Identification and characterisation of a novel acylpeptide hydrolase from Sulfolobus solfataricus: structural and functional insights.

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Marta Gogliettino

Full Text Available A novel acylpeptide hydrolase, named APEH-3(Ss, was isolated from the hypertermophilic archaeon Sulfolobus solfataricus. APEH is a member of the prolyl oligopeptidase family which catalyzes the removal of acetylated amino acid residues from the N terminus of oligopeptides. The purified enzyme shows a homotrimeric structure, unique among the associate partners of the APEH cluster and, in contrast to the archaeal APEHs which show both exo/endo peptidase activities, it appears to be a "true" aminopeptidase as exemplified by its mammalian counterparts, with which it shares a similar substrate specificity. Furthermore, a comparative study on the regulation of apeh gene expression, revealed a significant but divergent alteration in the expression pattern of apeh-3(Ss and apeh(Ss (the gene encoding the previously identified APEH(Ss from S. solfataricus, which is induced in response to various stressful growth conditions. Hence, both APEH enzymes can be defined as stress-regulated proteins which play a complementary role in enabling the survival of S. solfataricus cells under different conditions. These results provide new structural and functional insights into S. solfataricus APEH, offering a possible explanation for the multiplicity of this enzyme in Archaea.

The Dichotomy of the Poly(ADP-Ribose Polymerase-Like Thermozyme from Sulfolobus solfataricus

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Maria Rosaria Faraone Mennella

2018-01-01

Full Text Available The first evidence of an ADP-ribosylating activity in Archaea was obtained in Sulfolobus solfataricus(strain MT-4 where a poly(ADP-ribose polymerase (PARP-like thermoprotein, defined with the acronymous PARPSso, was found. Similarly to the eukaryotic counterparts PARPSso cleaves beta-nicotinamide adenine dinucleotide to synthesize oligomers of ADP-ribose; cross-reacts with polyclonal anti-PARP-1 catalytic site antibodies; binds DNA. The main differences rely on the molecular mass (46.5 kDa and the thermophily of PARPSso which works at 80 °C. Despite the biochemical properties that allow correlating it to PARP enzymes, the N-terminal and partial amino acid sequences available suggest that PARPSso belongs to a different group of enzymes, the DING proteins, an item discussed in detail in this review.This finding makes PARPSso the first example of a DING protein in Archaea and extends the existence of DING proteins into all the biological kingdoms. PARPSsohas a cell peripheral localization, along with the edge of the cell membrane. The ADP-ribosylation reaction is reverted by a poly(ADP-ribose glycohydrolase-like activity, able to use the eukaryotic poly(ADP-ribose as a substrate too. Here we overview the research of (ADP-ribosylation in Sulfolobus solfataricus in the past thirty years and discuss the features of PARPSso common with the canonical poly(ADP-ribose polymerases, and the structure fitting with that of DING proteins.

Structure of the heterotrimeric PCNA from Sulfolobus solfataricus

International Nuclear Information System (INIS)

Williams, Gareth J.; Johnson, Kenneth; Rudolf, Jana; McMahon, Stephen A.; Carter, Lester; Oke, Muse; Liu, Huanting; Taylor, Garry L.; White, Malcolm F.; Naismith, James H.

2006-01-01

The structure of the heterotrimeric PCNA complex from S. sulfataricus is reported to 2.3 Å. PCNA is a ring-shaped protein that encircles DNA, providing a platform for the association of a wide variety of DNA-processing enzymes that utilize the PCNA sliding clamp to maintain proximity to their DNA substrates. PCNA is a homotrimer in eukaryotes, but a heterotrimer in crenarchaea such as Sulfolobus solfataricus. The three proteins are SsoPCNA1 (249 residues), SsoPCNA2 (245 residues) and SsoPCNA3 (259 residues). The heterotrimeric protein crystallizes in space group P2 1 , with unit-cell parameters a = 44.8, b = 78.8, c = 125.6 Å, β = 100.5°. The crystal structure of this heterotrimeric PCNA molecule has been solved using molecular replacement. The resulting structure to 2.3 Å sheds light on the differential stabilities of the interactions observed between the three subunits and the specificity of individual subunits for partner proteins.

Redox stress proteins are involved in adaptation response of the hyperthermoacidophilic archaeon Sulfolobus solfataricus to nickel challenge

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Scaloni Andrea

2007-08-01

Full Text Available Abstract Background Exposure to nickel (Ni and its chemical derivatives has been associated with severe health effects in human. On the contrary, poor knowledge has been acquired on target physiological processes or molecular mechanisms of this metal in model organisms, including Bacteria and Archaea. In this study, we describe an analysis focused at identifying proteins involved in the recovery of the archaeon Sulfolobus solfataricus strain MT4 from Ni-induced stress. Results To this purpose, Sulfolobus solfataricus was grown in the presence of the highest nickel sulphate concentration still allowing cells to survive; crude extracts from treated and untreated cells were compared at the proteome level by using a bi-dimensional chromatography approach. We identified several proteins specifically repressed or induced as result of Ni treatment. Observed up-regulated proteins were largely endowed with the ability to trigger recovery from oxidative and osmotic stress in other biological systems. It is noteworthy that most of the proteins induced following Ni treatment perform similar functions and a few have eukaryal homologue counterparts. Conclusion These findings suggest a series of preferential gene expression pathways activated in adaptation response to metal challenge.

Gene content and organization of a 281-kbp contig from the genome of the extremely thermophilic archaeon, Sulfolobus solfataricus P2

NARCIS (Netherlands)

Charlebois, R.; Confalonieri, F.; Curtis, B.; Doolittle, W.F.; Duguet, M.; Erauso, G.; Faguy, D.; Gaasterland, T.; Garrett, R.A.; Gordon, P.; Kozera, C.; Medina, N.; Oost, van der J.; Peng, X.; Ragan, M.; She, Q.; Singh, R.K.

2000-01-01

The sequence of a 281-kbp contig from the crenarchaeote Sulfolobus solfataricus P2 was determined and analysed. Notable features in this region include 29 ribosomal protein genes, 12 tRNA genes (four of which contain archaeal-type introns), operons encoding enzymes of histidine biosynthesis,

A New Pepstatin-Insensitive Thermopsin-Like Protease Overproduced in Peptide-Rich Cultures of Sulfolobus solfataricus

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Marta Gogliettino

2014-02-01

Full Text Available In this study, we gain insight into the extracellular proteolytic system of Sulfolobus solfataricus grown on proteinaceous substrates, providing further evidence that acidic proteases were specifically produced in response to peptide-rich media. The main proteolytic component was the previously isolated SsMTP (Sulfolobus solfataricus multi-domain thermopsin-like protease, while the less abundant (named SsMTP-1 one was purified, characterized and identified as the sso1175 gene-product. The protein revealed a multi-domain organization shared with the cognate SsMTP with a catalytic domain followed by several tandemly-repeated motifs. Moreover, both enzymes were found spread across the Crenarchaeota phylum and belonging to the thermopsin family, although segregated into diverse phylogenetic clusters. SsMTP-1 showed a 75-kDa molecular mass and was stable in the temperature range 50–90 °C, with optimal activity at 70 °C and pH 2.0. Serine, metallo and aspartic protease inhibitors did not affect the enzyme activity, designating SsMTP-1 as a new member of the pepstatin-insensitive aspartic protease family. The peptide-bond-specificity of SsMTP-1 in the cleavage of the oxidized insulin B chain was uncommon amongst thermopsins, suggesting that it could play a distinct, but cooperative role in the protein degradation machinery. Interestingly, predictions of the transmembrane protein topology of SsMTP and SsMTP-1 strongly suggest a possible contribution in signal-transduction pathways.

Mutations and Rearrangements in the Genome of Sulfolobus solfataricus P2

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Redder, P.; Garrett, R. A.

2006-01-01

The genome of Sulfolobus solfataricus P2 carries a larger number of transposable elements than any other sequenced genome from an archaeon or bacterium and, as a consequence, may be particularly susceptible to rearrangement and change. In order to gain more insight into the natures and frequencies...... of different types of mutation and possible rearrangements that can occur in the genome, the pyrEF locus was examined for mutations that were isolated after selection with 5-fluoroorotic acid. About two-thirds of the 130 mutations resulted from insertions of mobile elements, including insertion sequence (IS...... deletions, insertions, and a duplication, were observed, and about one-fifth of the mutations occurred elsewhere in the genome, possibly in an orotate transporter gene. One mutant exhibited a 5-kb genomic rearrangement at the pyrEF locus involving a two-step IS element-dependent reaction, and its boundaries...

Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase

DEFF Research Database (Denmark)

Jensen, Kaj Frank; Hansen, Michael Riis; Jensen, Kristine Steen

2015-01-01

The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2, was subjected to crystallographic, kinetic and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5...

Mutational analysis of divalent metal ion binding in the active site of class II α-mannosidase from sulfolobus solfataricus

DEFF Research Database (Denmark)

Hansen, Dennis K.; Webb, Helen; Nielsen, Jonas Willum

2015-01-01

Mutational analysis of Sulfolobus solfataricus class II α-mannosidase was focused on side chains that interact with the hydroxyls of the-1 mannosyl of the substrate (Asp-534) or form ligands to the active site divalent metal ion (His-228 and His-533) judged from crystal structures of homologous e......, although less dramatically with some activating metal ions. No major differences in the pH dependence between wild-type and mutant enzymes were found in the presence of different metal ions. The pH optimum was 5, but enzyme instability was observed at pH...

Identification and sequence analysis of Sulfolobus solfataricus purE and purK genes

DEFF Research Database (Denmark)

Sørensen, Iben Schildt; Dandanell, Gert

1997-01-01

From a genomic library of Sulfolobus solfataricus DSM1617 we have isolated and identified the purEK locus. Two open reading frames are identified as homologs of the purE and purK purine biosynthetic genes in Escherichia coli. The C-terminus of purE overlaps with the N-terminus of purK. When either...... of the genes is expressed from an E. coli promoter they can complement the corresponding purE and purK mutations in E. coli. PurE seems to be more closely related to eubacteria than to other archaea and to eukaryotes. Also the purK gene, which has not yet been found in other archaea, is more closely related...

Specificities and pH profiles of adenine and hypoxanthine-guanine-xanthine phosphoribosyltransferases (nucleotide synthases) of the thermoacidophile archaeon Sulfolobus solfataricus

DEFF Research Database (Denmark)

Hansen, Michael Riis; Jensen, Kristine Steen; Rasmussen, Mads Skytte

2014-01-01

Two open reading frames in the genome of Sulfolobus solfataricus (SSO2341 and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2341 was annotated as a gene (gpT-1) encoding a 6-oxopurine...... phosphoribosyltransferase (PRTase), the protein product turned out to be a PRTase highly specific for adenine and we suggest that the reading frame should be renamed apT. The other reading frame SSO2424 (gpT-2) proved to be a true 6-oxopurine PRTase active with hypoxanthine, xanthine and guanine as substrates, and we.......5, while maximal activity with xanthine was observed at pH 7.5. We discuss likely reasons why SSO2341 in S. solfataricus and similar open reading frames in other Crenarchaeota could not be identified as genes encoding APRTase....

Biochemical, transcriptional and translational evidences of the phenol-meta-degradation pathway by the hyperthermophilic Sulfolobus solfataricus 98/2.

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Alexia Comte

Full Text Available Phenol is a widespread pollutant and a model molecule to study the biodegradation of monoaromatic compounds. After a first oxidation step leading to catechol in mesophilic and thermophilic microorganisms, two main routes have been identified depending on the cleavage of the aromatic ring: ortho involving a catechol 1,2 dioxygenase (C12D and meta involving a catechol 2,3 dioxygenase (C23D. Our work aimed at elucidating the phenol-degradation pathway in the hyperthermophilic archaea Sulfolobus solfataricus 98/2. For this purpose, the strain was cultivated in a fermentor under different substrate and oxygenation conditions. Indeed, reducing dissolved-oxygen concentration allowed slowing down phenol catabolism (specific growth and phenol-consumption rates dropped 55% and 39%, respectively and thus, evidencing intermediate accumulations in the broth. HPLC/Diode Array Detector and LC-MS analyses on culture samples at low dissolved-oxygen concentration (DOC  =  0.06 mg x L(-1 suggested, apart for catechol, the presence of 2-hydroxymuconic acid, 4-oxalocrotonate and 4-hydroxy-2-oxovalerate, three intermediates of the meta route. RT-PCR analysis on oxygenase-coding genes of S. solfataricus 98/2 showed that the gene coding for the C23D was expressed only on phenol. In 2D-DIGE/MALDI-TOF analysis, the C23D was found and identified only on phenol. This set of results allowed us concluding that S. solfataricus 98/2 degrade phenol through the meta route.

Dynamic fluorescence studies of beta-glycosidase mutants from Sulfolobus solfataricus: effects of single mutations on protein thermostability.

Science.gov (United States)

Bismuto, Ettore; Febbraio, Ferdinando; Limongelli, Simona; Briante, Raffaella; Nucci, Roberto

2003-04-01

Multiple sequence alignment on 73 proteins belonging to glycosyl hydrolase family 1 reveals the occurrence of a segment (83-124) in the enzyme sequences from hyperthermophilic archaea bacteria, which is absent in all the mesophilic members of the family. The alignment of the known three-dimensional structures of hyperthermophilic glycosidases with the known ones from mesophilic organisms shows a similar spatial organizations of beta-glycosidases except for this sequence segment whose structure is located on the external surface of each of four identical subunits, where it overlaps two alpha-helices. Site-directed mutagenesis substituting N97 or S101 with a cysteine residue in the sequence of beta-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus caused some changes in the structural and dynamic properties as observed by circular dichroism in far- and near-UV light, as well as by frequency domain fluorometry, with a simultaneous loss of thermostability. The results led us to hypothesize an important role of the sequence segment present only in hyperthermophilic beta-glycosidases, in the thermal adaptation of archaea beta-glycosidases. The thermostabilization mechanism could occur as a consequence of numerous favorable ionic interactions of the 83-124 sequence with the other part of protein matrix that becomes more rigid and less accessible to the insult of thermal-activated solvent molecules. Copyright 2003 Wiley-Liss, Inc.

Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus

DEFF Research Database (Denmark)

tang, T. H.; Polacek, N.; Zywicki, M.

2005-01-01

By generating a specialized cDNA library from the archaeon Sulfolobus solfataricus, we have identified 57 novel small non-coding RNA (ncRNA) candidates and confirmed their expression by Northern blot analysis. The majority was found to belong to one of two classes, either antisense or antisense...... elements by inhibiting expression of the transposase mRNA. Surprisingly, the class of antisense RNAs also contained RNAs complementary to tRNAs or sRNAs (small-nucleolar-like RNAs). For the antisense-box ncRNAs, the majority could be assigned to the class of C/D sRNAs, which specify 2'-O-methylation sites...... on rRNAs or tRNAs. Five C/D sRNAs of this group are predicted to target methylation at six sites in 13 different tRNAs, thus pointing to the widespread role of these sRNA species in tRNA modification in Archaea. Another group of antisense-box RNAs, lacking typical C/D sRNA motifs, was predicted...

Allosteric regulation of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermophilic archaeon Sulfolobus solfataricus

DEFF Research Database (Denmark)

Jensen, Kaj Frank; Arent, Susan; Larsen, Sine

2005-01-01

The upp gene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed in Escherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH 5.5 when...... assayed at 60 °C. Enzyme activity was strongly stimulated by GTP and inhibited by CTP. GTP caused an approximately 20-fold increase in the turnover number kcat and raised the Km values for 5-phosphoribosyl-1-diphosphate (PRPP) and uracil by two- and >10-fold, respectively. The inhibition by CTP...... was complex as it depended on the presence of the reaction product UMP. Neither CTP nor UMP were strong inhibitors of the enzyme, but when present in combination their inhibition was extremely powerful. Ligand binding analyses showed that GTP and PRPP bind cooperatively to the enzyme and that the inhibitors...

SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2

DEFF Research Database (Denmark)

Erdmann, Susanne; Shah, Shiraz Ali; Garrett, Roger Antony

2013-01-01

Organisms of the crenarchaeal order Sulfolobales carry complex CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune systems. These systems are modular and show extensive structural and functional diversity, especially in their interference complexes. The primary...... targets are an exceptional range of diverse viruses, many of which propagate stably within cells and follow lytic life cycles without producing cell lysis. These properties are consistent with the difficulty of activating CRISPR spacer uptake in the laboratory, but appear to conflict with the high...... complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2...

The Sso7d protein of Sulfolobus solfataricus: in vitro relationship among different activities

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Annamaria Guagliardi

2002-01-01

Full Text Available The physiological role of the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus is unknown. In vitro studies have shown that Sso7d promotes annealing of complementary DNA strands (Guagliardi et al. 1997, induces negative supercoiling (Lopez-Garcia et al. 1998, and chaperones the disassembly and renaturation of protein aggregates in an ATP hydrolysis-dependent manner (Guagliardi et al. 2000. In this study, we examined the relationships among the binding of Sso7d to double-stranded DNA, its interaction with protein aggregates, and its ATPase activity. Experiments with 1-anilinonaphthalene-8-sulfonic acid as probe demonstrated that exposed hydrophobic surfaces in Sso7d are responsible for interactions with protein aggregates and double-stranded DNA, whereas the site of ATPase activity has a non-hydrophobic character. The interactions of Sso7d with double-stranded DNA and with protein aggregates are mutually exclusive events, suggesting that the disassembly activity and the DNA-related activities of Sso7d may be competitive in vivo. In contrast, the hydrolysis of ATP by Sso7d is independent of the binding of Sso7d to double-stranded DNA or protein aggregates.

Sulfolobus turreted icosahedral virus c92 protein responsible for the formation of pyramid-like cellular lysis structures.

Science.gov (United States)

Snyder, Jamie C; Brumfield, Susan K; Peng, Nan; She, Qunxin; Young, Mark J

2011-07-01

Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system described for DNA bacteriophages. This study investigated the STIV gene products required for pyramid formation in its host Sulfolobus solfataricus. Overexpression of STIV open reading frame (ORF) c92 in S. solfataricus alone is sufficient to produce the pyramid-like lysis structures in cells. Gene disruption of c92 within STIV demonstrates that c92 is an essential protein for virus replication. Immunolocalization of c92 shows that the protein is localized to the cellular membranes forming the pyramid-like structures.

Role of tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase.

Science.gov (United States)

Pennacchio, Angela; Esposito, Luciana; Zagari, Adriana; Rossi, Mosè; Raia, Carlo A

2009-09-01

A mutant of the thermostable NAD(+)-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp --> Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD(+) and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 A resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.

Replication termination and chromosome dimer resolution in the archaeon Sulfolobus solfataricus.

Science.gov (United States)

Duggin, Iain G; Dubarry, Nelly; Bell, Stephen D

2011-01-05

Archaea of the genus Sulfolobus have a single-circular chromosome with three replication origins. All three origins fire in every cell in every cell cycle. Thus, three pairs of replication forks converge and terminate in each replication cycle. Here, we report 2D gel analyses of the replication fork fusion zones located between origins. These indicate that replication termination involves stochastic fork collision. In bacteria, replication termination is linked to chromosome dimer resolution, a process that requires the XerC and D recombinases, FtsK and the chromosomal dif site. Sulfolobus encodes a single-Xer homologue and its deletion gave rise to cells with aberrant DNA contents and increased volumes. Identification of the chromosomal dif site that binds Xer in vivo, and biochemical characterization of Xer/dif recombination revealed that, in contrast to bacteria, dif is located outside the fork fusion zones. Therefore, it appears that replication termination and dimer resolution are temporally and spatially distinct processes in Sulfolobus.

Screening alpha-glucosidase and alpha-amylase inhibitors from natural compounds by molecular docking in silico.

Science.gov (United States)

Jhong, Chien-Hung; Riyaphan, Jirawat; Lin, Shih-Hung; Chia, Yi-Chen; Weng, Ching-Feng

2015-01-01

The alpha-glucosidase inhibitor is a common oral anti-diabetic drug used for controlling carbohydrates normally converted into simple sugars and absorbed by the intestines. However, some adverse clinical effects have been observed. The present study seeks an alternative drug that can regulate the hyperglycemia by down-regulating alpha-glucosidase and alpha-amylase activity by molecular docking approach to screen the hyperglycemia antagonist against alpha-glucosidase and alpha-amylase activities from the 47 natural compounds. The docking data showed that Curcumin, 16-hydroxy-cleroda-3,13-dine-16,15-olide (16-H), Docosanol, Tetracosanol, Antroquinonol, Berberine, Catechin, Quercetin, Actinodaphnine, and Rutin from 47 natural compounds had binding ability towards alpha-amylase and alpha-glucosidase as well. Curcumin had a better biding ability of alpha-amylase than the other natural compounds. Analyzed alpha-glucosidase activity reveals natural compound inhibitors (below 0.5 mM) are Curcumin, Actinodaphnine, 16-H, Quercetin, Berberine, and Catechin when compared to the commercial drug Acarbose (3 mM). A natural compound with alpha-amylase inhibitors (below 0.5 mM) includes Curcumin, Berberine, Docosanol, 16-H, Actinodaphnine/Tetracosanol, Catechin, and Quercetin when compared to Acarbose (1 mM). When taken together, the implication is that molecular docking is a fast and effective way to screen alpha-glucosidase and alpha-amylase inhibitors as lead compounds of natural sources isolated from medicinal plants. © 2015 International Union of Biochemistry and Molecular Biology.

Sulfolobus Turreted Icosahedral Virus c92 Protein Responsible for the Formation of Pyramid-Like Cellular Lysis Structures

DEFF Research Database (Denmark)

Snyder, Jamie C; Brumfield, Susan K; Peng, Nan

2011-01-01

Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system desc...... disruption of c92 within STIV demonstrates that c92 is an essential protein for virus replication. Immunolocalization of c92 shows that the protein is localized to the cellular membranes forming the pyramid-like structures.......Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system...... described for DNA bacteriophages. This study investigated the STIV gene products required for pyramid formation in its host Sulfolobus solfataricus. Overexpression of STIV open reading frame (ORF) c92 in S. solfataricus alone is sufficient to produce the pyramid-like lysis structures in cells. Gene...

Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries

DEFF Research Database (Denmark)

Becker, F.; Schnorr, K.; Wilting, R.

2004-01-01

To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less beta-lactamase ('bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The 'bla gene was cloned into a bacteriophage MU...... minitransposon enabling translational fusions between 'bla and target genes. Fusion of TnSig in the correct reading frame to a protein carrying transmembrane domains or signal peptides resulted in ampicillin resistance of the corresponding clone. Prokaryotic gene libraries from the alkaliphilic bacterium...... Bacillus halodurans C125 and the hyperthermophilic archaeon Sulfolobus solfataricus P2 were tagged with TnSig. The genomic sequences, which are publicly available (EMBL BA000004 and EMBL AE006641), were used for rapid open reading frame (ORF) identification and prediction of protein localisation...

Cloning a cDNA for the lysosomal alpha-glucosidase

NARCIS (Netherlands)

KONINGS, A.; HUPKES, P.; Versteeg, R.; Grosveld, G.; Reuser, A.; Galjaard, H.

1984-01-01

Messenger RNA was isolated from monkey testes and size-fractionated on sucrose gradients. In vitro translation of these mRNA fractions resulted in nascent, labeled alpha-glucosidase that could be precipitated with anti human alpha-glucosidase antiserum. A cDNA library was constructed from the most

Potential antiradical and alpha-glucosidase inhibitors from Ecklonia maxima (Osbeck) Papenfuss.

Science.gov (United States)

Rengasamy, Kannan R R; Aderogba, Mutalib A; Amoo, Stephen O; Stirk, Wendy A; Van Staden, Johannes

2013-11-15

Alpha-glucosidase inhibitors play a potential role in the treatment of type 2 diabetes by delaying glucose absorption in the small intestine. Ecklonia maxima, a brown alga which grows abundantly on the west coast of South Africa, is used to produce alginate, animal feed, nutritional supplements and fertilizer. The crude aqueous methanol extract, four solvent fractions and three phlorotannins: 1,3,5-trihydroxybenezene (phloroglucinol) (1), dibenzo [1,4] dioxine-2,4,7,9-tetraol (2) and hexahydroxyphenoxydibenzo [1,4] dioxine (eckol) (3) isolated from E. maxima were evaluated for antiradical and alpha-glucosidase inhibitory activities. All the phlorotannins tested had strong antioxidant activities on DPPH free radicals with EC50 values ranging from 0.008 to 0.128μM. Compounds 2 and 3 demonstrated stronger antioxidant activity and an alpha-glucosidase inhibitory property than positive controls. These results suggest that E. maxima could be a natural source of potent antioxidants and alpha-glucosidase inhibitors. This study could facilitate effective utilization of E. maxima as an oral antidiabetic drug or functional food ingredient with a promising role in the formulation of medicines and nutrition supplements. Copyright © 2013 Elsevier Ltd. All rights reserved.

Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers

DEFF Research Database (Denmark)

Guðbergsdóttir, Sóley Ruth; Deng, Ling; Chen, Zhengjun

2011-01-01

The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching...... individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different...... protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers...

Nutrient Content, Phytonutrient Composition, Alpha Amylase, Alpha Glucosidase Inhibition Activity and Antioxidant Activity of the Stoechospermum Marginatum Collected in Pre Monsoon Season

OpenAIRE

Reka Palanivel; Thahira Banu Azeez; Seethalakshmi Muthaya

2017-01-01

The objective of this study was to investigate the nutrient content, phytonutrient composition, physicochemical properties, alpha amylase and alpha glucosidase inhibition activity and antioxidant activity of the brown algae Stoechospermum marginatum collected from Gulf of Mannar, Tamil Nadu, India in pre monsoon season (June- September, 2015). Six and eight hours of ethanol and aqueous extract of Stoechospermum marginatum were used for phytonutrient screening, alpha amylase, alpha glucosidase...

Compound K Production from Red Ginseng Extract by β-Glycosidase from Sulfolobus solfataricus Supplemented with α-L-Arabinofuranosidase from Caldicellulosiruptor saccharolyticus.

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Kyung-Chul Shin

Full Text Available Ginsenoside compound K (C-K is attracting a lot of interest because of its biological and pharmaceutical activities, including hepatoprotective, antitumor, anti-wrinkling, and anti-skin aging activities. C-K has been used as the principal ingredient in skin care products. For the effective application of ginseng extracts to the manufacture of cosmetics, the PPD-type ginsenosides in ginseng extracts should be converted to C-K by enzymatic conversion. For increased yield of C-K from the protopanaxadiol (PPD-type ginsenosides in red-ginseng extract (RGE, the α-L-arabinofuranoside-hydrolyzing α-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus (CS-abf was used along with the β-D-glucopyranoside/α-L-arabinopyranoside-hydrolyzing β-glycosidase from Sulfolobus solfataricus (SS-bgly because SS-bgly showed very low hydrolytic activity on the α-L-arabinofuranoside linkage in ginsenosides. The optimal reaction conditions for C-K production were as follows: pH 6.0, 80°C, 2 U/mL SS-bgly, 3 U/mL CS-abf, and 7.5 g/L PPD-type ginsenosides in RGE. Under these optimized conditions, SS-bgly supplemented with CS-abf produced 4.2 g/L C-K from 7.5 g/L PPD-type ginsenosides in 12 h without other ginsenosides, with a molar yield of 100% and a productivity of 348 mg/L/h. To the best of our knowledge, this is the highest concentration and productivity of C-K from ginseng extract ever published in literature.

Nutrient Content, Phytonutrient Composition, Alpha Amylase, Alpha Glucosidase Inhibition Activity and Antioxidant Activity of the Stoechospermum Marginatum Collected in Pre Monsoon Season

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Reka Palanivel

2017-03-01

Full Text Available The objective of this study was to investigate the nutrient content, phytonutrient composition, physicochemical properties, alpha amylase and alpha glucosidase inhibition activity and antioxidant activity of the brown algae Stoechospermum marginatum collected from Gulf of Mannar, Tamil Nadu, India in pre monsoon season (June- September, 2015. Six and eight hours of ethanol and aqueous extract of Stoechospermum marginatum were used for phytonutrient screening, alpha amylase, alpha glucosidase inhibition activity and antioxidant activity. From the results of the study it is understood that Stoechospermum marginatum contain a high amount of carbohydrate, protein, crude fiber and phytonutrients like tannin, flavonoid, saponin, alkaloid, terpenoids, steroid and total phenolic content. The physicochemical properties namely Water absorption and Swelling power were very promising. Alpha amylase and alpha glucosidase inhibition activity was recorded to be high in both aqueous and ethanol extracts of eight hour extraction than in extracts taken from six hours extraction. Antioxidant activity was detected using DPPH, FRAP, beta carotene scavenging and H2O2 assay and found to have a high radical scavenging activity. Stoechospermum marginatum possess a valuable amount of total phenolic content and other phytonutrients and physicochemical properties, it may the reason for the potential inhibition of alpha amylase, alpha glucosidase and antioxidant activity. It is concluded from the study that the brown algae may be incorporated into foods to enhance their nutritional and therapeutic value.

The Role of alpha-Glucosidase in Germinating Barley Grains

DEFF Research Database (Denmark)

Stanley, Duncan; Rejzek, Martin; Næsted, Henrik

2011-01-01

The importance of alpha-glucosidase in the endosperm starch metabolism of barley (Hordeum vulgare) seedlings is poorly understood. The enzyme converts maltose to glucose (Glc), but in vitro studies indicate that it can also attack starch granules. To discover its role in vivo, we took complementa...

Allosteric regulation and communication between subunits in uracil phosphoribosyltransferase from Sulfolobus solfataricus

DEFF Research Database (Denmark)

Arent, Susan; Harris, Pernille; Jensen, Kaj Frank

2005-01-01

organisms. To understand the allosteric regulation, crystal structures were determined for S. solfataricus UPRTase in complex with UMP and with UMP and the allosteric inhibitor CTP. Also, a structure with UMP bound in half of the active sites was determined. All three complexes form tetramers but reveal...... to rearrangements in the quaternary structure imply that this residue plays a major role in regulation of the enzyme and in communication between subunits. The ribose ring of UMP adopts alternative conformations in the cis and trans subunits of the UPRTase-UMP tetramer with associated differences...

Cloning and Molecular Characterization of an Alpha-Glucosidase (MalH) from the Halophilic Archaeon Haloquadratum walsbyi.

Science.gov (United States)

Cuebas-Irizarry, Mara F; Irizarry-Caro, Ricardo A; López-Morales, Carol; Badillo-Rivera, Keyla M; Rodríguez-Minguela, Carlos M; Montalvo-Rodríguez, Rafael

2017-11-21

We report the heterologous expression and molecular characterization of the first extremely halophilic alpha-glucosidase (EC 3.2.1.20) from the archaeon Haloquadratum walsbyi . A 2349 bp region ( Hqrw_2071 ) from the Hqr. walsbyi C23 annotated genome was PCR-amplified and the resulting amplicon ligated into plasmid pET28b(+), expressed in E. coli Rosetta cells, and the resulting protein purified by Ni-NTA affinity chromatography. The recombinant protein showed an estimated molecular mass of 87 kDa, consistent with the expected value of the annotated protein, and an optimal activity for the hydrolysis of α-PNPG was detected at 40 °C, and at pH 6.0. Enzyme activity values were the highest in the presence of 3 M NaCl or 3-4 M KCl. However, specific activity values were two-fold higher in the presence of 3-4 M KCl when compared to NaCl suggesting a cytoplasmic localization. Phylogenetic analyses, with respect to other alpha-glucosidases from members of the class Halobacteria, showed that the Hqr. walsbyi MalH was most similar (up to 41%) to alpha-glucosidases and alpha-xylosidases of Halorubrum . Moreover, computational analyses for the detection of functional domains, active and catalytic sites, as well as 3D structural predictions revealed a close relationship with an E. coli YicI-like alpha-xylosidase of the GH31 family. However, the purified enzyme did not show alpha-xylosidase activity. This narrower substrate range indicates a discrepancy with annotations from different databases and the possibility of specific substrate adaptations of halophilic glucosidases due to high salinity. To our knowledge, this is the first report on the characterization of an alpha-glucosidase from the halophilic Archaea, which could serve as a new model to gain insights into carbon metabolism in this understudied microbial group.

Cloning and Molecular Characterization of an Alpha-Glucosidase (MalH from the Halophilic Archaeon Haloquadratum walsbyi

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Mara F. Cuebas-Irizarry

2017-11-01

Full Text Available We report the heterologous expression and molecular characterization of the first extremely halophilic alpha-glucosidase (EC 3.2.1.20 from the archaeon Haloquadratum walsbyi. A 2349 bp region (Hqrw_2071 from the Hqr. walsbyi C23 annotated genome was PCR-amplified and the resulting amplicon ligated into plasmid pET28b(+, expressed in E. coli Rosetta cells, and the resulting protein purified by Ni-NTA affinity chromatography. The recombinant protein showed an estimated molecular mass of 87 kDa, consistent with the expected value of the annotated protein, and an optimal activity for the hydrolysis of α-PNPG was detected at 40 °C, and at pH 6.0. Enzyme activity values were the highest in the presence of 3 M NaCl or 3–4 M KCl. However, specific activity values were two-fold higher in the presence of 3–4 M KCl when compared to NaCl suggesting a cytoplasmic localization. Phylogenetic analyses, with respect to other alpha-glucosidases from members of the class Halobacteria, showed that the Hqr. walsbyi MalH was most similar (up to 41% to alpha-glucosidases and alpha-xylosidases of Halorubrum. Moreover, computational analyses for the detection of functional domains, active and catalytic sites, as well as 3D structural predictions revealed a close relationship with an E. coli YicI-like alpha-xylosidase of the GH31 family. However, the purified enzyme did not show alpha-xylosidase activity. This narrower substrate range indicates a discrepancy with annotations from different databases and the possibility of specific substrate adaptations of halophilic glucosidases due to high salinity. To our knowledge, this is the first report on the characterization of an alpha-glucosidase from the halophilic Archaea, which could serve as a new model to gain insights into carbon metabolism in this understudied microbial group.

Differential antibiotic sensitivity determined by the large ribosomal subunit in thermophilic archaea.

OpenAIRE

Ruggero, D; Londei, P

1996-01-01

Hybrid ribosomes obtained by mixing the ribosomal subunits of the extremely thermophilic archaea Sulfolobus solfataricus and Desulfurococcus mobilis were tested for their sensitivity to selected antibiotics. It is shown that structural differences in the large ribosomal subunits determine qualitatively and quantitatively the patterns of response to alpha-sarcin and paromomycin in these species.

Conditions for gene disruption by homologous recombination of exogenous DNA into the Sulfolobus solfataricus genome

OpenAIRE

Albers, Sonja-Verena; Driessen, Arnold J. M.

2008-01-01

The construction of directed gene deletion mutants is an essential tool in molecular biology that allows functional studies on the role of genes in their natural environment. For hyperthermophilic archaea, it has been difficult to obtain a reliable system to construct such mutants. However, during the past years, systems have been developed for Thermococcus kodakarensis and two Sulfolobus species, S. ac...

Alterations of the transcriptome of Sulfolobus acidocaldarius by exoribonuclease aCPSF2.

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Birgit Märtens

Full Text Available Recent studies identified a 5´ to 3´ exoribonuclease termed Sso-RNase J in the crenarchaeon Sulfolobus solfataricus (Sso, which has been reclassified to the aCPSF2 (archaeal cleavage and polyadenylation specificity factor 2 group of β-CASP proteins. In this study, the Sso-aCPSF2 orthologue of Sulfolobus acidocaldarius (Saci-aCPSF2 was functionally characterized. Like Sso-aCPSF2, Saci-aCPSF2 degrades RNA with 5´ to 3´ directionality in vitro. To address the biological significance of Saci-aCPSF2, a deletion mutant was constructed, and the influence of Saci-aCPSF2 on the transcriptome profile was assessed employing high throughput RNA sequencing. This analysis revealed 560 genes with differential transcript abundance, suggesting a considerable role of this enzyme in RNA metabolism. In addition, bioinformatic analyses revealed several transcripts that are preferentially degraded at the 5´ end. This was exemplarily verified for two transcripts by Northern-blot analyses, showing for the first time that aCPSF2 proteins play a role in 5' to 3' directional mRNA decay in the crenarchaeal clade of Archaea.

Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase.

Science.gov (United States)

Jensen, Kaj Frank; Hansen, Michael Riis; Jensen, Kristine Steen; Christoffersen, Stig; Poulsen, Jens-Christian Navarro; Mølgaard, Anne; Kadziola, Anders

2015-04-14

The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2 was subjected to crystallographic, kinetic, and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5-phosphoribosyl-α-1-pyrophosphate (PRPP) and adenine or the product adenosine monophosphate (AMP) or the inhibitor adenosine diphosphate (ADP) in each active site. The individual subunit adopts an overall structure that resembles a 6-oxopurine phosphoribosyltransferase (PRTase) more than known APRTases implying that APRT functionality in Crenarchaeotae has its evolutionary origin in this family of PRTases. Only the N-terminal two-thirds of the polypeptide chain folds as a traditional type I PRTase with a five-stranded β-sheet surrounded by helices. The C-terminal third adopts an unusual three-helix bundle structure that together with the nucleobase-binding loop undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site. The inhibitor ADP binds like the product AMP with both the α- and β-phosphates occupying the 5'-phosphoribosyl binding site. The enzyme shows activity over a wide pH range, and the kinetic and ligand binding properties depend on both pH and the presence/absence of phosphate in the buffers. A slow hydrolysis of PRPP to ribose 5-phosphate and pyrophosphate, catalyzed by the enzyme, may be facilitated by elements in the C-terminal three-helix bundle part of the protein.

ORF Alignment: NC_002754 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available ture Of Glcv, The ... Abc-Atpase Of The Glucose Abc Transporter From ... ... ... Sulfolobus Solfataricus pdb|1OXV|B Chain B, Crystal ... Structure Of Glcv, The Abc-Atpase Of Th...folobus Solfataricus ... pdb|1OXU|C Chain C, Crystal Structure Of Glcv, The ... Abc-Atpase Of ...tructure Of Glcv, The Abc-Atpase Of The Glucose Abc ... Transporter From S...ulfolobus Solfataricus pdb|1OXU|A ... Chain A, Crystal Structure Of Glcv, The Abc-Atpase Of ...

ORF Alignment: NC_006348 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available ture Of Glcv, The ... Abc-Atpase Of The Glucose Abc Transporter From ... ... ... Sulfolobus Solfataricus pdb|1OXV|B Chain B, Crystal ... Structure Of Glcv, The Abc-Atpase Of Th...folobus Solfataricus ... pdb|1OXU|C Chain C, Crystal Structure Of Glcv, The ... Abc-Atpase Of ...tructure Of Glcv, The Abc-Atpase Of The Glucose Abc ... Transporter From S...ulfolobus Solfataricus pdb|1OXU|A ... Chain A, Crystal Structure Of Glcv, The Abc-Atpase Of ...

ORF Alignment: NC_006350 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available ture Of Glcv, The ... Abc-Atpase Of The Glucose Abc Transporter From ... ... ... Sulfolobus Solfataricus pdb|1OXV|B Chain B, Crystal ... Structure Of Glcv, The Abc-Atpase Of Th...folobus Solfataricus ... pdb|1OXU|C Chain C, Crystal Structure Of Glcv, The ... Abc-Atpase Of ...tructure Of Glcv, The Abc-Atpase Of The Glucose Abc ... Transporter From S...ulfolobus Solfataricus pdb|1OXU|A ... Chain A, Crystal Structure Of Glcv, The Abc-Atpase Of ...

Alpha amylase and Alpha glucosidase inhibitory effects of aqueous stem extract of Salacia oblonga and its GC-MS analysis

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Gladis Raja Malar Chelladurai

2018-05-01

Full Text Available ABSTRACT Our present investigation deals with the phytochemical screening, estimation of total flavonoids, terpenoids and tannin contents to evaluate the anti-diabetic activities of Salacia oblonga stem followed by GC-MS analysis. It explores the natural compounds and the potential α-amylase and α-glucosidase inhibitory actions of stem extracts. The aqueous stem extract was selected from other extracts (ethanol, acetone, petroleum ether and chloroform for the in vitro study of anti-diabetic activity by alpha amylase and alpha glucosidase inhibitory assays. The stem extract was also analyzed by gas chromatography mass spectrometry to identify the natural chemical components. Phytochemical analysis of aqueous stem extract showed major classes of secondary metabolites such as phenols, flavonoids, alkaloids, terpenoids, tannins, saponins. The total flavonoid, terpenoid, and tannin contents were quantified as 19.82±0.06 mg QE/g, 96.2±0.20 mg/g and 11.25±0.03 mg TAE/g respectively. The percentage inhibition of assays showed maximum inhibitory effects (59.46±0.04% and 68.51±0.01% at a concentration of 100 mg/mL. The IC50 values of stem extract was found to be 73.56 mg/mL and 80.90 mg/mL for alpha amylase and alpha glucosidase inhibition. Fifteen chemical constituents were found by GC-MS analysis. This study suggest the aqueous stem extract of Salacia oblonga might be considered as potential source of bio active constituents with excellent antidiabetic activity.

A novel Sulfolobus non-conjugative extrachromosomal genetic element capable of integration into the host genome and spreading in the presence of a fusellovirus

DEFF Research Database (Denmark)

Wang, Ying; Duan, Zhenhong; Zhu, Haojun

2007-01-01

An integrative non-conjugative extrachromosomal genetic element, denoted as pSSVi, has been isolated from a Sulfolobus solfataricus P2 strain and was characterized. This genetic element is a double-stranded DNA of 5740 bp in size and contains eight open reading frames (ORFs). It resembles members....... Interestingly, pSSVi encodes an SSV-type integrase which probably catalyzes the integration of its genome into a specific site (a tRNA(Arg) gene) in the S. solfataricus P2 genome. Like pSSVx, pSSVi can be packaged into a spindle-like viral particle and spread with the help of SSV1 or SSV2. In addition, both SSV......1 and SSV2 appeared to replicate more efficiently in the presence of pSSVi. Given the versatile genetic abilities, pSSVi appears to be well suited for a role in horizontal gene transfer....

Augmenting the Genetic Toolbox for Sulfolobus islandicus with a Stringent Positive Selectable Marker for Agmatine Prototrophy

Science.gov (United States)

Cooper, Tara E.; Krause, David J.

2013-01-01

Sulfolobus species have become the model organisms for studying the unique biology of the crenarchaeal division of the archaeal domain. In particular, Sulfolobus islandicus provides a powerful opportunity to explore natural variation via experimental functional genomics. To support these efforts, we further expanded genetic tools for S. islandicus by developing a stringent positive selection for agmatine prototrophs in strains in which the argD gene, encoding arginine decarboxylase, has been deleted. Strains with deletions in argD were shown to be auxotrophic for agmatine even in nutrient-rich medium, but growth could be restored by either supplementation of exogenous agmatine or reintroduction of a functional copy of the argD gene from S. solfataricus P2 into the ΔargD host. Using this stringent selection, a robust targeted gene knockout system was established via an improved next generation of the MID (marker insertion and unmarked target gene deletion) method. Application of this novel system was validated by targeted knockout of the upsEF genes involved in UV-inducible cell aggregation formation. PMID:23835176

alpha-Glucosidase-albumin conjugates: effect of chronic administration in mice

International Nuclear Information System (INIS)

Allen, T.M.; Murray, L.; Bhardwaj, D.; Poznansky, M.J.

1985-01-01

Enzyme albumin conjugates have been proposed as a means of increasing the efficacy of enzyme use in vivo and decreasing immune response to the enzyme. Particulate drug carriers, however, have a pronounced tendency to localize in the mononuclear phagocyte (reticuloendothelial) system. The authors have examined in mice the effect on phagocytic index, tissue distribution and organ size of continued administration of conjugates of alpha-glucosidase with either homologous or heterologous albumin. Mice received 10 X 2-mg injections of bovine serum albumin (BSA) or mouse serum albumin (MSA), either free, polymerized or conjugated with alpha-glucosidase. Experiments involving BSA had to be terminated before the end of the experiment because of anaphylaxis, but these reactions were less severe to the polymerized albumin than to free albumin. Free BSA, BSA polymer and BSA-enzyme conjugates all caused a decrease in phagocytic index after six injections. Mice receiving MSA showed no evidence of anaphylaxis, but mice receiving six or more injections of free MSA, MSA polymer or MSA-enzyme conjugate had significantly decreased phagocytic indices as compared to controls. Phagocytic indices had returned to normal by 7 days after the final injection. Tissue distribution of 125 I-labeled albumin preparations was determined in either naive or chronically injected mice

Purification, enzymatic characterization, and nucleotide sequence of a high-isoelectric-point alpha-glucosidase from barley malt

DEFF Research Database (Denmark)

Frandsen, T P; Lok, F; Mirgorodskaya, E

2000-01-01

in the transition state complex. Mass spectrometry of tryptic fragments assigned the 92-kD protein to a barley cDNA (GenBank accession no. U22450) that appears to encode an alpha-glucosidase. A corresponding sequence (HvAgl97; GenBank accession no. AF118226) was isolated from a genomic phage library using a c......High-isoelectric-point (pI) alpha-glucosidase was purified 7, 300-fold from an extract of barley (Hordeum vulgare) malt by ammonium sulfate fractionation, ion-exchange, and butyl-Sepharose chromatography. The enzyme had high activity toward maltose (k(cat) = 25 s(-1)), with an optimum at pH 4...

Long-term intravenous treatment of Pompe disease with recombinant human alpha-glucosidase from milk.

Science.gov (United States)

Van den Hout, Johanna M P; Kamphoven, Joep H J; Winkel, Léon P F; Arts, Willem F M; De Klerk, Johannes B C; Loonen, M Christa B; Vulto, Arnold G; Cromme-Dijkhuis, Adri; Weisglas-Kuperus, Nynke; Hop, Wim; Van Hirtum, Hans; Van Diggelen, Otto P; Boer, Marijke; Kroos, Marian A; Van Doorn, Pieter A; Van der Voort, Edwin; Sibbles, Barbara; Van Corven, Emiel J J M; Brakenhoff, Just P J; Van Hove, Johan; Smeitink, Jan A M; de Jong, Gerard; Reuser, Arnold J J; Van der Ploeg, Ans T

2004-05-01

Recent reports warn that the worldwide cell culture capacity is insufficient to fulfill the increasing demand for human protein drugs. Production in milk of transgenic animals is an attractive alternative. Kilogram quantities of product per year can be obtained at relatively low costs, even in small animals such as rabbits. We tested the long-term safety and efficacy of recombinant human -glucosidase (rhAGLU) from rabbit milk for the treatment of the lysosomal storage disorder Pompe disease. The disease occurs with an estimated frequency of 1 in 40,000 and is designated as orphan disease. The classic infantile form leads to death at a median age of 6 to 8 months and is diagnosed by absence of alpha-glucosidase activity and presence of fully deleterious mutations in the alpha-glucosidase gene. Cardiac hypertrophy is characteristically present. Loss of muscle strength prevents infants from achieving developmental milestones such as sitting, standing, and walking. Milder forms of the disease are associated with less severe mutations and partial deficiency of alpha-glucosidase. In the beginning of 1999, 4 critically ill patients with infantile Pompe disease (2.5-8 months of age) were enrolled in a single-center open-label study and treated intravenously with rhAGLU in a dose of 15 to 40 mg/kg/week. Genotypes of patients were consistent with the most severe form of Pompe disease. Additional molecular analysis failed to detect processed forms of alpha-glucosidase (95, 76, and 70 kDa) in 3 of the 4 patients and revealed only a trace amount of the 95-kDa biosynthetic intermediate form in the fourth (patient 1). With the more sensitive detection method, 35S-methionine incorporation, we could detect low-level synthesis of -glucosidase in 3 of the 4 patients (patients 1, 2, and 4) with some posttranslation modification from 110 kDa to 95 kDa in 1 of them (patient 1). One patient (patient 3) remained totally deficient with both detection methods (negative for cross

Alpha-glucosidase inhibitory and antiplasmodial properties of terpenoids from the leaves of Buddleja saligna Willd

Czech Academy of Sciences Publication Activity Database

Chukwujekwu, J. C.; Rengasamy, K.R.R.; de Kock, C. A.; Smith, P. J.; Poštová Slavětínská, Lenka; van Staden, J.

2016-01-01

Roč. 31, č. 1 (2016), s. 63-66 ISSN 1475-6366 Institutional support: RVO:61388963 Keywords : alpha-glucosidase * antidiabetic * antiplasmodial * Buddleja saligna * terpenoids Subject RIV: CC - Organic Chemistry Impact factor: 4.293, year: 2016

An isozyme of acid alpha-glucosidase with reduced catalytic activity for glycogen.

OpenAIRE

Beratis, N G; LaBadie, G U; Hirschhorn, K

1980-01-01

Both the common and a variant isozyme of acid alpha-glucosidase have been purified from a heterozygous placenta with CM-Sephadex, ammonium sulfate precipitation, dialysis, Amicon filtration, affinity chromatography by Sephadex G-100, and DEAE-cellulose chromatography. Three and two activity peaks, from the common and variant isozymes, respectively, were obtained by DEAE-cellulose chromatography using a linear NaCl gradient. The three peaks of activity of the common isozyme were eluted with 0....

