    resembling various milk and plant galactooligosaccharides distinguishes the three GH42 members, Bga42A, Bga42B and Bga42C, encoded by the probiotic B. longum subsp. infantis ATCC 15697 and revealed the glycosyl residue at subsite +1 and its linkage to the terminal Gal at subsite −1 to be key specificity...

  9. Isolation of high-quality total RNA from rumen anaerobic bacteria and fungi, and subsequent detection of glycoside hydrolases.

    Science.gov (United States)

    Wang, Pan; Qi, Meng; Barboza, Perry; Leigh, Mary Beth; Ungerfeld, Emilio; Selinger, L Brent; McAllister, Tim A; Forster, Robert J

    2011-07-01

    The rumen is one of the most powerful fibrolytic fermentation systems known. Gene expression analyses, such as reverse transcription PCR (RT-PCR), microarrays, and metatranscriptomics, are techniques that could significantly expand our understanding of this ecosystem. The ability to isolate and stabilize representative RNA samples is critical to obtaining reliable results with these procedures. In this study, we successfully isolated high-quality total RNA from the solid phase of ruminal contents by using an improved RNA extraction method. This method is based on liquid nitrogen grinding of whole ruminal solids without microbial detachment and acid guanidinium - phenol - chloroform extraction combined with column purification. Yields of total RNA were as high as 150 µg per g of fresh ruminal content. The typical large subunit/small subunit rRNA ratio ranged from 1.8 to 2.0 with an RNA integrity number (Agilent Technologies) greater than 8.5. By eliminating the detachment step, the resulting RNA was more representative of the complete ecosystem. Our improved method removed a major barrier limiting analysis of rumen microbial function from a gene expression perspective. The polyA-tailed eukaryotic mRNAs obtained have successfully been applied to next-generation sequencing, and metatranscriptomic analysis of the solid fraction of rumen contents revealed abundant sequences related to rumen fungi.

  10. Structure of the Cyanuric Acid Hydrolase TrzD Reveals Product Exit Channel.

    Science.gov (United States)

    Bera, Asim K; Aukema, Kelly G; Elias, Mikael; Wackett, Lawrence P

    2017-03-27

    Cyanuric acid hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric acid, a stabilizer for the chlorine. Degradation of excess cyanuric acid is necessary to maintain chlorine disinfection in the waters. Cyanuric acid hydrolase opens the cyanuric acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric acid hydrolase from Acidovorax citrulli. The crystal structure at 2.19 Å resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas the two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric acid ring, and the mobile lysine guides products through the exit channel.

  11. Endogenous Turnover of Cyanogenic Glycosides in Plants

    DEFF Research Database (Denmark)

    Picmanova, Martina

    , there is strong evidence that CNglcs serve a no less significant purpose as a transport and storage form of reduced nitrogen which may be remobilized and recycled to balance the needs of primary metabolism during certain developmental events. Reduced nitrogen from CNglcs may be recovered either via HCN refixation...... revealed the formation of glycosides of amides, carboxylic acids and "anitriles", including their di- and triglycosides, evidently derived from CNglcs. Based on results common to the three phylogenetically unrelated plant species, a recycling endogenous turnover pathway for CNglcs was suggested in which...

  12. Flavononol Glycosides of Reseda arabica (Resedaceae

    Directory of Open Access Journals (Sweden)

    Djemaa Berrehal

    2012-07-01

    Full Text Available Five flavonol glycosides, kaempferol 3,7-di-O- α -L-rhamnopyranoside (1 , isorhamnetin 3,7-di-O- α -L-rhamnopyranoside (2 , kaempferol 3-O- β -D-glucopyranoside-7-O- α -L-rhamnopyranoside (3 , isorhamnetin 3-O- β -D-glucopyranoside-7-O- α -L-rhamnopyranoside (4, Kaempferol 3-O- β -xylopyranosyl-(1'''→2''-O- α -L-rhamnopyranoside-7-O- α -L-rhamnopyranoside (5, have been isolated from the aerial parts of Reseda arabica. Their structures were established on the basis of physical and spectroscopic analysis, and by comparison with the literature data.

  13. Three New Flavone Glycosides from Drymaria diandra Bl.

    Institute of Scientific and Technical Information of China (English)

    Zhong-Tao DING; Xue-Qiong YANG; Qiu-E CAO; Fei LI

    2005-01-01

    In order to find new structural and biologically active compounds, the constituents from the whole plant of Drymaria diandra B1. (Caryophyllaceae) were investigated and three new flavone glycosides,named drymariatins B (1), C (2), and D (3), were isolated by solvent partition, Si gel, sephadex LH-20, and Rp-18 column chromatography. Using spectroscopic methods, including two-dimensional nuclear magnetic resonance analysis, the structures of these compounds were elucidated as 6-C-(2-deoxy-β-D-fucopyranosyl)-5,7,4'-trihydroxyl-flavone, 6-C-(2-deoxy-β-D-fucopyranosyl)-7-O-(β-D-glucopyranosyl)-5,4'-dihydroxylflavone, and 6-C-(3-keto-β-digitoxopyranosyl)-7-O-(β-D-glucopyranosyl)-5,4'-dihydroxyl-flavone.

  14. Acetylated flavonoid glycosides potentiating NGF action from Scoparia dulcis.

    Science.gov (United States)

    Li, Yushan; Chen, Xigui; Satake, Masayuki; Oshima, Yasukatsu; Ohizumi, Yasushi

    2004-04-01

    Three new acetylated flavonoid glycosides, 5,6,4'-trihydroxyflavone 7-O-alpha-L-2,3-di-O-acetylrhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (1), apigenin 7-O-alpha-L-3-O-acetylrhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (2), and apigenin 7-O-alpha-L-2,3-di-O-acetylrhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (3), were isolated from Scoparia dulcis together with the known compound eugenyl beta-D-glucopyranoside (4). Their structures were elucidated by spectroscopic analyses. Compounds 2 and 3 showed an enhancing activity of nerve growth factor-mediated neurite outgrowth in PC12D cells.

  15. A new phenylethanoid glycoside from Orobanche cernua Loefling.

    Science.gov (United States)

    Qu, Zheng-yi; Zhang, Yu-wei; Zheng, Si-Wen; Yao, Chun-lin; Jin, Yin-ping; Zheng, Pei-he; Sun, Cheng-he; Wang, Ying-ping

    2016-01-01

    A novel phenylethanoid glycoside, 3'-O-methyl isocrenatoside (1), along with two known compounds, methyl caffeate (2) and protocatechuic aldehyde (3), were isolated from the fresh whole plant of Orobanche cernua Loefling. All the isolated compounds (1-3) were elucidated on the basis of spectroscopic analysis including IR, MS and NMR data. The cytotoxic activities of these compounds were evaluated. Results showed that 3'-O-methyl isocrenatoside (1) and methyl caffeate (2) exhibited significant cytotoxicity, with IC50 values of 71.89, 36.97 μg/mL and 32.32, 34.58 μg/mL against the B16F10 murine melanoma and Lewis lung carcinoma cell lines, respectively.

  16. Phytosteryl glycosides reduce cholesterol absorption: mechanisms in mice

    Science.gov (United States)

    Phytosteryl glycosides occur in natural foods but little is known about their metabolism and bioactivity. Purified acylated steryl glycosides (ASG) were compared with phytosteryl esters (PSE) in mice. Animals on a phytosterol-free diet received ASG or PSE by gavage in purified soybean oil along with...

  17. Acylated-oxypregnane glycosides from the roots of Asclepias syriaca.

    Science.gov (United States)

    Warashina, Tsutomu; Noro, Tadataka

    2009-02-01

    Twenty new pregnane glycosides were obtained from the roots of Asclepias syriaca L. (Asclepiadaceae). These glycosides were confirmed to contain ikemagenin, 12-O-nicotinoyllineolon, 5alpha,6-dihydroikemagenin, and 12-O-tigloylisolineolon, as their aglycones, using both spectroscopic and chemical methods.

  18. Steroidal glycosides from the roots of Asclepias curassavica.

    Science.gov (United States)

    Warashina, Tsutomu; Noro, Tadataka

    2008-03-01

    Twenty-six new acylated-oxypregnane glycosides were obtained along with three known cardenolide glycosides from the roots of Asclepias curassavica (Asclepiadaceae). The new compounds were confirmed to contain 12-O-benzoylsarcostin, 12-O-benzoyldeacylmetaplexigenin, kidjolanin, and 12-O-benzoyltayloron, and one new acylated-oxypregnane, 12-O-(E)-cinnamoyltayloron, as their aglycones, using both spectroscopic and chemical methods.

  19. Evaluating the Cancer Therapeutic Potential of Cardiac Glycosides

    Directory of Open Access Journals (Sweden)

    José Manuel Calderón-Montaño

    2014-01-01

    Full Text Available Cardiac glycosides, also known as cardiotonic steroids, are a group of natural products that share a steroid-like structure with an unsaturated lactone ring and the ability to induce cardiotonic effects mediated by a selective inhibition of the Na+/K+-ATPase. Cardiac glycosides have been used for many years in the treatment of cardiac congestion and some types of cardiac arrhythmias. Recent data suggest that cardiac glycosides may also be useful in the treatment of cancer. These compounds typically inhibit cancer cell proliferation at nanomolar concentrations, and recent high-throughput screenings of drug libraries have therefore identified cardiac glycosides as potent inhibitors of cancer cell growth. Cardiac glycosides can also block tumor growth in rodent models, which further supports the idea that they have potential for cancer therapy. Evidence also suggests, however, that cardiac glycosides may not inhibit cancer cell proliferation selectively and the potent inhibition of tumor growth induced by cardiac glycosides in mice xenografted with human cancer cells is probably an experimental artifact caused by their ability to selectively kill human cells versus rodent cells. This paper reviews such evidence and discusses experimental approaches that could be used to reveal the cancer therapeutic potential of cardiac glycosides in preclinical studies.

  20. Chemical and enzymatic hydrolysis of anthraquinone glycosides from Madder roots

    NARCIS (Netherlands)

    Derksen, G.C.H.; Naayer, M.; Beek, T.A. van; Capelle, A.; Haaksman, I.K.; Doren, H.A. van; Groot, Æ. de

    2003-01-01

    For the production of a commercially useful dye extract from madder, the glycoside ruberythric acid has to be hydrolysed to the aglycone alizarin which is the main dye component. An intrinsic problem is the simultaneous hydrolysis of the glycoside lucidin pritneveroside to the unwanted mutagenic

  1. Diarylheptanoid Glycosides of Morella salicifolia Bark

    Directory of Open Access Journals (Sweden)

    Edna Makule

    2017-12-01

    Full Text Available A methanolic extract of Morella salicifolia bark was fractionated by various chromatographic techniques yielding six previously unknown cyclic diarylheptanoids, namely, 7-hydroxymyricanol 5-O-β-d-glucopyranoside (1, juglanin B 3-O-β-d-glucopyranoside (2, 16-hydroxyjuglanin B 17-O-β-d-glucopyranoside (3, myricanone 5-O-β-d-gluco-pranosyl-(1→6-β-d-glucopyranoside (4, neomyricanone 5-O-β-d-glucopranosyl-(1→6-β-d-glucopyranoside (5, and myricanone 17-O-α-l-arabino-furanosyl-(1→6-β-d-glucopyranoside (6, respectively, together with 10 known cyclic diarylheptanoids. The structural diversity of the diarylheptanoid pattern in M. salicifolia resulted from varying glycosidation at C-3, C-5, and C-17 as well as from substitution at C-11 with hydroxy, carbonyl or sulfate groups, respectively. Structure elucidation of the isolated compounds was achieved on the basis of one- and two-dimensional nuclear magnetic resonance (NMR as well as high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS analyses. The absolute configuration of the glycosides was confirmed after hydrolysis and synthesis of O-(S-methyl butyrated (SMB sugar derivatives by comparison of their 1H-NMR data with those of reference sugars. Additionally, absolute configuration of diarylheptanoid aglycones at C-11 was determined by electronic circular dichroism (ECD spectra simulation and comparison with experimental CD spectra after hydrolysis.

  2. InterProScan Result: DC552514 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available 016286 Glycoside hydrolase, family 29, bacteria/metazoa/fungi Molecular Function: alpha-L-fucosidase activity (GO:0004560)|Biological Process: fucose metabolic process (GO:0006004) ...

  3. InterProScan Result: DY231230 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available 6286 Glycoside hydrolase, family 29, bacteria/metazoa/fungi Molecular Function: alpha-L-fucosidase activity (GO:0004560)|Biological Process: fucose metabolic process (GO:0006004) ...

  4. Antinociceptive and anti-inflammatory kaempferol glycosides from Sedum dendroideum.

    Science.gov (United States)

    De Melo, Giany O; Malvar, David do C; Vanderlinde, Frederico A; Rocha, Fabio F; Pires, Priscila Andrade; Costa, Elson A; de Matos, Lécia G; Kaiser, Carlos R; Costa, Sônia S

    2009-07-15

    To identify the compounds responsible for the antinociceptive and anti-inflammatory effects previously described for Sedum dendroideum, through bioassay-guided fractionation procedures. Antinociceptive activity was evaluated through mouse acetic acid-induced writhing model. The anti-inflammatory activity was assessed through croton oil-induced mouse ear oedema and carrageenan-induced peritonitis. The Sedum dendroideum juice afforded seven known flavonoids identified with basis on NMR data. The oral administration of the major kaempferol glycosides kaempferitrin [1] (17.29 micromol/kg), kaempferol 3-O-beta-glucopyranoside-7-O-alpha-rhamnopyranoside [2] (16.82 micromol/kg), kaempferol 3-O-neohesperidoside-7-O-alpha-rhamnopyranoside [3] (13.50 micromol/kg) or alpha-rhamnoisorobin [5] (23.13 micromol/kg) inhibited by 47.3%, 25.7%, 60.2% and 58.0%, respectively, the acetic acid-induced nociception (indomethacin: 27.95 micromol/kg, p.o.; 68.9%). Flavonoids 1, 2, 3 or 5, at the same doses, reduced by 39.5%, 46.5%, 35.6% and 33.3%, respectively, the croton oil-induced oedema (dexamethasone: 5.09 micromol/kg, s.c.; 83.7%) and impaired leukocyte migration by 42.9%, 46.3%, 50.4% and 49.6%, respectively (dexamethasone: 5.09 micromol/kg, s.c.; 66.1%). Our findings show that the major kaempferol glycosides may account for the renowned medicinal use of Sedum dendroideum against pain and inflammatory troubles.

  5. Novel microbial epoxide hydrolases for biohydrolysis of glycidyl derivatives

    Czech Academy of Sciences Publication Activity Database

    Kotík, Michael; Břicháč, Jiří; Kyslík, Pavel

    2005-01-01

    Roč. 120, - (2005), s. 364-375 ISSN 0168-1656 Institutional research plan: CEZ:AV0Z5020903 Keywords : screening * epoxide hydrolase * biotransformation Subject RIV: EE - Microbiology, Virology Impact factor: 2.687, year: 2005

  6. Effect of 1,25-dihydroxycholecalciferol and 1,25-dihydroxycholecalciferol glycoside on 2,3-diphosphoglycerate levels of the rat erythrocyte.

    Science.gov (United States)

    Skliar, M I; Fernandez, M C; Faienza, H; Orsatti, M B; Puche, R C; Boland, R L; Skliar, M I

    1980-12-01

    The erythrocytes of rats treated with 1, 25-dihydroxycholecalciferol or 1, 25-dihydroxycholecalciferol glycoside showed decreased levels of 2, 3-diphosphoglycerate. The same result has been obtained in vitro, indicating a direct effect of the sterol on the red cell. The glycoside is less active than the free sterol in vivo and more active in vitro. The decreased levels of diphosphoglycerate induced tissue hypoxia as shown by a higher plasma lactate/pyruvate ratio and a three fold increase in plasma erythropoietin concentration.

  7. Mining anaerobic digester consortia metagenomes for secreted carbohydrate active enzymes

    DEFF Research Database (Denmark)

    Wilkens, Casper; Busk, Peter Kamp; Pilgaard, Bo

    thermophilic and mesophilic ADs a wide variety of carbohydrate active enzyme functions were discovered in the metagenomic sequencing of the microbial consortia. The most dominating type of glycoside hydrolases were β-glucosidases (up to 27%), α-amylases (up to 10%), α-glucosidases (up to 8%), α......, and food wastes (Alvarado et al., 2014). The processes and the roles of the microorganisms that are involved in biomass conversion and methane production in ADs are still not fully understood. We are investigating thermophilic and mesophilic ADs that use wastewater surplus sludge for methane production...... was done with the Peptide Pattern Recognition (PPR) program (Busk and Lange, 2013), which is a novel non-alignment based approach that can predict function of e.g. CAZymes. PPR identifies a set of short conserved sequences, which can be used as a finger print when mining genomes for novel enzymes. In both...

  8. Increased amylosucrase activity and specificity, and identification of regions important for activity, specificity and stability through molecular evolution

    DEFF Research Database (Denmark)

    van der Veen, Bart A; Skov, Lars K; Potocki-Véronèse, Gabrielle

    2006-01-01

    Amylosucrase is a transglycosidase which belongs to family 13 of the glycoside hydrolases and transglycosidases, and catalyses the formation of amylose from sucrose. Its potential use as an industrial tool for the synthesis or modification of polysaccharides is hampered by its low catalytic...

  9. Use of full recovery hydrolasing equipment for facility decommissioning - 16325

    International Nuclear Information System (INIS)

    Martin, Scott A.; Adams, Scott R.

    2009-01-01

    The removal of surface contamination is a major challenge for nearly all nuclear facilities undergoing, or awaiting, decommissioning. Conventional means of surface decontamination can expose workers to unnecessary hazards, and are often not fit-for-purpose due to size constraints or weight restrictions. Additionally, conventional methods are not always easily deployed remotely due to their complexity or required services. The use of ultra high pressure water for surface decontamination, known as hydrolasing, is recognized as a technology which can be used in various applications requiring surface removal. Hydrolasing is an advantageous technology for many reasons including its versatility, overall simplicity and relative ease of remote deployment. For the nuclear industry, one of the largest challenges with regards to the use of hydrolasing is the requirement for the full recovery of the injected water and removed solids. For nonnuclear applications, there is often no requirement for recovery of the liquid and solid waste, which has led to few system designs which will recover the waste in full. S.A. Robotics' experience with the deployment of ultra high pressure water systems for nuclear applications has shown that full recovery of injected water and removed solids is achievable in both underwater and in-air applications. Innovative equipment and system design have allowed S.A. Robotics' hydrolasing systems to achieve near 100% solid and liquid recovery during concrete hydrolasing. This technology has been deployed for Fluor Hanford at Hanford's K-Basins, as well as for UKAEA as part of the Windscale Piles decommissioning project. The purpose of this paper is to provide a short description of the hydrolasing process and the associated waste issues, describe the unique design features of S.A. Robotics' hydrolasing systems which combat these issues, and provide an overview of two of the hydrolasing projects that S.A. Robotics has completed. (authors)

  10. Variation in the activities of late stage filaggrin processing enzymes, calpain-1 and bleomycin hydrolase, together with pyrrolidone carboxylic acid levels, corneocyte phenotypes and plasmin activities in non-sun-exposed and sun-exposed facial stratum corneum of different ethnicities.

    Science.gov (United States)

    Raj, N; Voegeli, R; Rawlings, A V; Summers, B; Munday, M R; Lane, M E

    2016-12-01

    Knowledge of the ethnic differences and effects of photodamage on the relative amounts of natural moisturizing factor (NMF) together with filaggrin processing enzymes in facial stratum corneum is limited. Our aim was to characterize the activities of calpain-1 (C-1), bleomycin hydrolase (BH) and the levels of pyrrolidone carboxylic acid (PCA) as a marker for total NMF levels and to relate them to plasmin activities and corneocyte maturation. Enzyme activities, PCA levels and corneocyte maturation were determined from facial tape strippings of photoexposed cheek and photoprotected post-auricular areas (PA) of healthy Caucasian (C), Black African (BA) and albino African (AA) female subjects living in South Africa. PCA concentration levels were of the order AA > BA > C subjects, and the highest activities of BH were present in the AA subjects. BH activities were greater on the photoexposed sites for the BA and C subjects, but they were only numerically elevated in the AA subjects. Photoprotected sites had an increase in C-1 activity in pigmented groups (C and BA), whereas in the AA subjects, the opposite was measured. Plasmin activities were greater on the cheek compared with the PA site for the AA and C subjects, but the activity was low in the BA subjects. In both test sites, the AA, but not the BA and C subjects, had smaller, parakeratotic and less mature corneocytes. Variation in PCA levels has been found for different ethnic groups in this study (AA > BA > C subjects). The values in the AA subjects are surprising as one might expect that the lack of pigmentation, and thereby increased photodamage, might lead to lower levels. Increased BH, but not C-1 activity, was observed in the AA subjects indicating that BH is associated with PCA production to a greater extent. Surprisingly, corneocyte maturation is still impaired with elevated PCA levels in AA subjects. The higher levels of plasmin and BH activities on the cheeks, especially for AA and C subjects, suggest

  11. Construction and characterisation of a genetically engineered Escherichia coli strain for the epoxide hydrolase-catalysed kinetic resolution of epoxides

    NARCIS (Netherlands)

    Visser, H.; Oliveira Vil Filho, de M.; Liese, A.; Weijers, C.A.G.M.; Verdoes, J.C.

    2003-01-01

    The Rhodotorula glutinis epoxide hydrolase, Eph1, was produced in the heterologous host Escherichia coli BL21(DE3) in order to develop a highly effective epoxide hydrolysis system. A 138-fold increase in Eph1 activity was found in cell extracts of the recombinant E. coli when compared to cell

  12. Characterization of an epoxide hydrolase from the Florida red tide dinoflagellate, Karenia brevis.

    Science.gov (United States)

    Sun, Pengfei; Leeson, Cristian; Zhi, Xiaoduo; Leng, Fenfei; Pierce, Richard H; Henry, Michael S; Rein, Kathleen S

    2016-02-01

    Epoxide hydrolases (EH, EC 3.3.2.3) have been proposed to be key enzymes in the biosynthesis of polyether (PE) ladder compounds such as the brevetoxins which are produced by the dinoflagellate Karenia brevis. These enzymes have the potential to catalyze kinetically disfavored endo-tet cyclization reactions. Data mining of K. brevis transcriptome libraries revealed two classes of epoxide hydrolases: microsomal and leukotriene A4 (LTA4) hydrolases. A microsomal EH was cloned and expressed for characterization. The enzyme is a monomeric protein with molecular weight 44kDa. Kinetic parameters were evaluated using a variety of epoxide substrates to assess substrate selectivity and enantioselectivity, as well as its potential to catalyze the critical endo-tet cyclization of epoxy alcohols. Monitoring of EH activity in high and low toxin producing cultures of K. brevis over a three week period showed consistently higher activity in the high toxin producing culture implicating the involvement of one or more EH in brevetoxin biosynthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Structure of HsaD, a steroid-degrading hydrolase, from Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Lack, Nathan; Lowe, Edward D.; Liu, Jie; Eltis, Lindsay D.; Noble, Martin E. M.; Sim, Edith; Westwood, Isaac M.

    2007-01-01

    The structure of HsaD, a carbon–carbon bond serine hydrolase involved in steroid catabolism that is critical for the survival of M. tuberculosis inside human macrophages, has been solved by X-ray crystallography. Data were collected at the Diamond Light Source in Oxfordshire, England: this paper describes one of the first structures determined at the new synchrotron. Tuberculosis is a major cause of death worldwide. Understanding of the pathogenicity of Mycobacterium tuberculosis has been advanced by gene analysis and has led to the identification of genes that are important for intracellular survival in macrophages. One of these genes encodes HsaD, a meta-cleavage product (MCP) hydrolase that catalyzes the hydrolytic cleavage of a carbon–carbon bond in cholesterol metabolism. This paper describes the production of HsaD as a recombinant protein and, following crystallization, the determination of its three-dimensional structure to 2.35 Å resolution by X-ray crystallography at the Diamond Light Source in Oxfordshire, England. To the authors’ knowledge, this study constitutes the first report of a structure determined at the new synchrotron facility. The volume of the active-site cleft of the HsaD enzyme is more than double the corresponding active-site volumes of related MCP hydrolases involved in the catabolism of aromatic compounds, consistent with the specificity of HsaD for steroids such as cholesterol. Knowledge of the structure of the enzyme facilitates the design of inhibitors

  14. ClbS Is a Cyclopropane Hydrolase That Confers Colibactin Resistance.

    Science.gov (United States)

    Tripathi, Prabhanshu; Shine, Emilee E; Healy, Alan R; Kim, Chung Sub; Herzon, Seth B; Bruner, Steven D; Crawford, Jason M

    2017-12-13

    Certain commensal Escherichia coli contain the clb biosynthetic gene cluster that codes for small molecule prodrugs known as precolibactins. Precolibactins are converted to colibactins by N-deacylation; the latter are postulated to be genotoxic and to contribute to colorectal cancer formation. Though advances toward elucidating (pre)colibactin biosynthesis have been made, the functions and mechanisms of several clb gene products remain poorly understood. Here we report the 2.1 Å X-ray structure and molecular function of ClbS, a gene product that confers resistance to colibactin toxicity in host bacteria and which has been shown to be important for bacterial viability. The structure harbors a potential colibactin binding site and shares similarity to known hydrolases. In vitro studies using a synthetic colibactin analog and ClbS or an active site residue mutant reveal cyclopropane hydrolase activity that converts the electrophilic cyclopropane of the colibactins into an innocuous hydrolysis product. As the cyclopropane has been shown to be essential for genotoxic effects in vitro, this ClbS-catalyzed ring-opening provides a means for the bacteria to circumvent self-induced genotoxicity. Our study provides a molecular-level view of the first reported cyclopropane hydrolase and support for a specific mechanistic role of this enzyme in colibactin resistance.

  15. Phenylethanoid Glycosides of Cistanche on menopausal syndrome model in mice

    Directory of Open Access Journals (Sweden)

    Shuo Tian

    2017-05-01

    Full Text Available Cistanche is the traditional and precious Chinese herbal, with two thousand years of use history in China. It has the effect on tonifying kidney, strong supplement to the liver and kidney, and replenishing essence and blood, known as the “desert ginseng”. Here, we explored the mechanism of Phenylethanoid Glycosides of Cistanche (PGC to the model mice of menopausal syndrome, as well as the therapeutic effect and characteristics of PGC to the menopausal syndrome. In this study, KM mice were reproduced by the complete resection of the ovaries on both sides of the back to establish the model mice of menopausal syndrome (MPS, and received distilled water or drugs, respectively. Model mice received distilled water. Mice received 200 mg/(kg day high doses of Phenylethanoid Glycosides of Cistanche (HPGC, and 100 mg/(kg day medium doses of Phenylethanoid Glycosides of Cistanche (MPGC, and 50 mg/(kg day low doses of Phenylethanoid Glycosides of Cistanche (LPGC. After 21 days, it could determine the number of independent activities and the number of standing, the latent period of first entering the dark room, and the electric number. It also calculated the viscera index of uterus, thymus, spleen, measured the levels of estradiol (E2, testosterone (T, luteinizing hormone (LH, and follicle-stimulating hormone (FSH in the serum. Furthermore, it observed the pathological changes of uterus, thymus, spleen and pituitary of mice. The results showed that behavioral indicators: Compared with the model group (MG, HPGC, MPGC, LPGC could increase the independent activities (P < 0.01; HPGC, MPGC could increase the number of standing, the latent period of first entering the dark room, and reduce the electric number (P < 0.01; LPGC could increase the number of standing (P < 0.05; Viscera index: Compared with MG, HPGC, MPGC could increase the viscera index of uterus, thymus, spleen (P < 0.01; LPGC could increase the viscera index of uterus (P < 0

  16. Effects of pregnane glycosides on food intake depend on stimulation of the melanocortin pathway and BDNF in an animal model.

    Science.gov (United States)

    Komarnytsky, Slavko; Esposito, Debora; Rathinasabapathy, Thirumurugan; Poulev, Alexander; Raskin, Ilya

    2013-02-27

    Pregnane glycosides appear to modulate food intake by possibly affecting the hypothalamic feeding circuits; however, the mechanisms of the appetite-regulating effect of pregnane glycosides remain obscure. Here, we show that pregnane glycoside-enriched extracts from swamp milkweed Asclepias incarnata at 25-100 mg/kg daily attenuated food intake (up to 47.1 ± 8.5% less than controls) and body weight gain in rats (10% for males and 9% for females, respectively) by activating melanocortin signaling and inhibiting gastric emptying. The major milkweed pregnane glycoside, ikemagenin, exerted its appetite-regulating effect by decreasing levels of agouti-related protein (0.6-fold) but not NPY satiety peptides. Ikemagenin treatment also increased secretion of brain-derived neurotropic factor (BDNF) downstream of melanocortin receptors in the hypothalamus (1.4-fold) and in the C6 rat glioma cell culture in vitro (up to 6-fold). These results support the multimodal effects of pregnane glycosides on feeding regulation, which depends on the activity of the melanocortin signaling pathway and BDNF.

  17. Recent development of phosphorylases possessing large potential for oligosaccharide synthesis

    DEFF Research Database (Denmark)

    Nakai, Hiroyuki; Kitaoka, Motomitsu; Svensson, Birte

    2013-01-01

    Phosphorylases are one group of carbohydrate active enzymes involved in the cleavage and formation of glycosidic linkages together with glycoside hydrolases and sugar nucleotide-dependent glycosyltransferases. Noticeably, the catalyzed phosphorolysis is reversible, making phosphorylases suitable...

  18. Inter-domain synergism is required for efficient feeding of cellulose chain into active site of cellobiohydrolase Cel7A

    DEFF Research Database (Denmark)

    Kont, Riin; Kari, Jeppe; Borch, Kim

    2016-01-01

    systems. TrCel7A consists of catalytic domain (CD) and a smaller carbohydrate binding module (CBM) connected through the glycosylated linker peptide. A tunnel shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two...... to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient......Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme...

  19. Preparative isolation and purification of five flavonoid glycosides and one benzophenone galloyl glycoside from Psidium guajava by high-speed counter-current chromatography (HSCCC).

    Science.gov (United States)

    Zhu, Yindi; Liu, Yue; Zhan, Ying; Liu, Lin; Xu, Yajuan; Xu, Tunhai; Liu, Tonghua

    2013-12-16

    Psidium guajava leaves have a diverse phytochemical composition including flavonoids, phenolics, meroterpenoids and triterpenes, responsible for the biological activities of the medicinal parts. In particular, flavonol glycosides show beneficial effects on type II diabetes mellitus. A simple and efficient HSCCC method has been developed for the preparative separation of five flavonoid glycosides and one diphenylmethane glycoside from P. guajava. A solvent system composed of n-hexane-ethyl acetate-methanol-water (0.7:4:0.8:4, v/v/v/v) was optimized for the separation. The upper phase was used as the stationary phase, and the lower phase was used as the mobile phase. Under the optimized conditions, hyperoside (15.3 mg), isoquercitrin (21.1 mg), reynoutrin (65.2 mg), quercetin-3-O-β-D-arabinopyranoside (71.7 mg), quercetin-3-O-α-L-arabinofuranoside (105.6 mg) and 2,4,6-trihydroxy-3,5-dimethylbenzophenone 4-O-(6''-O-galloyl)-β-D-glucopyranoside (98.4 mg) were separated from crude sample (19.8 g). The structures of all the isolates were identified by ESI-MS, 1H- and 13C-NMR analyses and their purities (>95%) were determined using HPLC.

  20. Preparative Isolation and Purification of Five Flavonoid Glycosides and One Benzophenone Galloyl Glycoside from Psidium guajava by High-Speed Counter-Current Chromatography (HSCCC

    Directory of Open Access Journals (Sweden)

    Yindi Zhu

    2013-12-01

    Full Text Available Psidium guajava leaves have a diverse phytochemical composition including flavonoids, phenolics, meroterpenoids and triterpenes, responsible for the biological activities of the medicinal parts. In particular, flavonol glycosides show beneficial effects on type II diabetes mellitus. A simple and efficient HSCCC method has been developed for the preparative separation of five flavonoid glycosides and one diphenylmethane glycoside from P. guajava. A solvent system composed of n-hexane–ethyl acetate–methanol–water (0.7:4:0.8:4, v/v/v/v was optimized for the separation. The upper phase was used as the stationary phase, and the lower phase was used as the mobile phase. Under the optimized conditions, hyperoside (15.3 mg, isoquercitrin (21.1 mg, reynoutrin (65.2 mg, quercetin-3-O-β-D-arabinopyranoside (71.7 mg, quercetin-3-O-α-L-arabinofuranoside (105.6 mg and 2,4,6-trihydroxy-3,5-dimethylbenzophenone 4-O-(6''-O-galloyl-β-D-glucopyranoside (98.4 mg were separated from crude sample (19.8 g. The structures of all the isolates were identified by ESI-MS, 1H- and 13C-NMR analyses and their purities (>95% were determined using HPLC.

  1. New steroidal glycosides from Tribulus terrestris L.

    Science.gov (United States)

    Chen, Gang; Liu, Tao; Lu, Xuan; Wang, Hai-Feng; Hua, Hui-Ming; Pei, Yue-Hu

    2012-01-01

    Two new steroidal glycosides were isolated from Tribulus terrestris L. Their structures were elucidated as 26-O-β-D-glucopyranosyl-5α-furostan-12-one-20(22)-ene-3β,23,26-triol-3-O-β-D-xylopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 3)]-β-D-glucopyranosyl-(1 → 4)-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-galactopyranoside (1) and 26-O-β-D-glucopyranosyl-5α-furostan-20(22)-ene-3β,23,26-triol-3-O-β-D-xylopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 3)]-β-D-glucopyranosyl-(1 → 4)-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-galactopyranoside (2) by spectroscopic methods including 1D and 2D NMR experiments.

  2. [A new phenethyl alcohol glycoside from Orobanche coerulescens].

    Science.gov (United States)

    Zhang, Qiang-Rong

    2017-03-01

    The constituents of the whole plant of Orobanche coerulescens were isolated and purified by using various column chromatographic techniques including D101, silica gel and ODS. The structures were identified by spectroscopic analyses including NMR and MS. A new phenylethanol glycoside was isolated from the whole plant of O. coerulescens, and was identified as 2-(3-methoxy-4-hydroxyphenyl)-ethanol-1-O- [(1→3)-O-α-L-rhamnopyranosyl-4, 6-O-di-feruloyl]-β-D-glucopyranoside, named as orobancheoside B. Through the antibacterial activity test, orobancheoside B was proved to have certain antibacterial activity, and be one of the main active components of O. coerulescens. The research result will laid a foundation for the medicinal materials and quality control research. Activity screening, broomrape orobancheoside B has certain antibacterial activity, as one of the main active components of O. coerulescens, and to constantly improve the quality of the medicinal materials laid a foundation. Copyright© by the Chinese Pharmaceutical Association.

  3. Structures and Biogenesis of Fallaxosides D4, D5, D6 and D7, Trisulfated Non-Holostane Triterpene Glycosides from the Sea Cucumber Cucumaria fallax

    Directory of Open Access Journals (Sweden)

    Alexandra S. Silchenko

    2016-07-01

    Full Text Available Four new trisulfated triterpene glycosides, fallaxosides D4 (1, D5 (2, D6 (3 and D7 (4 have been isolated from the sea cucumber Cucumaria fallax (Cucumariidae, Dendrochirotida. The structures of the glycosides have been elucidated by 2D NMR spectroscopy and HRESIMS. All the glycosides have the lanostane aglycones of a rare non-holostane type with 7(8-, 8(9- or 9(11-double bonds, one or two hydroxyl groups occupying unusual positions in the polycyclic nucleus and shortened or normal side chains. The pentasaccharide carbohydrate moieties of 1–4 have three sulfate groups. The cytotoxic activity of glycosides 1–4 against the ascite form of mouse Ehrlich carcinoma cells and mouse spleen lymphocytes and hemolytic activity against mouse erythrocytes have been studied.

  4. Epoxide hydrolase affects estrogen production in the human ovary.

    Science.gov (United States)

    Hattori, N; Fujiwara, H; Maeda, M; Fujii, S; Ueda, M

    2000-09-01

    To investigate the mechanisms of ovarian cell differentiation, we raised a new monoclonal antibody, HCL-3, which reacted with human luteal cells. It also reacted with human and porcine hepatocytes. The immunoaffinity-purified HCL-3 antigen from human corpora lutea (CL) was shown to be a 46-kDa protein. The N-terminal 22 amino acids of the 46-kDa protein from porcine liver exhibited high homology (82%) to human microsomal epoxide hydrolase (mEH). The purified HCL-3 antigen from human CL or porcine liver showed EH enzyme activity, confirming that HCL-3 antigen is identical to mEH, which is reported to detoxify the toxic substrates in the liver. In human follicles, mEH was immunohistochemically detected on granulosa and theca interna cells. In the menstrual and pregnant CL, mEH was also expressed on large and small luteal cells. A competitive inhibitor of EH, 1,2-epoxy-3,3,3-trichloropropane, inhibited the conversion of estradiol from testosterone by granulosa cells cultured in vitro, indicating the involvement of mEH in ovarian estrogen production. Because anticonvulsant sodium valproate and its analogues were reported to inhibit EH enzyme activity, these findings provide a new insight into the etiology of endocrine disorders that are frequently observed among epileptic patients taking anticonvulsant drugs.

  5. Structure of a Trypanosoma brucei α/β-hydrolase fold protein with unknown function

    International Nuclear Information System (INIS)

    Merritt, Ethan A.; Holmes, Margaret; Buckner, Frederick S.; Van Voorhis, Wesley C.; Quartly, Erin; Phizicky, Eric M.; Lauricella, Angela; Luft, Joseph; DeTitta, George; Neely, Helen; Zucker, Frank; Hol, Wim G. J.

    2008-01-01

    T. brucei gene Tb10.6k15.0140 codes for an α/β-hydrolase fold protein of unknown function. The 2.2 Å crystal structure shows that members of this sequence family retain a conserved Ser residue at the expected site of a catalytic nucleophile, but that trypanosomatid sequences lack structural homologs for the other expected residues of the catalytic triad. The structure of a structural genomics target protein, Tbru020260AAA from Trypanosoma brucei, has been determined to a resolution of 2.2 Å using multiple-wavelength anomalous diffraction at the Se K edge. This protein belongs to Pfam sequence family PF08538 and is only distantly related to previously studied members of the α/β-hydrolase fold family. Structural superposition onto representative α/β-hydrolase fold proteins of known function indicates that a possible catalytic nucleophile, Ser116 in the T. brucei protein, lies at the expected location. However, the present structure and by extension the other trypanosomatid members of this sequence family have neither sequence nor structural similarity at the location of other active-site residues typical for proteins with this fold. Together with the presence of an additional domain between strands β6 and β7 that is conserved in trypanosomatid genomes, this suggests that the function of these homologs has diverged from other members of the fold family

  6. Regulation of catalytic behaviour of hydrolases through interactions with functionalized carbon-based nanomaterials

    International Nuclear Information System (INIS)

    Pavlidis, Ioannis V.; Vorhaben, Torge; Gournis, Dimitrios; Papadopoulos, George K.; Bornscheuer, Uwe T.; Stamatis, Haralambos

    2012-01-01

    The interaction of enzymes with carbon-based nanomaterials (CBNs) is crucial for the function of biomolecules and therefore for the design and development of effective nanobiocatalytic systems. In this study, the effect of functionalized CBNs, such as graphene oxide (GO) and multi-wall carbon nanotubes (CNTs), on the catalytic behaviour of various hydrolases of biotechnological interest was monitored and the interactions between CBNs and proteins were investigated. The enzyme–nanomaterial interactions significantly affect the catalytic behaviour of enzymes, resulting in an increase up to 60 % of the catalytic efficiency of lipases and a decrease up to 30 % of the esterase. Moreover, the use of CNTs and GO derivatives, especially those that are amine-functionalized, led to increased thermal stability of most the hydrolases tested. Fluorescence and circular dichroism studies indicated that the altered catalytic behaviour of enzymes in the presence of CBNs arises from specific enzyme–nanomaterial interactions, which can lead to significant conformational changes. In the case of lipases, the conformational changes led to a more active and rigid structure, while in the case of esterases this led to destabilization and unfolding. Kinetic and spectroscopic studies indicated that the extent of the interactions between CBNs and hydrolases can be mainly controlled by the functionalization of nanomaterials than by their geometry.