An isozyme of acid alpha-glucosidase with reduced catalytic activity for glycogen.

Science.gov (United States)

Beratis, N G; LaBadie, G U; Hirschhorn, K

1980-03-01

Both the common and a variant isozyme of acid alpha-glucosidase have been purified from a heterozygous placenta with CM-Sephadex, ammonium sulfate precipitation, dialysis, Amicon filtration, affinity chromatography by Sephadex G-100, and DEAE-cellulose chromatography. Three and two activity peaks, from the common and variant isozymes, respectively, were obtained by DEAE-cellulose chromatography using a linear NaCl gradient. The three peaks of activity of the common isozyme were eluted with 0.08, 0.12, and 0.17 M NaCl, whereas the two peaks of the variant, with 0.01 and 0.06 M NaCl. The pH optimum and thermal denaturation at 57 degrees C were the same in all enzyme peaks of both isozymes. Rabbit antiacid alpha-glucosidase antibodies produced against the common isozyme were found to cross-react with both peaks of the variant isozyme. The two isozymes shared antigenic identity and had similar Km's with maltose as substrate. Normal substrate saturation kinetics were observed with the common isozyme when glycogen was the substrate, but the variant produced an S-shaped saturation curve indicating a phase of negative and positive cooperativity at low and high glycogen concentrations, respectively. The activity of the variant was only 8.6% and 19.2% of the common isozyme when assayed with nonsaturating and saturating concentrations of glycogen, respectively. A similar rate of hydrolysis of isomaltose by both isozymes was found indicating that the reduced catalytic activity of the variant isozyme toward glycogen is not the result of a reduced ability of this enzyme to cleave the alpha-1,6 linkages of glycogen.

Synergisms in Alpha-glucosidase Inhibition and Antioxidant Activity of Camellia sinensis L. Kuntze and Eugenia uniflora L. Ethanolic Extracts

Science.gov (United States)

Vinholes, Juliana; Vizzotto, Márcia

2017-01-01

Background: Camellia sinensis, the most consumed and popular beverages worldwide, and Eugenia uniflora, a Brazilian native species, have been already confirmed to have beneficial effects in the treatment of diabetes mellitus. However, their potential acting together against an enzyme linked to this pathology has never been exploited. Objective: The aim of this study was to evaluate the inhibitory properties of individual and combined ethanolic extracts of the leaves of C. sinensis and E. uniflora over alpha-glucosidase, a key digestive enzyme used on the Type 2 diabetes mellitus (T2DM) control. In addition, their inhibitory activity against 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) and peroxyl radicals was also assayed. Materials and Methods: Enzyme inhibition and antioxidant potential were assessed based on in vitro assays. Total phenolic compounds, carotenoids, and chlorophylls A and B were achieved using spectrophotometric methods. Results: E. uniflora was almost 40 times more active on alpha-glucosidase than C. sinensis and combined extracts showed a significant synergistic effect with an obtained IC50 value almost 5 times lower than the theoretical value. C. sinensis extract was twice more active than E. uniflora concerning DPPH•, in contrast, E. uniflora was almost 10 times more effective than C. sinensis on inhibition of peroxyl radicals with a significant synergistic effect for combined extracts. The extracts activities may be related with their phytochemicals, mainly phenolic compounds, and chlorophylls. Conclusion: Combined C. sinensis and E. uniflora ethanolic extracts showed synergistic effect against alpha-glucosidase and lipid peroxidation. These herbal combinations can be used to control postprandial hyperglycemia and can also provide antioxidant defenses to patients with T2DM. SUMMARY Alfa-glucosidase and antioxidant Interaction between Camellia sinensis L. Kuntze and Eugenia uniflora L. ethanolic extracts was investigated.Extracts showed

Synergisms in Alpha-glucosidase Inhibition and Antioxidant Activity of Camellia sinensis L. Kuntze and Eugenia uniflora L. Ethanolic Extracts.

Science.gov (United States)

Vinholes, Juliana; Vizzotto, Márcia

2017-01-01

Camellia sinensis , the most consumed and popular beverages worldwide, and Eugenia uniflora , a Brazilian native species, have been already confirmed to have beneficial effects in the treatment of diabetes mellitus. However, their potential acting together against an enzyme linked to this pathology has never been exploited. The aim of this study was to evaluate the inhibitory properties of individual and combined ethanolic extracts of the leaves of C. sinensis and E. uniflora over alpha-glucosidase, a key digestive enzyme used on the Type 2 diabetes mellitus (T2DM) control. In addition, their inhibitory activity against 2,2-diphenyl-1-picrylhydrazyl radical (DPPH • ) and peroxyl radicals was also assayed. Enzyme inhibition and antioxidant potential were assessed based on in vitro assays. Total phenolic compounds, carotenoids, and chlorophylls A and B were achieved using spectrophotometric methods. E. uniflora was almost 40 times more active on alpha-glucosidase than C. sinensis and combined extracts showed a significant synergistic effect with an obtained IC 50 value almost 5 times lower than the theoretical value. C. sinensis extract was twice more active than E. uniflora concerning DPPH • , in contrast, E. uniflora was almost 10 times more effective than C. sinensis on inhibition of peroxyl radicals with a significant synergistic effect for combined extracts. The extracts activities may be related with their phytochemicals, mainly phenolic compounds, and chlorophylls. Combined C. sinensis and E. uniflora ethanolic extracts showed synergistic effect against alpha-glucosidase and lipid peroxidation. These herbal combinations can be used to control postprandial hyperglycemia and can also provide antioxidant defenses to patients with T2DM. Alfa-glucosidase and antioxidant Interaction between Camellia sinensis L. Kuntze and Eugenia uniflora L. ethanolic extracts was investigated.Extracts showed synergistic effect over alpha-glucosidase and peroxyl radicals

In vitro alpha glucosidase inhibition and free-radical scavenging activity of propolis from Thai stingless bees in mangosteen orchard

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Boonyadist Vongsak

Full Text Available ABSTRACTThe chemical component and biological activity of propolis depend on flora area of bee collection and bee species. In the study, the propolis from three stingless bee species, Lepidotrigona ventralis Smith, Lepidotrigona terminata Smith, and Tetragonula pagdeni Schwarz, was collected in the same region of mangosteen garden from Thailand. Total phenolic content, alpha glucosidase inhibitory effect, and free-radical scavenging activity using FRAP, ABTS, DPPH assays were determined. The most potent activity of propolis extract was investigated for bioactive compounds and their quantity. The ethanol extract of T. pagdeni propolis had the highest total phenolic content 12.83 ± 0.72 g of gallic acid equivalents in 100 g of the extract, and the strongest alpha glucosidase inhibitory effect with the IC50 of 70.79 ± 6.44 µg/ml. The free-radical scavenging activity evaluated by FRAP, ABTS, DPPH assays showed the FRAP value of 279.70 ± 20.55 µmol FeSO4 equivalent/g extract and the IC50 of 59.52 ± 10.76 and 122.71 ± 11.76 µg/ml, respectively. Gamma- and alpha-mangostin from T. pagdeni propolis extract were isolated and determined for the biological activity. Gamma-mangostin exhibited the strongest activity for both alpha glucosidase inhibitory effect and free-radical scavenging activity. Using HPLC quantitative analysis method, the content of gamma- and alpha-mangostin in the extract was found to be 0.94 ± 0.01 and 2.77 ± 0.08% (w/w, respectively. These findings suggested that T. pagdeni propolis may be used as a more suitable raw material for nutraceutical and pharmaceutical products and these mangostin derivatives as markers.

Human acid alpha-glucosidase from rabbit milk has therapeutic effect in mice with glycogen storage disease type II

NARCIS (Netherlands)

A.G.A. Bijvoet (Agnes); A.J.J. Reuser (Arnold); H. van Hirtum (Hans); M.A. Kroos (Marian); E.H. van de Kamp; O. Schoneveld; P. Visser (Pim); J.P. Brakenhoff (Just); M. Weggeman (Miranda); E.J.J.M. van Corven (Emiel); A.T. van der Ploeg (Ans)

1999-01-01

textabstractPompe's disease or glycogen storage disease type II (GSDII) belongs to the family of inherited lysosomal storage diseases. The underlying deficiency of acid alpha-glucosidase leads in different degrees of severity to glycogen storage in heart, skeletal

Alpha-glucosidase inhibitors for patients with type 2 diabetes: results from a Cochrane systematic review and meta-analysis.

NARCIS (Netherlands)

Laar, F.A. van de; Lucassen, P.L.B.J.; Akkermans, R.P.; Lisdonk, E.H. van de; Rutten, G.E.H.M.; Weel, C. van

2005-01-01

OBJECTIVE: To review the effects of monotherapy with alpha-glucosidase inhibitors (AGIs) for patients with type 2 diabetes, with respect to mortality, morbidity, glycemic control, insulin levels, plasma lipids, body weight, and side effects. RESEARCH DESIGN AND METHODS: We systematically searched

ORF Alignment: NC_002754 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available otydyl ... transferase [imported] - Sulfolobus solfataricus ... Length = 393 ... Query: 2 ... KAVVLAAGKGERLEPITHTRPKPFVPV...LETPLILRHXXXXXXXXXXXXXXXXXXXXDYFK 61 ... KAVVLAAGKGERLEPITHTRPKPFVPVLETPLILRH ... ... ... DYFK Sbjct: 1 ... KAVVLAAGKGERLEPITHTRPKPFVPVLETPLILRHIRILKKYINKI

Treatment with acarbose, an alpha-glucosidase inhibitor, reduces increased albumin excretion in streptozotocin-diabetic rats.

Science.gov (United States)

Cohen, M P; Vasselli, J R; Neuman, R G; Witt, J

1995-10-01

1. We examined the effect of the alpha-glucosidase inhibitor acarbose on urinary albumin excretion (UAE) in streptozotocin diabetic rats. 2. Treatment with acarbose for 8 weeks after induction of diabetes prevented the significant increase in UAE observed in untreated diabetic rats relative to nondiabetic controls. 3. Acarbose significantly reduced integrated glycemia, which correlated with albumin excretion rates, and exerts a salutary effect on diabetic renal dysfunction.

A Bac Library and Paired-PCR Approach to Mapping and Completing the Genome Sequence of Sulfolobus Solfataricus P2

DEFF Research Database (Denmark)

She, Qunxin; Confalonieri, F.; Zivanovic, Y.

2000-01-01

The original strategy used in the Sulfolobus solfatnricus genome project was to sequence non overlapping, or minimally overlapping, cosmid or lambda inserts without constructing a physical map. However, after only about two thirds of the genome sequence was completed, this approach became counter......-productive because there was a high sequence bias in the cosmid and lambda libraries. Therefore, a new approach was devised for linking the sequenced regions which may be generally applicable. BAC libraries were constructed and terminal sequences of the clones were determined and used for both end mapping and PCR...

Antioxidant, cytotoxic and alpha-glucosidase inhibition activities from the Mexican berry "Anacahuita" (Cordia boissieri).

Science.gov (United States)

Viveros-Valdez, Ezequiel; Jaramillo-Mora, Carlos; Oranday-Cardenas, Azucena; Mordn-Martinez, Javier; Carranza-Rosales, Pilar

2016-09-01

This study describes the total phenolic and flavonoid content as well as cytotoxic, alpha-glucosidase inhibition and antiradical/antioxidant potential of extracts obtained from the edible fruits of Cordia boissieri, which is widely distributed throughout northeastern Mexico. Phenolic and flavonoid content were evaluated by means of the Folin-Ciocalteu method and aluminum chloride colorimetric assay respectively. The antiradical/antioxidant activity was determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and Trolox Equivalent Antioxidant Capacity (TEAC) assays. Cytotoxic activity was assessed by means of human cancer cell lines (MCF-7 and HeLa), alpha-glucosidase inhibition was determined by colorimetric assay using p-Nitrophenyl a-D-glucopyranoside (PNPG) as a substrate. Results indicate that extract of C. boissieri fruit has a good antioxidant potential to show a EC₅₀: 137.76 ± 35 ptg/mL and 65 ±2 ltM/g in the DPPH and TEAC assays respectively, inhibitor of the enzyme alpha-glu- cosidase involved in sugar uptake (ICSO: 215.20 ± 35 μg/ mL), cytotoxic activities against MCF-7 (IC50: 310 ± 42 μg/mL) and HeLa (IC₅₀0: 450.4 ±21μgg/mL) cancer cell lines as well as an important phenolic content with 230 t 23 mg/1OOg and 54±11 mg100g g of phenols and flavonoids totals respectively. These results point towards an interesting potential for the fruits of C. boissieri as chemopreventive properties and expand the possibilities.

Correlation between L-Carnitine and alpha-1, 4-glucosidase activity in the semen of normal, infertile and vasectomized men.

Science.gov (United States)

Tremblay, R R; Chapdelaine, P; Roy, R; Thabet, M

1982-01-01

The semen content of L-carnitine and of alpha 1, 4-glucosidase has been measured in subjects consulting for evaluation of their fertility. A close correlation (r=0.684) was found between both parameters over the range of azoospermia to normal zoospermia. A significant number of patients with oligo or azoospermia displayed normal values of L-carnitine and of alpha-1, 4-glucosidase while approximately 50% showed levels in the low spectrum of vasectomized men. On the basis of these findings, an obstructive pathology at epididymal or vas deferens level was established by vasography and/or bilateral scrotal exploration in 9 patients with azoospermia. These 2 epididymal markers might thus be useful in the hands of the practicing andrologist who has to determine precisely the site of a dysfunction in the reproductive system which leads to infertility.

Repetitive postprandial hyperglycemia increases cardiac ischemia/reperfusion injury: prevention by the alpha-glucosidase inhibitor acarbose.

Science.gov (United States)

Frantz, Stefan; Calvillo, Laura; Tillmanns, Jochen; Elbing, Inka; Dienesch, Charlotte; Bischoff, Hilmar; Ertl, Georg; Bauersachs, Johann

2005-04-01

Protective effects of the alpha-glucosidase inhibitor acarbose have been reported for various diabetic complications. In the STOP-NIDDM study, even patients without overt diabetes, but with impaired glucose tolerance, had a reduction in cardiovascular events when treated with acarbose. Therefore, we investigated the effect of repetitive postprandial hyperglycemia on the cardiac ischemia/reperfusion injury in vivo. Mice were treated daily by single applications of placebo, sucrose (4 g/kg body weight), or sucrose + acarbose (10 mg/kg body weight) by gavage for 7 days. Acarbose treatment significantly reduced the sucrose-induced increase in plasma glucose concentration. Subsequently, animals underwent 30 min of ischemia by coronary artery ligation and 24 h of reperfusion in vivo. In the sucrose group, ischemia/reperfusion damage was significantly increased (infarct/area at risk, placebo vs. sucrose, 38.8+/-7.5% vs. 62.2+/-4.8%, P<0.05). This was prevented by acarbose treatment (infarct/area at risk 30.7+/-7.2%). While myocardial inflammation was similar in all groups, oxidative stress as indicated by a significant increase in lipid peroxides was enhanced in the sucrose, but not in the sucrose + acarbose group. In summary, repetitive postprandial hyperglycemia increases ischemia/reperfusion damage. This effect can be prevented by treatment with the alpha-glucosidase inhibitor acarbose.

Genus-specific protein binding to the large clusters of DNA repeats (short regularly spaced repeats) present in Sulfolobus genomes

DEFF Research Database (Denmark)

Peng, Xu; Brügger, Kim; Shen, Biao

2003-01-01

terminally modified and corresponds to SSO454, an open reading frame of previously unassigned function. It binds specifically to DNA fragments carrying double and single repeat sequences, binding on one side of the repeat structure, and producing an opening of the opposite side of the DNA structure. It also...... recognizes both main families of repeat sequences in S. solfataricus. The recombinant protein, expressed in Escherichia coli, showed the same binding properties to the SRSR repeat as the native one. The SSO454 protein exhibits a tripartite internal repeat structure which yields a good sequence match...... with a helix-turn-helix DNA-binding motif. Although this putative motif is shared by other archaeal proteins, orthologs of SSO454 were only detected in species within the Sulfolobus genus and in the closely related Acidianus genus. We infer that the genus-specific protein induces an opening of the structure...

Recombinant human acid alpha-glucosidase: high level production in mouse milk, biochemical characteristics, correction of enzyme deficiency in GSDII KO mice

NARCIS (Netherlands)

A.G.A. Bijvoet (Agnes); M.A. Kroos (Marian); F.R. Pieper (Frank); M. Van der Vliet (Martin); H.A. de Boer (Herman); A.T. van der Ploeg (Ans); M.Ph. Verbeet (Martin); A.J.J. Reuser (Arnold)

1998-01-01

textabstractGlycogen storage disease type II (GSDII) is caused by lysosomal acid alpha-glucosidase deficiency. Patients have a rapidly fatal or slowly progressive impairment of muscle function. Enzyme replacement therapy is under investigation. For large-scale, cost-effective

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....

Divergent clinical outcomes of alpha-glucosidase enzyme replacement therapy in two siblings with infantile-onset Pompe disease treated in the symptomatic or pre-symptomatic state

OpenAIRE

Matsuoka, Takashi; Miwa, Yoshiyuki; Tajika, Makiko; Sawada, Madoka; Fujimaki, Koichiro; Soga, Takashi; Tomita, Hideshi; Uemura, Shigeru; Nishino, Ichizo; Fukuda, Tokiko; Sugie, Hideo; Kosuga, Motomichi; Okuyama, Torayuki; Umeda, Yoh

2016-01-01

Pompe disease is an autosomal recessive, lysosomal glycogen storage disease caused by acid ?-glucosidase deficiency. Infantile-onset Pompe disease (IOPD) is the most severe form and is characterized by cardiomyopathy, respiratory distress, hepatomegaly, and skeletal muscle weakness. Untreated, IOPD generally results in death within the first year of life. Enzyme replacement therapy (ERT) with recombinant human acid alpha glucosidase (rhGAA) has been shown to markedly improve the life expectan...

Impaired performance of skeletal muscle in alpha-glucosidase knockout mice

NARCIS (Netherlands)

Hesselink, R.P; Gorselink, M.; Schaart, G.; Wagenmakers, A.J.M.; Kamphoven, G.; Reuser, A.J.J.; Vusse, van der G.J.; Drost, M.R.

2002-01-01

Glycogen storage disease type II (GSD II) is an inherited progressive muscle disease in which lack of functional acid -glucosidase (AGLU) results in lysosomal accumulation of glycogen. We report on the impact of a null mutation of the acid -glucosidase gene (AGLU-/-) in mice on the force production

Bacteria as source of diglycosidase activity: Actinoplanes missouriensis produces 6-O-alpha-l-rhamnosyl-beta-d-glucosidase active on flavonoids

Czech Academy of Sciences Publication Activity Database

Neher, B.D.; Mazzaferro, L.S.; Kotík, Michael; Oyhenart, J.; Halada, Petr; Křen, Vladimír; Breccia, J.D.

2016-01-01

Roč. 100, č. 7 (2016), s. 3061-3070 ISSN 0175-7598 R&D Projects: GA MŠk(CZ) LD13042; GA MŠk(CZ) LD15085; GA MŠk 7AMB13AR005 Institutional support: RVO:61388971 Keywords : alpha-L-rhamnosidase * beta-D-glucosidase * Inverting glycosidase Subject RIV: CE - Biochemistry Impact factor: 3.420, year: 2016

Study of the role of epididymal alpha-glucosidase in the fertility of male rats by the administration of the enzyme inhibitor castanospermine.

Science.gov (United States)

Yeung, C H; Cooper, T G

1994-11-01

The activity of epididymal alpha-glucosidase in adult rats was rapidly suppressed to histochemically undetectable levels within 2 days by the continuous release of the enzyme inhibitor castanospermine via a peritoneal osmotic pump at a rate of 100-200 nmol h-1. It was established that mating activities overnight depleted 72% of the spermatozoa in the distal cauda, which was replenished in 2 days, and that fertility began to decline 3 weeks after efferent duct ligation. Male rats of proven mating proficiency and fertility were treated with castanospermine, or buffered saline as control, for up to 30 days and enzyme inhibition was confirmed at the end of treatment by histochemistry. Fertility was normal at the first mating test on day 7, significantly decreased at the second mating on day 9, but recovered in a stepwise manner at subsequent matings on days 12 and 14. Delaying the third mating until day 25 did not sustain the transient subfertility. However, prolonging sperm storage in the distal cauda epididymides and preventing replenishment with freshly matured spermatozoa, by efferent duct ligation for 14 days performed on day 15 during castanospermine administration, caused a decrease in fertility and a change in the kinematics of epididymal spermatozoa of the castanospermine-treated group. In control rats, binding of epididymal spermatozoa to Vicia faba, a lectin specific for glucose and glucosamine, and mannose and mannosamine residues, decreased from the proximal caput to the distal corpus coincident with the increase in alpha-glucosidase activity on the epithelial brush border. Lectin binding then increased in the cauda where enzyme activity was absent. However, castanospermine treatment did not significantly alter this binding profile. The findings suggest that epididymal alpha-glucosidase does not play a crucial role in the development of sperm fertilizing capacity, but may be involved in the preparation of spermatozoa for storage.

Alpha-glucosidase inhibitory effect and inorganic constituents of Phyllanthus amarus Schum. & Thonn. ash

Directory of Open Access Journals (Sweden)

Malinee Wongnawa

2014-10-01

Full Text Available This study investigated the -glucosidase inhibitory effect and determined the concentration of some inorganic constituents in P. amarus ash. Oral glucose and sucrose tolerance test were performed on normal mice. In vitro -glucosidase inhibitory activity was evaluated by using yeast a-glucosidase. The element concentrations were measured by inductively coupled plasma (ICP spectroscopy. Single oral administration of P. amarus ash did not show antihyperglycemic effect after glucose administration, but decreased blood glucose level after sucrose administration. The ash showed -glucosidase inhibitory activity in vitro with IC50 of 982 mg/mL. The concentrations of K, Ca, Mg, Mn, Fe, Zn, Cu, Pb, Cr, Ni and Co in P. amarus ash were 35049.80±340.64, 3337.24±52.10, 1368.52±13.29, 90.81±1.34, 87.68±1.15, 18.28±0.22, 4.69±0.07, 1.07±0.15, 0.29±0.03, 0.20±0.04 and 0.10±0.02 mg/g, respectively. These results indicate that the antihyperglycemic effect of P. amarus ash might be partly due to the -glucosidase inhibitory activity of the inorganic constituents.

Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment

NARCIS (Netherlands)

Brouns, S.J.J.; Walther, J.; Snijders, A.P.; Werken, van de H.J.G.; Willemen, H.L.D.M.; Worm, P.; Vos, de M.G.; Andersson, A.; Lundgren, M.; Mazon, H.F.; Heuvel, van den R.H.H.; Nilsson, P.; Salmon, L.; Vos, de W.M.; Wright, P.C.; Bernander, R.; Oost, van der J.

2006-01-01

The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for D-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to

Purification, crystallization and preliminary X-ray diffraction analysis of an archaeal ABC-ATPase

NARCIS (Netherlands)

Verdon, Grégory; Albers, Sonja-V.; Dijkstra, Bauke W.; Driessen, Arnold J.M.; Thunnissen, Andy-Mark W.H.

2002-01-01

In the archaeon Sulfolobus solfataricus glucose uptake is mediated by an ABC transport system. The ABC-ATPase of this transporter (GlcV) has been overproduced in Escherichia coli and purified. Crystals of GlcV suitable for data collection were obtained in the absence of nucleotide by microseeding

Two novel conjugative plasmids from a single strain of Sulfolobus

NARCIS (Netherlands)

Erauso, G.; Stedman, K.M.; Werken, van de H.J.G.; Zillig, W.; Oost, van der J.

2006-01-01

Two conjugative plasmids (CPs) were isolated and characterized from the same 'Sulfolobus islandicus' strain, SOG2/4, The plasmids were separated from each other and transferred into Sulfolobus soltataricus. One has a high copy number and is not stable (pSOG1) whereas the other has a low copy number

alpha-Glucosidase inhibition (acarbose) fails to enhance secretion of glucagon-like peptide 1 (7-36 amide) and to delay gastric emptying in Type 2 diabetic patients

DEFF Research Database (Denmark)

Hücking, K; Kostic, Z; Pox, C

2005-01-01

AIM: Acarbose is able to enhance GLP-1 release and delay gastric emptying in normal subjects. The effect of alpha-glucosidase inhibition on GLP-1 has been less evident in Type 2 diabetic patients. The aim of this study was to investigate the possible influence of acarbose on GLP-1 release and gas...