  7. Regulation of catalytic behaviour of hydrolases through interactions with functionalized carbon-based nanomaterials

    Energy Technology Data Exchange (ETDEWEB)

    Pavlidis, Ioannis V. [University of Ioannina, Laboratory of Biotechnology, Department of Biological Applications and Technologies (Greece); Vorhaben, Torge [Institute of Biochemistry, Greifswald University, Department of Biotechnology and Enzyme Catalysis (Germany); Gournis, Dimitrios [University of Ioannina, Department of Materials Science and Engineering (Greece); Papadopoulos, George K. [Epirus Institute of Technology, Laboratory of Biochemistry and Biophysics, Faculty of Agricultural Technology (Greece); Bornscheuer, Uwe T. [Institute of Biochemistry, Greifswald University, Department of Biotechnology and Enzyme Catalysis (Germany); Stamatis, Haralambos, E-mail: hstamati@cc.uoi.gr [University of Ioannina, Laboratory of Biotechnology, Department of Biological Applications and Technologies (Greece)

    2012-05-15

    The interaction of enzymes with carbon-based nanomaterials (CBNs) is crucial for the function of biomolecules and therefore for the design and development of effective nanobiocatalytic systems. In this study, the effect of functionalized CBNs, such as graphene oxide (GO) and multi-wall carbon nanotubes (CNTs), on the catalytic behaviour of various hydrolases of biotechnological interest was monitored and the interactions between CBNs and proteins were investigated. The enzyme-nanomaterial interactions significantly affect the catalytic behaviour of enzymes, resulting in an increase up to 60 % of the catalytic efficiency of lipases and a decrease up to 30 % of the esterase. Moreover, the use of CNTs and GO derivatives, especially those that are amine-functionalized, led to increased thermal stability of most the hydrolases tested. Fluorescence and circular dichroism studies indicated that the altered catalytic behaviour of enzymes in the presence of CBNs arises from specific enzyme-nanomaterial interactions, which can lead to significant conformational changes. In the case of lipases, the conformational changes led to a more active and rigid structure, while in the case of esterases this led to destabilization and unfolding. Kinetic and spectroscopic studies indicated that the extent of the interactions between CBNs and hydrolases can be mainly controlled by the functionalization of nanomaterials than by their geometry.

  8. Oxidoreductases provide a more generic response to metallic stressors (Cu and Cd) than hydrolases in soil fungi: new ecotoxicological insights.

    Science.gov (United States)

    Lebrun, Jérémie D; Demont-Caulet, Nathalie; Cheviron, Nathalie; Laval, Karine; Trinsoutrot-Gattin, Isabelle; Mougin, Christian

    2016-02-01

    The present study investigates the effect of metals on the secretion of enzymes from 12 fungal strains maintained in liquid cultures. Hydrolases (acid phosphatase, β-glucosidase, β-galactosidase, and N-acetyl-β-glucosaminidase) and ligninolytic oxidoreductases (laccase, Mn, and lignin peroxidases) activities, as well as biomass production, were measured in culture fluids from fungi exposed to Cu or Cd. Our results showed that all fungi secreted most of the selected hydrolases and that about 50% of them produced a partial oxidative system in the absence of metals. Then, exposure of fungi to metals led to the decrease in biomass production. At the enzymatic level, Cu and Cd modified the secretion profiles of soil fungi. The response of hydrolases to metals was contrasted and complex and depended on metal, enzyme, and fungal strain considered. By contrast, the metals always stimulated the activity of ligninolytic oxidoreductases in fungal strains. In some of them, oxidoreductases were specifically produced following metal exposure. Fungal oxidoreductases provide a more generic response than hydrolases, constituting thus a physiological basis for their use as biomarkers of metal exposure in soils.

  9. Influence of phytoecdysteroids and plants steroidal glycosides on the lifespan and stress resistance of drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Mikhail Vyacheslavovich Shaposhnikov

    2014-12-01

    Full Text Available Background. Elucidation of the molecular mechanisms of effects of the active substances of plant adaptogens is a topical area of researches. Materials and methods. We studied the effect of herbal substances containing phytoecdysteroids (20-hydroxyecdysone and inokosterone of Serratula coronata L. or steroidal glycosides (dioscin and protodioscine of Trigonella foenum-graecum L. on the expression level of stress response genes (genes of heat shock proteins, DNA repair, antioxidant defense and apoptosis, stressresistanse (paraquat, starvation, hyperthermia and lifespan of Drosophila melanogaster. Results. The studied herbal substances upregulated genes of antioxidant defense mechanisms (Sod1, but downregulated the DNA repair (XPF and Rad51 and apoptosis (Hid genes. At the same time herbal substances induced weak adaptogenic and antiaging effects. Conclusion. Our results demonstrate that the herbal substances containing phytoecdysteroids and steroidal glycosides change the expression level of stress-response genes and activate mechanisms of hormesis.

  10. Two new acylated flavonol glycosides from Mimosa pigra L. leaves sub-family Mimosoideae

    Directory of Open Access Journals (Sweden)

    Chinedu J. Okonkwo

    2016-12-01

    Conclusion: Myricetin, quercetin and their glycoside derivatives are strong antioxidants; and elicit cytotoxic effect on human cancer cell lines among other pharmacological activities. The isolation of acylated flavonoids in M. pigra provided an important insight on the evolutionary trend of the medicinal plant. While the dominance of flavonols, may account for the various ethnomedicinal uses of the herb and the mechanism and mode of its confirmed pharmacological actions.

  11. Generation and characterization of epoxide hydrolase 3 (EPHX3-deficient mice.

    Directory of Open Access Journals (Sweden)

    Samantha L Hoopes

    Full Text Available Cytochrome P450 (CYP epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs, which play an important role in blood pressure regulation, protection against ischemia-reperfusion injury, angiogenesis, and inflammation. Epoxide hydrolases metabolize EETs to their corresponding diols (dihydroxyeicosatrienoic acids; DHETs which are biologically less active. Microsomal epoxide hydrolase (EPHX1, mEH and soluble epoxide hydrolase (EPHX2, sEH were identified >30 years ago and are capable of hydrolyzing EETs to DHETs. A novel epoxide hydrolase, EPHX3, was recently identified by sequence homology and also exhibits epoxide hydrolase activity in vitro with a substrate preference for 9,10-epoxyoctadecamonoenoic acid (EpOME and 11,12-EET. EPHX3 is highly expressed in the skin, lung, stomach, esophagus, and tongue; however, its endogenous function is unknown. Therefore, we investigated the impact of genetic disruption of Ephx3 on fatty acid epoxide hydrolysis and EET-related physiology in mice. Ephx3-/- mice were generated by excising the promoter and first four exons of the Ephx3 gene using Cre-LoxP methodology. LC-MS/MS analysis of Ephx3-/- heart, lung, and skin lysates revealed no differences in endogenous epoxide:diol ratios compared to wild type (WT. Ephx3-/- mice also exhibited no change in plasma levels of fatty acid epoxides and diols relative to WT. Incubations of cytosolic and microsomal fractions prepared from Ephx3-/- and WT stomach, lung, and skin with synthetic 8,9-EET, 11,12-EET, and 9,10-EpOME revealed no significant differences in rates of fatty acid diol formation between the genotypes. Ephx3-/- hearts had similar functional recovery compared to WT hearts following ischemia/reperfusion injury. Following intranasal lipopolysaccharide (LPS exposure, Ephx3-/- mice were not different from WT in terms of lung histology, bronchoalveolar lavage fluid cell counts, or fatty acid epoxide and diol levels. We conclude that genetic

  12. Microfluidic glycosyl hydrolase screening for biomass-to-biofuel conversion.

    Science.gov (United States)

    Bharadwaj, Rajiv; Chen, Zhiwei; Datta, Supratim; Holmes, Bradley M; Sapra, Rajat; Simmons, Blake A; Adams, Paul D; Singh, Anup K

    2010-11-15

    The hydrolysis of biomass to fermentable sugars using glycosyl hydrolases such as cellulases and hemicellulases is a limiting and costly step in the conversion of biomass to biofuels. Enhancement in hydrolysis efficiency is necessary and requires improvement in both enzymes and processing strategies. Advances in both areas in turn strongly depend on the progress in developing high-throughput assays to rapidly and quantitatively screen a large number of enzymes and processing conditions. For example, the characterization of various cellodextrins and xylooligomers produced during the time course of saccharification is important in the design of suitable reactors, enzyme cocktail compositions, and biomass pretreatment schemes. We have developed a microfluidic-chip-based assay for rapid and precise characterization of glycans and xylans resulting from biomass hydrolysis. The technique enables multiplexed separation of soluble cellodextrins and xylose oligomers in around 1 min (10-fold faster than HPLC). The microfluidic device was used to elucidate the mode of action of Tm_Cel5A, a novel cellulase from hyperthermophile Thermotoga maritima . The results demonstrate that the cellulase is active at 80 °C and effectively hydrolyzes cellodextrins and ionic-liquid-pretreated switchgrass and Avicel to glucose, cellobiose, and cellotriose. The proposed microscale approach is ideal for quantitative large-scale screening of enzyme libraries for biomass hydrolysis, for development of energy feedstocks, and for polysaccharide sequencing.

  13. Heterologous expression of the methyl carbamate-degrading hydrolase MCD.

    Science.gov (United States)

    Naqvi, Tatheer; Cheesman, Matthew J; Williams, Michelle R; Campbell, Peter M; Ahmed, Safia; Russell, Robyn J; Scott, Colin; Oakeshott, John G

    2009-10-26

    The methyl carbamate-degrading hydrolase (MCD) of Achromobacter WM111 has considerable potential as a pesticide bioremediation agent. However this potential has been unrealisable until now because of an inability to express MCD in heterologous hosts such as Escherichia coli. Herein, we describe the first successful attempt to express appreciable quantities of MCD in active form in E. coli, and the subsequent characterisation of the heterologously expressed material. We find that the properties of this material closely match the previously reported properties of MCD produced from Achromobacter WM111. This includes the presence of two distinct forms of the enzyme that we show are most likely due to the presence of two functional translational start sites. The purified enzyme catalyses the hydrolysis of a carbamate (carbaryl), a carboxyl ester (alpha-naphthyl acetate) and a phophotriester (dimethyl umbelliferyl phosphate) and it is relatively resistant to thermal and solvent-mediated denaturation. The robust nature and catalytic promiscuity of MCD suggest that it could be exploited for various biotechnological applications.

  14. Ubiquitin C-Terminal Hydrolase L1 in Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Jennifer Hurst-Kennedy

    2012-01-01

    Full Text Available Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1, aka PGP9.5 is an abundant, neuronal deubiquitinating enzyme that has also been suggested to possess E3 ubiquitin-protein ligase activity and/or stabilize ubiquitin monomers in vivo. Recent evidence implicates dysregulation of UCH-L1 in the pathogenesis and progression of human cancers. Although typically only expressed in neurons, high levels of UCH-L1 have been found in many nonneuronal tumors, including breast, colorectal, and pancreatic carcinomas. UCH-L1 has also been implicated in the regulation of metastasis and cell growth during the progression of nonsmall cell lung carcinoma, colorectal cancer, and lymphoma. Together these studies suggest UCH-L1 has a potent oncogenic role and drives tumor development. Conversely, others have observed promoter methylation-mediated silencing of UCH-L1 in certain tumor subtypes, suggesting a potential tumor suppressor role for UCH-L1. In this paper, we provide an overview of the evidence supporting the involvement of UCH-L1 in tumor development and discuss the potential mechanisms of action of UCH-L1 in oncogenesis.

  15. A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes

    DEFF Research Database (Denmark)

    Pierce, Brian; Wittrup Agger, Jane; Zhang, Zhenghong

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty......-four lytic polysaccharide monooxygenases (LPMOs) expressed in Trichoderma reesei were evaluated for their ability to oxidize the complex polysaccharides in soybean spent flakes, an abundant and industrially relevant substrate. TrCel61A, a soy-polysaccharide-active AA9 LPMO from T. reesei, was used...... as a benchmark in this evaluation. In total, seven LPMOs demonstrated activity on pretreated soy spent flakes, with the products from enzymatic treatments evaluated using mass spectrometry and high performance anion exchange chromatography. The hydrolytic boosting effect of the top-performing enzymes...

  16. Brominated Oxylipins and Oxylipin Glycosides from Red Sea Corals

    Czech Academy of Sciences Publication Activity Database

    Řezanka, Tomáš; Dembitsk, V. M.

    - (2003), s. 309-316 ISSN 1434-193X Institutional research plan: CEZ:AV0Z5020903 Keywords : glycosides * natural products * oxylipins Subject RIV: EE - Microbiology, Virology Impact factor: 2.227, year: 2003

  17. Phenolic glycosides from sugar maple (Acer saccharum) bark.

    Science.gov (United States)

    Yuan, Tao; Wan, Chunpeng; González-Sarrías, Antonio; Kandhi, Vamsikrishna; Cech, Nadja B; Seeram, Navindra P

    2011-11-28

    Four new phenolic glycosides, saccharumosides A-D (1-4), along with eight known phenolic glycosides, were isolated from the bark of sugar maple (Acer saccharum). The structures of 1-4 were elucidated on the basis of spectroscopic data analysis. All compounds isolated were evaluated for cytotoxicity effects against human colon tumorigenic (HCT-116 and Caco-2) and nontumorigenic (CCD-18Co) cell lines.

  18. Molecular and biochemical characterization of a novel intracellular invertase from Aspergillus niger with transfructosylating activity

    NARCIS (Netherlands)

    Goosen, C.; Yuan, X.L.; Munster, J.M. van; Ram, A.F.J.; Maarel, M.J.E.C. van der; Dijkhuizen, L.

    2007-01-01

    A novel subfamily of putative intracellular invertase enzymes (glycoside hydrolase family 32) has previously been identified in fungal genomes. Here, we report phylogenetic, molecular, and biochemical characteristics of SucB, one of two novel intracellular invertases identified in Aspergillus niger.

  19. Antitrypanosomal isothiocyanate and thiocarbamate glycosides from Moringa peregrina.

    Science.gov (United States)

    Ayyari, Mahdi; Salehi, Peyman; Ebrahimi, Samad Nejad; Zimmermann, Stefanie; Portmann, Lena; Krauth-Siegel, R Luise; Kaiser, Marcel; Brun, Reto; Rezadoost, Hassan; Rezazadeh, Shamsali; Hamburger, Matthias

    2014-01-01

    O-Methyl (1), O-ethyl (2), and O-butyl (3) 4-[(α-L-rhamnosyloxy) benzyl] thiocarbamate (E), along with 4-(α-L-rhamnosyloxy) benzyl isothiocyanate (4) have been isolated from the aerial parts of Moringa peregrina. The compounds were tested for in vitro activity against Trypanosoma brucei rhodesiense and cytotoxicity in rat skeletal myoblasts (L6 cells). The most potent compound was 4 with an IC50 of 0.10 µM against T.b. rhodesiense and a selectivity index of 73, while the thiocarbamate glycosides 1, 2, and 3 showed only moderate activity. Intraperitoneal administration of 50 mg/kg body weight/day of 4 in the T.b. rhodesiense STIB 900 acute mouse model revealed significant in vivo toxicity. Administration of 10 mg/kg body weight/day resulted in a 95% reduction of parasitemia on day 7 postinfection, but did not cure the animals. Because of its high in vitro activity and its ability to irreversibly inhibit trypanothione reductase, an attractive parasite-specific target enzyme, 4-[(α-L-rhamnosyloxy) benzyl] isothiocyanate (4), can be considered as a lead structure for the development and characterization of novel antitrypanosomal drugs. Georg Thieme Verlag KG Stuttgart · New York.

  20. Cytotoxicity of cardenolides and cardenolide glycosides from Asclepias curassavica.

    Science.gov (United States)

    Li, Jun-Zhu; Qing, Chen; Chen, Chang-Xiang; Hao, Xiao-Jiang; Liu, Hai-Yang

    2009-04-01

    A new cardenolide, 12beta,14beta-dihydroxy-3beta,19-epoxy-3alpha-methoxy-5alpha-card-20(22)-enolide (6), and a new doubly linked cardenolide glycoside, 12beta-hydroxycalotropin (13), together with eleven known compounds, coroglaucigenin (1), 12beta-hydroxycoroglaucigenin (2), calotropagenin (3), desglucouzarin (4), 6'-O-feruloyl-desglucouzarin (5), calotropin (7), uscharidin (8), asclepin (9), 16alpha-hydroxyasclepin (10), 16alpha-acetoxycalotropin (11), and 16alpha-acetoxyasclepin (12), were isolated from the aerial part of ornamental milkweed, Asclepias curassavica and chemically elucidated through spectral analyses. All the isolates were evaluated for their cytotoxic activity against HepG2 and Raji cell lines. The results showed that asclepin (9) had the strongest cytotoxic activity with an IC(50) value of 0.02 microM against the two cancer cell lines and the new compound 13 had significant cytotoxic activity with IC(50) values of 0.69 and 1.46 microM, respectively.

  1. Synthetic Study on the Relationship Between Structure and Sweet Taste Properties of Steviol Glycosides

    Directory of Open Access Journals (Sweden)

    Grant Dubois

    2012-04-01

    Full Text Available The structure activity relationship between the C16-C17 methylene double bond on the aglycone of steviol glycosides and the corresponding impact on their sweet taste has been reported here for the first time. It has been observed that converting stevioside and rebaudioside A to their corresponding ketones by switching the doubly bonded methylene on C-17 for a ketone group actually removes the sweet taste properties of these molecules completely. Regenerating the original molecules tends to restore the sweet taste of both the steviol glycosides. Thus this C16-C17 methylene double bond in rebaudioside A and stevioside can be regarded as a pharmacophore essential for the sweetness property of these molecules.

  2. Mitochondrial NUDIX hydrolases: A metabolic link between NAD catabolism, GTP and mitochondrial dynamics.

    Science.gov (United States)

    Long, Aaron; Klimova, Nina; Kristian, Tibor

    2017-10-01

    NAD + catabolism and mitochondrial dynamics are important parts of normal mitochondrial function and are both reported to be disrupted in aging, neurodegenerative diseases, and acute brain injury. While both processes have been extensively studied there has been little reported on how the mechanisms of these two processes are linked. This review focuses on how downstream NAD + catabolism via NUDIX hydrolases affects mitochondrial dynamics under pathologic conditions. Additionally, several potential targets in mitochondrial dysfunction and fragmentation are discussed, including the roles of mitochondrial poly(ADP-ribose) polymerase 1(mtPARP1), AMPK, AMP, and intra-mitochondrial GTP metabolism. Mitochondrial and cytosolic NUDIX hydrolases (NUDT9α and NUDT9β) can affect mitochondrial and cellular AMP levels by hydrolyzing ADP- ribose (ADPr) and subsequently altering the levels of GTP and ATP. Poly (ADP-ribose) polymerase 1 (PARP1) is activated after DNA damage, which depletes NAD + pools and results in the PARylation of nuclear and mitochondrial proteins. In the mitochondria, ADP-ribosyl hydrolase-3 (ARH3) hydrolyzes PAR to ADPr, while NUDT9α metabolizes ADPr to AMP. Elevated AMP levels have been reported to reduce mitochondrial ATP production by inhibiting the adenine nucleotide translocase (ANT), allosterically activating AMPK by altering the cellular AMP: ATP ratio, and by depleting mitochondrial GTP pools by being phosphorylated by adenylate kinase 3 (AK3), which uses GTP as a phosphate donor. Recently, activated AMPK was reported to phosphorylate mitochondria fission factor (MFF), which increases Drp1 localization to the mitochondria and promotes mitochondrial fission. Moreover, the increased AK3 activity could deplete mitochondrial GTP pools and possibly inhibit normal activity of GTP-dependent fusion enzymes, thus altering mitochondrial dynamics. Published by Elsevier Ltd.

  3. Simultaneous determination of iridoid glycosides, phenethylalcohol glycosides and furfural derivatives in Rehmanniae Radix by high performance liquid chromatography coupled with triple-quadrupole mass spectrometry

    DEFF Research Database (Denmark)

    Xu, Jun; Wu, Jie; Zhu, Ling-Ying

    2012-01-01

    In this study, a sensitive and selective method for simultaneously quantifying eight major components (four iridoid glycosides, three phenethylalcohol glycosides and one furfural derivative) of Rehmanniae Radix by high performance liquid chromatography coupled with triple-quadrupole mass spectrom......In this study, a sensitive and selective method for simultaneously quantifying eight major components (four iridoid glycosides, three phenethylalcohol glycosides and one furfural derivative) of Rehmanniae Radix by high performance liquid chromatography coupled with triple-quadrupole mass...

  4. Iridoid glycoside biosynthesis in Penstemon secundiflorus. Another H-5, H-9 trans iridoid glycoside

    DEFF Research Database (Denmark)

    Krull, Robert E.; Stermitz, Frank R.; Franzyk, Henrik

    1998-01-01

    Isolation and characterization of the new iridoid 10-hydroxy-(5 alpha H)-6-epidihydrocornin from Penstemon secundiflorus (Scrophulariaceae) is described. In biosynthetic experiments, deoxyloganic acid was incorporated into the transfused iridoid glycosides (5 alpha H)-6-epidihydrocornin and 10-hy......-hydroxy-(5 alpha H)-6-epidihydrocornin in P. secundiflorus. Formation of the trans-fused compounds is therefore a late event in the biosynthesis and does not occur during iridoid formation by cyclization of the open chain monoterpene precursor. In the same plant, 8-epideoxyloganic acid...

  5. Optimization of the fermentation conditions and substrate specifity of mycelium-bound ester hydrolases of Aspergillus oryzae Cs007

    Directory of Open Access Journals (Sweden)

    de Hong Yan

    2015-01-01

    Full Text Available In order to improve mycelium-bound ester hydrolases activities of Aspergillus oryzae Cs007, the main production conditions were investigated. The ester hydrolases activities were simultaneously determined by titration assay and spectrophotometric assay methods, using olive oil and p-nitrophenyl esters as substrates, respectively. The optimum carbon source and nitrogen source were olive oil and peptone, with the concentrations of 1% and 2.2%, respectively. The effects of carbon source, nitrogen source and their concentrations on the production of enzymes were identical when the enzymes activities were assayed by the two methods. The mycelium-bound enzymes showed hydrolytic activity toward all the tested p-nitrophenyl esters, triglycerides and fatty acid ethyl esters. But it showed greater preference for long-chain triglycerides and short-chain p-nitrophenyl esters.

  6. New acylated flavonoid glycosides from flowers of Aerva javanica.

    Science.gov (United States)

    Mussadiq, Sara; Riaz, Naheed; Saleem, Muhammad; Ashraf, Muhammad; Ismail, Tayaba; Jabbar, Abdul

    2013-07-01

    Chromatographic purification of ethyl acetate soluble fraction of the methanolic extract of the flowers of Aerva javanica yielded three new acylated flavone glycosides: kaempferol-3-O-β-d-[4‴-E-p-coumaroyl-α-l-rhamnosyl(1 → 6)]-galactoside (1), kaempferol-3-O-β-d-[4‴-E-p-coumaroyl-α-l-rhamnosyl(1 → 6)]-(3″-E-p-coumaroyl)galactoside (2), and kaempferol-3-O-β-d-[4‴-E-p-coumaroyl-α-l-rhamnosyl(1 → 6)]-(4″-E-p-coumaroyl)galactoside (3), along with p-coumaric acid (4), caffeic acid (5), gallic acid (6), eicosanyl-trans-p-coumarate (7), hexadecyl ferulate (8), and hexacosyl ferulate (9). The compounds 1-9 were characterized using 1D ((1)H, (13)C) and 2D NMR (HMQC, HMBC, and COSY) spectroscopy and mass spectrometry (EI-MS, HR-EI-MS, FAB-MS, and HR-FAB-MS) and in comparison with the reported data in the literature. Compound 1 showed weak inhibitory activity against enzymes, such as acetylcholinesterase, butyrylcholinesterase, and lipoxygenase with IC50 values 205.1, 304.1, and 212.3 μM, respectively, whereas compounds 2 and 3 were only weakly active against the enzyme acetylcholinesterase.

  7. Screening Brazilian Macrophomina phaseolina isolates for alkaline lipases and other extracellular hydrolases.

    Science.gov (United States)

    Schinke, Claudia; Germani, José C

    2012-03-01

    Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipases. Hydrolase detection and growth rate determination were done on citric pectin, gelatin, casein, soluble starch, and olive oil as substrates. Ten isolates were found to be active on all substrates tested. The most commonly detected enzymes were pectinases, amylases, and lipases. The growth rate on pectin was significantly higher (P media identified CMM 2105, CMM 1091, and PEL as the fastest-growing isolates. The lipase activity of four isolates grown on olive oil was followed for 4 days by measuring the activity in the cultivation broth. The specific lipolytic activity of isolate PEL was significantly higher at 96 h (130 mU mg protein(-1)). The broth was active at 37 °C, pH 8, indicating the potential utility of the lipases of this isolate in mild alkaline detergents. There was a strong and positive correlation (0.86) between radial growth rate and specific lipolytic activity.

  8. Carbohydrate-active enzymes in Trichoderma harzianum: a bioinformatic analysis bioprospecting for key enzymes for the biofuels industry.

    Science.gov (United States)

    Ferreira Filho, Jaire Alves; Horta, Maria Augusta Crivelente; Beloti, Lilian Luzia; Dos Santos, Clelton Aparecido; de Souza, Anete Pereira

    2017-10-12

    Trichoderma harzianum is used in biotechnology applications due to its ability to produce powerful enzymes for the conversion of lignocellulosic substrates into soluble sugars. Active enzymes involved in carbohydrate metabolism are defined as carbohydrate-active enzymes (CAZymes), and the most abundant family in the CAZy database is the glycoside hydrolases. The enzymes of this family play a fundamental role in the decomposition of plant biomass. In this study, the CAZymes of T. harzianum were identified and classified using bioinformatic approaches after which the expression profiles of all annotated CAZymes were assessed via RNA-Seq, and a phylogenetic analysis was performed. A total of 430 CAZymes (3.7% of the total proteins for this organism) were annotated in T. harzianum, including 259 glycoside hydrolases (GHs), 101 glycosyl transferases (GTs), 6 polysaccharide lyases (PLs), 22 carbohydrate esterases (CEs), 42 auxiliary activities (AAs) and 46 carbohydrate-binding modules (CBMs). Among the identified T. harzianum CAZymes, 47% were predicted to harbor a signal peptide sequence and were therefore classified as secreted proteins. The GH families were the CAZyme class with the greatest number of expressed genes, including GH18 (23 genes), GH3 (17 genes), GH16 (16 genes), GH2 (13 genes) and GH5 (12 genes). A phylogenetic analysis of the proteins in the AA9/GH61, CE5 and GH55 families showed high functional variation among the proteins. Identifying the main proteins used by T. harzianum for biomass degradation can ensure new advances in the biofuel production field. Herein, we annotated and characterized the expression levels of all of the CAZymes from T. harzianum, which may contribute to future studies focusing on the functional and structural characterization of the identified proteins.

  9. Zebrafish chemical screening reveals the impairment of dopaminergic neuronal survival by cardiac glycosides.

    Directory of Open Access Journals (Sweden)

    Yaping Sun

    Full Text Available Parkinson's disease is a neurodegenerative disorder characterized by the prominent degeneration of dopaminergic (DA neurons among other cell types. Here we report a first chemical screen of over 5,000 compounds in zebrafish, aimed at identifying small molecule modulators of DA neuron development or survival. We find that Neriifolin, a member of the cardiac glycoside family of compounds, impairs survival but not differentiation of both zebrafish and mammalian DA neurons. Cardiac glycosides are inhibitors of Na(+/K(+ ATPase activity and widely used for treating heart disorders. Our data suggest that Neriifolin impairs DA neuronal survival by targeting the neuronal enriched Na(+/K(+ ATPase α3 subunit (ATP1A3. Modulation of ionic homeostasis, knockdown of p53, or treatment with antioxidants protects DA neurons from Neriifolin-induced death. These results reveal a previously unknown effect of cardiac glycosides on DA neuronal survival and suggest that it is mediated through ATP1A3 inhibition, oxidative stress, and p53. They also elucidate potential approaches for counteracting the neurotoxicity of this valuable class of medications.

  10. Studies on whole cell fluorescence-based screening for epoxide hydrolases and Baeyer-Villiger monooxygenases

    International Nuclear Information System (INIS)

    Bicalho, Beatriz; Chen, Lu S.; Marsaioli, Anita J.; Grognux, Johann; Reymond, Jean-Louis

    2004-01-01

    Biocatalysis reactions were performed on microtiter plates (200 μL) aiming at the utilization of fluorogenic substrates (100 μmol L -1 ) for rapid whole cell screening for epoxide hydrolases (EHs) and Baeyer-Villiger monooxygenases (BVMOs). A final protocol was achieved for EHs, with 3 new enzymatic sources being detected (Agrobacterium tumefaciens, Pichia stipitis, Trichosporom cutaneum). The fluorogenic assay for BVMO did not work as expected. However, an approach to possible variables involved (aeration; pH) provided the first detection of a BVMO activity in T. cutaneum. (author)

  11. Enantioselectivity of a recombinant epoxide hydrolase from Agrobacterium radiobacter

    NARCIS (Netherlands)

    Lutje Spelberg, Jeffrey H.; Rink, Rick; Kellogg, Richard M.; Janssen, Dick B.

    1998-01-01

    The recombinant epoxide hydrolase from Agrobacterium radiobacter AD1 was used to obtain enantiomerically pure epoxides by means of a kinetic resolution. Epoxides such as styrene oxide and various derivatives thereof and phenyl glycidyl ether were obtained in high enantiomeric excess and in

  12. Properties of epoxide hydrolase from the yeast Rhodotorula glutinis

    NARCIS (Netherlands)

    Ariës-Kronenburg, N.A.E.

    2002-01-01

    Epoxide hydrolases are ubiquitous enzymes that can be found in nearly all living organisms. Some of the enzymes play an important role in detoxifying xenobiotic and metabolic compounds. Others are important in the growth of organisms like

  13. Further characterization of intestinal lactase/phlorizin hydrolase

    DEFF Research Database (Denmark)

    Skovbjerg, H; Norén, O; Sjöström, H

    1982-01-01

    Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis in the pres......Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis...... in the presence of SDS. Pig lactase/phlorizin hydrolase was shown to have the same quaternary structure as the human enzyme, i.e., built up of two polypeptides of the same molecular weight (160000). In addition to hydrolyzing lactose, phlorizin and a number of synthetic substrates, both the human and the pig...... membranes (basolateral and intracellular membranes) exhibited in SDS-polyacrylamide gel electrophoresis the same size of constituent polypeptides and the same catalytic and immunological properties as a normal brush border lactase/phlorizin hydrolase....

  14. Six new C21 steroidal glycosides from Asclepias curassavica L.

    Science.gov (United States)

    Li, Jun-Zhu; Liu, Hai-Yang; Lin, Yi-Ju; Hao, Xiao-Jiang; Ni, Wei; Chen, Chang-Xiang

    2008-07-01

    Six new C(21) steroidal glycosides, named curassavosides A-F (3-8), were obtained from the aerial parts of Asclepias curassavica (Asclepiadaceae), along with two known oxypregnanes, 12-O-benzoyldeacylmetaplexigenin (1) and 12-O-benzoylsarcostin (2). By spectroscopic methods, the structures of the six new compounds were determined as 12-O-benzoyldeacylmetaplexigenin 3-O-beta-D-oleandropyranosyl-(1-->4)-beta-D-digitoxopyranoside (3), 12-O-benzoylsarcostin 3-O-beta-D-oleandropyranosyl-(1-->4)-beta-D-digitoxopyranoside (4), sarcostin 3-O-beta-D-oleandropyranosyl-(1-->4)-beta-D-canaropyranosyl-(1-->4)-beta-D-oleandropyranosyl-(1-->4)-beta-D-digitoxopyranoside (5), sarcostin 3-O-beta-D-oleandropyranosyl-(1-->4)-beta-D-canaropyranosyl-(1-->4)-beta-D-canaropyranosyl-(1-->4)-beta-D-digitoxopyranoside (6), 12-O-benzoyldeacylmetaplexigenin 3-O-beta-D-glucopyranosyl-(1-->4)-beta-D-oleandropyranosyl-(1-->4)-beta-D-canaropyranosyl-(1-->4)-beta-d-oleandropyranosyl-(1-->4)-beta-D-digitoxopyranoside (7), and 12-O-benzoylsarcostin 3-O-beta-D-glucopyranosyl-(1-->4)-beta-D-oleandropyranosyl-(1-->4)-beta-d-canaropyranosyl-(1-->4)-beta-D-oleandropyranosyl-(1-->4)-beta-D-digitoxopyranoside (8), respectively. All compounds (1-8) were tested for in vitro cytotoxicity; only compound 3 showed weak inhibitory activity against Raji and AGZY cell lines.

  15. Effects of synthetic glycosides on steroid balance in Macaca fascicularis

    International Nuclear Information System (INIS)

    Malinow, M.R.; Elliott, W.H.; McLaughlin, P.; Upson, B.

    1987-01-01

    The predominantly beta-anomer of diosgenin glucoside (DG) was synthesized and its effects on cholesterol homeostasis were tested in monkeys. Cynomolgus macaques (Macaca fascicularis) were fed, during two 3-week periods, a semipurified diet with 0.1% cholesterol and a similar ration containing 1% DG, respectively. A Chow diet was given for 5 weeks between the experimental periods. Cholesterol and bile acid balance were analyzed during the last week of each semipurified diet. Diosgenin glucoside reduced cholesterolemia from 292 mg/dl to 172 mg/dl, decreased intestinal absorption of exogenous cholesterol from 62.4% to 26.0%, and increased secretion of endogenous cholesterol from -0.8 to 93.5 mg/day. The fecal excretion of neutral steroids rose from 40.7 to 157.3 mg/day; that of bile acids changed, nonsignificantly, from 23.1 to 16.0 mg/day. The cholesterol balance was -44 mg/day in the control period, and 88 mg/day in the DG-fed animals. No toxic signs were observed. Thus, when long-term studies demonstrate that the glucoside is well tolerated, DG and other synthetic glycosides with similar activities may be of use in the management of hypercholesterolemia and atherosclerosis

  16. On the phosphorylase activity of GH3 enzymes: A β-N-acetylglucosaminidase from Herbaspirillum seropedicae SmR1 and a glucosidase from Saccharopolyspora erythraea.

    Science.gov (United States)

    Ducatti, Diogo R B; Carroll, Madison A; Jakeman, David L

    2016-11-29

    A phosphorolytic activity has been reported for beta-N-acetylglucosaminidases from glycoside hydrolase family 3 (GH3) giving an interesting explanation for an unusual histidine as catalytic acid/base residue and suggesting that members from this family may be phosphorylases [J. Biol. Chem. 2015, 290, 4887]. Here, we describe the characterization of Hsero1941, a GH3 beta-N-acetylglucosaminidase from the endophytic nitrogen-fixing bacterium Herbaspirillum seropedicae SmR1. The enzyme has significantly higher activity against pNP-beta-D-GlcNAcp (K m  = 0.24 mM, k cat  = 1.2 s -1 , k cat /K m  = 5.0 mM -1 s -1 ) than pNP-beta-D-Glcp (K m  = 33 mM, k cat  = 3.3 × 10 -3 s -1 , k cat /K m  = 9 × 10 -4  mM -1 s -1 ). The presence of phosphate failed to significantly modify the kinetic parameters of the reaction. The enzyme showed a broad aglycone site specificity, being able to hydrolyze sugar phosphates beta-D-GlcNAc 1P and beta-D-Glc 1P, albeit at a fraction of the rate of hydrolysis of aryl glycosides. GH3 beta-glucosidase EryBI, that does not have a histidine as the general acid/base residue, also hydrolyzed beta-D-Glc 1P, at comparable rates to Hsero1941. These data indicate that Hsero1941 functions primarily as a hydrolase and that phosphorolytic activity is likely adventitious. The prevalence of histidine as a general acid/base residue is not predictive, nor correlative, with GH3 beta-N-acetylglucosaminidases having phosphorolytic activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Extracellular Xylanolytic and Pectinolytic Hydrolase Production by Aspergillus flavus Isolates Contributes to Crop Invasion

    Directory of Open Access Journals (Sweden)

    Jay E. Mellon

    2015-08-01

    Full Text Available Several atoxigenic Aspergillus flavus isolates, including some being used as biocontrol agents, and one toxigenic isolate were surveyed for the ability to produce extracellular xylanolytic and pectinolytic hydrolases. All of the tested isolates displayed good production of endoxylanases when grown on a medium utilizing larch xylan as a sole carbon substrate. Four of the tested isolates produced reasonably high levels of esterase activity, while the atoxigenic biocontrol agent NRRL 21882 isolate esterase level was significantly lower than the others. Atoxigenic A. flavus isolates 19, 22, K49, AF36 (the latter two are biocontrol agents and toxigenic AF13 produced copious levels of pectinolytic activity when grown on a pectin medium. The pectinolytic activity levels of the atoxigenic A. flavus 17 and NRRL 21882 isolates were significantly lower than the other tested isolates. In addition, A. flavus isolates that displayed high levels of pectinolytic activity in the plate assay produced high levels of endopolygalacturonase (pectinase P2c, as ascertained by isoelectric focusing electrophoresis. Isolate NRRL 21882 displayed low levels of both pectinase P2c and pectin methyl esterase. A. flavus appears capable of producing these hydrolytic enzymes irrespective of aflatoxin production. This ability of atoxigenic isolates to produce xylanolytic and pectinolytic hydrolases mimics that of toxigenic isolates and, therefore, contributes to the ability of atoxigenic isolates to occupy the same niche as A. flavus toxigenic isolates.

  18. Cloning, expression and mutation of a triazophos hydrolase gene from Burkholderia sp. SZL-1.

    Science.gov (United States)

    Zhang, Hao; Li, Qiang; Guo, Su-Hui; Cheng, Ming-Gen; Zhao, Meng-Jun; Hong, Qing; Huang, Xing

    2016-06-01

    Triazophos is a broad-spectrum and highly effective insecticide, and the residues of triazophos have been frequently detected in the environment. A triazophos-degrading bacterium, Burkholderia sp. SZL-1, was isolated from a long-term triazophos-polluted soil. Strain SZL-1 could hydrolyze triazophos to 1-phenyl-3-hydroxy-1,2,4-triazole, which was further utilized as the carbon sources for growth. The triazophos hydrolase gene trhA, cloned from strain SZL-1, was expressed and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. TrhA is 55 kDa and displays maximum activity at 25°C, pH 8.0. This enzyme still has nearly 60% activity at the range of 15°C-50°C for 30 min. TrhA was mutated by sequential error prone PCR and screened for improved activity for triazophos degradation. One purified variant protein (Val89-Gly89) named TrhA-M1 showed up to 3-fold improvement in specific activity against triazophos, and the specificity constants of Kcat and Kcat/Km for TrhA-M1 were improved up to 2.3- and 8.28-fold, respectively, compared to the wild-type enzyme. The results in this paper provided potential material for the contaminated soil remediation and hydrolase genetic structure research. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. GH32 family activity: a topological approach through protein contact networks.

    Science.gov (United States)

    Cimini, Sara; Di Paola, Luisa; Giuliani, Alessandro; Ridolfi, Alessandra; De Gara, Laura

    2016-11-01

    The application of Protein Contact Networks methodology allowed to highlight a novel response of border region between the two domains to substrate binding. Glycoside hydrolases (GH) are enzymes that mainly hydrolyze the glycosidic bond between two carbohydrates or a carbohydrate and a non-carbohydrate moiety. These enzymes are involved in many fundamental and diverse biological processes in plants. We have focused on the GH32 family, including enzymes very similar in both sequence and structure, each having however clear specificities of substrate preferences and kinetic properties. Structural and topological differences among proteins of the GH32 family have been here identified by means of an emerging approach (Protein Contact network, PCN) based on the formalization of 3D structures as contact networks among amino-acid residues. The PCN approach proved successful in both reconstructing the already known functional domains and in identifying the structural counterpart of the properties of GH32 enzymes, which remain uncertain, like their allosteric character. The main outcome of the study was the discovery of the activation upon binding of the border (cleft) region between the two domains. This reveals the allosteric nature of the enzymatic activity for all the analyzed forms in the GH32 family, a character yet to be highlighted in biochemical studies. Furthermore, we have been able to recognize a topological signature (graph energy) of the different affinity of the enzymes towards small and large substrates.

  20. Mutants of Streptomyces coeruleorubidus impaired in the biosynthesis of daunomycinone glycosides and related metabolites

    International Nuclear Information System (INIS)

    Blumauerova, M.; Stajner, K.; Pokorny, V.; Hostalek, Z.; Vanek, Z.

    1978-01-01

    Mutants of Streptomyces coeruleorubidus, blocked in the biosynthesis of anthracycline antibiotics of the daunomycine complex, were isolated from the production strains after treatment with UV light, γ-radiation, nitrous acid, and after natural selection; according to their different biosynthetic activity the mutants were divided into five phenotypic groups. Mutants of two of these groups produced compounds that had not yet been described in Streptomyces coeruleorubidus (aklavinone, 7-deoxyaklavinone, zeta-rhodomycinone and glycosides of epsilon-rhodomycinone). The mutants differed from the parent strains and also mutually in morphological characteristics but no direct correlation between these changes and the biosynthetic activity could be observed in most cases. (author)

  1. The impact of nonpolar lipids on the regulation of the steryl ester hydrolases Tgl1p and Yeh1p in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Klein, Isabella; Korber, Martina; Athenstaedt, Karin; Daum, Günther

    2017-12-01

    In the yeast Saccharomyces cerevisiae degradation of steryl esters is catalyzed by the steryl ester hydrolases Tgl1p, Yeh1p and Yeh2p. The two steryl ester hydrolases Tgl1p and Yeh1p localize to lipid droplets, a cell compartment storing steryl esters and triacylglycerols. In the present study we investigated regulatory aspects of these two hydrolytic enzymes, namely the gene expression level, protein amount, stability and enzyme activity of Tgl1p and Yeh1p in strains lacking both or only one of the two major nonpolar lipids, steryl esters and triacylglycerols. In a strain lacking both nonpolar lipids and consequently lipid droplets, Tgl1p as well as Yeh1p were present at low amount, became highly unstable compared to wild-type cells, and lost their enzymatic activity. Under these conditions both steryl ester hydrolases were retained in the endoplasmic reticulum. The lack of steryl esters alone was not sufficient to cause an altered intracellular localization of Tgl1p and Yeh1p. Surprisingly, the stability of Tgl1p and Yeh1p was markedly reduced in a strain lacking triacylglycerols, but their capacity to mobilize steryl esters remained unaffected. We also tested a possible cross-regulation of Tgl1p and Yeh1p by analyzing the behavior of each hydrolase in the absence of its counterpart steryl ester hydrolases. In summary, this study demonstrates a strong regulation of the two lipid droplet associated steryl ester hydrolases Tgl1p and Yeh1p due to the presence/absence of their host organelle. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Cytotoxic cardiac glycosides and other compounds from Asclepias syriaca.