The Effects of Temperature and Growth Phase on the Lipidomes of Sulfolobus islandicus and Sulfolobus tokodaii

DEFF Research Database (Denmark)

Jensen, Sara Munk; Neesgaard, Vinnie Lund; Skjoldbjerg, Sandra Landbo Nedergaard

2015-01-01

at three different temperatures, with samples withdrawn during lag, exponential, and stationary phases. Three abundant tetraether lipid classes and one diether lipid class were monitored. Beside the expected increase in the number of cyclopentane moieties with higher temperature in both archaea, we......The functionality of the plasma membrane is essential for all organisms. Adaption to high growth temperatures imposes challenges and Bacteria, Eukarya, and Archaea have developed several mechanisms to cope with these. Hyperthermophilic archaea have earlier been shown to synthesize tetraether...... membrane lipids with an increased number of cyclopentane moieties at higher growth temperatures. Here we used shotgun lipidomics to study this effect as well as the influence of growth phase on the lipidomes of Sulfolobus islandicus and Sulfolobus tokodaii for the first time. Both species were cultivated...

Free Radical Scavenging and Alpha/Beta-glucosidases Inhibitory Activities of Rambutan (Nephelium lappaceum L. Peel Extract

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Wahyu Widowati

2015-12-01

Full Text Available BACKGROUND: Diabetes mellitus (DM is associated with oxidative reaction and hyperglycemic condition. Human body has an antioxidant defense system toward free radical, but overproduction of free radical causing imbalance condition between the free radical and the antioxidant defense in the body that lead to several diseases, including DM. Glucosidase is an enzyme that hydrolize carbohydrates causing increase of blood glucose level, so by inhibiting this enzyme blood glucose level in plasma could be effectively decreased. Rambutan (Nephelium lappaceum L. peel has been reported to have many potential roles, such as antioxidant and anti-glycemia. Therefore our current study was conducted to evaluate possible effectivity of Rambutan peel to scavenge free radical and to inhibit α- and β-glucosidases. METHODS: Rambutan peel extraction (RPE was performed based on maceration method. Geraniin was used as control. For antioxidant study, 2,2-diphenyl-1- picrylhydrazyl (DPPH free radical scavenging test was performed. For glucosidase inhibitory activity study,  α- and β-glucosidases inhibitory activity tests were performed. Results were analyzed for median of Inhibitory Concentration (IC50. RESULTS: The scavenging activity of RPE was comparable with Geraniin. Meanwhile, the α-glucosidase inhibitory activity of RPE was higher than the one of Geraniin. The α-glucosidase-inhibitory-activity IC50 of RPE and Geraniin were 0.106±0.080 μg/ml and 16.12±0.29 μg/ml, respectively. The β-glucosidase inhibitory activity of RPE was also higher than the one of Geraniin. The β-glucosidase-inhibitory-activity IC50 of RPE and Geraniin were 7.02±0.99 μg/ml and 19.81±0.66 μg/ml, respectively. CONCLUSIONS: Since RPE showed comparable free radical scavenging activity with Geraniin and higher α- and β-glucosidases inhibitory activities than Geraniin, RPE could be suggested as a promising antioxidant and antiglycemic agent.  KEYWORDS

A genetic study of SSV1, the prototypical fusellovirus.

Directory of Open Access Journals (Sweden)

Eric eIverson

2012-06-01

Full Text Available Viruses of thermophilic Archaea are unique in both their structures and genomic sequences. The most widespread and arguably best studied are the lemon-shaped fuselloviruses. The spindle-shaped virus morphology is unique to Archaea but widespread therein. The best studied fusellovirus is SSV1 from Beppu Japan, which infects Sulfolobus solfataricus. Very little is known about the function of the genes in the SSV1 genome. Recently we have developed genetic tools to analyze these genes. In this study, we have deleted three SSV1 open reading frames ranging from completely conserved to poorly conserved: VP2, d244, and b129. Deletion of the universally conserved ORF b129, which encodes a predicted transcriptional regulator, results in loss of infectivity. Deletion of the poorly-conserved predicted DNA binding protein gene VP2 yields viable virus that is indistinguishable from wild-type Deletion of the well-conserved ORF d244 that encodes a predicted nuclease yields viable virus. However infection of Sulfolobus solfataricus with virus lacking ORF d244 dramatically retards host growth, compared to the wild-type virus.

Alpha-glucosidase inhibitory and antiplasmodial properties of terpenoids from the leaves of Buddleja saligna Willd.

Science.gov (United States)

Chukwujekwu, Jude C; Rengasamy, Kannan R R; de Kock, Carmen A; Smith, Peter J; Slavětínská, Lenka Poštová; van Staden, Johannes

2016-01-01

In our continuing search for biologically active natural product(s) of plant origin, Buddleja saligna, a South African medicinal plant, was screened in line with its traditional use for antidiabetic (yeast alpha glucosidase inhibitory) and antiplasmodial (against a chloroquine sensitive strain of Plasmodium falciparum (NF54)) activities. The hexane fraction showed the most promising activity with regards to its antidiabetic (IC(50) = 260 ± 0.112 µg/ml) and antiplasmodial (IC(50) = 8.5 ± 1.6 µg/ml) activities. Using activity guided fractionation three known terpenoids (betulonic acid, betulone and spinasterol) were isolated from this species for the first time. The compounds displayed varying levels of biological activities (antidiabetic: 27.31 µg/ml ≥ IC(50) ≥ 5.6 µg/ml; antiplasmodial: 14 µg/ml ≥ IC(50) ≥ 2 µg/ml) with very minimal toxicity.

The Role of α-Glucosidase in Germinating Barley Grains1[W][OA

Science.gov (United States)

Stanley, Duncan; Rejzek, Martin; Naested, Henrik; Smedley, Mark; Otero, Sofía; Fahy, Brendan; Thorpe, Frazer; Nash, Robert J.; Harwood, Wendy; Svensson, Birte; Denyer, Kay; Field, Robert A.; Smith, Alison M.

2011-01-01

The importance of α-glucosidase in the endosperm starch metabolism of barley (Hordeum vulgare) seedlings is poorly understood. The enzyme converts maltose to glucose (Glc), but in vitro studies indicate that it can also attack starch granules. To discover its role in vivo, we took complementary chemical-genetic and reverse-genetic approaches. We identified iminosugar inhibitors of a recombinant form of an α-glucosidase previously discovered in barley endosperm (ALPHA-GLUCOSIDASE97 [HvAGL97]), and applied four of them to germinating grains. All four decreased the Glc-to-maltose ratio in the endosperm 10 d after imbibition, implying inhibition of maltase activity. Three of the four inhibitors also reduced starch degradation and seedling growth, but the fourth did not affect these parameters. Inhibition of starch degradation was apparently not due to inhibition of amylases. Inhibition of seedling growth was primarily a direct effect of the inhibitors on roots and coleoptiles rather than an indirect effect of the inhibition of endosperm metabolism. It may reflect inhibition of glycoprotein-processing glucosidases in these organs. In transgenic seedlings carrying an RNA interference silencing cassette for HvAgl97, α-glucosidase activity was reduced by up to 50%. There was a large decrease in the Glc-to-maltose ratio in these lines but no effect on starch degradation or seedling growth. Our results suggest that the α-glucosidase HvAGL97 is the major endosperm enzyme catalyzing the conversion of maltose to Glc but is not required for starch degradation. However, the effects of three glucosidase inhibitors on starch degradation in the endosperm indicate the existence of unidentified glucosidase(s) required for this process. PMID:21098673

Extremophiles survival to simulated space conditions: an astrobiology model study.

Science.gov (United States)

Mastascusa, V; Romano, I; Di Donato, P; Poli, A; Della Corte, V; Rotundi, A; Bussoletti, E; Quarto, M; Pugliese, M; Nicolaus, B

2014-09-01

In this work we investigated the ability of four extremophilic bacteria from Archaea and Bacteria domains to resist to space environment by exposing them to extreme conditions of temperature, UV radiation, desiccation coupled to low pressure generated in a Mars' conditions simulator. All the investigated extremophilic strains (namely Sulfolobus solfataricus, Haloterrigena hispanica, Thermotoga neapolitana and Geobacillus thermantarcticus) showed a good resistance to the simulation of the temperature variation in the space; on the other hand irradiation with UV at 254 nm affected only slightly the growth of H. hispanica, G. thermantarcticus and S. solfataricus; finally exposition to Mars simulated condition showed that H. hispanica and G. thermantarcticus were resistant to desiccation and low pressure.

Inhibitory activities of Moringa oleifera leaf extract against α-glucosidase enzyme in vitro

Science.gov (United States)

Natsir, H.; Wahab, A. W.; Laga, A.; Arif, A. R.

2018-03-01

Alpha-glucosidase is a key enzyme in the final process of breaking carbohydrates into glucose. Inhibition of α-glucosidase affected more absorption of glucose, so it can reduce hyperglycemia condition. The aims of this study is to determine the effectiveness of inhibition wet and dried Moringa oleifera leaf extract through α-glucosidase activity in vitro. The effectiveness study of inhibition on the activity of α-glucosidase enzyme obtained from white glutinous rice (Oryza sativa glutinosa) was carried out using wet and dried kelor leaf extract of 13% (w/v) with 10 mM α-D-glucopyranoside (PNPG) substrate. A positive control used 1% acarbose and substrate without addition of extract was a negative control. Inhibitory activity was measured using spectrophotometers at a wavelength of 400 nm. The result showed that the inhibition activity against α-glucosidase enzyme of dried leaf extract, wet leaf extract and acarbose was 81,39%, 83,94%, and 95,4%, respectively on pH 7,0. The effectiveness inhibition of the wet Moringa leaf extract was greater than the dried leaf extract. The findings suggest that M. oleifera leaf has the potential to be developed as an alternative food therapy for diabetics.

Lactic Acid Bacteria Producing Inhibitor of Alpha Glucosidase Isolated from Ganyong (Canna Edulis) and Kimpul (Xanthosoma sagittifolium)

Science.gov (United States)

Nurhayati, Rifa; Miftakhussolikhah; Frediansyah, Andri; Lailatul Rachmah, Desy

2017-12-01

Type 2 diabetes is a disease that caused by the failure of insulin secretion by the beta cells of the pancreas and insulin resistance in peripheral levels. One therapy for diabetics is by inhibiting the activity of α-glucosidase. Lactic acid bacteria have the ability to inhibit of α-glucosidase activity. The aims of this research was to isolation and screening of lactic acid bacteria from ganyong tuber (Canna Edulis) and kimpul tuber (Xanthosoma sagittifolium), which has the ability to inhibit the activity of α-glucosidase. Eightteen isolates were identified as lactic acid bacteria and all of them could inhibit the activity of α-glukosidase. The GN 8 isolate was perform the highest inhibition acivity.

Structure-Based Mutagenesis of Sulfolobus Turreted Icosahedral Virus B204 Reveals Essential Residues in the Virion-Associated DNA-Packaging ATPase.

Science.gov (United States)

Dellas, Nikki; Snyder, Jamie C; Dills, Michael; Nicolay, Sheena J; Kerchner, Keshia M; Brumfield, Susan K; Lawrence, C Martin; Young, Mark J

2015-12-23

Sulfolobus turreted icosahedral virus (STIV), an archaeal virus that infects the hyperthermoacidophile Sulfolobus solfataricus, is one of the most well-studied viruses of the domain Archaea. STIV shares structural, morphological, and sequence similarities with viruses from other domains of life, all of which are thought to belong to the same viral lineage. Several of these common features include a conserved coat protein fold, an internal lipid membrane, and a DNA-packaging ATPase. B204 is the ATPase encoded by STIV and is thought to drive packaging of viral DNA during the replication process. Here, we report the crystal structure of B204 along with the biochemical analysis of B204 mutants chosen based on structural information and sequence conservation patterns observed among members of the same viral lineage and the larger FtsK/HerA superfamily to which B204 belongs. Both in vitro ATPase activity assays and transfection assays with mutant forms of B204 confirmed the essentiality of conserved and nonconserved positions. We also have identified two distinct particle morphologies during an STIV infection that differ in the presence or absence of the B204 protein. The biochemical and structural data presented here are not only informative for the STIV replication process but also can be useful in deciphering DNA-packaging mechanisms for other viruses belonging to this lineage. STIV is a virus that infects a host from the domain Archaea that replicates in high-temperature, acidic environments. While STIV has many unique features, there exist several striking similarities between this virus and others that replicate in different environments and infect a broad range of hosts from Bacteria and Eukarya. Aside from structural features shared by viruses from this lineage, there exists a significant level of sequence similarity between the ATPase genes carried by these different viruses; this gene encodes an enzyme thought to provide energy that drives DNA packaging into

The Identification of Sulfolobus Species from a Biocorrosion of Wastewater Pipes

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Shakila Motamedi

2011-01-01

Full Text Available One of the species which has a prominent role in the biocorrosion of wastewater pipes is Sulfolobus bacterium. This bacterium is among the iron oxidizing bacteria. Sulfolobus is from the archae bacteria genus, which provides its needed energy by oxidation of sulfur in warm and acidic environments. In this research, the water sample exist in the water supply pipelines at Sarcheshmeh copper mine was studied, sulfur corrosive bacteria were separated and identified and their biochemical activity was researched and studied and it was found that the target species are in fact Sulfolobus archae bacteria which have different enzymatic activities. Due to these enzymatic activities, a considerable amount of sulfur is accumulated on the cell surface. Based on various incubation environments, as a result of sulfur accumulation, Sulfolubos archae bacteria were identified, and after doing isolation and purification methods, number of colonies have been decrease from 10-1 dilution to 10-10 dilution and in sample with 10-1 dilution sample 200 colonies has been reported and in plate with 10-10 dilution no colonies has been observed.

Increased glucose metabolism and alpha-glucosidase inhibition in Cordyceps militaris water extract-treated HepG2 cells

Science.gov (United States)

Kim, Dae Jung; Kang, Yun Hwan; Kim, Kyoung Kon; Kim, Tae Woo; Park, Jae Bong

2017-01-01

BACKGROUND/OBJECTIVES Recent living condition improvements, changes in dietary habits, and reductions in physical activity are contributing to an increase in metabolic syndrome symptoms including diabetes and obesity. Through such societal developments, humankind is continuously exposed to metabolic diseases such as diabetes, and the number of the victims is increasing. This study investigated Cordyceps militaris water extract (CMW)-induced glucose uptake in HepG2 cells and the effect of CMW treatment on glucose metabolism. MATERIALS/METHODS Colorimetric assay kits were used to determine the glucokinase (GK) and pyruvate dehydrogenase (PDH) activities, glucose uptake, and glycogen content. Either RT-PCR or western blot analysis was performed for quantitation of glucose transporter 2 (GLUT2), hepatocyte nuclear factor 1 alpha (HNF-1α), phosphatidylinositol 3-kinase (PI3k), protein kinase B (Akt), phosphorylated AMP-activated protein kinase (pAMPK), phosphoenolpyruvate carboxykinase, GK, PDH, and glycogen synthase kinase 3 beta (GSK-3β) expression levels. The α-glucosidase inhibitory activities of acarbose and CMW were evaluated by absorbance measurement. RESULTS CMW induced glucose uptake in HepG2 cells by increasing GLUT2 through HNF-1α expression stimulation. Glucose in the cells increased the CMW-induced phosphorylation of AMPK. In turn, glycolysis was stimulated, and glyconeogenesis was inhibited. Furthermore, by studying the mechanism of action of PI3k, Akt, and GSK-3β, and measuring glycogen content, the study confirmed that the glucose was stored in the liver as glycogen. Finally, CMW resulted in a higher level of α-glucosidase inhibitory activity than that from acarbose. CONCLUSION CMW induced the uptake of glucose into HepG2 cells, as well, it induced metabolism of the absorbed glucose. It is concluded that CMW is a candidate or potential use in diabetes prevention and treatment. PMID:28584574

Alpha-glucosidase inhibitor, acarbose, improves glycamic control and reduces body weight in type 2 diabetes: Findings on indian patients from the pooled data analysis

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Sanjay Kalra

2013-01-01

Full Text Available Alpha-glucosidase inhibitors are widely used especially in Asian countries as a treatment option for type 2 diabetes patients with high postprandial glycemia (PPG. The higher carbohydrate in the Indian diets lead to greater prandial glycemic excursion, increased glucosidase, and incretin activity in the gut and may need special therapeutic strategies to tackle these glucose peaks. This is the subgroup analysis of Indian subjects who participated in the GlucoVIP study that investigated the effectiveness and tolerability of acarbose as add-on or monotherapy in a range of patients with type 2 diabetes mellitus. A total of 1996 Indian patients were included in the effectiveness analysis. After 12.5 weeks (mean, the mean change in 2-hour PPG from baseline was −74.4 mg/dl, mean HbA1c decreased by -1.0%, and mean fasting blood glucose decreased by -37.9 mg/dl. The efficacy of acarbose was rated "very good" or "good" in 91.1% of patients, and tolerability as "very good" or "good" in 88.0% of patients. The results of this observational study suggest that acarbose was effective and well tolerated in the Indian patients with T2DM.

CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties

DEFF Research Database (Denmark)

Lillestøl, Reidun K; Shah, Shiraz Ali; Brügger, Kim

2009-01-01

Summary CRISPRs of Sulfolobus fall into three main families based on their repeats, leader regions, associated cas genes, and putative recognition sequences on viruses and plasmids. Spacer sequence matches to different viruses and plasmids of the Sulfolobales revealed some bias particularly...... for family III CRISPRs. Transcription occurs on both strands of the five repeat-clusters of Sulfolobus acidocaldarius and a repeat-cluster of the conjugative plasmid pKEF9. Leader strand transcripts cover whole repeat-clusters and are processed mainly from the 3'-end, within repeats, yielding heterogeneous...

Sulfolobus Replication Factor C stimulates the activity of DNA Polymerase B1

DEFF Research Database (Denmark)

Xing, Xuanxuan; Zhang, Likui; Guo, Li

2014-01-01

the hyperthermophilic archaea of the genus Sulfolobus physically interacts with DNA polymerase B1 (PolB1) and enhances both the polymerase and 3'-5' exonuclease activities of PolB1 in an ATP-independent manner. Stimulation of the PolB1 activity by RFC is independent of the ability of RFC to bind DNA but is consistent...... with the ability of RFC to facilitate DNA binding by PolB1 through protein-protein interaction. These results suggest that Sulfolobus RFC may play a role in recruiting DNA polymerase for efficient primer extension, in addition to clamp loading, during DNA replication....

Antioxidant rich grape pomace extract suppresses postprandial hyperglycemia in diabetic mice by specifically inhibiting alpha-glucosidase

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Hogan Shelly

2010-08-01

Full Text Available Abstract Background Postprandial hyperglycemia is an early defect of type 2 diabetes and one of primary anti-diabetic targets. Treatment of postprandial hyperglycemia can be achieved by inhibiting intestinal α-glucosidase, the key enzyme for oligosaccharide digestion and further glucose absorption. Grape pomace is winemaking byproduct rich in bioactive food compounds such as phenolic antioxidants. This study evaluated the anti-diabetic potential of two specific grape pomace extracts by determining their antioxidant and anti-postprandial hyperglycemic activities in vitro and in vivo. Methods The extracts of red wine grape pomace (Cabernet Franc and white wine grape pomace (Chardonnay were prepared in 80% ethanol. An extract of red apple pomace was included as a comparison. The radical scavenging activities and phenolic profiles of the pomace extracts were determined through the measurement of oxygen radical absorbance capacity, DPPH radical scavenging activity, total phenolic content and flavonoids. The inhibitory effects of the pomace extracts on yeast and rat intestinal α-glucosidases were determined. Male 6-week old C57BLKS/6NCr mice were treated with streptozocin to induce diabetes. The diabetic mice were then treated with vehicle or the grape pomace extract to determine whether the oral intake of the extract can suppress postprandial hyperglycemia through the inhibition of intestinal α-glucosidases. Results The red grape pomace extract contained significantly higher amounts of flavonoids and phenolic compounds and exerted stronger oxygen radical absorbance capacity than the red apple pomace extract. Both the grape pomace extracts but not the apple pomace extract exerted significant inhibition on intestinal α-glucosidases and the inhibition appears to be specific. In the animal study, the oral intake of the grape pomace extract (400 mg/kg body weight significantly suppressed the postprandial hyperglycemia by 35% in streptozocin

Antioxidant rich grape pomace extract suppresses postprandial hyperglycemia in diabetic mice by specifically inhibiting alpha-glucosidase.

Science.gov (United States)

Hogan, Shelly; Zhang, Lei; Li, Jianrong; Sun, Shi; Canning, Corene; Zhou, Kequan

2010-08-27

Postprandial hyperglycemia is an early defect of type 2 diabetes and one of primary anti-diabetic targets. Treatment of postprandial hyperglycemia can be achieved by inhibiting intestinal α-glucosidase, the key enzyme for oligosaccharide digestion and further glucose absorption. Grape pomace is winemaking byproduct rich in bioactive food compounds such as phenolic antioxidants. This study evaluated the anti-diabetic potential of two specific grape pomace extracts by determining their antioxidant and anti-postprandial hyperglycemic activities in vitro and in vivo. The extracts of red wine grape pomace (Cabernet Franc) and white wine grape pomace (Chardonnay) were prepared in 80% ethanol. An extract of red apple pomace was included as a comparison. The radical scavenging activities and phenolic profiles of the pomace extracts were determined through the measurement of oxygen radical absorbance capacity, DPPH radical scavenging activity, total phenolic content and flavonoids. The inhibitory effects of the pomace extracts on yeast and rat intestinal α-glucosidases were determined. Male 6-week old C57BLKS/6NCr mice were treated with streptozocin to induce diabetes. The diabetic mice were then treated with vehicle or the grape pomace extract to determine whether the oral intake of the extract can suppress postprandial hyperglycemia through the inhibition of intestinal α-glucosidases. The red grape pomace extract contained significantly higher amounts of flavonoids and phenolic compounds and exerted stronger oxygen radical absorbance capacity than the red apple pomace extract. Both the grape pomace extracts but not the apple pomace extract exerted significant inhibition on intestinal α-glucosidases and the inhibition appears to be specific. In the animal study, the oral intake of the grape pomace extract (400 mg/kg body weight) significantly suppressed the postprandial hyperglycemia by 35% in streptozocin-induced diabetic mice following starch challenge. This is the

Beta-glucosidase variants and polynucleotides encoding same

Science.gov (United States)

Wogulis, Mark; Harris, Paul; Osborn, David

2017-06-27

The present invention relates to beta-glucosidase variants, e.g. beta-glucosidase variants of a parent Family GH3A beta-glucosidase from Aspergillus fumigatus. The present invention also relates to polynucleotides encoding the beta-glucosidase variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the beta-glucosidase variants.

Preliminary phytochemical screening and alpha-glucosidase inhibitory activity of Philippine taro (Colocasia esculenta (L.) Schott var. PSB-VG #9)

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Lebosada, Richemae Grace R.; Librando, Ivy L.

2017-01-01

The study was conducted to determine the anti-hyperglycemic property in terms of α-glucosidase inhibitory activity of the various parts (corm, leaf and petiole) of Colocasia esculenta (L.) Schott var. PSB-VG #9. Each of the plant parts were extracted with 95% ethanol and concentrated using a rotary evaporator at 40 °C. The crude extracts were screened for the presence of alkaloids, flavonoids, glycosides and saponins using Thin Layer Chromatography. The α-glucosidase inhibitory activity of the crude extracts (50 mg/L) were assayed spectrophotometrically using a microplate reader. The results of the phytochemical screening revealed the presence of alkaloids, flavonoids, and saponins in the leaf part while flavonoids and saponins were detected in the petiole and only saponins were present in the corm. The assay showed that the percentage α-glucosidase inhibition of the 50 mg/L ethanolic crude extract of the corm, leaves and petiole of C. esculenta are 68.03, 71.64 and 71.39%, respectively. Statistical analysis shows significant differences in the α-glucosidase inhibition among the various plant parts. It can be concluded that the ethanolic crude extracts of the different parts of C. esculenta (L.) Schott var. PSB-VG #9 exhibited inhibitory activity against α-glucosidase and the presence of phytochemicals like alkaloids, flavonoids and saponins may have contributed greatly to the inhibitory activity of the plant extract and can be further subjected for isolation of the therapeutically active compounds with antidiabetes potency.

Genetic technologies for extremely thermophilic microorganisms of Sulfolobus, the only genetically tractable genus of crenarchaea.