    Science.gov (United States)

    Araya, Juan J; Kindscher, Kelly; Timmermann, Barbara N

    2012-03-23

    Phytochemical investigation of the dried biomass of Asclepias syriaca afforded five new compounds (1-5), along with 19 known structures. Overall, the secondary metabolites isolated and identified from this plant showed a wide structural diversity including pentacyclic triterpenes, cardiac glycosides, flavonoid glycosides, lignans, a phenylethanoid, and a glycosylated megastigmane. In addition, the isolates were tested against the cancer breast cell line Hs578T, and those showing IC(50) values lower than 50 μM (1 and 6-9) were further investigated in three additional breast cancer cell lines (MCF-7, T47D, and Sk-Br-3) and the normal breast cell line Hs578Bst.

  3. Radiation-induced O-glycoside bond scission in carbohydrates

    International Nuclear Information System (INIS)

    Kisel', R.M.

    2005-01-01

    Regularities in formation of products resulting from O-glycoside bond cleavage on radiolysis of aqueous solutions of (-methyl-D-glucopyranoside (I), 3-O-methylglucopyranose (II), maltose and lactose were studied. Oxygen and quinones were shown to inhibit radiation-induced homolytic destruction processes taking place in glycosides. The data obtained in this study enabled the authors to demonstrate an important role played by fragmentation reaction of C-2 radicals generated from the starting substances in formation of final radiolysis products. (authors)

  4. Phylogenetic diversity and environment-specific distributions of glycosyl hydrolase family 10 xylanases in geographically distant soils.

    Directory of Open Access Journals (Sweden)

    Guozeng Wang

    Full Text Available BACKGROUND: Xylan is one of the most abundant biopolymers on Earth. Its degradation is mediated primarily by microbial xylanase in nature. To explore the diversity and distribution patterns of xylanase genes in soils, samples of five soil types with different physicochemical characters were analyzed. METHODOLOGY/PRINCIPAL FINDINGS: Partial xylanase genes of glycoside hydrolase (GH family 10 were recovered following direct DNA extraction from soil, PCR amplification and cloning. Combined with our previous study, a total of 1084 gene fragments were obtained, representing 366 OTUs. More than half of the OTUs were novel (identities of <65% with known xylanases and had no close relatives based on phylogenetic analyses. Xylanase genes from all the soil environments were mainly distributed in Bacteroidetes, Proteobacteria, Acidobacteria, Firmicutes, Actinobacteria, Dictyoglomi and some fungi. Although identical sequences were found in several sites, habitat-specific patterns appeared to be important, and geochemical factors such as pH and oxygen content significantly influenced the compositions of xylan-degrading microbial communities. CONCLUSION/SIGNIFICANCE: These results provide insight into the GH 10 xylanases in various soil environments and reveal that xylan-degrading microbial communities are environment specific with diverse and abundant populations.

  5. Development and Properties of a Wax Ester Hydrolase in the Cotyledons of Jojoba Seedlings 1

    Science.gov (United States)

    Huang, Anthony H. C.; Moreau, Robert A.; Liu, Kitty D. F.

    1978-01-01

    The activity of a wax ester hydrolase in the cotyledons of jojoba (Simmondsia chinensis) seedlings increased drastically during germination, parallel to the development of the gluconeogenic process. The enzyme at its peak of development was obtained in association with the wax body membrane, and its properties were studied. It had an optimal activity at alkaline pH (8.5-9). The apparent Km value for N-methylindoxylmyristate was 93 μM. It was stable at 40 C for 30 min but was inactivated at higher temperature. Various divalent cations and ethylenediaminetetraacetate had little effect on the activity. p-Chloromercuribenzoate was a strong inhibitor of the enzyme activity, and its effect was reversed by subsequent addition of dithiothreitol. It had a broad substrate specificity with highest activities on monoglycerides, wax esters, and the native substrate (jojoba wax). PMID:16660288

  6. Development and properties of a wax ester hydrolase in the cotyledons of jojoba seedlings.

    Science.gov (United States)

    Huang, A H; Moreau, R A; Liu, K D

    1978-03-01

    The activity of a wax ester hydrolase in the cotyledons of jojoba (Simmondsia chinensis) seedlings increased drastically during germination, parallel to the development of the gluconeogenic process. The enzyme at its peak of development was obtained in association with the wax body membrane, and its properties were studied. It had an optimal activity at alkaline pH (8.5-9). The apparent K(m) value for N-methylindoxylmyristate was 93 muM. It was stable at 40 C for 30 min but was inactivated at higher temperature. Various divalent cations and ethylenediaminetetraacetate had little effect on the activity. p-Chloromercuribenzoate was a strong inhibitor of the enzyme activity, and its effect was reversed by subsequent addition of dithiothreitol. It had a broad substrate specificity with highest activities on monoglycerides, wax esters, and the native substrate (jojoba wax).

  7. Structure of XC6422 from Xanthomonas campestris at 1.6 Å resolution: a small serine α/β-hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Chao-Yu; Chin, Ko-Hsin [Institute of Biochemistry, National Chung-Hsing University, Taichung 40227,Taiwan (China); Chou, Chia-Cheng; Wang, Andrew H.-J. [Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei,Taiwan (China); Core Facility for Protein Crystallography, Academia Sinica, Nankang, Taipei,Taiwan (China); Chou, Shan-Ho, E-mail: shchou@nchu.edu.tw [Institute of Biochemistry, National Chung-Hsing University, Taichung 40227,Taiwan (China)

    2006-06-01

    The crystal structure of a conserved hypothetical protein from X. campestris has been determined to a resolution of 1.6 Å. The determined X. campestris structure shows that it belongs to the superfamily of serine α/β hydrolase, with an extra strand preceding the first β-strand to lead to extensive subunit interactions in the crystal. XC6422 is a conserved hypothetical protein from Xanthomonas campestris pathovar campestris (Xcc), a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. The protein consists of 220 amino acids and its structure has been determined to 1.6 Å resolution using the multi-wavelength anomalous dispersion (MAD) method. Although it has very low sequence identity to protein sequences in the PDB (less than 20%), the determined structure nevertheless shows that it belongs to the superfamily of serine α/β-hydrolases, with an active site that is fully accessible to solvent owing to the absence of a lid domain. Modelling studies with the serine esterase inhibitor E600 indicate that XC6422 adopts a conserved Ser-His-Asp catalytic triad common to this superfamily and has a preformed oxyanion hole for catalytic activation. These structural features suggest that XC6422 is most likely to be a hydrolase active on a soluble ester or a small lipid. An extra strand preceding the first β-strand in the canonical α/β-hydrolase fold leads to extensive subunit interactions between XC6422 monomers, which may explain why XC6422 crystals of good diffraction quality can grow to dimensions of up to 1.5 mm in a few days.

  8. Lysophosphatidylcholine hydrolases of human erythrocytes, lymphocytes, and brain: Sensitive targets of conserved specificity for organophosphorus delayed neurotoxicants

    International Nuclear Information System (INIS)

    Vose, Sarah C.; Holland, Nina T.; Eskenazi, Brenda; Casida, John E.

    2007-01-01

    Brain neuropathy target esterase (NTE), associated with organophosphorus (OP)-induced delayed neuropathy, has the same OP inhibitor sensitivity and specificity profiles assayed in the classical way (paraoxon-resistant, mipafox-sensitive hydrolysis of phenyl valerate) or with lysophosphatidylcholine (LysoPC) as the substrate. Extending our earlier observation with mice, we now examine human erythrocyte, lymphocyte, and brain LysoPC hydrolases as possible sensitive targets for OP delayed neurotoxicants and insecticides. Inhibitor profiling of human erythrocytes and lymphocytes gave the surprising result of essentially the same pattern as with brain. Human erythrocyte LysoPC hydrolases are highly sensitive to OP delayed neurotoxicants, with in vitro IC 50 values of 0.13-85 nM for longer alkyl analogs, and poorly sensitive to the current OP insecticides. In agricultural workers, erythrocyte LysoPC hydrolyzing activities are similar for newborn children and their mothers and do not vary with paraoxonase status but have high intersample variation that limits their use as a biomarker. Mouse erythrocyte LysoPC hydrolase activity is also of low sensitivity in vitro and in vivo to the OP insecticides whereas the delayed neurotoxicant ethyl n-octylphosphonyl fluoride inhibits activity in vivo at 1-3 mg/kg. Overall, inhibition of blood LysoPC hydrolases is as good as inhibition of brain NTE as a predictor of OP inducers of delayed neuropathy. NTE and lysophospholipases (LysoPLAs) both hydrolyze LysoPC, yet they are in distinct enzyme families with no sequence homology and very different catalytic sites. The relative contributions of NTE and LysoPLAs to LysoPC hydrolysis and clearance from erythrocytes, lymphocytes, and brain remain to be defined

  9. Poly(aspartic acid) (PAA) hydrolases and PAA biodegradation: current knowledge and impact on applications.

    Science.gov (United States)

    Hiraishi, Tomohiro

    2016-02-01

    Thermally synthesized poly(aspartic acid) (tPAA) is a bio-based, biocompatible, biodegradable, and water-soluble polymer that has a high proportion of β-Asp units and equivalent moles of D- and L-Asp units. Poly(aspartic acid) (PAA) hydrolase-1 and hydrolase-2 are tPAA biodegradation enzymes purified from Gram-negative bacteria. PAA hydrolase-1 selectively cleaves amide bonds between β-Asp units via an endo-type process, whereas PAA hydrolase-2 catalyzes the exo-type hydrolysis of the products of tPAA hydrolysis by PAA hydrolase-1. The novel reactivity of PAA hydrolase-1 makes it a good candidate for a biocatalyst in β-peptide synthesis. This mini-review gives an overview of PAA hydrolases with emphasis on their biochemical and functional properties, in particular, PAA hydrolase-1. Functionally related enzymes, such as poly(R-3-hydroxybutyrate) depolymerases and β-aminopeptidases, are compared to PAA hydrolases. This mini-review also provides findings that offer an insight into the catalytic mechanisms of PAA hydrolase-1 from Pedobacter sp. KP-2.

  10. Steviol glycosides in purified stevia leaf extract sharing the same metabolic fate.

    Science.gov (United States)

    Purkayastha, Sidd; Markosyan, Avetik; Prakash, Indra; Bhusari, Sachin; Pugh, George; Lynch, Barry; Roberts, Ashley

    2016-06-01

    The safety of steviol glycosides is based on data available on several individual steviol glycosides and on the terminal absorbed metabolite, steviol. Many more steviol glycosides have been identified, but are not yet included in regulatory assessments. Demonstration that these glycosides share the same metabolic fate would indicate applicability of the same regulatory paradigm. In vitro incubation assays with pooled human fecal homogenates, using rebaudiosides A, B, C, D, E, F and M, as well as steviolbioside and dulcoside A, at two concentrations over 24-48 h, were conducted to assess the metabolic fate of various steviol glycoside classes and to demonstrate that likely all steviol glycosides are metabolized to steviol. The data show that glycosidic side chains containing glucose, rhamnose, xylose, fructose and deoxy-glucose, including combinations of α(1-2), β-1, β(1-2), β(1-3), and β(1-6) linkages, were degraded to steviol mostly within 24 h. Given a common metabolite structure and a shared metabolic fate, safety data available for individual steviol glycosides can be used to support safety of purified steviol glycosides in general. Therefore, steviol glycosides specifications adopted by the regulatory authorities should include all steviol glycosides belonging to the five groups of steviol glycosides and a group acceptable daily intake established. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Molecular Cloning and Nucleotide Sequence of the Gene Encoding the Major Peptidoglycan Hydrolase of Lactococcus lactis, a Muramidase Needed for Cell Separation

    NARCIS (Netherlands)

    Buist, Girbe; Kok, Jan; Leenhouts, Kees J.; Dabrowska, Magdalena; Venema, Gerhardus; Haandrikman, Alfred J.

    A gene of Lactococcus lactis subsp, cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells,

  12. Inhibitory effects of Citrus hassaku extract and its flavanone glycosides on melanogenesis.

    Science.gov (United States)

    Itoh, Kimihisa; Hirata, Noriko; Masuda, Megumi; Naruto, Shunsuke; Murata, Kazuya; Wakabayashi, Keitaro; Matsuda, Hideaki

    2009-03-01

    The 50% ethanolic extract (CH-ext) obtained from the unripe fruit of Citrus hassaku exhibited significant tyrosinase inhibitory activity. The CH-ext showed antioxidant activity, such as superoxide dismutase (SOD)-like activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity. Activity-guided fractionation of the CH-ext indicated that flavanone glycoside-rich fractions showed potent tyrosinase inhibitory activity. Further examination revealed that the tyrosinase inhibitory activity and antioxidant activity of the CH-ext were attributable to naringin and neohesperidin, respectively. The CH-ext showed inhibition of melanogenesis without any effects on cell proliferation in cultured murine B16 melanoma cells after glucosamine exposure. The topical application of the CH-ext to the dorsal skin of brownish guinea pigs showed in vivo preventive effects against UVB-induced pigmentation.

  13. Crystallization of mouse S-adenosyl-l-homocysteine hydrolase

    International Nuclear Information System (INIS)

    Ishihara, Masaaki; Kusakabe, Yoshio; Ohsumichi, Tsuyoshi; Tanaka, Nobutada; Nakanishi, Masayuki; Kitade, Yukio; Nakamura, Kazuo T.

    2010-01-01

    Mouse S-adenosyl-l-homocysteine hydrolase has been crystallized in the presence of the reaction product adenosine. Diffraction data to 1.55 Å resolution were collected using synchrotron radiation. S-Adenosyl-l-homocysteine hydrolase (SAHH; EC 3.3.1.1) catalyzes the reversible hydrolysis of S-adenosyl-l-homocysteine to adenosine and l-homocysteine. For crystallographic investigations, mouse SAHH (MmSAHH) was overexpressed in bacterial cells and crystallized using the hanging-drop vapour-diffusion method in the presence of the reaction product adenosine. X-ray diffraction data to 1.55 Å resolution were collected from an orthorhombic crystal form belonging to space group I222 with unit-cell parameters a = 100.64, b = 104.44, c = 177.31 Å. Structural analysis by molecular replacement is in progress

  14. Evaluation of fish models of soluble epoxide hydrolase inhibition.

    OpenAIRE

    Newman, J W; Denton, D L; Morisseau, C; Koger, C S; Wheelock, C E; Hinton, D E; Hammock, B D

    2001-01-01

    Substituted ureas and carbamates are mechanistic inhibitors of the soluble epoxide hydrolase (sEH). We screened a set of chemicals containing these functionalities in larval fathead minnow (Pimphales promelas) and embryo/larval golden medaka (Oryzias latipes) models to evaluate the utility of these systems for investigating sEH inhibition in vivo. Both fathead minnow and medaka sEHs were functionally similar to the tested mammalian orthologs (murine and human) with respect to substrate hydrol...

  15. IMMOBILIZATION OF TANNIN ACYL HYDROLASE FROM ASPERGILLUS NIGER

    OpenAIRE

    B. Lenin Kumar*, N. Lokeswari and D. Sriramireddy

    2013-01-01

    ABSTRACT: Tannin acyl hydrolase, commonly referred to as tannase (E.C. 3.1.1.20), an inducible extra-cellular enzyme produced by a number of animals, plants and microbes. In this investigation, tannase production under solid-state fermentation by using Aspergillus niger and the waste residue of cashew husk was used as substrate for obtaining the desired fermented product. Microbial tannase is more stable than tannase from other sources like plants or animals. Tannase from fungal sources are r...

  16. Glycosidation of Methanol with Ribose: An Interdisciplinary Undergraduate Laboratory Experiment

    Science.gov (United States)

    Simon, Erin; Cook, Katie; Pritchard, Meredith R.; Stripe, Wayne; Bruch, Martha; Bendinskas, Kestutis

    2010-01-01

    This exercise provides students hands-on experience with the topics of glycosidation, hemiacetal and acetal formation, proton nuclear magnetic resonance ([superscript 1]H NMR) spectroscopy, and kinetic and thermodynamic product formation. In this laboratory experiment, the methyl acetal of ribose is synthesized, and the kinetic and thermodynamic…

  17. A new C-methylated flavonoid glycoside from Pinus densiflora.

    Science.gov (United States)

    Jung, M J; Choi, J H; Chung, H Y; Jung, J H; Choi, J S

    2001-12-01

    A new C-methyl flavonol glycoside, 5,7,8,4'-tetrahydroxy-3-methoxy-6-methylflavone 8-O-beta-D-glucopyranoside (1), has been isolated from the needles of Pinus densiflora, together with kaempferol 3-O-beta-(6"-acetyl)-galactopyranoside.

  18. Investigations of Reactive Carbohydrates in Glycosidic Bond Formation and Degradation

    DEFF Research Database (Denmark)

    Heuckendorff, Mads

    The overall objective of the research described in this thesis was to explore the field of glycosidic bond formation and degradation. In more detail, the objective was to do further research in the field of highly reactive glycosyl donors. New ways of making highly reactive donors were explored...

  19. Bottom-up elucidation of glycosidic bond stereochemistry

    DEFF Research Database (Denmark)

    Gray, Christopher J.; Schindler, Baptiste; Migas, Lukasz G.

    2017-01-01

    a particular challenge. Here, we show that "memory" of anomeric configuration is retained following gas-phase glycosidic bond fragmentation during tandem mass spectrometry (MS(2)). These findings allow for integration of MS(2) with ion mobility spectrometry (IM-MS(2)) and lead to a strategy to distinguish α...

  20. A New Flavone C-Glycoside from Gentiana lutea

    OpenAIRE

    Sachiko, Yamada; Rie, Kakuda; Yasunori, Yaoita; Masao, Kikuchi; Tohoku Pharmaceutical University; Tohoku Pharmaceutical University; Tohoku Pharmaceutical University; Tohoku Pharmaceutical University

    2005-01-01

    A new flavone C-glycoside, 6"-O-β-D-xylopyranosylisosaponarin (1), was isolated, together with four known compounds from the rhizomes and roots of Gentiana lutea. The structure of the new compound was elucidated on the basis of spectral data.

  1. (Anti)mutagenic and immunomodulatory properties of quercetin glycosides

    Czech Academy of Sciences Publication Activity Database

    Valentová, Kateřina; Šíma, Petr; Rybková, Z.; Křižan, Jiří; Malachová, K.; Křen, Vladimír

    2016-01-01

    Roč. 96, č. 5 (2016), s. 1492-1499 ISSN 0022-5142 R&D Projects: GA ČR(CZ) GAP301/11/0767; GA MŠk(CZ) LD14096 Institutional support: RVO:61388971 Keywords : quercetin glycosides * (anti)mutagenicity * mice Subject RIV: EE - Microbiology, Virology Impact factor: 2.463, year: 2016

  2. Reagents for the assay of cardenolide glycosides and aglycones

    International Nuclear Information System (INIS)

    Wilkinson, S.

    1976-01-01

    Some novel reagents are described for use in the radioimmunoassay of the 3-glycone derivatives of cardenolides (cardiac glycosides) and more especially digoxin, digitoxin, gitoxin, periplocin and lanatosides. Using these reagents these cardenolides and their derivatives may be assayed both in aqueous solution and in urine. A method is also described for performing such assays, including a suitable kit. (U.K.)

  3. Phenylpropanoid glycosides in Italian Orobanche spp., sect. Orobanche.

    Science.gov (United States)

    Serafini, M; Corazzi, G; Poli, F; Piccin, A; Tomassini, L; Foddai, S

    2005-09-01

    We studied the occurrence of phenylpropanoid glycosides (PhG) in five species of the genus Orobanche L., collected in the Latium region of Italy. The presence of orobanchoside and verbascoside in all four species confirms that these PhGs are taxonomic markers of the genus. The results suggest that O. gracilis form. citrina could be a diverse entity.

  4. A new phenylpropanoid glycoside from Jasminum subtriplinerve Blume.

    Science.gov (United States)

    Huong, Nguyen Thi Hong; Cu, Nguyen Khac Quynh; Quy, Trinh Van; Zidorn, Christian; Ganzera, Markus; Stuppner, Hermann

    2008-01-01

    From the ethyl acetate extract of the aerial parts of Jasminum subtriplinerve Blume (Oleaceae), 6'-O-menthiafoloylverbascoside (1), rutin (2), isoverbascoside (4), isooleoverbascoside (6), apiosylverbascoside (7), astragalin (9), isoquercitrin (10), and verbascoside (11) were isolated. Their structures were elucidated by extensive MS and NMR spectroscopy. Amongst 6'-O-menthiafoloylverbascoside (1) is a new phenylpropanoid glycoside.

  5. Three flavonol glycosides from Ricinus communis | Aqil | Bulletin of ...

    African Journals Online (AJOL)

    Bulletin of the Chemical Society of Ethiopia. Journal Home · ABOUT THIS JOURNAL · Advanced Search · Current Issue · Archives · Journal Home > Vol 11, No 1 (1997) >. Log in or Register to get access to full text downloads. Username, Password, Remember me, or Register. Three flavonol glycosides from Ricinus ...

  6. A New Phenyl Ethyl Glycoside from the Twigs of Acer tegmentosum.

    Science.gov (United States)

    Park, Seonju; Lee, Hwa Young; Nhiem, Nguyen Xuan; Lee, Taek Hwan; Kim, Nanyoung; Cho, Seung Hun; Kim, Seung Hyun

    2015-07-01

    One new phenyl ethyl glycoside, 2-(4-hydroxyphenyl)ethyl-O-α-L-arabinofuranosyl-(1 --> 6)-O-β-D-glucopyranoide (1) and 11 known compounds (2-12) were isolated from the twigs of Acer tegmentosum. Compound 6 showed potent anti-neuroinflammatory activity against the LPS-stimulated BV-2 microglial cells with tNO production of 25.0 ± 2.5 μM and TNF-α concentration of 617.6 ± 47.1 pg/mL at 30 μM.

  7. Cardiac Glycoside Glucoevatromonoside Induces Cancer Type-Specific Cell Death

    Directory of Open Access Journals (Sweden)

    Naira F. Z. Schneider

    2018-03-01

    Full Text Available Cardiac glycosides (CGs are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV out of 46 CGs for its low nanomolar anti-lung cancer activity. GEV presented reduced toxicity toward non-cancerous cell types (lung MRC-5 and PBMC and high-affinity binding to the Na+/K+-ATPase α subunit, assessed by computational docking. GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A549 cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy. In the presence of GEV, markers of autophagic flux (e.g. LC3I-II conversion were impacted, even in presence of bafilomycin A1. Cell death induction remained unaffected by calpain, cathepsin, parthanatos, or necroptosis inhibitors. Interestingly, GEV triggered caspase-dependent apoptosis in U937 acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. Differential cell cycle modulation by this CG led to a G2/M arrest, cyclin B1 and p53 downregulation in A549, but not in U937 cells. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings in vivo by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities.

  8. Separation of three anthraquinone glycosides including two isomers by preparative high-performance liquid chromatography and high-speed countercurrent chromatography from Rheum tanguticum Maxim. ex Balf.

    Science.gov (United States)

    Chen, Tao; Li, Hongmei; Zou, Denglang; Liu, Yongling; Chen, Chen; Zhou, Guoying; Li, Yulin

    2016-08-01

    Anthraquinone glycosides, such as chrysophanol 1-O-β-d-glucoside, chrysophanol 8-O-β-d-glucoside, and physion 8-O-β-d-glucoside, are the accepted important active components of Rheum tanguticum Maxim. ex Balf. due to their pharmacological properties: antifungal, antimicrobial, cytotoxic, and antioxidant activities. However, an effective method for the separation of the above-mentioned anthraquinone glycosides from this herb is not currently available. Especially, greater difficulty existed in the separation of the two isomers chrysophanol 1-O-β-d-glucoside and chrysophanol 8-O-β-d-glucoside. This study demonstrated an efficient strategy based on preparative high-performance liquid chromatography and high-speed countercurrent chromatography for the separation of the above-mentioned anthraquinone glycosides from Rheum tanguticum Maxim.ex Balf. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Identification of the chain-dispersing peptidoglycan hydrolase LytB of Streptococcus gordonii.

    Directory of Open Access Journals (Sweden)

    Riccardo Arrigucci

    Full Text Available Bacterial cell division ends with the separation of the daughter cells, a process that requires peptidoglycan hydrolases (PGHs. Bacteria lacking cell separating PGHs are impaired in cell separation with the formation of long chains or clusters. We identified a gene in Streptococcus gordonii encoding for a putative glucosaminidase (lytB. The lytB isogenic mutant grew in long bacterial chains and resulted in impaired biofilm formation. Purified recombinant LytB showed a murolytic activity on Micrococcus lysodeikticus cell suspension and was able to disperse the long chains of the mutant, restoring the wild type diplococci/short chain phenotype. LytB protein was localized only in culture supernatant cell fraction of S. gordonii, and co-cultures of wild type and lytB mutant showed a significant reduction of bacterial chain length, indicating that LytB is a secreted enzyme. Our results demonstrate that LytB is a secreted peptidoglycan hydrolase required for S. gordonii cell separation.

  10. Malbranchea cinnamomea: A thermophilic fungal source of catalytically efficient lignocellulolytic glycosyl hydrolases and metal dependent enzymes.

    Science.gov (United States)

    Mahajan, Chhavi; Basotra, Neha; Singh, Surender; Di Falco, Marcos; Tsang, Adrian; Chadha, B S

    2016-01-01

    This study reports thermophilic fungus Malbranchea cinnamomea as an important source of lignocellulolytic enzymes. The secretome analysis using LC-MS/MS orbitrap showed that fungus produced a spectrum of glycosyl hydrolases (cellulase/hemicellulase), polysaccharide lyases (PL) and carbohydrate esterases (CE) in addition to cellobiose dehydrogenase (CDH) indicating the presence of functional classical and oxidative cellulolytic mechanisms. The protein fractions in the secretome resolved by ion exchange chromatography were analyzed for ability to hydrolyze alkali treated carrot grass (ATCG) in the presence of Mn(2+)/Cu(2+). This strategy in tandem with peptide mass fingerprinting led to identification of metal dependent protein hydrolases with no apparent hydrolytic activity, however, showed 5.7 folds higher saccharification in presence of Mn(2+). Furthermore, adding different protein fractions to commercial cellulase (Novozymes: Cellic CTec2) resulted in enhanced hydrolysis of ATCG ranging between 1.57 and 3.43 folds indicating the enzymes from M. cinnamomea as catalytically efficient. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Flavonoid glycosides from Olax mannii: Structure elucidation and effect on the nuclear factor kappa B pathway.

    Science.gov (United States)

    Okoye, Festus B C; Sawadogo, Wamtinga Richard; Sendker, Jandirk; Aly, Amal H; Quandt, Bettina; Wray, Victor; Hensel, Andreas; Esimone, Charles O; Debbab, Abdessamad; Diederich, Marc; Proksch, Peter

    2015-12-24

    Olax mannii Oliv. (Olacaceae) is among the many medicinal plants used in Nigeria for the ethnomedicinal management of both cancer and inflammation. Such plants represent potential sources of innovative therapeutic agents for the treatment of cancer and other malignant disorders. While the majority of medicinal plants exert their anticancer effects by direct cytotoxicity on tumor cells, it is important that other mechanisms through which these plants can exhibit anticancer effects are investigated. Preliminary studies indicated that Olax mannii leaves are rich sources of novel flavonoid glycosides. The detailed chemistry as well the mechanisms through which these flavonoid constituents may exert their cancer chemo-preventive and therapeutic effects are, however, not yet investigated. The aim of this study is to carry out a detailed chemical investigation of Olax mannii leaves and the effects of the isolated constituents on the nuclear factor kappa B (NF-κB) pathway. A methanol leaf extract was subjected to various chromatographic separations to achieve isolation of flavonoid glycosides and the structures of the isolated compounds were elucidated by a combination of 1D and 2D NMR and high resolution mass spectrometry. Biological activities were assessed by measurement of cellular viability and proliferation using quantitative IncuCyte videomicroscopy, trypan blue staining and by quantification of the number of metabolically active K562 cells based on quantitation of ATP. The effect of the compounds on the inhibition of the NF-κB pathway as well as toxicity towards peripheral blood mononuclear cells to evaluate differential toxicity was also assayed. Chemical investigation of the methanol leaf extract of the plant material led to the isolation of three new flavonoid triglycosides, kaempferol 3-O-[α-D-apiofuranosyl-(1 → 2)-α-L-arabinofuranoside]-7-O-α-L-rhamnopyranoside (1), kaempferol 3-O-[β-D-glucopyranosyl-(1 → 2)-α-L-arabinofuranoside]-7-O

  12. Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Deacon, Ashley M.; Wilson, Ian A.

    2015-09-15

    ABSTRACT

    Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.

    IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural

  13. Stevia rebaudiana ethanolic extract exerts better antioxidant properties and antiproliferative effects in tumour cells than its diterpene glycoside stevioside.

    Science.gov (United States)

    López, Víctor; Pérez, Sergio; Vinuesa, Arturo; Zorzetto, Christian; Abian, Olga

    2016-04-01

    Steviol glycosides are currently being used as natural sweeteners by the food industry and Stevia rebaudiana has long been used as a sweet plant in South America for patients suffering from diabetes. In this study, a Stevia rebaudiana ethanolic extract (SREE) was prepared, analysed and tested for antioxidant activity in terms of free radical scavenging properties and antiproliferative effects in cervix (HeLa), pancreatic (MiaPaCa-2) and colonic (HCT116) cancer cells. The antiproliferative mechanism was confirmed by testing the effects on cyclin D1-CDK4. Bioassays were also performed for the diterpene glycoside stevioside. Our results demonstrate that the extract acts as an antioxidant being able to scavenge free radicals, but this activity was not due to stevioside. The extract also induced cell death in the three cell lines, being more active against cervix cancer cells (HeLa); however, the concentration of stevioside needed to produce antiproliferative effects was higher than the amount of steviol glycosides found in a lower dose of extract inducing cell death. In addition, the extract clearly inhibited CDK4 whereas stevioside did not, concluding that the antiproliferative activity of stevia may be due to inhibition of cyclin-dependent kinases performed by other compounds of the extract.

  14. ENZYMATIC KINETIC STUDY HYDROLASE FROM CITRUS

    Directory of Open Access Journals (Sweden)

    Israel Hernández

    2015-09-01

    Full Text Available In this paper the degrading activity of enzymes derived from orange peels (Citrus x sinensis, grapefruit (Citrus paradise and pineapple (Ananas comosus on the organic matter in wastewater is evaluated. This activity is measured indirectly by quantifying the biochemical oxygen demand (COD before and after degradation process based on a period of time using the HACH DR / 2010, and then the kinetic study was performed by the differential method and integral with the experimental data, obtaining a reaction order of 1 to pectinase (orange, and order 2 for bromelain (pineapple.

  15. Protective effect of total phenylethanoid glycosides from Monochasma savatieri Franch on myocardial ischemia injury.

    Science.gov (United States)

    Shi, Mengfan; He, Wenjun; Liu, Yanli; Li, Xiaoran; Yang, Shilin; Xu, Qiongming

    2013-11-15

    The present study was designed to investigate the cardioprotective effect of total phenylethanoid glycosides from Monochasma savatieri Franch (TPG). The data showed that there were mainly four phenylethanoid glycosides isolated and identified from TPG. TPG significantly increased cells viability and inhibited morphological changes on H9c2 cardiomyocytes induced by H2O2 or Na2S2O4. In addition, TPG significantly decreased T-wave elevation and histopathological changes of heart tissues in myocardial infracted rats induced by isoproterenol. It also significantly reduced the infarct size induced by ligating the coronary artery in rats, increased the activities of antioxidative enzymes superoxide dismutase (SOD), the content of glutathione (GSH), and decreased the leakage of lactic dehydrogenase (LDH), the activities of creatine kinase (CK) and the content of maleic dialdehyde (MDA). In conclusion, these results suggested that TPG from Monochasma savatieri Franch might be developed as new natural medicine or food additives with effects of prevention of coronary artery disease due to its significant antioxidant activity. Copyright © 2013 Elsevier GmbH. All rights reserved.

  16. ETHANOL PRECIPITATION OF GLYCOSYL HYDROLASES PRODUCED BY Trichoderma harzianum P49P11

    Directory of Open Access Journals (Sweden)

    M. A. Mariño

    2015-06-01

    Full Text Available AbstractThis study aimed to concentrate glycosyl hydrolases produced by Trichoderma harzianum P49P11 by ethanol precipitation. The variables tested besides ethanol concentration were temperature and pH. The precipitation with 90% (v/v ethanol at pH 5.0 recovered more than 98% of the xylanase activity, regard less of the temperature (5.0, 15.0, or 25.0 °C. The maximum recovery of cellulase activity as FPase was 77% by precipitation carried out at this same pH and ethanol concentration but at 5.0 °C. Therefore, ethanol precipitation can be considered to be an efficient technique for xylanase concentration and, to a certain extent, also for the cellulase complex.

  17. Studies on sterol-ester hydrolase from Fusarium oxysporum. I. Partial purification and properties.

    Science.gov (United States)

    Okawa, Y; Yamaguchi, T

    1977-05-01

    1. A search for a long chain fatty acyl sterol-ester hydrolase in microorganisms led to the isolation from soil of five strains belonging to Fusarium sp. which produced strong activity in the culture medium. 2. The cholesterol esterase from Fusarium oxysporum IGH-2 was purified about 270-fold by means of CaCl2 precipitation and Sephadex G-75 column chromatography. 3. The cholesterol esterase was activated by adekatol and Triton X-100. It was inhibited by lecithin and lysolecithin, and completely inactivated by heat treatment (60 degrees C for 30 min, at pH 7.0). 4. The optimum pH of the enzyme was found to be around 7.0. 5. Among various cholesterol esters tested, cholesterol linoleate was the most suitable substrate. 6. Cholesterol esters in serum were also hydrolyzed by this enzyme.

  18. Ability of human oral microbiota to produce wine odorant aglycones from odourless grape glycosidic aroma precursors.

    Science.gov (United States)

    Muñoz-González, Carolina; Cueva, Carolina; Ángeles Pozo-Bayón, M; Victoria Moreno-Arribas, M

    2015-11-15

    Grape aroma precursors are odourless glycosides that represent a natural reservoir of potential active odorant molecules in wines. Since the first step of wine consumption starts in the oral cavity, the processing of these compounds in the mouth could be an important factor in influencing aroma perception. Therefore, the objective of this work has been to evaluate the ability of human oral microbiota to produce wine odorant aglycones from odourless grape glycosidic aroma precursors previously isolated from white grapes. To do so, two methodological approaches involving the use of typical oral bacteria or the whole oral microbiota isolated from human saliva were followed. Odorant aglycones released in the culture mediums were isolated and analysed by HS-SPME-GC/MS. Results showed the ability of oral bacteria to hydrolyse grape aroma precursors, releasing different types of odorant molecules (terpenes, benzenic compounds and lipid derivatives). The hydrolytic activity seemed to be bacteria-dependent and was subject to large inter-individual variability. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Enzymatic Biosynthesis of Novel Resveratrol Glucoside and Glycoside Derivatives

    OpenAIRE

    Pandey, Ramesh Prasad; Parajuli, Prakash; Shin, Ju Yong; Lee, Jisun; Lee, Seul; Hong, Young-Soo; Park, Yong Il; Kim, Joong Su; Sohng, Jae Kyung

    2014-01-01

    A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d- and l-sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α-d-glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3-O-β-d-glucoside, resveratrol 4′-O-β-d-glucoside, resveratr...

  20. A Chalcone Glycoside from the Fruits of Sorbus commixta Hedl.

    Directory of Open Access Journals (Sweden)

    Kyu Yun Chai

    2009-12-01

    Full Text Available Sorbus commixta Hedl. (Rosaceae has been traditionally used in oriental countries for the treatment of asthma and other bronchial disorders. In this study, a chalcone glycoside was isolated from the ethyl acetate extract of the fruits of this plant. The compound was identified as neosakuranin based on the spectroscopic analysis and comparion with literature data. This is the first report of isolation of neosakuranin from Sorbus commixta.

  1. A new lignan glycoside from the rhizomes of Imperata cylindrica.

    Science.gov (United States)

    Lee, Dae-Young; Han, Kyung-Min; Song, Myoung-Chong; Lee, Do-Gyeong; Rho, Yeong-Deok; Baek, Nam-In

    2008-01-01

    A new lignan glycoside, 6-acetyl-1-[1,3-(4,4'-dihydroxy-3,3'-dimethoxy-beta-truxinyl)-beta-d-fructofuranosyl]-alpha-d-glucopyranoside (1), named impecyloside, was isolated from the rhizomes of Imperata cylindrica. The structure of the compound was determined by spectroscopic data including FABMS, UV, IR, 1H NMR and 13C NMR (DEPT) and 2D NMR (COSY, HSQC, HMBC).

  2. Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases.

    Science.gov (United States)

    Wheeler, Richard; Turner, Robert D; Bailey, Richard G; Salamaga, Bartłomiej; Mesnage, Stéphane; Mohamad, Sharifah A S; Hayhurst, Emma J; Horsburgh, Malcolm; Hobbs, Jamie K; Foster, Simon J

    2015-07-28

    Most bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology of Staphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan. Understanding bacterial growth and division is a fundamental problem, and knowledge in this area underlies the treatment of many infectious diseases. Almost all bacteria are surrounded by a macromolecule of peptidoglycan that encloses the cell and maintains shape, and bacterial cells must increase the size of this molecule in order to enlarge themselves. This requires not only the insertion of new peptidoglycan monomers, a process targeted by antibiotics, including penicillin, but also breakage of existing bonds, a potentially hazardous activity for the cell. Using Staphylococcus aureus, we have identified a set of enzymes that are critical for cellular enlargement. We

  3. S-Inosyl-L-Homocysteine Hydrolase, a Novel Enzyme Involved in S-Adenosyl-L-Methionine Recycling.

    Science.gov (United States)

    Miller, Danielle; Xu, Huimin; White, Robert H

    2015-07-01

    S-Adenosyl-L-homocysteine, the product of S-adenosyl-L-methionine (SAM) methyltransferases, is known to be a strong feedback inhibitor of these enzymes. A hydrolase specific for S-adenosyl-L-homocysteine produces L-homocysteine, which is remethylated to methionine and can be used to regenerate SAM. Here, we show that the annotated S-adenosyl-L-homocysteine hydrolase in Methanocaldococcus jannaschii is specific for the hydrolysis and synthesis of S-inosyl-L-homocysteine, not S-adenosyl-L-homocysteine. This is the first report of an enzyme specific for S-inosyl-L-homocysteine. As with S-adenosyl-L-homocysteine hydrolase, which shares greater than 45% sequence identity with the M. jannaschii homologue, the M. jannaschii enzyme was found to copurify with bound NAD(+) and has Km values of 0.64 ± 0.4 mM, 0.0054 ± 0.006 mM, and 0.22 ± 0.11 mM for inosine, L-homocysteine, and S-inosyl-L-homocysteine, respectively. No enzymatic activity was detected with S-adenosyl-L-homocysteine as the substrate in either the synthesis or hydrolysis direction. These results prompted us to redesignate the M. jannaschii enzyme an S-inosyl-L-homocysteine hydrolase (SIHH). Identification of SIHH demonstrates a modified pathway in this methanogen for the regeneration of SAM from S-adenosyl-L-homocysteine that uses the deamination of S-adenosyl-L-homocysteine to form S-inosyl-L-homocysteine. In strictly anaerobic methanogenic archaea, such as Methanocaldococcus jannaschii, canonical metabolic pathways are often not present, and instead, unique pathways that are deeply rooted on the phylogenetic tree are utilized by the organisms. Here, we discuss the recycling pathway for S-adenosyl-L-homocysteine, produced from S-adenosyl-L-methionine (SAM)-dependent methylation reactions, which uses a hydrolase specific for S-inosyl-L-homocysteine, an uncommon metabolite. Identification of the pathways and the enzymes involved in the unique pathways in the methanogens will provide insight into the

  4. Dysregulation of soluble epoxide hydrolase and lipidomic profiles in anorexia nervosa

    KAUST Repository

    Shih, P. B.