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Peng, Nan; Han, Wenyuan; Li, Yingjun; Liang, Yunxiang; She, Qunxin

2017-04-01

Archaea represents the third domain of life, with the information-processing machineries more closely resembling those of eukaryotes than the machineries of the bacterial counterparts but sharing metabolic pathways with organisms of Bacteria, the sister prokaryotic phylum. Archaeal organisms also possess unique features as revealed by genomics and genome comparisons and by biochemical characterization of prominent enzymes. Nevertheless, diverse genetic tools are required for in vivo experiments to verify these interesting discoveries. Considerable efforts have been devoted to the development of genetic tools for archaea ever since their discovery, and great progress has been made in the creation of archaeal genetic tools in the past decade. Versatile genetic toolboxes are now available for several archaeal models, among which Sulfolobus microorganisms are the only genus representing Crenarchaeota because all the remaining genera are from Euryarchaeota. Nevertheless, genetic tools developed for Sulfolobus are probably the most versatile among all archaeal models, and these include viral and plasmid shuttle vectors, conventional and novel genetic manipulation methods, CRISPR-based gene deletion and mutagenesis, and gene silencing, among which CRISPR tools have been reported only for Sulfolobus thus far. In this review, we summarize recent developments in all these useful genetic tools and discuss their possible application to research into archaeal biology by means of Sulfolobus models.

Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene vp3.

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Iverson, Eric A; Goodman, David A; Gorchels, Madeline E; Stedman, Kenneth M

2017-05-15

Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae , where >90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1), which infects Sulfolobus solfataricus in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all open reading frames. We demonstrate that almost half of the SSV1 genes are not essential for infectivity, and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology. IMPORTANCE Most of the putative genes in the spindle-shaped archaeal hyperthermophile fuselloviruses have no sequences that are clearly similar to characterized genes. In order to determine which of these SSV genes are important for function, we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known fuselloviruses, vp3 , allows the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of

The 60 kDa heat shock proteins in the hyperthermophilic archaeon Sulfolobus shibatae.

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Kagawa, H K; Osipiuk, J; Maltsev, N; Overbeek, R; Quaite-Randall, E; Joachimiak, A; Trent, J D

1995-11-10

One of the most abundant proteins in the hyperthermophilic archaeon Sulfolobus shibatae is the 59 kDa heat shock protein (TF55) that is believed to form a homo-oligomeric double ring complex structurally similar to the bacterial chaperonins. We discovered a second protein subunit in the S. shibatae ring complex (referred to as alpha) that is stoichiometric with TF55 (renamed beta). The gene and flanking regions of alpha were cloned and sequenced and its inferred amino acid sequence has 54.4% identity and 74.4% similarity to beta. Transcription start sites for both alpha and beta were mapped and three potential transcription regulatory regions were identified. Northern analyses of cultures shifted from normal growth temperatures (70 to 75 degrees C) to heat shock temperatures (85 to 90 degrees C) indicated that the levels of alpha and beta mRNAs increased during heat shock, but at all temperatures their relative proportions remained constant. Monitoring protein synthesis by autoradiography of total proteins from cultures pulse labeled with L(-)[35S]methionine at normal and heat shock temperatures indicated significant increases in alpha and beta synthesis during heat shock. Under extreme heat shock conditions (> or = 90 degrees C) alpha and beta appeared to be the only two proteins synthesized. The purified alpha and beta subunits combined to form high molecular mass complexes with similar mobilities on native polyacrylamide gels to the complexes isolated directly from cells. Equal proportions of the two subunits gave the greatest yield of the complex, which we refer to as a "rosettasome". It is argued that the rosettasome consists of two homo-oligomeric rings; one of alpha and the other of beta. Polyclonal antibodies against alpha and beta from S. shibatae cross-reacted with proteins of similar molecular mass in 10 out of the 17 archaeal species tested, suggesting that the two rosettasome proteins are highly conserved among the archaea. The archaeal sequences were

Backbone assignment of the little finger domain of a Y-family DNA polymerase.

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Ma, Dejian; Fowler, Jason D; Suo, Zucai

2011-10-01

Sulfolobus solfataricus DNA polymerase IV (Dpo4), a prototype Y-family DNA polymerase, contains a unique little finger domain besides a catalytic core. Here, we report the chemical shift assignments for the backbone nitrogens, α and β carbons, and amide protons of the little finger domain of Dpo4. This work and our published backbone assignment for the catalytic core provide the basis for investigating the conformational dynamics of Dpo4 during catalysis using solution NMR spectroscopy.

Pactamycin binding site on archaebacterial and eukaryotic ribosomes

International Nuclear Information System (INIS)

Tejedor, F.; Amils, R.; Ballesta, J.P.G.

1987-01-01

The presence of a photoreactive acetophenone group in the protein synthesis inhibitor pactamycin and the possibility of obtaining active iodinated derivatives that retain full biological activity allow the antibiotic binding site on Saccharomyces cerevisiae and archaebacterium Sulfolobus solfataricus ribosomes to be photoaffinity labeled. Four major labeled proteins have been identified in the yeast ribosomes, i.e., YS10, YS18, YS21/24, and YS30, while proteins AL1a, AS10/L8, AS18/20, and AS21/22 appeared as radioactive spots in S. solfataricus. There seems to be a correlation between some of the proteins labeled in yeast and those previously reported in Escherichia coli indicating that the pactamycin binding sites of both species, which are in the small subunit close to the initiation factors and mRNA binding sites, must have similar characteristics

Structural and Functional Characterization of an Archaeal Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Complex for Antiviral Defense (CASCADE)

DEFF Research Database (Denmark)

Lintner, Nathanael G; Kerou, Melina; Brumfield, Susan K

2011-01-01

In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA....... The Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5......a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2...

Phytochemical Screening, Polyphenolic Content and Alpha ...

African Journals Online (AJOL)

traditionally in the management of diabetes mellitus and in the treatment of wounds and stomach ache. In this study, phytochemical screening, total phenolic contents and alpha-glucosidase ... Parkinson's and Alzheimer's diseases (Di Matteo and Esposito, 2003) as well as inflammation and problems caused by cell and ...

Crenarchaeal biofilm formation under extreme conditions.

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Andrea Koerdt

Full Text Available BACKGROUND: Biofilm formation has been studied in much detail for a variety of bacterial species, as it plays a major role in the pathogenicity of bacteria. However, only limited information is available for the development of archaeal communities that are frequently found in many natural environments. METHODOLOGY: We have analyzed biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii. We established a microtitre plate assay adapted to high temperatures to determine how pH and temperature influence biofilm formation in these organisms. Biofilm analysis by confocal laser scanning microscopy demonstrated that the three strains form very different communities ranging from simple carpet-like structures in S. solfataricus to high density tower-like structures in S. acidocaldarius in static systems. Lectin staining indicated that all three strains produced extracellular polysaccharides containing glucose, galactose, mannose and N-acetylglucosamine once biofilm formation was initiated. While flagella mutants had no phenotype in two days old static biofilms of S. solfataricus, a UV-induced pili deletion mutant showed decreased attachment of cells. CONCLUSION: The study gives first insights into formation and development of crenarchaeal biofilms in extreme environments.

Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius.

Science.gov (United States)

Pennacchio, Angela; Giordano, Assunta; Pucci, Biagio; Rossi, Mosè; Raia, Carlo A

2010-03-01

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

Competitive inhibitor of cellular alpha-glucosidases protects mice from lethal dengue virus infection

OpenAIRE

Chang, Jinhong; Schul, Wouter; Yip, Andy; Xu, Xiaodong; Guo, Ju-Tao; Block, Timothy M.

2011-01-01

Dengue virus infection causes diseases in people, ranging from the acute febrile illness Dengue fever, to life-threatening Dengue Hemorrhagic Fever/Dengue Shock Syndrome. We previously reported that a host cellular α-glucosidases I and II inhibitor, imino sugar CM-10-18, potently inhibited dengue virus replication in cultured cells, and significantly reduced viremia in dengue virus infected AG129 mice. In this report we show that CM-10-18 also significantly protects mice from death and/or dis...

Molecular analysis of the UV-inducible pili operon from Sulfolobus acidocaldarius

NARCIS (Netherlands)

Wolferen, Marleen van; Ajon, Małgorzata; Driessen, Arnold J.M.; Albers, Sonja-Verena

2013-01-01

Upon ultraviolet (UV) stress, hyperthermophilic Sulfolobus species show a highly induced transcription of a gene cluster responsible for pili biogenesis: the UV-inducible pili operon (ups operon). This operon is involved in UV-induced pili assembly, cellular aggregation, and subsequent DNA exchange

Novel RepA-MCM proteins encoded in plasmids pTAU4, pORA1 and pTIK4 from Sulfolobus neozealandicus

DEFF Research Database (Denmark)

Greve, B.; Jensen, S.; Phan, H.

2005-01-01

Three plasmids isolated from the crenarchaeal thermoacidophile Sulfolobus neozealandicus were characterized. Plasmids pTAU4 (7,192 bp), pORA1 (9,689 bp) and pTIK4 (13,638 bp) show unusual properties that distinguish them from previously characterized cryptic plasmids of the genus Sulfolobus. Plas...

[The isolation and characterization of beta-glucosidase gene and beta-glucosidase of Trichoderma viride]: Progress report

International Nuclear Information System (INIS)

Stafford, D.W.

1983-01-01

Our project was to isolate and characterize the enzyme β-glucosidase and to clone and characterize the β-glucosidase gene; our goal is to clone and characterize each of the cellulase genes from Trichoderma. The induction of the Trichoderma reesei cellulase complex by cellulose and by the soluble inducer, sophorose, has been demonstrated. Although the induction of the cellulase complex has previously been well documented, the induction of β-glucosidase had been questioned. 49 refs., 6 figs., 2 tabs

Characterization of the Sulfolobus host-SSV2 virus interaction

DEFF Research Database (Denmark)

Contursi, P.; Jensen, S.; Aucelli, T.

2006-01-01

The Sulfolobus spindle virus, SSV2, encodes a tyrosine integrase which furthers provirus formation in host chromosomes. Consistently with the prediction made during sequence analysis, integration was found to occur in the downstream half of the tRNAGly (CCC) gene. In this paper we report the find......The Sulfolobus spindle virus, SSV2, encodes a tyrosine integrase which furthers provirus formation in host chromosomes. Consistently with the prediction made during sequence analysis, integration was found to occur in the downstream half of the tRNAGly (CCC) gene. In this paper we report...... during the growth of the natural host REY15/4, the cellular content of SSV2 DNA remains fairly low throughout the incubation of the foreign host. The accumulation of episomal DNA in the former case cannot be traced to decreased packaging activity because of a simultaneous increase in the virus titre...... in the medium. In addition, the interaction between SSV2 and its natural host is characterized by the concurrence of host growth inhibition and the induction of viral DNA replication. When this virus-host interaction was investigated using S. islandicus REY15A, a strain which is closely related to the natural...

Synthesis of (E)-N'-[1-(2,4-Dihydroxyphenyl)ethylidene]substituted hydrazides as possible alpha-glucosidase and butyrylcholinesterase Inhibitors

International Nuclear Information System (INIS)

Abbasi, M.A.; Shah, S.A.H.; Siddiqui, S.Z.; Khan, K.M.

2017-01-01

In the current research work, (E)-N'-[1-(2,4-dihydroxyphenyl)ethylidene]substituted hydrazides were synthesized in a couple of steps and their enzyme inhibition potential was analyzed. Firstly 2,4-hydroxyacetophenone (1) was reacted with hydrated hydrazine (2) under stirring to yield (E)-4-(1-hydrazonoethyl)benzene-1,3-diol (3) which was further reacted with different acid halides, (4a-i) to afford (E)-[1-(2,4-dihydroxyphenyl)ethylidene]substituted hydrazides (5a-i). These synthesized compounds were characterized by EI-MS, 1H-NMR spectral techniques and were also evaluated against a-glucosidase and butyrylcholinesterase enzymes. The synthesized compounds were found to be acceptable inhibitors of a-glucosidase and decent inhibition against butyrylcholinesterase. (author)

Genetic manipulation in Sulfolobus islandicus and functional analysis of DNA repair genes

DEFF Research Database (Denmark)

Zhang, Changyi; Tian, Bin; Li, Suming

2013-01-01

Recently, a novel gene-deletion method was developed for the crenarchaeal model Sulfolobus islandicus, which is a suitable tool for addressing gene essentiality in depth. Using this technique, we have investigated functions of putative DNA repair genes by constructing deletion mutants and studying...

KAPASITAS ANTIOKSIDAN DAN INHIBITOR ALFA GLUKOSIDASE EKSTRAK UMBI BAWANG DAYAK [Antioxidant and Alpha-Glucosidase Inhibitory Properties of Bawang Dayak Bulb Extracts

Directory of Open Access Journals (Sweden)

Andi Early Febrinda*

2013-12-01

Full Text Available Bawang dayak (Eleutherine palmifolia is an indigenous plant in Borneo traditionally used by Dayak tribes to treat any kind of degenerative deseases including diabetes mellitus. The purpose of this research was to measure antioxidant and antidiabetic capacities of water and ethanolic extracts of bawang dayak bulb. Parameters evaluated in this research were phytochemical screening, total phenolics, flavonoid content, DPPH free-radical scavenging activity, and alpha glucosidase inhibiting (AGI activity. The result showed that the total phenolics and flavonoid content in bawang dayak ethanolic extract (217.71 mg GAE/g and 65.35 mg QE/g were higher than that of the water extract (139.93 mg GAE/g and 16.95 mg QE/g. The ethanolic extract also had higher antioxidant and AGI activities (IC50 112 and 241 ppm than that of the water extract (IC50 526 and 505 ppm. In addition, the IC50 values for AGI in bawang dayak ethanolic extract was lower than acarbose which is known as a commercial antidiabetic agent.

Identification and functional verification of archaeal-type phosphoenolpyruvate carboxylase, a missing link in archaeal central carbohydrate metabolism.

Science.gov (United States)

Ettema, Thijs J G; Makarova, Kira S; Jellema, Gera L; Gierman, Hinco J; Koonin, Eugene V; Huynen, Martijn A; de Vos, Willem M; van der Oost, John

2004-11-01

Despite the fact that phosphoenolpyruvate carboxylase (PEPC) activity has been measured and in some cases even purified from some Archaea, the gene responsible for this activity has not been elucidated. Using sensitive sequence comparison methods, we detected a highly conserved, uncharacterized archaeal gene family that is distantly related to the catalytic core of the canonical PEPC. To verify the predicted function of this archaeal gene family, we cloned a representative from the hyperthermophilic acidophile Sulfolobus solfataricus and functionally produced the corresponding enzyme as a fusion with the Escherichia coli maltose-binding protein. The purified fusion protein indeed displayed highly thermostable PEPC activity. The structural and biochemical properties of the characterized archaeal-type PEPC (atPEPC) from S. solfataricus are in good agreement with previously reported biochemical analyses of other archaeal PEPC enzymes. The newly identified atPEPC, with its distinct properties, constitutes yet another example of the versatility of the enzymes of the central carbon metabolic pathways in the archaeal domain.

Doubling Power Output of Starch Biobattery Treated by the Most Thermostable Isoamylase from an Archaeon Sulfolobus tokodaii.

Science.gov (United States)

Cheng, Kun; Zhang, Fei; Sun, Fangfang; Chen, Hongge; Percival Zhang, Y-H

2015-08-20

Biobattery, a kind of enzymatic fuel cells, can convert organic compounds (e.g., glucose, starch) to electricity in a closed system without moving parts. Inspired by natural starch metabolism catalyzed by starch phosphorylase, isoamylase is essential to debranch alpha-1,6-glycosidic bonds of starch, yielding linear amylodextrin - the best fuel for sugar-powered biobattery. However, there is no thermostable isoamylase stable enough for simultaneous starch gelatinization and enzymatic hydrolysis, different from the case of thermostable alpha-amylase. A putative isoamylase gene was mined from megagenomic database. The open reading frame ST0928 from a hyperthermophilic archaeron Sulfolobus tokodaii was cloned and expressed in E. coli. The recombinant protein was easily purified by heat precipitation at 80 (o)C for 30 min. This enzyme was characterized and required Mg(2+) as an activator. This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 (o)C in the presence of 0.5 mM MgCl2, suitable for simultaneous starch gelatinization and isoamylase hydrolysis. The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated.

Glycogen-bound polyphosphate kinase from the archaebacterium Sulfolobus acidocaldarius.

OpenAIRE

Skórko, R; Osipiuk, J; Stetter, K O

1989-01-01

Glycogen-bound polyphosphate kinase has been isolated from a crude extract of Sulfolobus acidocaldarius by isopycnic centrifugation in CsCl. Divalent cations (Mn2+ greater than Mg2+) stimulated the reaction. The enzyme does not require the presence of histones for its activity; it is inhibited strongly by phosphate and slightly by fluoride. The protein from the glycogen complex migrated in a sodium dodecyl sulfate-polyacrylamide gel as a 57-kilodalton protein band; after isoelectric focusing ...

Synthèses enzymatiques de néoglucoconjugués catalysées par l'alpha-glucosidase purifiée de la blatte Periplaneta americana (Linnaeus

Directory of Open Access Journals (Sweden)

Kamenan A.

2005-01-01

Full Text Available Enzymatic synthesis of neoglucoconjugates by purified α-glucosidase from cockroach Periplaneta americana (Linnaeus. Cockroach Periplaneta americana (Linnaeus contains in his digestive tract an acid (pH 5,0 and mesophile (50°C α-glucosidase. This enzyme, purified to homogeneity, hydrolyses highly maltose, sucrose and p-nitrophenyl-α-Dglucopyranoside. The ability of α-glucosidase from cockroach purified to homogeneity to catalyse transglucosylation reactions was tested using maltose and saccharose as glucosyl donors and 2-phenylethanol and phenol as acceptors. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glucosyl donors and acceptors. The yields in transglucosylation reactions at 37 °C were very high and could attain 67% and 48% with 2-phenylethanol and phenol respectively as glucosyl acceptors. This α-glucosidase hydrolyzed the products formed. It seems that the products formed were the phenylethyl-α-D-glucoside and phenyl-α-D-glucoside. These results suggest that α- glucosidase from cockroach is an exoglucosidase which catalyse the splitting of the α-glucosyl residue from the non reducing terminal of the substrate to liberate α-glucose. This comportment indicates that this enzyme operated by a mechanism involving the retention of the anomeric configuration. On the basis of this work, α-glucosidase from P. americana appears to be a valuable tool for the preparation of α-neoglucoconjugates.

Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase.

Science.gov (United States)

Liew, Li Phing; Lim, Zun Yi; Cohen, Matan; Kong, Ziqing; Marjavaara, Lisette; Chabes, Andrei; Bell, Stephen D

2016-11-01

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Elucidating the transcription cycle of the UV-inducible hyperthermophilic archaeal virus SSV1 by DNA microarrays

International Nuclear Information System (INIS)

Froels, Sabrina; Gordon, Paul M.K.; Panlilio, Mayi Arcellana; Schleper, Christa; Sensen, Christoph W.

2007-01-01

The spindle-shaped Sulfolobus virus SSV1 was the first of a series of unusual and uniquely shaped viruses isolated from hyperthermophilic Archaea. Using whole-genome microarrays we show here that the circular 15.5 kb DNA genome of SSV1 exhibits a chronological regulation of its transcription upon UV irradiation, reminiscent to the life cycles of bacteriophages and eukaryotic viruses. The transcriptional cycle starts with a small UV-specific transcript and continues with early transcripts on both its flanks. The late transcripts appear after the onset of viral replication and are extended to their full lengths towards the end of the approximately 8.5 h cycle. While we detected only small differences in genome-wide analysis of the host Sulfolobus solfataricus comparing infected versus uninfected strains, we found a marked difference with respect to the strength and speed of the general UV response of the host. Models for the regulation of the virus cycle, and putative functions of genes in SSV1 are presented

Factor requirements for transcription in the Archaeon Sulfolobus shibatae.

Science.gov (United States)

Qureshi, S A; Bell, S D; Jackson, S P

1997-05-15

Archaea (archaebacteria) constitute a domain of life that is distinct from Bacteria (eubacteria) and Eucarya (eukaryotes). Although archaeal cells share many morphological features with eubacteria, their transcriptional apparatus is more akin to eukaryotic RNA polymerases I, II and III than it is to eubacterial transcription systems. Thus, in addition to possessing a 10 subunit RNA polymerase and a homologue of the TATA-binding protein (TBP), Archaea possess a polypeptide termed TFB that is homologous to eukaryotic TFIIB. Here, we investigate the factor requirements for transcription of several promoters of the archaeon Sulfolobus shibatae and its associated virus SSV. Through in vitro transcription and immunodepletion, we demonstrate that S. shibatae TBP, TFB and RNA polymerase are not complexed tightly with one another and that each is required for efficient transcription of all promoters tested. Furthermore, full transcription is restored by supplementing respective depleted extracts with recombinant TBP or TFB, indicating that TBP-associated factors or TFB-associated factors are not required. Indeed, gel-filtration suggests that Sulfolobus TBP and TFB are not associated stably with other proteins. Finally, all promoters analysed are transcribed accurately and efficiently in an in vitro system comprising recombinant TBP and TFB, together with essentially homogeneous preparation of RNA polymerase. Transcription in Archaea is therefore fundamentally homologous to that in eukaryotes, although factor requirements appear to be much less complex.

Isolation of Extracellular Polymeric Substances from Biofilms of the Thermoacidophilic Archaeon Sulfolobus acidocaldarius.

Science.gov (United States)

Jachlewski, Silke; Jachlewski, Witold D; Linne, Uwe; Bräsen, Christopher; Wingender, Jost; Siebers, Bettina

2015-01-01

Extracellular polymeric substances (EPS) are the major structural and functional components of microbial biofilms. The aim of this study was to establish a method for EPS isolation from biofilms of the thermoacidophilic archaeon, Sulfolobus acidocaldarius, as a basis for EPS analysis. Biofilms of S. acidocaldarius were cultivated on the surface of gellan gum-solidified Brock medium at 78°C for 4 days. Five EPS extraction methods were compared, including shaking of biofilm suspensions in phosphate buffer, cation-exchange resin (CER) extraction, and stirring with addition of EDTA, crown ether, or NaOH. With respect to EPS yield, impact on cell viability, and compatibility with subsequent biochemical analysis, the CER extraction method was found to be the best suited isolation procedure resulting in the detection of carbohydrates and proteins as the major constituents and DNA as a minor component of the EPS. Culturability of CER-treated cells was not impaired. Analysis of the extracellular proteome using two-dimensional gel electrophoresis resulted in the detection of several hundreds of protein spots, mainly with molecular masses of 25-116 kDa and pI values of 5-8. Identification of proteins suggested a cytoplasmic origin for many of these proteins, possibly released via membrane vesicles or biofilm-inherent cell lysis during biofilm maturation. Functional analysis of EPS proteins, using fluorogenic substrates as well as zymography, demonstrated the activity of diverse enzyme classes, such as proteases, lipases, esterases, phosphatases, and glucosidases. In conclusion, the CER extraction method, as previously applied to bacterial biofilms, also represents a suitable method for isolation of water soluble EPS from the archaeal biofilms of S. acidocaldarius, allowing the investigation of composition and function of EPS components in these types of biofilms.

Isolation of extracellular polymeric substances from biofilms of the thermoacidophilic archaeon Sulfolobus acidocaldarius

Directory of Open Access Journals (Sweden)

Silke eJachlewski

2015-08-01

Full Text Available Extracellular polymeric substances (EPS are the major structural and functional components of microbial biofilms. The aim of this study was to establish a method for EPS isolation from biofilms of the thermoacidophilic archaeon Sulfolobus acidocaldarius as a basis for EPS analysis. Biofilms of S. acidocaldarius were cultivated on the surface of gellan gum-solidified Brock medium at 78 °C for 4 days. Five EPS extraction methods were compared, including shaking of biofilm suspensions in phosphate buffer, cation-exchange resin (CER extraction and stirring with addition of EDTA, crown ether or NaOH. With respect to EPS yield, impact on cell viability and compatibility with subsequent biochemical analysis, the CER extraction method was found to be the best suited isolation procedure resulting in the detection of carbohydrates and proteins as the major constituents and DNA as a minor component of the EPS. Culturability of CER-treated cells was not impaired. Analysis of the extracellular proteome using two-dimensional gel electrophoresis resulted in the detection of several hundredshundred of protein spots, mainly with molecular masses of 25 kDa to 116 kDa and pI values of 5 to 8. Identification of proteins suggested a cytoplasmic origin for many of these proteins, possibly released via membrane vesicles or biofilm-inherent cell lysis during biofilm maturation. Functional analysis of EPS proteins, using fluorogenic substrates as well as zymography, demonstrated the activity of diverse groups of enzymes such as proteases, lipases, esterases, phosphatases and glucosidases. In conclusion, the CER extraction method, as previously applied to bacterial biofilms, also represents a suitable method for isolation of water soluble EPS from the archaeal biofilms of S. acidocaldarius, allowing the investigation of composition and function of EPS components in these types of biofilms.

Enzyme assay, cloning and sequencing of novel β-glucosidase ...

African Journals Online (AJOL)

Bioinformatics studies also suggested that the cloned β-glucosidases share some characteristics with their bacterial counterparts. The findings in this study highlight the increasing need for more information on β-glucosidase structure and function. Keywords: Aspergillus niger, β-glucosidase, cellulase, PCR, sequencing, ...

Combination of alpha-glucosidase inhibitor and ribavirin for the treatment of Dengue virus infection in vitro and in vivo

Science.gov (United States)

Chang, Jinhong; Schul, Wouter; Butters, Terry D.; Yip, Andy; Liu, Boping; Goh, Anne; Lakshminarayana, Suresh B.; Alonzi, Dominic; Reinkensmeier, Gabriele; Pan, Xiaoben; Qu, Xiaowang; Weidner, Jessica M.; Wang, Lijuan; Yu, Wenquan; Borune, Nigel; Kinch, Mark A.; Rayahin, Jamie E.; Moriarty, Robert; Xu, Xiaodong; Shi, Pei-Yong; Guo, Ju-Tao; Block, Timothy M.

2010-01-01

Cellular α-glucosidases I and II are enzymes that sequentially trim the three terminal glucoses in the N-linked oligosaccharides of viral envelope glycoproteins. This process is essential for the proper folding of viral glycoproteins and subsequent assembly of many enveloped viruses, including dengue virus (DENV). Imino sugars are substrate mimics of α-glucosidases I and II. In this report, we show that two oxygenated alkyl imino sugar derivatives, CM-9-78 and CM-10-18, are potent inhibitors of both α-glucosidases I and II in vitro and in treated animals, and efficiently inhibit DENV infection of cultured human cells. Pharmacokinetic studies reveal that both compounds are well tolerated at doses up to 100mg/kg in rats and have favorable pharmacokinetic properties and bioavailability in mice. Moreover, we showed that oral administration of either CM-9-78 or CM-10-18 reduces the peak viremia of DENV in mice. Interestingly, while treatment of DENV infected mice with ribavirin alone did not reduce the viremia, combination therapy of ribavirin with sub-effective dose of CM-10-18 demonstrated a significantly enhanced antiviral activity, as indicated by a profound reduction of the viremia. Our findings thus suggest that combination therapy of two broad-spectrum antiviral agents may provide a practically useful approach for the treatment of DENV infection. PMID:21073903

Glycogen-bound polyphosphate kinase from the archaebacterium Sulfolobus acidocaldarius.

Science.gov (United States)

Skórko, R; Osipiuk, J; Stetter, K O

1989-09-01

Glycogen-bound polyphosphate kinase has been isolated from a crude extract of Sulfolobus acidocaldarius by isopycnic centrifugation in CsCl. Divalent cations (Mn2+ greater than Mg2+) stimulated the reaction. The enzyme does not require the presence of histones for its activity; it is inhibited strongly by phosphate and slightly by fluoride. The protein from the glycogen complex migrated in a sodium dodecyl sulfate-polyacrylamide gel as a 57-kilodalton protein band; after isoelectric focusing it separated into several spots in the pH range of 5.6 to 6.7.

Phytochemicals Content, Antioxidant and α-Glucosidase Inhibition Activity of Bouea Macrophylla Griff Seed Extract

International Nuclear Information System (INIS)

Zainah Adam; Hazlina Ahmad Hassali; Rosniza Razali

2016-01-01

Bouea macrophylla Griff or locally known as kundang is one of the common fruit plant available in Malaysia. This plant from Anacardiaceae family is native to Southeast Asia particularly in Malaysia, Thailand and Indonesia. Medicinal values of this plant is not yet been explored. The present study was done to evaluate phytochemicals constituents in B. macrophylla seed extract qualitatively and quantitatively. Biological evaluations focusing on antioxidant and α-glucosidase inhibition were also performed. Qualitative phytochemicals screening revealed the presence of anthraquinones, terpenoids, flavanoids, tannins, alkaloids, glycosides, reducing sugar, steroids, triterpenes, phenolic, coumarine and proteins in B. macrophylla seed extract. Quantitative determination showed that B. macrophylla seed extract contains high amount of phenolic compounds (689.17±37.50 mg GAE/ g extract), but low amount of flavonoids (2.78±0.01 mg QE/ g extract), suggesting that most of the phenolics in B. macrophylla seed extract were non-flavonoids. Antioxidant assays showed that the extract possesses strong reducing power and DPPH radical scavenging activity (IC_5_0: 4.73±0.51 μg/ ml). These activities were almost comparable to that of vitamin C. α-Glucosidase inhibition study showed that the extract inhibited alpha-glucosidase activity potently with the IC_5_0 value of 0.55±0.04 mg/ ml, suggesting the ability of the plant to delay glucose absorption in small intestine, hence reduces hyperglycemia in diabetic condition. Potent antioxidant and α-glucosidase inhibitory activity of the extract might be attributed to the presence of high amount of phenolic compounds. In conclusion, this study showed that B. macrophylla seed extract contains various phytochemicals, possess strong antioxidant property and showed promising antidiabetic activity. These results indicate that B. macrophylla might have the potential to be developed as new pharmacological agent targeting on oxidative stress

C68 from the Sulfolobus islandicus plasmid-virus pSSVx is a novel member of the AbrB-like transcription factor family

DEFF Research Database (Denmark)

Contursi, Patrizia; D'Ambrosio, Katia; Pirone, Luciano

2011-01-01

The genetic element pSSVx from Sulfolobus islandicus, strain REY15/4, is a hybrid between a plasmid and a fusellovirus. This plasmid-virus hybrid infects several species of the hyperthermophilic acidophilic crenarchaeon Sulfolobus. The open reading frame orfc68 of pSSVx encodes a 7.7 kDa protein...... factors, such as AbrB from Bacillus subtilis. Nevertheless, C68 constitutes a novel representative of this family because it shows several peculiar structural and functional features....

In vivo assay to identify bacteria with β-glucosidase activity

Directory of Open Access Journals (Sweden)

Erwin Strahsburger

2017-11-01

Conclusion: This in vivo β-glucosidase assay can be used as an enzymatic test on living cells without cell disruption. The method is simple, quantitative, and recommended, especially in studies screening for bacteria not only with β-glucosidase activity but also with high β-glucosidase activity.

α-Glucosidase inhibitory effect of resveratrol and piceatannol.

Science.gov (United States)

Zhang, Albert J; Rimando, Agnes M; Mizuno, Cassia S; Mathews, Suresh T

2017-09-01

Dietary polyphenols have been shown to inhibit α-glucosidase, an enzyme target of some antidiabetic drugs. Resveratrol, a polyphenol found in grapes and wine, has been reported to inhibit the activity of yeast α-glucosidase. This triggered our interest to synthesize analogs and determine their effect on mammalian α-glucosidase activity. Using either sucrose or maltose as substrate resveratrol, piceatannol and 3'-hydroxypterostilbene showed strong inhibition of mammalian α-glucosidase activity; pinostilbene, cis-desoxyrhapontigenin and trans-desoxyrhapontigenin had moderate inhibition. Compared to acarbose (IC 50 3-13 μg/ml), piceatannol and resveratrol inhibited mammalian α-glucosidase to a lesser extent (IC 50 14-84 and 111-120 μg/ml, respectively). 3'-Hydroxypterostilbene (IC 50 105-302 μg/ml) was 23-35-fold less potent than acarbose. We investigated the effect of piceatannol and resveratrol on postprandial blood glucose response in high-fat-fed C57Bl/6 mice. Animals administered resveratrol (30 mg/kg body weight [BW]) or piceatannol (14 mg/kg BW) 60 min prior to sucrose or starch loading had a delayed absorption of carbohydrates, resulting in significant lowering of postprandial blood glucose concentrations, similar to the antidiabetic drug acarbose, while no significant effect was observed with the glucose-loaded animals. Our studies demonstrate that the dietary polyphenols resveratrol and piceatannol lower postprandial hyperglycemia and indicate that inhibition of intestinal α-glucosidase activity may be a potential mechanism contributing to their antidiabetic property. Copyright © 2017 Elsevier Inc. All rights reserved.

Glucosidase: microbial production and effect on enzymatic hydrolysis of cellulose

Energy Technology Data Exchange (ETDEWEB)

Sternberg, D

1977-01-01

The enzymic conversion of cellulose is catalyzed by a multiple enzyme system. The Trichoderma enzyme system has insufficient ..beta..-glucosidase (EC 3.2.1.21) activity for the practical saccharification of cellulose. Aspergillus niger and A. phoenicis were superior producers of ..beta.. glucosidase and a method for production of this enzyme in liquid culture is presented. When Trichoderma cellulase preparations are supplemented with ..beta.. glucosidase from Aspergullus during practical saccharifications glucose is the predominant product and the rate of saccharification is significantly increased. The stimulatory effect of ..beta.. glucosidase appears to be due to the removal of inhibitory levels of cellobiose.

Production and characterization of β-glucosidase from Gongronella ...

African Journals Online (AJOL)

Among the enzymes of the cellulolytic complex, β-glucosidases are noteworthy due to the possibility of their application in different industrial processes, such as production of biofuels, winemaking, and development of functional foods. This study aimed to evaluate the production and characterization of β-glucosidase from ...

Trichoderma .beta.-glucosidase

Science.gov (United States)

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2006-01-03

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

Characteristics of β-glucosidase production by Paecilomyces variotii ...

African Journals Online (AJOL)

This study reports the potential application of Paecilomyces variotii immobilized in calcium alginate beads as a sensing element in the analysis of boric acid. In the presence of boric acid, β-glucosidase production of P. variotii was inhibited and the changes of β-glucosidase concentration were correlated to the ...

Phytochemical Screening, Alpha-Glucosidase Inhibition, Antibacterial and Antioxidant Potential of Ajuga bracteosa Extracts.

Science.gov (United States)

Hafeez, Kokab; Andleeb, Saiqa; Ghousa, Tahseen; Mustafa, Rozina G; Naseer, Anum; Shafique, Irsa; Akhter, Kalsoom

2017-01-01

Ajuga bracteosa, a medicinal herb, is used by local community to cure a number of diseases such as inflammation, jaundice bronchial asthma, cancer and diabetes. The aim of present work was to evaluate the antioxidant potential, in vitro antidiabetic and antimicrobial effects of A. bracteosa. n-hexane, ethyl acetate, chloroform, acetone, methanol and aqueous extracts of Ajuga bracteosa roots, were prepared via maceration. Antibacterial activity was carried out by agar well diffusion method. Quantitative and qualitative phytochemical screening was done. The antioxidant activity was determined by iron (II) chelating activity, iron reducing power, DPPH, and ABTS free radical scavenging methods, Antidiabetic activity was evaluated through inhibition of α-glucosidase assay. Phytochemical analysis showed the presence of phenols, flavonoids, tannins, saponins, quinines, terpenoids, xanthoproteins, glycosides, carbohydrates, steroids, phytosterols and amino acids. DPPH and ABTS potential values were recorded as 61.92% to 88.84% and 0.11% to 38.82%, respectively. Total phenolic and total flavonoid contents were expressed as gallic acid and rutin equivalents. Total iron content was expressed as FeSO4 equivalents. Chloroform and n-hexane extracts showed significant enzyme inhibition potential with IC50 values of 29.92 μg/ml and 131.7 μg/ml respectively. Aqueous extract showed maximum inhibition of E. coli, S. typhimurium, E. amnigenus, S. pyogenes, and S. aureus, (18.0±1.0 mm, 12.5±0.7 mm, 17.0±0.0 mm, 11.0±0.0 mm and 15.3±2.0 mm mm), respectively. Similarly, n-hexane extract showed maximum inhibition of E. coli, E. amnigenus, S. aureus (11.6±1.5 mm; 11.3±1.5 mm; 13.3±0.5 mm). This study also shows that n-hexane, chloroform, ethyl acetate and aqueous extracts of A. bracteosa root possess α-glucosidase inhibitory activities and therefore it may be used as hypoglycemic agents in the management of postprandial hyperglycemia. Ajuga bracteosa root extracts may provide a

Purification and properties of two /beta/-glucosidases isolated from Aspergillus niger

Energy Technology Data Exchange (ETDEWEB)

Witte, K.; Wartenberg, A.

1989-01-01

The cellulase complex of the fungus Aspergillus niger (strain CBS 554.65=ATCC 16 888) was fractionated by gel filtration yielding six pronounced peaks. Only proteins from the fraction corresponding to the first peak (96 kDa) showed /beta/-glucosidase activity vs. the substrate 4-nitrophenyl-/beta/-D-glucopyranoside (pNPG). These proteins have been fractionated by chromatofocusing, yielding two /beta/-glucosidases (I and II) which are shown to be homogeneous in isoelectric focusing experiments (pI=4.6 and 3.8, respectively). Kinetic experiments with pNPG, MU-glucopyranoside and cellobiose revealed that both types of /beta/-glucosidases behave like aryl-/beta/-glucosidases, /beta/-Glucosidase-I acting on pNPG exhibits a split kinetics characterized by high and low substrate-concentration kinetics which are differentiated by different values of V and of K/sub m/. In addition, /beta/-glucosidase-II is shown to be an exo-glucohydrolase as deduced from experiments with MU-cellobiopyranoside. Experimental features should be emphasized; usual soft-gel ion-exchange materials did not work in the chromatofocusing separation of the two /beta/-glucosidases, in contrast to the 10 /mu/-Si 500=DEAE exchange material (Serva) typically used in HPLC-experiments. Furthermore, protein content determinations based on different procedures yielded widely differing values. (orig.).

Oxindole based oxadiazole hybrid analogs: Novel α-glucosidase inhibitors.

Science.gov (United States)

Taha, Muhammad; Imran, Syahrul; Rahim, Fazal; Wadood, Abdul; Khan, Khalid Mohammed

2018-02-01

Inhibition of α-glucosidase is an effective strategy for controlling post-prandial hyperglycemia in diabetic patients. Beside these α-glucosidase inhibitors has been also used as anti-obesity and anti-viral drugs. Keeping in view the greater importance of α-glucosidase inhibitors here in this study we are presenting oxindole based oxadiazoles hybrid analogs (1-20) synthesis, characterized by different spectroscopic techniques including 1 H NMR and EI-MS and their α-glucosidase inhibitory activity. All compounds were found potent inhibitors for the enzyme with IC 50 values ranging between 1.25 ± 0.05 and 268.36 ± 4.22 µM when compared with the standard drug acarbose having IC 50 value 895.09 ± 2.04 µM. Our study identifies novel series of potent α-glucosidase inhibitors and further investigation on this may led to the lead compounds. A structure activity relationship has been established for all compounds. The interactions of the active compounds and enzyme active site were established with the help of molecular docking studies. Copyright © 2017 Elsevier Inc. All rights reserved.

Association of. beta. -glucosidase with intact cells of thermoactinomyces

Energy Technology Data Exchange (ETDEWEB)

Haegerdal, B; Harris, H; Pye, E K

1979-03-01

The location of the ..beta..-glucosidase activity in a whole culture broth of the thermophilic organism Thermoactinomyces has been studied. Little ..beta..-glucosidase activity was found in the culture filtrate, while the culture solids contained the major part of the activity of the whole culture broth. The activity does not appear to be adsorbed to the culture solids; rather there is evidence that it is an intracellular soluble enzyme(s). The pH and temperature optima for a crude ..beta..-glucosidase preparation were determined to be pH 6.5 and 50 to 55/sup 0/C. Enzyme activity studies indicate that the same enzyme(s) accounts for the ..beta..-glucosidase and the cellobiase activities. The validity of using the filter paper activity of culture filtrates from Thermoactinomyces to predict the total saccharification of cellulosic materials to glucose is discussed.

The distribution of active β-glucosidase-producing microbial communities in composting.

Science.gov (United States)

Zang, Xiangyun; Liu, Meiting; Wang, Han; Fan, Yihong; Zhang, Haichang; Liu, Jiawen; Xing, Enlu; Xu, Xiuhong; Li, Hongtao

2017-12-01

The composting ecosystem is a suitable source for the discovery of novel microorganisms and secondary metabolites. Cellulose degradation is an important part of the global carbon cycle, and β-glucosidases complete the final step of cellulose hydrolysis by converting cellobiose to glucose. This work analyzes the succession of β-glucosidase-producing microbial communities that persist throughout cattle manure - rice straw composting, and evaluates their metabolic activities and community advantage during the various phases of composting. Fungal and bacterial β-glucosidase genes belonging to glycoside hydrolase families 1 and 3 (GH1 and GH3) amplified from DNA were classified and gene abundance levels were analyzed. The major reservoirs of β-glucosidase genes were the fungal phylum Ascomycota and the bacterial phyla Firmicutes, Actinobacteria, Proteobacteria, and Deinococcus-Thermus. This indicates that a diverse microbial community utilizes cellobiose. The succession of dominant bacteria was also detected during composting. Firmicutes was the dominant bacteria in the thermophilic phase of composting; there was a shift to Actinomycetes in the maturing stage. Proteobacteria accounted for the highest proportions during the heating and thermophilic phases of composting. By contrast, the fungal phylum Ascomycota was a minor microbial community constituent in thermophilic phase of composting. Combined with the analysis of the temperature, cellulose degradation rate and the carboxymethyl cellulase and β-glucosidase activities showed that the bacterial GH1 family β-glucosidase genes make greater contribution in cellulose degradation at the later thermophilic stage of composting. In summary, even GH1 bacteria families β-glucosidase genes showing low abundance in DNA may be functionally important in the later thermophilic phase of composting. The results indicate that a complex community of bacteria and fungi expresses β-glucosidases in compost. Several β-glucosidase

Characteristics of alpha-glucosidase production from recombinant Aspergillus oryzae by membrane-surface liquid culture in comparison with various cultivation methods.

Science.gov (United States)

Morita, Masakazu; Shimamura, Hiroko; Ishida, Natsuko; Imamura, Koreyoshi; Sakiyama, Takaharu; Nakanishi, Kazuhiro

2004-01-01

alpha-Glucosidase was produced using recombinant Aspergillus oryzae by membrane-surface liquid culture (MSLC), a method previously developed by the authors and the results compared with other methods, including shaking flask culture (SFC), agar-plate culture (APC), culture on urethane sponge supports (USC), and liquid surface culture (LSC) to determine possible reasons for the advantageous features of MSLC. When yeast extract was used as a nitrogen source, the amount of enzyme produced by MSLC was 5 or more times higher than those for SFC and LSC, but similar to that using APC. Enzyme production in USC was slightly lower than in MSLC and APC. Cell growth was similar irrespective of the cultivation method used. When NaNO3, a typical inorganic nitrogen source was used, enzyme production in all the cultures was lower than that using yeast extract. However, even using NaNO3, the amount of the enzyme produced by MSLC was 8 to 20 times higher than those by SFC, APC, USC, and LSC. Although cell growth using NaNO3 was similar to that for yeast extract in MSLC, it was markedly decreased in SFC, APC, and LSC. The reason for the difference in enzyme productivity for various cultivation methods using yeast extract and NaNO3 as a nitrogen source is discussed, on the basis of the experimental findings. The role of the oxygen transfer effect and gene expression levels in enzyme production were also examined.

Major and minor crRNA annealing sites facilitate low stringency DNA protospacer binding prior to Type I-A CRISPR-Cas interference in Sulfolobus

DEFF Research Database (Denmark)

Mousaei, Marzieh; Deng, Ling; She, Qunxin

2016-01-01

The stringency of crRNA-protospacer DNA base pair matching required for effective CRISPR-Cas interference is relatively low in crenarchaeal Sulfolobus species in contrast to that required in some bacteria. To understand its biological significance we studied crRNA-protospacer interactions...... in Sulfolobus islandicus REY15A which carries multiple, and functionally diverse, interference complexes. A range of mismatches were introduced into a vector-borne protospacer that was identical to spacer 1 of CRISPR locus 2, with a cognate CCN PAM sequence. Two important crRNA annealing regions were identified...

Extraction, isolation and NMR data of the tetraether lipid calditoglycerocaldarchaeol (GDNT) from Sulfolobus metallicus harvested from a bioleaching reactor

CSIR Research Space (South Africa)

Bode, ML

2008-08-01

Full Text Available The successful extraction and isolation of the hydrolysed tetraether lipid calditoglycerocaldarchaeol (GDNT) from Sulfolobus etallicus, a key thermophilic bioleaching archaeon, is described. The archaeal biomasswas recovered directly from a...

Divergent clinical outcomes of alpha-glucosidase enzyme replacement therapy in two siblings with infantile-onset Pompe disease treated in the symptomatic or pre-symptomatic state.

Science.gov (United States)

Matsuoka, Takashi; Miwa, Yoshiyuki; Tajika, Makiko; Sawada, Madoka; Fujimaki, Koichiro; Soga, Takashi; Tomita, Hideshi; Uemura, Shigeru; Nishino, Ichizo; Fukuda, Tokiko; Sugie, Hideo; Kosuga, Motomichi; Okuyama, Torayuki; Umeda, Yoh

2016-12-01

Pompe disease is an autosomal recessive, lysosomal glycogen storage disease caused by acid α-glucosidase deficiency. Infantile-onset Pompe disease (IOPD) is the most severe form and is characterized by cardiomyopathy, respiratory distress, hepatomegaly, and skeletal muscle weakness. Untreated, IOPD generally results in death within the first year of life. Enzyme replacement therapy (ERT) with recombinant human acid alpha glucosidase (rhGAA) has been shown to markedly improve the life expectancy of patients with IOPD. However, the efficacy of ERT in patients with IOPD is affected by the presence of symptoms and cross-reactive immunologic material (CRIM) status. We have treated two siblings with IOPD with ERT at different ages: the first was symptomatic and the second was asymptomatic. The female proband (Patient 1) was diagnosed with IOPD and initiated ERT at 4 months of age. Her younger sister (Patient 2) was diagnosed with IOPD at 10 days of age and initiated ERT at Day 12. Patient 1, now 6 years old, is alive but bedridden, and requires 24-hour invasive ventilation due to gradually progressive muscle weakness. In Patient 2, typical symptoms of IOPD, including cardiac failure, respiratory distress, progressive muscle weakness, hepatomegaly and myopathic facial features were largely absent during the first 12 months of ERT. Her cardiac function and mobility were well-maintained for the first 3 years, and she had normal motor development. However, she developed progressive hearing impairment and muscle weakness after 3 years of ERT. Both siblings have had low anti-rhGAA immunoglobulin G (IgG) antibody titers during ERT and have tolerated the treatment well. These results suggest that initiation of ERT during the pre-symptomatic period can prevent and/or attenuate the progression of IOPD, including cardiomyopathy, respiratory distress, and muscle weakness for first several years of ERT. However, to improve the long-term efficacy of ERT for IOPD, new strategies

Cinnamic acid amides from Tribulus terrestris displaying uncompetitive α-glucosidase inhibition.

Science.gov (United States)

Song, Yeong Hun; Kim, Dae Wook; Curtis-Long, Marcus J; Park, Chanin; Son, Minky; Kim, Jeong Yoon; Yuk, Heung Joo; Lee, Keun Woo; Park, Ki Hun

2016-05-23

The α-glucosidase inhibitory potential of Tribulus terrestris extracts has been reported but as yet the active ingredients are unknown. This study attempted to isolate the responsible metabolites and elucidate their inhibition mechanism of α-glucosidase. By fractionating T. terristris extracts, three cinnamic acid amide derivatives (1-3) were ascertained to be active components against α-glucosidase. The lead structure, N-trans-coumaroyltyramine 1, showed significant inhibition of α-glucosidase (IC50 = 0.42 μM). Moreover, all active compounds displayed uncompetitive inhibition mechanisms that have rarely been reported for α-glucosidase inhibitors. This kinetic behavior was fully demonstrated by showing a decrease of both Km and Vmax, and Kik/Kiv ratio ranging between 1.029 and 1.053. We progressed to study how chemical modifications to the lead structure 1 may impact inhibition. An α, β-unsaturation carbonyl group and hydroxyl group in A-ring of cinnamic acid amide emerged to be critical functionalities for α-glucosidase inhibition. The molecular modeling study revealed that the inhibitory activities are tightly related to π-π interaction as well as hydrogen bond interaction between enzyme and inhibitors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Beta-Glucosidases from a new Aspergillus species can substitute commercial beta-glucosidases for saccharification of lignocellulosic biomass

Energy Technology Data Exchange (ETDEWEB)

Sorensen, Annette; Lubeck, Peter Stephensen; Lubeck, Mette; Teller, Philip Johan; Kiaer Ahring, Birgitte

2011-07-01

Exploitation of lignocellulosic biomasses for the production of biofuels and biochemicals gives a promising alternative to the world's limited fossil energy resources. Cellulose is of great interest in terms of producing sugars for biofuels and biochemicals, since its hydrolysis product, glucose, can readily be fermented into ethanol or converted into high-value chemicals. The hydrolysis of cellulose involves the synergistic action of cellobiohydrolases, endoglucanases and B-glucosidases, and B-glucosidases is key in ensuring final glucose release and the decrease of the accumulation of cellobiose and shorter cellodextrins, known as product inhibitors of the cellobiohydrolases. The aim of the present work was to search for efficient B-glucosidase-producing fungi using a screening strategy based on wheat bran as fermentation substrate. The fungi selected originated from several different countries and fungal fermentation broth were compared with an onsite enzyme production in mind. The broth of the best strain was tested against commercial enzyme preparations based on enzyme kinetics and it proved to be a valid substitute.