    2015-03-31

    Individuals with anorexia nervosa (AN) restrict eating and become emaciated. They tend to have an aversion to foods rich in fat. Because epoxide hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product, soluble epoxide hydrolase (sEH), converts bioactive epoxides of polyunsaturated fatty acid (PUFA) to the corresponding diols, lipidomic and metabolomic targets of EPHX2 were assessed to evaluate the biological functions of EPHX2 and their role in AN. Epoxide substrates of sEH and associated oxylipins were measured in ill AN, recovered AN and gender- and race-matched controls. PUFA and oxylipin markers were tested as potential biomarkers for AN. Oxylipin ratios were calculated as proxy markers of in vivo sEH activity. Several free- and total PUFAs were associated with AN diagnosis and with AN recovery. AN displayed elevated n-3 PUFAs and may differ from controls in PUFA elongation and desaturation processes. Cytochrome P450 pathway oxylipins from arachidonic acid, linoleic acid, alpha-linolenic acid and docosahexaenoic acid PUFAs are associated with AN diagnosis. The diol:epoxide ratios suggest the sEH activity is higher in AN compared with controls. Multivariate analysis illustrates normalization of lipidomic profiles in recovered ANs. EPHX2 influences AN risk through in vivo interaction with dietary PUFAs. PUFA composition and concentrations as well as sEH activity may contribute to the pathogenesis and prognosis of AN. Our data support the involvement of EPHX2-associated lipidomic and oxylipin dysregulations in AN, and reveal their potential as biomarkers to assess responsiveness to future intervention or treatment.

  5. NMR Characterization of Flavanone Naringenin 7-O-Glycoside Diastereomer

    Directory of Open Access Journals (Sweden)

    SUN Li-juan

    2017-12-01

    Full Text Available To discriminate R and S flavanone glycoside using NMR, the mixture of R and S naringenin 7-O-glycoside was first isolated from Gleditsia sinensis. 1H and 13C NMR data of the mixture were recorded with 1H NMR, 13C NMR, 1H-1H COSY, 1H-13C HSQC and 1H-13C HMBC in DMSO-d6 solution. The two diastereomers were then separated with chiral chromatographic isolation, with their absolute configurations determined by circular dichroism. To avoid the disturbance of protons from glucose residues to dihydroflavonoid, 1H NMR spectra were acquired for pure R and S naringenin 7-O-glycoside and their mixture in CD3CN. The two diastereomers showed the largest proton chemical shift differences at the end group of glucose residue (H-1" with a chemical shift difference of 9.4 Hz. The OH-5 proton showed a chemical shift difference of 5.8 Hz. The chemical shift of the three protons on ring C were all influenced by configuration.

  6. Glycosidically bound flavor compounds of cape gooseberry (Physalis peruviana L.).

    Science.gov (United States)

    Mayorga, H; Knapp, H; Winterhalter, P; Duque, C

    2001-04-01

    The bound volatile fraction of cape gooseberry (Physalis peruviana L.) fruit harvested in Colombia has been examined by HRGC and HRGC-MS after enzymatic hydrolysis using a nonselective pectinase (Rohapect D5L). Forty bound volatiles could be identified, with 21 of them being reported for the first time in cape gooseberry. After preparative isolation of the glycosidic precursors on XAD-2 resin, purification by multilayer coil countercurrent chromatography and HPLC of the peracetylated glycosides were carried out. Structure elucidation by NMR, ESI-MS/MS, and optical rotation enabled the identification of (1S,2S)-1-phenylpropane-1,2-diol 2-O-beta-D-glucopyranoside (1) and p-menth-4(8)-ene-1,2-diol 1-O-alpha-L-arabinopyranosyl-(1-6)-beta-D-glucopyranoside (2). Both glycosides have been identified for the first time in nature. They could be considered as immediate precursors of 1-phenylpropane-1,2-diol and p-menth-4(8)-ene-1,2-diol, typical volatiles found in the fruit of cape gooseberry.

  7. Resolution of D- and L-glucoses by chiral N-octyl-beta-D-glycoside-Cu(II) complex adsorbed at the gas/liquid interface of small bubbles

    NARCIS (Netherlands)

    Sakai, M.; Miyazawa, K.; Jitsumatsu, H.; Kamio, K.; Mitsuiki, S.; Toh, N.; Sugihara, G.; Norde, W.

    2010-01-01

    A new technique of the jet drop method (JDM) was applied to a chiral molecular discrimination of optically active D- or L-glucose (guest) by chiral N-octyl-beta-D-glycoside (O beta DG)-Cu(II) complex (host) at the gas/liquid interface of small bubbles. The discrimination of glucoses as the guests is

  8. Identification and characterization of carprofen as a multitarget fatty acid amide hydrolase/cyclooxygenase inhibitor.

    Science.gov (United States)

    Favia, Angelo D; Habrant, Damien; Scarpelli, Rita; Migliore, Marco; Albani, Clara; Bertozzi, Sine Mandrup; Dionisi, Mauro; Tarozzo, Glauco; Piomelli, Daniele; Cavalli, Andrea; De Vivo, Marco

    2012-10-25

    Pain and inflammation are major therapeutic areas for drug discovery. Current drugs for these pathologies have limited efficacy, however, and often cause a number of unwanted side effects. In the present study, we identify the nonsteroidal anti-inflammatory drug carprofen as a multitarget-directed ligand that simultaneously inhibits cyclooxygenase-1 (COX-1), COX-2, and fatty acid amide hydrolase (FAAH). Additionally, we synthesized and tested several derivatives of carprofen, sharing this multitarget activity. This may result in improved analgesic efficacy and reduced side effects (Naidu et al. J. Pharmacol. Exp. Ther.2009, 329, 48-56; Fowler, C. J.; et al. J. Enzyme Inhib. Med. Chem.2012, in press; Sasso et al. Pharmacol. Res.2012, 65, 553). The new compounds are among the most potent multitarget FAAH/COX inhibitors reported so far in the literature and thus may represent promising starting points for the discovery of new analgesic and anti-inflammatory drugs.

  9. Selective inhibition of plant serine hydrolases by agrochemicals revealed by competitive ABPP.

    Science.gov (United States)

    Kaschani, Farnusch; Nickel, Sabrina; Pandey, Bikram; Cravatt, Benjamin F; Kaiser, Markus; van der Hoorn, Renier A L

    2012-01-15

    Organophosphate and -phosphonates and their thio derivatives are often used in agroindustry as herbicides and insecticides, but their potential off-targets in the plant are poorly investigated. Here, we use competitive activity-based protein profiling (ABPP) of serine hydrolases (SHs) to detect targets of these agrochemicals and other compounds in Arabidopsis thaliana. Using broad-range and specific probes, and by overexpression of various SHs in planta, we are able to confirm eight SH-compound interactions, including selective inhibition of carboxylesterase CXE12, prolyloligopeptidase, methylesterase MES2 and tripeptidyl peptidase TPP2. These observations can be used for the design of novel probes and selective inhibitors and may help to assess physiological effects of agrochemicals on crop plants. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Discovery and characterization of thermophilic limonene-1,2-epoxide hydrolases from hot spring metagenomic libraries

    DEFF Research Database (Denmark)

    Ferrandi, Erica Elisa; Sayer, Christopher; Isupov, Michail N.

    2015-01-01

    thermophilic sources, have higher optimal temperatures and apparent melting temperatures than Re-LEH. The new LEH enzymes have been crystallized and their structures solved to high resolution in the native form and in complex with the inhibitor valpromide for Tomsk-LEH and poly(ethylene glycol) for CH55-LEH......,2-epoxide hydrolase (LEH) family of enzymes. These two LEHs (Tomsk-LEH and CH55-LEH) show EH activities towards different epoxide substrates, differing in most cases from those previously identified for Rhodococcus erythropolis (Re-LEH) in terms of stereoselectivity. Tomsk-LEH and CH55-LEH, both from....... The structural analysis has provided insights into the LEH mechanism, substrate specificity and stereoselectivity of these new LEH enzymes, which has been supported by mutagenesis studies....

  11. Salicylic Acid Treatment Increases the Levels of Triterpene Glycosides in Black Cohosh (Actaea Racemosa) Rhizomes.

    Science.gov (United States)

    De Capite, Annette; Lancaster, Tyler; Puthoff, David

    2016-01-01

    Black cohosh (Actaea racemosa) serves as the host plant for the Appalachian azure butterfly, Celastrina neglectamajor. Overharvesting of Black cohosh for the dietary supplement industry may result in its extirpation, and may also cause the elimination of the dependent butterfly. One way to increase or maintain the number of host plants in forested environments would be to reduce the number harvested, for example by increasing the levels of the desired metabolites in Black cohosh rhizomes. The secondary metabolites actein and deoxyactein are triterpene glycosides and are among the compounds associated with the putative activity of Black cohosh extracts. Acetein and deoxyacetein are used to standardize Black cohosh supplements. To gain an understanding of mechanisms that may control actein and deoxyactein accumulation, Black cohosh rhizomes were treated with exogenous salicylic acid, jasmonic acid, or ethylene, or were mechanically wounded. Salicylic acid treatment significantly increased the levels of actein and deoxyactein in the rhizome of Black cohosh, suggesting that the synthesis of triterpene glycosides is controlled in part by salicylic acid. Using salicylic acid or related chemicals to increase the levels of actein and deoxyactein in rhizomes may help supply the supplement industry and, simultaneously, help conserve Black cohosh and species dependent upon it.

  12. A comparison of flavonoid glycosides by electrospray tandem mass spectrometry

    Science.gov (United States)

    March, Raymond E.; Lewars, Errol G.; Stadey, Christopher J.; Miao, Xiu-Sheng; Zhao, Xiaoming; Metcalfe, Chris D.

    2006-01-01

    A comparison is presented of product ion mass spectra of protonated and deprotonated molecules of kaempferol-3-O-glucoside, quercitin-3-O-glucoside (isoquercitrin), quercitin-3-O-galactoside (hyperoin), apigenin-7-O-glucoside, luteolin-7-O-glucoside, genistein-7-O-glucoside, naringenin-7-O-glucoside (prunin), luteolin-4'-O-glucoside, luteolin-6-C-glucoside (homoorientin, known also as isoorientin), apigenin-8-C-glucoside (vitexin), and luteolin-8-C-glucoside (orientin) together with the product ion mass spectrum of deprotonated kaempferol-7-O-glucoside. All isomeric ions were distinguishable on the basis of their product ion mass spectra. For protonated 3-O-, 7-O-, and 4'-O-glycosides at a collision energy of 46-47 eV, homolytic cleavage of the O-glycosidic bond yielded aglycon Y+ ions, whereas in deprotonated 3-O-, 7-O-, and 4'-O-glycosides, heterolytic and homolytic cleavage of the O-glycosidic bond yielded radical aglycon (Y-H)- and aglycon (Y-) ions. In each case, fragmentation of either the glycan or the aglycon or both was observed. For 6-C- and 8-C-glycosides at a collision energy of 46-47 eV, fragmentation was restricted almost exclusively to the glycan. For luteolin-6-C-glucoside, the integrity of the aglycon structure is preserved at the expense of the glycan for which some 30 fragmentations were observed. Breakdown curves were determined as a function of collision energy for protonated and deprotonated luteolin-6-C-glucoside. An attempt has been made to rationalize the product ion mass spectra derived from C-O- and C-C-luteolin glucosides in terms of computed structures that indicate significant intramolecular hydrogen bonding and rotation of the B-ring to form a coplanar luteolin structure. It is proposed that protonated and deprotonated luteolin-6-C-glucoside may afford examples of cooperative interactive bonding that plays a major role in directing fragmentation.

  13. Catalytic properties of IgMs with amylolytic activity isolated from patients with multiple sclerosis.

    Science.gov (United States)

    Ivanen, Dina R; Kulminskaya, Anna A; Shabalin, Konstantin A; Isaeva-Ivanova, Luydmila V; Ershova, Nadezhda A; Saveliev, Andrew N; Nevinsky, Gregory A; Neustroev, Kirill N

    2004-08-01

    Recently, amylolytic activity was detected in IgMs isolated from the sera of the patients with multiple sclerosis. All purified samples of IgM were electrophoretically homogenous and did not contain any co-purified a-amylase and a-glucosidase activities, in accordance with a set of criteria developed for abzymes. The amylolytic activity of abzymes was studied in the hydrolysis of p-nitrophenyl a-D-maltooligosaccharides with different degrees of polymerization from 1 to 8 by TLC and reverse-phase HPLC techniques. All IgM samples isolated from 54 patients with clinically definite multiple sclerosis demonstrated hydrolytic activity towards the above artificial substrates. The Michaelis constant values (Km) in the hydrolysis of p-nitrophenyl a-D-maltoheptaoside were in the range of 10 p-nitrophenyl or p-nitrophenyl a-D-glucosides, thus indicating the presence of an a-D-glucosidase activity. For a number of the investigated samples, specific amylolytic activity increased depending on the length of substrates (from p-nitrophenyl maltopentaoside to p-nitrophenyl maltohexaoside); for other IgMs, the opposite dependence was observed. All IgMs studied did not exhibit any other glycoside hydrolase activities toward p-nitrophenyl glycoside substrates. Abzyme fractions from different donors demonstrated catalytic heterogeneity in Michaelis-Menten parameters and different modes of action in the hydrolysis of p-nitrophenyl maltooligosaccharides. Enzymatic properties of the IgMs tested varied from human a-amylases. All investigated abzyme samples did not show transglycosylating ability.

  14. Degradation of Polyester Polyurethane by Bacterial Polyester Hydrolases

    Directory of Open Access Journals (Sweden)

    Juliane Schmidt

    2017-02-01

    Full Text Available Polyurethanes (PU are widely used synthetic polymers. The growing amount of PU used industrially has resulted in a worldwide increase of plastic wastes. The related environmental pollution as well as the limited availability of the raw materials based on petrochemicals requires novel solutions for their efficient degradation and recycling. The degradation of the polyester PU Impranil DLN by the polyester hydrolases LC cutinase (LCC, TfCut2, Tcur1278 and Tcur0390 was analyzed using a turbidimetric assay. The highest hydrolysis rates were obtained with TfCut2 and Tcur0390. TfCut2 also showed a significantly higher substrate affinity for Impranil DLN than the other three enzymes, indicated by a higher adsorption constant K. Significant weight losses of the solid thermoplastic polyester PU (TPU Elastollan B85A-10 and C85A-10 were detected as a result of the enzymatic degradation by all four polyester hydrolases. Within a reaction time of 200 h at 70 °C, LCC caused weight losses of up to 4.9% and 4.1% of Elastollan B85A-10 and C85A-10, respectively. Gel permeation chromatography confirmed a preferential degradation of the larger polymer chains. Scanning electron microscopy revealed cracks at the surface of the TPU cubes as a result of enzymatic surface erosion. Analysis by Fourier transform infrared spectroscopy indicated that the observed weight losses were a result of the cleavage of ester bonds of the polyester TPU.

  15. Purification and Characterization of Tannin Acyl Hydrolase from Aspergillus niger ATCC 16620

    Directory of Open Access Journals (Sweden)

    Abdulhameed Sabu

    2005-01-01

    Full Text Available Tannin acyl hydrolase produced extracellularly by the fungal strain Aspergillus niger ATTC 16620 in solid state fermentation was purified from the cell free culture broth by ammonium sulphate fractionation followed by DEAE–Sephadex A-50 chromatography. SDS-PAGE analysis indicated that the enzyme protein molecular mass was 168 kDa. Enzyme activity was stable up to the temperature of 40 °C and the enzyme activity was optimal at pH=6. Tannase activity was maximal at 0.01 M concentration of the substrate. The addition of metal ions like Zn2+, Mn2+, Cu2+, Ca2+, Mg2+and Fe2+ inhibited the enzyme activity. Only K+ ions enhanced tannase activity, and an activity of 4.31 U/mL was reported here. Enzyme activity was maximal after 15–20 min of incubation time, with an activity of 3.9 U/mL. Km was found to be 1.03 mM and Vmax=4.25 mmol/min. Since the enzyme is active over a wide range of pH and temperature it could find potential use in the food-processing industry.

  16. The rate of uptake of cardiac glycosides into human cultured cells and the effects of chloroquine on it.

    Science.gov (United States)

    Algharably, N; Owler, D; Lamb, J F

    1986-10-15

    HeLa cells grown on Petri dishes were either pulse labelled with various cardiac glycosides or grown in low concentrations of them for up to 2 days; either in the presence of chloroquine or not. The cells were then homogenised and the cell free homogenate layered on a continuous sucrose gradient; and the glycoside content and that of various markers measured. In another series of experiments HeLa cells were grown on plastic beads under the above conditions and then the content of glycosides and of some marker enzymes measured. The rate of internalisation of ouabain, digoxin and digitoxin from the plasma membrane preparation produced by the bead method is at 9% hr-1, similar to the rate of loss of digoxin and digitoxin from whole cells but much faster than that of ouabain. In the sucrose gradient experiments it was found that [3H]ouabain, digoxin and digitoxin all initially co-distribute with the plasma membrane marker, 5'-nucleotidase, and then leave this fraction of the homogenate at a fast rate when kept at 37 degrees, to co-distribute with the lysosomal marker, beta-hexosaminidase. At 2 degrees the ouabain remains co-distributed with the plasma membrane marker. The rate of transfer is estimated to be some 90% hr-1, much faster than previously thought. Chloroquine causes an increased retention of digoxin and digitoxin in the lysosomal fraction of the homogenate. These results are best explained by supposing that the sodium pump-glycoside complex rapidly enters a region of the peripheral cytoplasm, and that this region then controls the subsequent exit of digoxin and digitoxin from the cell. The main barrier for ouabain occurs at a stage later than this. The consequences of this model on other aspects of pump activity is discussed.

  17. High production of succinyl isoflavone glycosides by Bacillus licheniformis ZSP01 resting cells in aqueous miscible organic medium.

    Science.gov (United States)

    Zhang, Sen; Chen, Guoguang; Chu, Jianlin; Wu, Bin; He, Bingfang

    2015-01-01

    To achieve efficient production of succinyldaidzin and succinylgenistin, resting cells of a solvent-stable strain Bacillus licheniformis ZSP01 were used to react with pure isoflavones or soybean flour extract in a reaction medium with 10% dimethyl sulfoxide. Strikingly, 0.8 mM daidzein, 0.8 mM genistein, 2.0 mM daidzin, and 2.0 mM genistin were transformed to succinyl isoflavone glycosides in 27 H (yield >90%). The soybean flour extract (6.1%, w/v) contained 0.32 mM daidzein, 0.84 mM daidzin, 0.38 mM genistein, and 1.04 mM genistin. Over 95% of total isoflavones (daidzein, daidzin, genistein, and genistin) in the soybean flour extract were converted to succinyl isoflavone glycosides after 27 H. Strain ZSP01 shows both high glycosylation and succinylation activities. These results suggest that B. licheniformis ZSP01 could be useful for the efficient production of succinyl soybean isoflavone glycosides. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  18. [Soil hydrolase characteristics in late soil-thawing period in subalpine/alpine forests of west Sichuan].

    Science.gov (United States)

    Tan, Bo; Wu, Fu-Zhong; Yang, Wan-Qin; Yu, Sheng; Yang, Yu-Lian; Wang, Ao

    2011-05-01

    Late soil-thawing period is a critical stage connecting winter and growth season. The significant temperature fluctuation at this stage might have strong effects on soil ecological processes. In order to understand the soil biochemical processes at this stage in the subalpine/alpine forests of west Sichuan, soil samples were collected from the representative forests including primary fir forest, fir and birch mixed forest, and secondary fir forest in March 5-April 25, 2009, with the activities of soil invertase, urease, and phosphatase (neutral, acid and alkaline phosphatases) measured. In soil frozen period, the activities of the three enzymes in test forests still kept relatively higher. With the increase of soil temperature, the activities of hydrolases at the early stage of soil-thawing decreased rapidly after a sharp increase, except for neutral phosphatease. Thereafter, there was an increase in the activities of urease and phosphatase. Relative to soil mineral layer, soil organic layer had higher hydrolase activity in late soil-thawing period, and showed more obvious responses to the variation of soil temperature.

  19. Overexpression of fatty acid amide hydrolase induces early flowering in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Neal D. Teaster

    2012-02-01

    Full Text Available N-Acylethanolamines (NAEs are bioactive lipids derived from the hydrolysis of the membrane phospholipid N-acylphosphatidylethanolamine (NAPE. In animal systems this reaction is part of the endocannabinoid signaling pathway, which regulates a variety of physiological processes. The signaling function of NAE is terminated by fatty acid amide hydrolase (FAAH, which hydrolyzes NAE to ethanolamine and free fatty acid. Our previous work in Arabidopsis thaliana showed that overexpression of AtFAAH (At5g64440 lowered endogenous levels of NAEs in seeds, consistent with its role in NAE signal termination. Reduced NAE levels were accompanied by an accelerated growth phenotype, increased sensitivity to abscisic acid (ABA, enhanced susceptibility to bacterial pathogens, and early flowering. Here we investigated the nature of the early flowering phenotype of AtFAAH overexpression. AtFAAH overexpressors flowered several days earlier than wild type and AtFAAH knockouts under both non-inductive short day (SD and inductive long day (LD conditions. Microarray analysis revealed that the FLOWERING LOCUS T (FT gene, which plays a major role in regulating flowering time, and one target MADS box transcription factor, SEPATALLA3 (SEP3, were elevated in AtFAAH overexpressors. Furthermore, AtFAAH overexpressors, with the early flowering phenotype had lower endogenous NAE levels in leaves compared to wild type prior to flowering. Exogenous application of NAE 12:0, which was reduced by up to 30% in AtFAAH overexpressors, delayed the onset of flowering in wild type plants. We conclude that the early flowering phenotype of AtFAAH overexpressors is, in part, explained by elevated FT gene expression resulting from the enhanced NAE hydrolase activity of AtFAAH, suggesting that NAE metabolism may participate in floral signaling pathways.