Purification and Partial Characterization of β-Glucosidase in Chayote (Sechium edule

Directory of Open Access Journals (Sweden)

Sergio Espíndola Mateos

2015-10-01

Full Text Available β-Glucosidase (EC 3.2.1.21 is a prominent member of the GH1 family of glycoside hydrolases. The properties of this β-glucosidase appear to include resistance to temperature, urea, and iodoacetamide, and it is activated by 2-ME, similar to other members. β-Glucosidase from chayote (Sechium edule was purified by ionic-interchange chromatography and molecular exclusion chromatography. Peptides detected by LC-ESI-MS/MS were compared with other β-glucosidases using the BLAST program. This enzyme is a 116 kDa protein composed of two sub-units of 58 kDa and shows homology with Cucumis sativus β-glucosidase (NCBI reference sequence XP_004154617.1, in which seven peptides were found with relative masses ranging from 874.3643 to 1587.8297. The stability of β-glucosidase depends on an initial concentration of 0.2 mg/mL of protein at pH 5.0 which decreases by 33% in a period of 30 h, and then stabilizes and is active for the next 5 days (pH 4.0 gives similar results. One hundred μg/mL β-D-glucose inhibited β-glucosidase activity by more than 50%. The enzyme had a Km of 4.88 mM with p-NPG and a Kcat of 10,000 min−1. The optimal conditions for the enzyme require a pH of 4.0 and a temperature of 50 °C.

Purification and Partial Characterization of β-Glucosidase in Chayote (Sechium edule).

Science.gov (United States)

Mateos, Sergio Espíndola; Cervantes, Carlos Alberto Matías; Zenteno, Edgar; Slomianny, Marie-Christine; Alpuche, Juan; Hernández-Cruz, Pedro; Martínez-Cruz, Ruth; Canseco, Maria Del Socorro Pina; Pérez-Campos, Eduardo; Rubio, Manuel Sánchez; Mayoral, Laura Pérez-Campos; Martínez-Cruz, Margarito

2015-10-23

β-Glucosidase (EC 3.2.1.21) is a prominent member of the GH1 family of glycoside hydrolases. The properties of this β-glucosidase appear to include resistance to temperature, urea, and iodoacetamide, and it is activated by 2-ME, similar to other members. β-Glucosidase from chayote (Sechium edule) was purified by ionic-interchange chromatography and molecular exclusion chromatography. Peptides detected by LC-ESI-MS/MS were compared with other β-glucosidases using the BLAST program. This enzyme is a 116 kDa protein composed of two sub-units of 58 kDa and shows homology with Cucumis sativus β-glucosidase (NCBI reference sequence XP_004154617.1), in which seven peptides were found with relative masses ranging from 874.3643 to 1587.8297. The stability of β-glucosidase depends on an initial concentration of 0.2 mg/mL of protein at pH 5.0 which decreases by 33% in a period of 30 h, and then stabilizes and is active for the next 5 days (pH 4.0 gives similar results). One hundred μg/mL β-D-glucose inhibited β-glucosidase activity by more than 50%. The enzyme had a Km of 4.88 mM with p-NPG and a Kcat of 10,000 min(-1). The optimal conditions for the enzyme require a pH of 4.0 and a temperature of 50 °C.

Dicty_cDB: Contig-U04013-1 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available pid:none) Flavobacterium psychrophilum JIP... 98 3e-19 EU678894_1( EU678894 |pid:none) Bacillus intermedius ...46 3e-04 2 ( EH672780 ) CCIL10412.b1_G11.ab1 CCI(LMS) chicory Cichorium i... 46 3e-04 2 ( EJ368820 ) 1092963715450 Global-Ocean-Sampl...41 |pid:none) Sulfolobus solfataricus P2, com... 89 2e-16 AH1770( AH1770 ) conserved hypothetical protein lin2710 [imported...AY653733 ) Acanthamoeba polyphaga mimivirus, complete genome. 62 3e-05 1 ( EH678404 ) CCIL3410.b1_D14.ab1 CC...ngs S... 44 6.8 1 >( AC116963 ) Dictyostelium discoideum chromosome 2 map 4657875-4914984 strain AX4, compl

Pycnalin, a new α-glucosidase inhibitor from Acer pycnanthum.

Science.gov (United States)

Ogawa, Ai; Miyamae, Yusaku; Honma, Atsushi; Koyama, Tomoyuki; Yazawa, Kazunaga; Shigemori, Hideyuki

2011-01-01

A new compound, pycnalin (1), together with four known compounds, ginnalins A (2), B (3), C (4), and 3,6-di-O-galloyl-1,5-anhydro-D-glucitol (3,6-di-GAG) (5), were isolated from Acer pycnanthum. The structure of 1 was determined on the basis of 2D-NMR spectral data and synthesis of 1. Pycnalin (1) is the first 1,5-anhydro-D-mannitol linked to a gallic acid, while compounds 2-5 were 1,5-anhydro-D-glucitol linked to gallic acids. All compounds were tested in vitro for α-glucosidase inhibitory and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities. Pycnalin (1) exhibited moderate α-glucosidase inhibitory activity as well as free radical scavenging activity. Ginnalin A (2) and 3,6-di-GAG (5), which have two galloyl groups, exhibited potent α-glucosidase inhibition, compared to those of other compounds 1, 3, and 4 containing a galloyl group. These results suggest that α-glucosidase inhibition is influenced by the number of galloyl groups.

Covalent immobilization of β-glucosidase on magnetic particles for lignocellulose hydrolysis.

Science.gov (United States)

Alftrén, Johan; Hobley, Timothy John

2013-04-01

β-Glucosidase hydrolyzes cellobiose to glucose and is an important enzyme in the consortium used for hydrolysis of cellulosic and lignocellulosic feedstocks. In the present work, β-glucosidase was covalently immobilized on non-porous magnetic particles to enable re-use of the enzyme. It was found that particles activated with cyanuric chloride and polyglutaraldehyde gave the highest bead-related immobilized enzyme activity when tested with p-nitrophenyl-β-D-glucopyranoside (104.7 and 82.2 U/g particles, respectively). Furthermore, the purified β-glucosidase preparation from Megazyme gave higher bead-related enzyme activities compared to Novozym 188 (79.0 and 9.8 U/g particles, respectively). A significant improvement in thermal stability was observed for immobilized enzyme compared to free enzyme; after 5 h (at 65 °C), 36 % of activity remained for the former, while there was no activity in the latter. The performance and recyclability of immobilized β-glucosidase on more complex substrate (pretreated spruce) was also studied. It was shown that adding immobilized β-glucosidase (16 U/g dry matter) to free cellulases (8 FPU/g dry matter) increased the hydrolysis yield of pretreated spruce from ca. 44 % to ca. 65 %. In addition, it was possible to re-use the immobilized β-glucosidase in the spruce and retain activity for at least four cycles. The immobilized enzyme thus shows promise for lignocellulose hydrolysis.

α-Amylase and α-glucosidase inhibitory effects of Sclerocarya birrea ...

African Journals Online (AJOL)

Inhibition of intestinal α-amylase and α-glucosidase is an important strategy to control post-prandial hyperglycemia associated with type 2 diabetes mellitus. In vitro inhibitory effects of crude Sclerocarya birrea stem bark (SBSB) extracts against human urinary α-amylase and Bacillus steatothermophilus α-glucosidase were ...

Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

NARCIS (Netherlands)

Albers, S.-V.; Jonuscheit, M.; Dinkelaker, S.; Urich, T.; Kletzin, A.; Tampé, R.; Driessen, A.J.M.; Schleper, C.

Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for

α-/β-Glucosidase and α-Amylase Inhibitory Activities of Roselle (Hibiscus sabdariffa L. Ethanol Extract

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Marisca Evalina Gondokesumo

2017-03-01

Full Text Available Background: Diabetes mellitus is a metabolic disease, characterized by hyperglycemia due to disturbance in both insulin secretion and function. One of theurapeutic approaches is to reduce blood glucose levels by inhbiting α-/β-glucosidase and α-amylase involved in carbohydrate digestion. Thus, inhibition of these enzymes play important role in the treatment of diabetes mellitus. Roselle (Hibiscus sabdariffa L. has been known to have several medicinal properties and potency as an antidiabetics agents. This reseacrh aimed to observe antidiabetic properties of roselle ethanol extract (REE towards α-glucosidase, β-glucosidase and α-amylase. Materials and Methods: REE was done with maceration technique using diluent of 70% ethanol. Antidiabetic properties were measured by inhibitory activity of α-amylase, α-glucosidase and β-glucosidase. Results: REE was able to inhibit α-/β-glucosidase and α-amylase in the highest concentration with inhibition percentage of 72.68, 47.34 and 73.08% respectively, and were comparable with Acarbose of 81.49, 50.97, 73.08%. The median inhibitory concentration (IC50 of α-/β-glucosidase and α-amylase of REE were 15.81, 41.77, 18.09 μg/mL respectively, and Acarbose were 9.45, 22.57, 3.64 μg/mL respectively. Conclusions: REE inhibits α-/β-glucosidase and α-amylase. Keywords: Roselle, Acarbose, α-glucosidase, β-glucosidase, α-amylase, antidiabetic

Genome Analyses of Icelandic Strains of Sulfolobus islandicus, Model Organisms for Genetic and Virus-Host Interaction Studies

DEFF Research Database (Denmark)

Guo, Li; Brügger, Kim; Liu, Chao

2011-01-01

The genomes of two Sulfolobus islandicus strains obtained from Icelandic solfataras were sequenced and analyzed. Strain REY15A is a host for a versatile genetic toolbox. It exhibits a genome of minimal size, is stable genetically, and is easy to grow and manipulate. Strain HVE10/4 shows a broad h...

Adsorption, immobilization, and activity of beta-glucosidase on different soil colloids.

Science.gov (United States)

Yan, Jinlong; Pan, Genxing; Li, Lianqing; Quan, Guixiang; Ding, Cheng; Luo, Ailan

2010-08-15

For a better understanding of enzyme stabilization and the subsequent catalytic process in a soil environment, the adsorption, immobilization, and activity of beta-glucosidase on various soil colloids from a paddy soil were studied. The calculated parameters maximum adsorption capacity (q(0)) for fine soil colloids ranged from 169.6 to 203.7 microg mg(-1), which was higher than coarse soil colloids in the range of 81.0-94.6 microg mg(-1), but the lower adsorption affinity (K(L)) was found on fine soil colloids. The percentages of beta-glucosidase desorbed from external surfaces of the coarse soil colloids (27.6-28.5%) were higher than those from the fine soil colloids (17.5-20.2%). Beta-glucosidase immobilized on the coarse inorganic and organic soil colloids retained 72.4% and 69.8% of activity, respectively, which indicated the facilitated effect of soil organic matter in the inhibition of enzyme activity. The residual activity for the fine soil clay is 79-81%. After 30 days of storage at 40 degrees C the free beta-glucosidase retained 66.2% of its initial activity, whereas the soil colloidal particle-immobilized enzyme retained 77.1-82.4% of its activity. The half-lives of free beta-glucosidase appeared to be 95.9 and 50.4 days at 25 and 40 degrees C. Immobilization of beta-glucosidase on various soil colloids enhanced the thermal stability at all temperatures, and the thermal stability was greatly affected by the affinity between the beta-glucosidase molecules and the surface of soil colloidal particles. Due to the protective effect of supports, soil colloidal particle-immobilized enzymes were less sensitive to pH and temperature changes than free enzymes. Data obtained in this study are helpful for further research on the enzymatic mechanisms in carbon cycling and soil carbon storage. Copyright 2010 Elsevier Inc. All rights reserved.

Factors affecting Archaeal Lipid Compositions of the Sulfolobus Species

Science.gov (United States)

He, L.; Han, J.; Wei, Y.; Lin, L.; Wei, Y.; Zhang, C.

2010-12-01

Temperature is the best known variable affecting the distribution of the archaeal glycerol dibiphytanyl glycerol tetraethers (GDGTs) in marine and freshwater systems. Other variables such as pH, ionic strength, or bicarbonate concentration may also affect archaeal GDGTs in terrestrial systems. Studies of pure cultures can help us pinpoint the specific effects these variables may have on archaeal lipid distribution in natural environments. In this study, three Sulfolobus species (HG4, HB5-2, HB9-6) isolated from Tengchong hot springs (pH 2-3, temperature 73-90°C) in China were used to investigate the effects of temperature, pH, substrate, and type of strain on the composition of GDGTs. Results showed that increase in temperature had negative effects on the relative contents of GDGT-0 (no cyclopentyl rings), GDGT-1 (one cyclopentyl ring), GDGT-2 and GDGT-3 but positive effects on GDGT-4, GDGT-4', GDGT-5 and GDGT-5'. Increase in pH, on the other hand, had negative effects on GDGT-0, GDGT-1, GDGT-4', GDGT-5 and GDGT-5', and positive effects on GDGT-3 and GDGT-4. GDGT-2 remained relatively constant with changing pH. When the HG4 was grown on different substrates, GDGT-5 was five time more abundant in sucrose-grown cultures than in yeast extract- or sulfur- grown cultures, suggesting that carbohydrates may stimulate the production of GDGT-5. For all three species, the ring index (average number of rings) of GDGTs correlated positively with incubation temperature. In HG4, ring index was much lower at optimal pH (3.5) than at other pH values. Ring index of HB5-2 or HB9-6 is higher than that of HG4, suggesting that speciation may affect the degree of cyclization of GDGT of the Sulfolobus. These results indicate that individual archaeal lipids respond differently to changes in environmental variables, which may be also species specific.

Oral delivery of Acid Alpha Glucosidase epitopes expressed in plant chloroplasts suppresses antibody formation in treatment of Pompe mice.

Science.gov (United States)

Su, Jin; Sherman, Alexandra; Doerfler, Phillip A; Byrne, Barry J; Herzog, Roland W; Daniell, Henry

2015-10-01

Deficiency of acid alpha glucosidase (GAA) causes Pompe disease in which the patients systemically accumulate lysosomal glycogen in muscles and nervous systems, often resulting in infant mortality. Although enzyme replacement therapy (ERT) is effective in treating patients with Pompe disease, formation of antibodies against rhGAA complicates treatment. In this report, we investigated induction of tolerance by oral administration of GAA expressed in chloroplasts. Because full-length GAA could not be expressed, N-terminal 410-amino acids of GAA (as determined by T-cell epitope mapping) were fused with the transmucosal carrier CTB. Tobacco transplastomic lines expressing CTB-GAA were generated through site-specific integration of transgenes into the chloroplast genome. Homoplasmic lines were confirmed by Southern blot analysis. Despite low-level expression of CTB-GAA in chloroplasts, yellow or albino phenotype of transplastomic lines was observed due to binding of GAA to a chloroplast protein that has homology to mannose-6 phosphate receptor. Oral administration of the plant-made CTB-GAA fusion protein even at 330-fold lower dose (1.5 μg) significantly suppressed immunoglobulin formation against GAA in Pompe mice injected with 500 μg rhGAA per dose, with several-fold lower titre of GAA-specific IgG1 and IgG2a. Lyophilization increased CTB-GAA concentration by 30-fold (up to 190 μg per g of freeze-dried leaf material), facilitating long-term storage at room temperature and higher dosage in future investigations. This study provides the first evidence that oral delivery of plant cells is effective in reducing antibody responses in ERT for lysosomal storage disorders facilitating further advances in clinical investigations using plant cell culture system or in vitro propagation. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

α-Glucosidase inhibitory activity of selected Malaysian plants

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Dzatil Awanis Mohd Bukhari

2017-01-01

Full Text Available Diabetes is a common metabolic disease indicated by unusually high plasma glucose level that can lead to major complications such as diabetic neuropathy, retinopathy, and cardiovascular diseases. One of the effective therapeutic managements of the disease is to reduce postprandial hyperglycemia through inhibition of α-glucosidase, a carbohydrate-hydrolyzing enzyme to retard overall glucose absorption. In recent years, a plenty of research works have been conducted looking for novel and effective α-glucosidase inhibitors (AGIs from natural sources as alternatives for the synthetic AGI due to their unpleasant side effects. Plants and herbs are rich with secondary metabolites that have massive pharmaceutical potential. Besides, studies showed that phytochemicals such as flavonoids, alkaloids, terpenoids, anthocyanins, glycosides, and phenolic compounds possess significant inhibitory activity against α-glucosidase enzyme. Malaysia is a tropical country that is rich with medicinal herbs. In this review, we focus on eight Malaysian plants with the potential as AGI to develop a potential functional food or lead compounds against diabetes.

α-Glucosidase Inhibitory Activity of Selected Malaysian Plants.

Science.gov (United States)

Mohd Bukhari, Dzatil Awanis; Siddiqui, Mohammad Jamshed; Shamsudin, Siti Hadijah; Rahman, Md Mukhlesur; So'ad, Siti Zaiton Mat

2017-01-01

Diabetes is a common metabolic disease indicated by unusually high plasma glucose level that can lead to major complications such as diabetic neuropathy, retinopathy, and cardiovascular diseases. One of the effective therapeutic managements of the disease is to reduce postprandial hyperglycemia through inhibition of α-glucosidase, a carbohydrate-hydrolyzing enzyme to retard overall glucose absorption. In recent years, a plenty of research works have been conducted looking for novel and effective α-glucosidase inhibitors (AGIs) from natural sources as alternatives for the synthetic AGI due to their unpleasant side effects. Plants and herbs are rich with secondary metabolites that have massive pharmaceutical potential. Besides, studies showed that phytochemicals such as flavonoids, alkaloids, terpenoids, anthocyanins, glycosides, and phenolic compounds possess significant inhibitory activity against α-glucosidase enzyme. Malaysia is a tropical country that is rich with medicinal herbs. In this review, we focus on eight Malaysian plants with the potential as AGI to develop a potential functional food or lead compounds against diabetes.

A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus

DEFF Research Database (Denmark)

Deng, Ling; Garrett, Roger Antony; Shah, Shiraz Ali

2013-01-01

Recent studies on CRISPR-based adaptive immune systems have revealed extensive structural and functional diversity of the interference complexes which often coexist intracellularly. The archaeon Sulfolobus islandicus REY15A encodes three interference modules, one of type IA and two of type IIIB...... targeting. A rationale is provided for the intracellular coexistence of the different interference systems in S.¿islandicus REY15A which cooperate functionally by sharing a single Cas6 protein for crRNA processing and utilize crRNA products from identical CRISPR spacers....

Fungal Beta-Glucosidases: A Bottleneck in Industrial Use of Lignocellulosic Materials

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Peter S. Lübeck

2013-09-01

Full Text Available Profitable biomass conversion processes are highly dependent on the use of efficient enzymes for lignocellulose degradation. Among the cellulose degrading enzymes, beta-glucosidases are essential for efficient hydrolysis of cellulosic biomass as they relieve the inhibition of the cellobiohydrolases and endoglucanases by reducing cellobiose accumulation. In this review, we discuss the important role beta-glucosidases play in complex biomass hydrolysis and how they create a bottleneck in industrial use of lignocellulosic materials. An efficient beta-glucosidase facilitates hydrolysis at specified process conditions, and key points to consider in this respect are hydrolysis rate, inhibitors, and stability. Product inhibition impairing yields, thermal inactivation of enzymes, and the high cost of enzyme production are the main obstacles to commercial cellulose hydrolysis. Therefore, this sets the stage in the search for better alternatives to the currently available enzyme preparations either by improving known or screening for new beta-glucosidases.

Triterpenes as uncompetitive inhibitors of α-glucosidase from flowers of Punica granatum L.

Science.gov (United States)

Salah El Dine, Riham; Ma, Qiong; Kandil, Zeinab A; El-Halawany, Ali M

2014-01-01

The α-glucosidase and maltase inhibitory effects of Punica granatum L. flowers (PGF) were investigated. The methanol extract (PGFMe), n-hexane extract (PGFH), chloroform extract (PGFC) and the remaining water fraction (PGFW) were assayed for their α-glucosidase and maltase inhibitory effects. PGFW showed potent α-glucosidase inhibition with IC₅₀ of 0.8 μg/mL followed by PGFMe (IC₅₀ of 4.0 μg/mL) then PGFC (IC₅₀ of 5.21 μg/mL) in comparison to acarbose (0.9 μM). Due to its selectivity towards α-glucosidase, PGFC was subjected to bioactivity-guided isolation of its main active constituents. Five known compounds (1-5) were identified as β-sitosterol (1), oleanolic acid (2), ursolic acid (3), p-coumaric acid (4) and apigenin (5). Ursolic and oleanolic acids showed potent α-glucosidase inhibition (IC₅₀ of 39.0 and 35.0 μM, respectively), while they did not show significant maltase inhibition. Kinetic study using the double Lineweaver-Burk plot revealed that ursolic acid uncompetitively inhibited α-glucosidase in comparison with acarbose as a competitive inhibitor.

In vitro anti-diabetic activity of flavonoids and pheophytins from Allophylus cominia Sw . on PTP1B, DPPIV, alpha-glucosidase and alpha-amylase enzymes.

Science.gov (United States)

Semaan, D G; Igoli, J O; Young, L; Marrero, E; Gray, A I; Rowan, E G

2017-05-05

Ethno-botanical information from diabetic patients in Cuba led to the identification of Allophylus cominia as a possible source of new drugs for the treatment of type 2 diabetes mellitus (T2-DM). Chemical characterization of the extracts from A. cominia was carried out using chromatographic and spectroscopic methods. The extracts were tested for their activity on PTP1B, DPPIV, α-glucosidase enzymes and α-amylase. The flavonoid rich fractions from A. cominia inhibited DPPIV enzyme (75.3±2.33%) at 30µg/ml and produced a concentration-dependent inhibition against DPPIV with a Ki value of 2.6µg/ml. At 30µg/ml, flavonoids and pheophytins extracts significantly inhibited PTP1B enzyme (100±2.6% and 68±1% respectively). The flavonoids, pheophytin A and pheophytin B fractions showed significant concentration-dependent inhibition against PTP1B with Ki values of 3µg/ml, 0.64µg/ml and 0.88µg/ml respectively. At 30µg/ml, the flavonoid fraction significantly inhibited α-glucosidase enzyme (86±0.3%) in a concentration-dependent pattern with a Ki value of 2µg/ml. None of the fractions showed significant effects on α-amylase. Fatty acids, tannins, pheophytins A and B, and a mixture of flavonoids were detected in the methanolic extract from A. cominia. The identified flavonoids were mearnsitrin, quercitrin, quercetin-3-alloside, and naringenin-7-glucoside. The pharmacological effects of the extracts from A. cominia earlier observed in experimental diabetic models was confirmed in this study. Thus a new drug or formulation for the treatment of T2-DM could be developed from A. cominia. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Fruit Wines Inhibitory Activity Against α-Glucosidase.