  20. New chalcanonol glycoside from the seeds of saw palmetto: antiproliferative and antioxidant effects.

    Science.gov (United States)

    Abdel Bar, Fatma M

    2015-01-01

    A new chalcanonol glycoside dimer, bis-O-[(I-4') → (II-6')]-α-hydroxyphloretin-2'-O-β-glucoside (1), in addition to six known compounds, namely (-)-epicatechin (2) and (-)-epiafzelechin (3), 4-hydroxybenzoic acid (4), protocatechuic acid (5), methylgallate (6), β-sitosterol (7) and β-sitosterol-3-O-glucoside (8), was isolated from the seeds of saw palmetto. The structures of the isolated compounds were established from the analysis of their MS and 1D and 2D NMR spectroscopic data. The antiproliferative activities of the isolated compounds towards PC3, the human prostate cancer cells were investigated. Amongst the isolated compounds, the new compound and the sterolic derivatives showed antiproliferative effects. Screening of the antioxidant effects of the isolated compounds by 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid radical assay revealed that the isolated phenolics were active free radical scavengers.

  1. Improved yields of daunomycinone glycosides in developmental mutants of Streptomyces coeruleorubidus

    International Nuclear Information System (INIS)

    Blumauerova, M.; Pokorny, V.; Stastna, J.; Hostalek, Z.; Vanek, Z.

    1978-01-01

    When improving Streptomyces coeruleorubidus JA 10092, a producer of antibiotics of the daunomycinone complex, the most active variants were found among isolates of morphological types bld-1 (with a suppressed production of the aerial mycelium on organic media containing glucose) and whi (with an asporogenic aerial mycelium on glucose media and with the bald phenotype on media containing starch). Submerged cultures of the whi mutants produced increased quantities of daunomycinone glycosides in the antibiotic complex, the amount of free anthracyclinones being simultaneously decreased. The whi strains differed from the wild type also in higher demands for aeration, concentration of glucose and in an increased production capacity in starch media. The overall antibiotic activity increased more than 40 times after a six-step selection (application of UV light, γ-radiation, nitrous acid and natural spreads) combined with an altered fermentation technology. (author)

  2. Perillanolides A and B, new monoterpene glycosides from the leaves of Perilla frutescens

    Directory of Open Access Journals (Sweden)

    Yang Liu

    Full Text Available Abstract Two new monoterpene glycosides, perillanolides A and B, together with a known compound reported from the genus Perilla for the first time were isolated and characterized from the leaves of Perilla frutescens (L. Britton, Lamiaceae, a garnish and colorant for foods as well as commonly used for traditional medicine. The structures of the isolated compounds were elucidated on the basis of extensive spectroscopic evidences derived from nuclear magnetic resonance experiments, mass spectrometry and by comparing their physical and spectroscopic data of literature. These compounds, together with the previously isolated secondary metabolites of this species, were investigated for their inhibitory effects on xanthine oxidase in vitro. Of the compounds, luteolin showed the strongest inhibitory activity with an IC50 value of 2.18 µM. Esculetin and scutellarein moderately inhibited the enzyme, while perillanolides A and B, and 4-(3,4-dihydroxybenzoyloxymethylphenyl-O-β-D-glucopyranoside exerted weak activities.

  3. Utilization of quercetin and quercetin glycosides from onion (Allium cepa L.) solid waste as an antioxidant, urease and xanthine oxidase inhibitors.

    Science.gov (United States)

    Nile, Shivraj Hariram; Nile, Arti Shivraj; Keum, Young Soo; Sharma, Kavita

    2017-11-15

    This study aimed to determine the flavonol glycosides from onion solid waste (OSW) using HPLC analysis, with antioxidant and enzyme inhibitory activities. We found considerable amount of quercetin-4'-O-monoglucoside (QMG: 254.85), quercetin-3,4'-O-diglucoside (QDG: 162.34), quercetin (Q: 60.44), and isorhamnetin-3-glucoside (IMG: 23.92) (mg/100g) dry weight (DW) of OSW. For OSW, the methanol and ethanol showed the strongest antioxidant activities, followed by ethyl acetate, chloroform, and n-hexane extracts. Among the flavonols, Q and QDG possessed higher antioxidant activities. OSW and flavonol glycosides displayed significant enzyme inhibitory activity, with IC 50 values ranging from 12.5±0.11 to 32.5±0.28 for OSW, 8.2±0.07 to 16.8±0.02 for flavonol glycosides, and 4.2±0.05μg/mL for thiourea (positive control) towards urease; while 15.2±0.8 to 35.8±0.2 (μg/mL) for OSW, 10.5±0.06 to 20.8±0.05 (μg/mL) for flavonol glycosides, and 6.5±0.05μg/mL for allopurinol (positive control) towards xanthine oxidase, respectively. The OSW and flavonol glycosides may thus be considered as potential antioxidant and antigout agents. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. A novel strategy for target profiling analysis of bioactive phenylethanoid glycosides in Plantago medicinal plants using ultra-performance liquid chromatography coupled with tandem quadrupole mass spectrometry.

    Science.gov (United States)

    Qi, Meng; Xiong, Aizhen; Geng, Fang; Yang, Li; Wang, Zhengtao

    2012-06-01

    Phenylethanoid glycosides are a group of phenolic compounds with diverse biological activities such as hypotensive, diuretic, and hypoglycemic effects. In this study, a target profiling analysis approach using ultra-performance liquid chromatography coupled with tandem quadrupole mass spectrometry (MS) was established on the basis of parent ion scanning for m/z 161, the characteristic product ion for phenylethanoid glycosides. It was successfully employed to discriminate the chemical composition of phenylethanoid glycosides between Plantaginis Herba and Plantaginis Semen, two medicinal parts of Plantago plants, which are widely used as herbal medicine in China. Totally, 34 phenylethanoid glycosides were characterized and tentatively identified by their retention times, MS, and tandem quadrupole MS (MS/MS) data. Combined with chemometrics analysis of principal component analysis and orthogonal projection to latent structural discriminate analysis, eight of them, especially acteoside and plantamajoside, were picked out and contributed to the chemical distinction between Plantaginis Herba and Plantaginis Semen, which might be responsible for the differences in diuretic and hypotensive effects between the two medicinal parts. This new approach for target profiling provides not only a novel idea for specific analysis of active chemical constituents in the same type, but also a promising and reference method for quality evaluation of traditional Chinese medicines. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Hepatotoxicity of kaurene glycosides from Xanthium strumarium L. fruits in mice.

    Science.gov (United States)

    Wang, Yang; Han, Ting; Xue, Li-Ming; Han, Ping; Zhang, Qiao-Yan; Huang, Bao-Kang; Zhang, Hong; Ming, Qian-Liang; Peng, Wei; Qin, Lu-Ping

    2011-06-01

    The fruit of Xanthium strumarium L. (Cang-Er-Zi) is a traditional Chinese medicine that is used in curing nasal diseases and headache according to the Chinese Pharmacopoeia. However, clinical utilization of Xanthium strumarium is relatively limited because of its toxicity. The present investigation was carried out to evaluate the toxic effects on acute liver injury in mice of the two kaurene glycosides (atractyloside and carbxyatractyloside), which are main toxic constituents isolated from Fructus Xanthii on acute liver injury in mice. Histopathological examinations revealed that there were not obviously visible injury in lungs, heart, spleen, and the central nervous system in the mice by intraperitoneal injection of atractyloside (ATR, at the doses 50,125 and 200 mg/kg) and carbxyatractyloside (CATR, at the doses 50,100 and 150 mg/kg) for 5 days. However, it revealed extensive liver injuries compared with the normal group. In the determination of enzyme levels in serum, intraperitoneal injection of ATR and CATR resulted in significantly elevated serum alanine aminotransferase (ALT), asparate aminotransferase (AST), alkaline phosphatase (ALP) activities compared to controls. In the hepatic oxidative stress level, antioxidant-related enzyme activity assays showed that ATR and CATR administration significantly increased hepatic malondialdehyde (MDA) concentration, as well as decreased superoxide dismutase (SOD), catalase (CAT) activities and glutathione (GSH) concentration, and this was in good agreement with the results of serum aminotransferase activity and histopathological examinations. Taken together, our results demonstrate that kaurene glycosides induce hepatotoxicity in mice by way of its induction of oxidative stress as lipid peroxidation in liver, which merited further studies. Therefore, these toxic constituents explain, at least in part, the hepatotoxicity of X. strumarium L. in traditional medicine.

  6. Steroidal glycosides from the bulbs of Easter lily (Lilium longiflorum Thunb.) promote dermal fibroblast migration in vitro.

    Science.gov (United States)

    Esposito, Debora; Munafo, John P; Lucibello, Teresa; Baldeon, Manuel; Komarnytsky, Slavko; Gianfagna, Thomas J

    2013-07-09

    Preparations derived from bulbs of various Lilium species have been used to promote the healing of skin abrasions, sores and burns and to aid in healing wounds in Traditional Chinese and Greco-Roman Medicine. To evaluate fractionated Easter lily bulb extracts and their steroidal glycosides (1-5) for the promotion of dermal fibroblast migration in vitro, a model for the early events in wound healing. An activity-guided screening approach was used by coupling sequential solvent extraction, gel permeation chromatography (GPC), and semi-preparative reverse-phase high performance liquid chromatography (RP-HPLC) with an in vitro dermal fibroblast migration assay. Cytotoxicity was evaluated with methyl thiazole tetrazolium (MTT). To gain insight into the mode of action of the steroidal glycosides, nitric oxide (NO) production, and expression of genes for transforming growth factor beta-1 (TGF-β) and its receptors were evaluated. Fractionated bulb extracts and the two isolated steroidal glycoalkaloids (1) and (2) induced NO production and TGF-β receptor I mRNA expression in fibroblast cell culture. In a cytotoxicity assay, steroidal glycosides (1) and (3) had IC50 values of 8.2 and 8.7 µM, but the natural acetylation of the C-6″' hydroxy of the terminal glucose unit in (2) resulted in a 3-fold decrease in cell cytotoxicity when compared with (1). Results from the dermal fibroblast migration assay revealed that the steroidal glycoalkaloids (1) and (2), and the furostanol saponin (3) promoted fibroblast migration from the range of 23.7±5.7 to 37.7±5.1%, as compared with the control. Collectively, our data demonstrate that the steroidal glycosides present in Easter lily bulbs induce, at least in part, the observed dermal fibroblast migration activity of the bulb extracts. This is the first evidence that steroidal glycosides from Lilium longiflorum may potentially play a role in the wound healing process and may provide a scientific basis for the historical use of lily

  7. Purification, crystallization and preliminary X-ray analysis of a thermostable glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08

    NARCIS (Netherlands)

    Rohman, Ali; van Oosterwijk, Niels; Kralj, Slavko; Dijkhuizen, Lubbert; Dijkstra, Bauke W.; Puspaningsih, Ni Nyoman Tri

    2007-01-01

    The main enzymes involved in xylan-backbone hydrolysis are endo-1,4-beta-xylanase and beta-xylosidase. beta-Xylosidase converts the xylo-oligosaccharides produced by endo-1,4-beta-xylanase into xylose monomers. The beta-xylosidase from the thermophilic Geobacillus thermoleovorans IT-08, a member of

  8. Additional New Minor Cucurbitane Glycosides from Siraitia grosvenorii

    Directory of Open Access Journals (Sweden)

    Indra Prakash

    2014-03-01

    Full Text Available Continuous phytochemical studies of the crude extract of Luo Han Guo (Siraitia grosvenorii furnished three additional new cucurbitane triterpene glycosides, namely 11-deoxymogroside V, 11-deoxyisomogroside V, and 11-deoxymogroside VI. The structures of all the isolated compounds were characterized on the basis of extensive NMR and mass spectral data as well as hydrolysis studies. The complete 1H- and 13C-NMR spectral assignments of the three unknown compounds are reported for the first time based on COSY, TOCSY, HSQC, and HMBC spectroscopic data.

  9. A New Iridoid Glycoside from the Roots of Dipsacus asper

    Directory of Open Access Journals (Sweden)

    Zhonglin Yang

    2012-02-01

    Full Text Available A new iridoid glycoside, named loganic acid ethyl ester (1, together with five known compounds: chlorogenic acid (2, caffeic acid (3, loganin (4, cantleyoside (5 and syringaresinol-4′,4′′-O-bis-β-D-glucoside (6 were isolated from the roots of Dipsacus asper. The structure of compound 1 was elucidated on the basis of detailed spectroscopic analyses. Lignan is isolated from Dipsacaceae species for the first time. Compounds 1, 4 and 5 had moderate neuroprotective effects against the Aβ25–35 induced cell death in PC12 cells.

  10. Radiation-induced glycoside bond breaking in cellulose methyl ethers

    International Nuclear Information System (INIS)

    Petryaev, E.P.; Boltromeyuk, V.V.; Kovalenko, N.I.; Shadyro, O.I.

    1988-01-01

    Radiation-induced destruction of cellulose methyl ethers of different degree of esterification in aqueous solutions with and without acceptors: (N 2 O, O 2 , H 2 O + , Co(2), Cu(2)) is investigated. It is established that OH radicals make main contribution into radiolytic transformations of cellulose ethers in aqueous solutions. Reactions of radicals with free valency on carbon atoms containing secondary nonsubstituted hydroxyl groups lead also to glycoside bond breaking besides the reaction of β-fragmentation and hydrolysis of radicals with an unpaired electron localized near C 1 , C 4 , C 5 aroms

  11. Radioimmunoassay method for the determination of cardiotonic glycosides

    International Nuclear Information System (INIS)

    1975-01-01

    A kit method for the in vitro determination of digoxin and digitoxin is described. The blood serum is mixed with the reagent which consists of an aqueous buffer solution containing a radiolabeled hapten for the glycoside. Antiserum with specific antibodies is added and the mixture is incubated. Thereafter, a thin strip of membrane mainly consisting of an ionexchanger is brought into contact with the mixture to separate the antibody bound hapten from the unbound hapten. The ratios of both are determined by counting the radioactive hapten

  12. Chemotaxonomy of Plantago. Iridoid glucosides and caffeoyl phenylethanoid glycosides

    DEFF Research Database (Denmark)

    Rønsted, N.; Göbel, E.; Franzyk, Henrik

    2000-01-01

    Data for 34 species of Plantago (Plantaginaceae), including subgen. Littorella (=Littorella uniflora), have been collected with regard to their content of iridoid glucosides and caffeoyl phenylethanoid glycosides (CPGs). In the present work, 21 species were investigated for the first time and man...... in the family. Finally, the close relationship between Plantago and Veronica suggested by chloroplast DNA sequence analysis, could be corroborated by the common occurrence of the rare 8,9-unsaturated iridoids in these two genera. (C) 2000 Elsevier Science Ltd. All rights reserved....

  13. A New Flavonoid Glycoside from Salix denticulata Aerial Parts

    Directory of Open Access Journals (Sweden)

    Amita Bamola

    2009-09-01

    Full Text Available Abstract: A new flavonoid glycoside (1 has been isolated from the aerial parts of Salix denticulata (Salicaceae together with five known compounds, β-sitosterol, 2,6-dihydroxy- 4-methoxy acetophenone, eugenol-1-O-β-D-glucopyranoside, 1-O-β-D-(3’-benzoyl salicyl alcohol and luteolin-7-O-β-D-glucopyranosyl-(1-6-glucopyranoside. The structure of 1 was elucidated as 2’,5-dihydroxy-3’-methoxyflavone-7-O-β-D-glucopyranoside by means of chemical and spectral data including 2D NMR studies.

  14. Aurantoside K, a New Antifungal Tetramic Acid Glycoside from a Fijian Marine Sponge of the Genus Melophlus

    Directory of Open Access Journals (Sweden)

    Rohitesh Kumar

    2012-01-01

    Full Text Available A new tetramic acid glycoside, aurantoside K, was isolated from a marine sponge belonging to the genus Melophlus. The structure of the compound was elucidated on the basis of spectroscopic analysis (1H NMR, 1H–1H COSY, HSQC, and HMBC, as well as high-resolution ESILCMS. Aurantoside K did not show any significant activity in antimalarial, antibacterial, or HCT-116 cytotoxicity assays, but exhibited a wide spectrum of antifungal activity against wild type Candida albicans, amphotericin-resistant C. albicans, Cryptococcus neoformans, Aspergillus niger, Penicillium sp., Rhizopus sporangia and Sordaria sp.

  15. Development and application of a fluorescent glucose uptake assay for the high-throughput screening of non-glycoside SGLT2 inhibitors.

    Science.gov (United States)

    Wu, Szu-Huei; Yao, Chun-Hsu; Hsieh, Chieh-Jui; Liu, Yu-Wei; Chao, Yu-Sheng; Song, Jen-Shin; Lee, Jinq-Chyi

    2015-07-10

    Sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors are of current interest as a treatment for type 2 diabetes. Efforts have been made to discover phlorizin-related glycosides with good SGLT2 inhibitory activity. To increase structural diversity and better understand the role of non-glycoside SGLT2 inhibitors on glycemic control, we initiated a research program to identify non-glycoside hits from high-throughput screening. Here, we report the development of a novel, fluorogenic probe-based glucose uptake system based on a Cu(I)-catalyzed [3+2] cycloaddition. The safer processes and cheaper substances made the developed assay our first priority for large-scale primary screening as compared to the well-known [(14)C]-labeled α-methyl-D-glucopyranoside ([(14)C]-AMG) radioactive assay. This effort culminated in the identification of a benzimidazole, non-glycoside SGLT2 hit with an EC50 value of 0.62 μM by high-throughput screening of 41,000 compounds. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. [Substrate specificities of bile salt hydrolase 1 and its mutants from Lactobacillus salivarius].

    Science.gov (United States)

    Bi, Jie; Fang, Fang; Qiu, Yuying; Yang, Qingli; Chen, Jian

    2014-03-01

    In order to analyze the correlation between critical residues in the catalytic centre of BSH and the enzyme substrate specificity, seven mutants of Lactobacillus salivarius bile salt hydrolase (BSH1) were constructed by using the Escherichia coli pET-20b(+) gene expression system, rational design and site-directed mutagenesis. These BSH1 mutants exhibited different hydrolytic activities against various conjugated bile salts through substrate specificities comparison. Among the residues being tested, Cys2 and Thr264 were deduced as key sites for BSH1 to catalyze taurocholic acid and glycocholic acid, respectively. Moreover, Cys2 and Thr264 were important for keeping the catalytic activity of BSH1. The high conservative Cys2 was not the only active site, other mutant amino acid sites were possibly involved in substrate binding. These mutant residues might influence the space and shape of the substrate-binding pockets or the channel size for substrate passing through and entering active site of BSH1, thus, the hydrolytic activity of BSH1 was changed to different conjugated bile salt.

  17. Regulation of synaptic structure by ubiquitin C-terminal hydrolase L1.

    Science.gov (United States)

    Cartier, Anna E; Djakovic, Stevan N; Salehi, Afshin; Wilson, Scott M; Masliah, Eliezer; Patrick, Gentry N

    2009-06-17

    Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We found that UCH-L1 activity is rapidly upregulated by NMDA receptor activation, which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of presynaptic and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1-inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling, most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner.

  18. Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites.

    Science.gov (United States)

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-05-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  19. Compositional profile of α/β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

    Science.gov (United States)

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-01-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. PMID:25171437

  20. Two new ent-kaurane-type diterpene glycosides from zucchini (Cucurbita pepo L.) seeds.

    Science.gov (United States)

    Kikuchi, Takashi; Ando, Hiromi; Maekawa, Ken-Ichiro; Arie, Hiroki; Yamada, Takeshi; Tanaka, Reiko

    2015-12-01

    Two new ent-kaurane diterpene glycosides; 12α-(β-d-glucopyranosyloxy)-7β-hydroxykaurenolide (1) and 7β-(β-d-glucopyranosyloxy)-12α-hydroxykaurenolide (2), a new steroid; (24S)-stigmasta-7,22E,25-trien-3-one (12), and known compounds (3-11, 13-14) were isolated from zucchini (Cucurbita pepo L.) seeds. The absolute structures of 1 and 2 were determined by acid hydrolysis and application of a modified Moscher's method. Furthermore, isolated compounds (1-14), and a derivative, 1a, were evaluated for their inhibitory effects on macrophage activation by an inhibitory assay of nitric oxide (NO) production. Copyright © 2015 Elsevier B.V. All rights reserved.