Science.gov (United States)

Cakar, Uros; Grozdanic, Nada; Petrovic, Aleksandar; Pejin, Boris; Nastasijevic, Branislav; Markovic, Bojan; Dordevic, Brizita

2017-01-01

Fruit wines are well known for their profound health-promoting properties including both enzyme activations and inhibitions. They may act preventive in regard to diabetes melitus and other chronic diseases. Potential α-glucosidase inhibitory activity of fruit wines made from blueberry, black chokeberry, blackberry, raspberry and sour cherry was the subject of this study. In order to increase the alcohol content due to enriched extraction of total phenolics, sugar was added in the fruit pomace of the half of the examined fruit wine samples. Compared with acarbose used as a positive control (IC50 = 73.78 µg/mL), all fruit wine samples exhibited higher α-glucosidase inhibitory activity. Indeed, blueberry wine samples stood out, both prepared with IC50 = 24.14 µg/mL, lyophilised extract yield 3.23% and without IC50 = 46.39 µg/mL, lyophilised extract yield 2.89% and with addition of sugar before fermentation. Chlorogenic acid predominantly contributed to α-glucosidase inhibitory activity of the blueberry, black chokeberry and sour cherry wine samples. However, ellagic acid, a potent α-glucosidase inhibitor possessing a planar structure, only slightly affected the activity of the blueberry wine samples, due to the lower concentration. In addition to this, molecular docking study of chlorogenic acid pointed out the importance of binding energy (-8.5 kcal/mol) for the inhibition of the enzyme. In summary, fruit wines made from blueberry should be primarily taken into consideration as a medicinal food targeting diabetes mellitus type 2 in the early stage, if additional studies would confirm their therapeutic potential for the control of postprandial hyperglycemia. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Characterization of recombinant high pI Barley α-Glucosidase

DEFF Research Database (Denmark)

Næsted, Henrik; Svensson, Birte

(MacGregor A.W.). Here we present the recent results of the expression and characterization of the recombinant full length barley high pI α-glucosidase in Pichia Pastoris. In order to facilitate in the range of mg protein yield, a clone representing an N-terminal hexa histidine tagged recombinant form...... of the enzyme was grown under high cell-density fermentation conditions. This approach enabled a successful protein expression profile under the highly sensitive methanol utilization phase of the fermentation procedure. The enzyme was purified using a four step purification strategy. Interestingly, the purified...... enzyme exhibits a higher molecular mass than expected from its primary sequence when applied on SDS-PAGE, indicating a possible post translational modification of the recombinant α-glucosidase. Preliminary enzyme kinetic analysis has demonstrated that the purified α-glucosidase is “fully” active when...

The structural and functional contributions of β-glucosidase-producing microbial communities to cellulose degradation in composting.

Science.gov (United States)

Zang, Xiangyun; Liu, Meiting; Fan, Yihong; Xu, Jie; Xu, Xiuhong; Li, Hongtao

2018-01-01

Compost habitats sustain a vast ensemble of microbes that engender the degradation of cellulose, which is an important part of global carbon cycle. β-Glucosidase is the rate-limiting enzyme of degradation of cellulose. Thus, analysis of regulation of β-glucosidase gene expression in composting is beneficial to a better understanding of cellulose degradation mechanism. Genetic diversity and expression of β-glucosidase-producing microbial communities, and relationships of cellulose degradation, metabolic products and the relative enzyme activity during natural composting and inoculated composting were evaluated. Compared with natural composting, adding inoculation agent effectively improved the degradation of cellulose, and maintained high level of the carboxymethyl cellulose (CMCase) and β-glucosidase activities in thermophilic phase. Gene expression analysis showed that glycoside hydrolase family 1 (GH1) family of β-glucosidase genes contributed more to β-glucosidase activity in the later thermophilic phase in inoculated compost. In the cooling phase of natural compost, glycoside hydrolase family 3 (GH3) family of β-glucosidase genes contributed more to β-glucosidase activity. Intracellular β-glucosidase activity played a crucial role in the regulation of β-glucosidase gene expression, and upregulation or downregulation was also determined by extracellular concentration of glucose. At sufficiently high glucose concentrations, the functional microbial community in compost was altered, which may contribute to maintaining β-glucosidase activity despite the high glucose content. This research provides an ecological functional map of microorganisms involved in carbon metabolism in cattle manure-rice straw composting. The performance of the functional microbial groups in the two composting treatments is different, which is related to the cellulase activity and cellulose degradation, respectively.

SwissProt search result: AK066051 [KOME

Lifescience Database Archive (English)

Full Text Available AK066051 J013051B02 (P10253) Lysosomal alpha-glucosidase precursor (EC 3.2.1.20) (A...cid maltase) (Aglucosidase alfa) [Contains: 76 kDa lysosomal alpha-glucosidase; 70 kDa lysosomal alpha-glucosidase] LYAG_HUMAN 1e-89 ...

SwissProt search result: AK243062 [KOME

Lifescience Database Archive (English)

Full Text Available AK243062 J100014O03 (P10253) Lysosomal alpha-glucosidase precursor (EC 3.2.1.20) (A...cid maltase) (Aglucosidase alfa) [Contains: 76 kDa lysosomal alpha-glucosidase; 70 kDa lysosomal alpha-glucosidase] LYAG_HUMAN 3e-72 ...

SwissProt search result: AK121014 [KOME

Lifescience Database Archive (English)

Full Text Available AK121014 J023048A03 (P10253) Lysosomal alpha-glucosidase precursor (EC 3.2.1.20) (A...cid maltase) (Aglucosidase alfa) [Contains: 76 kDa lysosomal alpha-glucosidase; 70 kDa lysosomal alpha-glucosidase] LYAG_HUMAN 6e-81 ...

SwissProt search result: AK121588 [KOME

Lifescience Database Archive (English)

Full Text Available AK121588 J033037N10 (P10253) Lysosomal alpha-glucosidase precursor (EC 3.2.1.20) (A...cid maltase) (Aglucosidase alfa) [Contains: 76 kDa lysosomal alpha-glucosidase; 70 kDa lysosomal alpha-glucosidase] LYAG_HUMAN 1e-126 ...

SwissProt search result: AK063966 [KOME

Lifescience Database Archive (English)

Full Text Available AK063966 001-124-A04 (P10253) Lysosomal alpha-glucosidase precursor (EC 3.2.1.20) (...Acid maltase) (Aglucosidase alfa) [Contains: 76 kDa lysosomal alpha-glucosidase; 70 kDa lysosomal alpha-glucosidase] LYAG_HUMAN 1e-145 ...

SwissProt search result: AK105449 [KOME

Lifescience Database Archive (English)

Full Text Available AK105449 001-125-B12 (P10253) Lysosomal alpha-glucosidase precursor (EC 3.2.1.20) (...Acid maltase) (Aglucosidase alfa) [Contains: 76 kDa lysosomal alpha-glucosidase; 70 kDa lysosomal alpha-glucosidase] LYAG_HUMAN 1e-142 ...

SwissProt search result: AK102309 [KOME

Lifescience Database Archive (English)

Full Text Available AK102309 J033090B18 (P10253) Lysosomal alpha-glucosidase precursor (EC 3.2.1.20) (A...cid maltase) (Aglucosidase alfa) [Contains: 76 kDa lysosomal alpha-glucosidase; 70 kDa lysosomal alpha-glucosidase] LYAG_HUMAN 1e-145 ...

SwissProt search result: AK110088 [KOME

Lifescience Database Archive (English)

Full Text Available AK110088 002-160-G07 (P10253) Lysosomal alpha-glucosidase precursor (EC 3.2.1.20) (...Acid maltase) (Aglucosidase alfa) [Contains: 76 kDa lysosomal alpha-glucosidase; 70 kDa lysosomal alpha-glucosidase] LYAG_HUMAN 1e-62 ...

SwissProt search result: AK121428 [KOME

Lifescience Database Archive (English)

Full Text Available AK121428 J023136H14 (P10253) Lysosomal alpha-glucosidase precursor (EC 3.2.1.20) (A...cid maltase) (Aglucosidase alfa) [Contains: 76 kDa lysosomal alpha-glucosidase; 70 kDa lysosomal alpha-glucosidase] LYAG_HUMAN 6e-54 ...

Diversification and specialization of β-glucosidases in the catabolism of hydroxynitrile glucosides in Lotus japonicus

DEFF Research Database (Denmark)

Lai, Daniela

that involves specific β-glucosidases. If plant tissue is disrupted, cyanogenic glucosides come into contact with these β-glucosidases and are hydrolyzed, which results in the release of hydrogen cyanide gas. The work reported in this thesis is focused on the β-glucosidases that activated hydroxynitrile...... glucosides in the model plant Lotus japonicus. The work highlights how closely related β-glucosidases have evolved distinct substrate specificities and differential expression patterns to serve distinct physiological and ecological roles....

Biosynthesis of beta-glucosidase by Aspergillus niger a-5

Energy Technology Data Exchange (ETDEWEB)

Atev, A.; Panayotov, C.; Bubareva, L.; Benadova, R.; Kolev, E.

1984-01-01

Aspergillus niger A-5 produced beta-glucosidase, exocellobihydrolase (C1 enzyme) and endo-1, 4-beta-glucanase (Cx enzyme) in a culture medium containing farm residues of plant origin: wheat straw, ground maize stalks, wheat bran, and micricell as substrates. Maize stalk and wheat bran were the best inducers of the cellulase complex. Intensive aeration stimulated growth and enzyme synthesis. The highest beta-glucosidase activity (54 units/mL) was observed after 96 h of cultivation.

Alpha-glucosidase Inhibitory and Antioxidant Potential of Antidiabetic Herb Alternanthera sessilis: Comparative Analyses of Leaf and Callus Solvent Fractions.

Science.gov (United States)

Chai, Tsun-Thai; Khoo, Chee-Siong; Tee, Chong-Siang; Wong, Fai-Chu

2016-01-01

Alternanthera sessilis is a medicinal herb which is consumed as vegetable and used as traditional remedies of various ailments in Asia and Africa. This study aimed to investigate the antiglucosidase and antioxidant activity of solvent fractions of A. sessilis leaf and callus. Leaf and callus methanol extracts were fractionated to produce hexane, chloroform, ethyl acetate, butanol, and water fractions. Antiglucosidase and 1,1-diphenyl-2-picrylhydrazyl scavenging activities as well as total phenolic (TP), total flavonoid (TF), and total coumarin (TC) contents were evaluated. Lineweaver-Burk plot analysis was performed on leaf and callus fractions with the strongest antiglucosidase activity. Leaf ethyl acetate fraction (LEF) had the strongest antiglucosidase (EC 50 0.55 mg/mL) and radical scavenging (EC 50 10.81 μg/mL) activity among leaf fractions. Callus ethyl acetate fraction (CEF) and chloroform fraction had the highest antiglucosidase (EC 50 0.25 mg/mL) and radical scavenging (EC 50 34.12 μg/mL) activity, respectively, among callus fractions. LEF and CEF were identified as noncompetitive and competitive α-glucosidase inhibitors, respectively. LEF and CEF had greater antiglucosidase activity than acarbose. Leaf fractions had higher phytochemical contents than callus fractions. LEF had the highest TP, TF, and TC contents. Antiglucosidase and antioxidant activities of leaf fractions correlated with phytochemical contents. LEF had potent antiglucosidase activity and concurrent antioxidant activity. CEF had the highest antiglucosidase activity among all fractions. Callus culture is a promising tool for enhancing production of potent α-glucosidase inhibitors. Leaf ethyl acetate fraction (LEF) had the strongest antiglucosidase (EC 50 0.55 mg/mL) and radical scavenging (EC 50 10.81 μg/mL) activity among leaf fractionsCallus ethyl acetate fraction (CEF) and chloroform fraction had the highest antiglucosidase (EC 50 0.25 mg/mL) and radical scavenging (EC 50 34.12 �

Edible seaweed as future functional food: Identification of α-glucosidase inhibitors by combined use of high-resolution α-glucosidase inhibition profiling and HPLC-HRMS-SPE-NMR.

Science.gov (United States)

Liu, Bingrui; Kongstad, Kenneth T; Wiese, Stefanie; Jäger, Anna K; Staerk, Dan

2016-07-15

Crude chloroform, ethanol and acetone extracts of nineteen seaweed species were screened for their antioxidant and α-glucosidase inhibitory activity. Samples showing more than 60% α-glucosidase inhibitory activity, at a concentration of 1 mg/ml, were furthermore investigated using high-resolution α-glucosidase inhibition profiling combined with high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HR-bioassay/HPLC-HRMS-SPE-NMR). The results showed Ascophyllum nodosum and Fucus vesicolosus to be rich in antioxidants, equaling a Trolox equivalent antioxidant capacity of 135 and 108 mM Troloxmg(-1) extract, respectively. HR-bioassay/HPLC-HRMS-SPE-NMR showed the α-glucosidase inhibitory activity of A. nodosum, F. vesoculosus, Laminaria digitata, Laminaria japonica and Undaria pinnatifida to be caused by phlorotannins as well as fatty acids - with oleic acid, linoleic acid and eicosapentaenoic acid being the most potent with IC50 values of 0.069, 0.075 and 0.10 mM, respectively, and showing a mixed-type inhibition mode. Copyright © 2016 Elsevier Ltd. All rights reserved.

β-Glucosidases from the Fungus Trichoderma: An Efficient Cellulase Machinery in Biotechnological Applications

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Pragya Tiwari

2013-01-01

Full Text Available β-glucosidases catalyze the selective cleavage of glucosidic linkages and are an important class of enzymes having significant prospects in industrial biotechnology. These are classified in family 1 and family 3 of glycosyl hydrolase family. β-glucosidases, particularly from the fungus Trichoderma, are widely recognized and used for the saccharification of cellulosic biomass for biofuel production. With the rising trends in energy crisis and depletion of fossil fuels, alternative strategies for renewable energy sources need to be developed. However, the major limitation accounts for low production of β-glucosidases by the hyper secretory strains of Trichoderma. In accordance with the increasing significance of β-glucosidases in commercial applications, the present review provides a detailed insight of the enzyme family, their classification, structural parameters, properties, and studies at the genomics and proteomics levels. Furthermore, the paper discusses the enhancement strategies employed for their utilization in biofuel generation. Therefore, β-glucosidases are prospective toolbox in bioethanol production, and in the near future, it might be successful in meeting the requirements of alternative renewable sources of energy.

Drug: D01665 [KEGG MEDICUS

Lifescience Database Archive (English)

Full Text Available D01665 Drug Voglibose (JP17/USAN/INN); Basen (TN) ... C10H21NO7 D01665.gif ... Antidiabetic... agent ... DG01663 ... alpha-Glucosidase inhibitor ... DG01803 ... Antidiabetic, alpha-glucosidase inhibitor Uncla...ssified ... DG02044 ... Hypoglycemics ... DG01803 ... Antidiabetic, alpha-glucosidase inhibitor Therapeutic category: 3

Drug: D00625 [KEGG MEDICUS

Lifescience Database Archive (English)

Full Text Available D00625 Drug Miglitol (JP17/USAN/INN); Glyset (TN) ... C8H17NO5 D00625.gif ... Antidiabetic... agent ... DG01663 ... alpha-Glucosidase inhibitor ... DG01803 ... Antidiabetic, alpha-glucosidase inhibitor Unclas...sified ... DG02044 ... Hypoglycemics ... DG01803 ... Antidiabetic, alpha-glucosidase inhibitor Same as: C07708 Therapeu

Acquired thermotolerance and heat shock in the extremely thermophilic archaebacterium Sulfolobus sp. strain B12.

Science.gov (United States)

Trent, J D; Osipiuk, J; Pinkau, T

1990-03-01

The extreme thermophile Sulfolobus sp. strain B12 exhibits an acquired thermotolerance response. Thus, survival of cells from a 70 degrees C culture at the lethal temperature of 92 degrees C was enhanced by as much as 6 orders of magnitude over a 2-h period if the culture was preheated to 88 degrees C for 60 min or longer before being exposed to the lethal temperature. In eubacteria and eucaryotes, acquired thermotolerance correlates with the induced synthesis of a dozen or so proteins known as heat shock proteins. In this Sulfolobus species, it correlates with the preferential synthesis of primarily one major protein (55 kilodaltons) and, to a much lesser extent, two minor proteins (28 and 35 kilodaltons). Since the synthesis of all other proteins was radically reduced and these proteins were apparently not degraded or exported, their relative abundance within the cell increased during the time the cells were becoming thermotolerant. They could not yet be related to known heat shock proteins. In immunoassays, they were not cross-reactive with antibodies against heat shock proteins from Escherichia coli (DnaK and GroE), which are highly conserved between eubacteria and eucaryotes. However, it appears that if acquired thermotolerance depends on the synthesis of protective proteins, then in this extremely thermophilic archaebacterium it depends primarily on one protein.

Identifying and characterizing the most significant β-glucosidase of the novel species Aspergillus saccharolyticus

Energy Technology Data Exchange (ETDEWEB)

Sorensen, Anette; Ahring, Birgitte K.; Lubeck, Mette; Ubhayasekera, Wimal; Bruno, Kenneth S.; Culley, David E.; Lubeck, Peter S.

2012-08-20

A newly discovered fungal species, Aspergillus saccharolyticus, was found to produce a culture broth rich in beta-glucosidase activity. In this present work, the main beta-glucosidase of A. saccharolyticus responsible for the efficient hydrolytic activity was identified, isolated, and characterized. Ion exchange chromatography was used to fractionate the culture broth, yielding fractions with high beta-glucosidase activity and only one visible band on an SDS-PAGE gel. Mass spectrometry analysis of this band gave peptide matches to beta-glucosidases from aspergilli. Through a PCR approach using degenerate primers and genome walking, a 2919 base pair sequence encoding the 860 amino acid BGL1 polypeptide was determined. BGL1 of A. saccharolyticus has 91% and 82% identity with BGL1 from Aspergillus aculeatus and BGL1 from Aspergillus niger, respectively, both belonging to Glycoside hydrolase family 3. Homology modeling studies suggested beta-glucosidase activity with preserved retaining mechanism and a wider catalytic pocket compared to other beta-glucosidases. The bgl1 gene was heterologously expressed in Trichoderma reesei QM6a, purified, and characterized by enzyme kinetics studies. The enzyme can hydrolyze cellobiose, pNPG, and cellodextrins. The enzyme showed good thermostability, was stable at 50°C, and at 60°C it had a half-life of approximately 6 hours.

Process development studies for the production of β-glucosidase from Aspergillus phoenicis

Energy Technology Data Exchange (ETDEWEB)

Howell, Mary Jane [Univ. of California, Berkeley, CA (United States); Wilke, C. R. [Univ. of California, Berkeley, CA (United States)

1978-09-01

This work is concerned with the production of β-glucosidase from Aspergillus phoenicis for use in the enzymatic hydrolysis of cellulose. Kinetic growth data indicate that two distinct periods of growth exist. The observed growth kinetics result from a biochemical differentiation of the filament which is independent of the substrate concentration. The optimum temperature for cell mass and β-glucosidase production was found to be 30°C. The optimum pH for β-glucosidase production is 5 and the highest specific cell growth rate was observed when the growth medium was controlled at pH 4.5. The most economical substrate was 0.75 g/l of Solka Floc, a spruce wood pulp, plus 0.25 g/l of Trichoderma viride cellulase, required because A. phoenicis does not produce all the enzymes required to solubilize cellulose. When freeze-dried A. phoenicis enzyme was added to the hydrolysis of acid treated corn stover by Tricoderma viride cellulase, the total sugar yield was increased by 4 g/l of hydrolysate over the yield of 20 g/l obtained without β-glucosidase addition. In addition, the cellobiose, which accounted for about 10% of the sugar concentration, was converted to glucose, a more widely useable product. Preliminary designs of several processes for the production of β-glucosidase were made. The most economical processes were continuous production schemes. Ball milling was the most cost effective method, but the use of an elevated temperature stage was economical enough to warrant further study. The cost of production of β-glucosidase was found to be too high to justify its addition to a process for enzymatically hydrolyzing cellulose at this time.

Identification of PTP1B and α-Glucosidase Inhibitory Serrulatanes from Eremophila spp. by Combined use of Dual High-Resolution PTP1B and α-Glucosidase Inhibition Profiling and HPLC-HRMS-SPE-NMR.

Science.gov (United States)

Wubshet, Sileshi G; Tahtah, Yousof; Heskes, Allison M; Kongstad, Kenneth T; Pateraki, Irini; Hamberger, Björn; Møller, Birger L; Staerk, Dan

2016-04-22

According to the International Diabetes Federation, type 2 diabetes (T2D) has reached epidemic proportions, affecting more than 382 million people worldwide. Inhibition of protein tyrosine phosphatase-1B (PTP1B) and α-glucosidase is a recognized therapeutic approach for management of T2D and its associated complications. The lack of clinical drugs targeting PTP1B and side effects of the existing α-glucosidase drugs, emphasize the need for new drug leads for these T2D targets. In the present work, dual high-resolution PTP1B and α-glucosidase inhibition profiles of Eremophila gibbosa, E. glabra, and E. aff. drummondii "Kalgoorlie" were used for pinpointing α-glucosidase and/or PTP1B inhibitory constituents directly from the crude extracts. A subsequent targeted high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR) analysis and preparative-scale HPLC isolation led to identification of 21 metabolites from the three species, of which 16 were serrulatane-type diterpenoids (12 new) associated with either α-glucosidase and/or PTP1B inhibition. This is the first report of serrulatane-type diterpenoids as potential α-glucosidase and/or PTP1B inhibitors.

Isolation and Characterization of an α-Glucosidase Inhibitor from Musa spp. (Baxijiao Flowers

Directory of Open Access Journals (Sweden)

Zhanwu Sheng

2014-07-01

Full Text Available The use of α-glucosidase inhibitors is considered to be an effective strategy in the treatment of diabetes. Using a bioassay-guided fractionation technique, five Bacillus stearothermophilus α-glucosidase inhibitors were isolated from the flowers of Musa spp. (Baxijiao. Using NMR spectroscopy analysis they were identified as vanillic acid (1, ferulic acid (2, β-sitosterol (3, daucosterol (4 and 9-(4′-hydroxyphenyl-2-methoxyphenalen-1-one (5. The half maximal inhibitory concentration (IC50 values of compounds 1–5 were 2004.58, 1258.35, 283.67, 247.35 and 3.86 mg/L, respectively. Compared to a known α-glucosidase inhibitor (acarbose, IC50 = 999.31 mg/L, compounds 3, 4 and 5 showed a strong α-glucosidase inhibitory effect. A Lineweaver-Burk plot indicated that compound 5 is a mixed-competitive inhibitor, while compounds 3 and 4 are competitive inhibitors. The inhibition constants (Ki of compounds 3, 4 and 5 were 20.09, 2.34 and 4.40 mg/L, respectively. Taken together, these data show that the compounds 3, 4 and 5 are potent α-glucosidase inhibitors.

Thermophilic archaeal enzymes and applications in biocatalysis.

Science.gov (United States)

Littlechild, Jennifer A

2011-01-01

Thermophilic enzymes have advantages for their use in commercial applications and particularly for the production of chiral compounds to produce optically pure pharmaceuticals. They can be used as biocatalysts in the application of 'green chemistry'. The thermophilic archaea contain enzymes that have already been used in commercial applications such as the L-aminoacylase from Thermococcus litoralis for the resolution of amino acids and amino acid analogues. This enzyme differs from bacterial L-aminoacylases and has similarities to carboxypeptidases from other archaeal species. An amidase/γ-lactamase from Sulfolobus solfataricus has been used for the production of optically pure γ-lactam, the building block for antiviral carbocyclic nucleotides. This enzyme has similarities to the bacterial signature amidase family. An alcohol dehydrogenase from Aeropyrum pernix has been used for the production of optically pure alcohols and is related to the zinc-containing eukaryotic alcohol dehydrogenases. A transaminase and a dehalogenase from Sulfolobus species have also been studied. The archaeal transaminase is found in a pathway for serine synthesis which is found only in eukaryotes and not in bacteria. It can be used for the asymmetric synthesis of homochiral amines of high enantioselective purity. The L-2-haloacid dehalogenase has applications both in biocatalysis and in bioremediation. All of these enzymes have increased thermostability over their mesophilic counterparts.

Properties of native and immobilised preparations of. beta. -D-glucosidase from Aspergillus niger

Energy Technology Data Exchange (ETDEWEB)

Woodward, J.; Wohlpart, D.L.

1982-01-01

The enzyme ..beta..-D-glucosidase from Aspergillus niger has been immobilised through its carbohydrate moiety on concanavalin A-Sepharose and on cyanogen bromide-activated Sepharose after aminoalkylation of the carbohydrate side chains of the enzyme. For comparison, the enzyme was also immobilised on microcrystalline cellulose through its protein moiety. High retention of activity and a decrease in K/sub m/ and V/sub max/ were observed when ..beta..-D-glucosidase was immobilised by these methods. An increase in the thermal stability of the immobilised ..beta..-D-glucosidase preparations over the soluble enzyme was achieved if it was treated with glutaraldehyde before its adsorption on concanavalin A-Sepharose or if the enzyme immobilised on cyanogen bromide-activated Sepharose was subsequently treated with glutaralydehyde. Treatment of ..beta..-D-glucosidase immobilised on microcrystalline cellulose with glutaraldehyde hardy increased its thermal stability over the soluble enzyme.

Properties of native and immobilised preparations of beta-d-glucosidase from Aspergillus niger

Energy Technology Data Exchange (ETDEWEB)

Woodward, J.; Wohlpart, D.L.

1982-04-01

The enzyme beta-glucosidase from Aspergillus niger has been immobilised through its carbohydrate moiety on concanavalin A-Sepharose and on cyanogen bromide-activated Sepharose after aminoalkylation of the carbohydrate side chains of the enzyme. For comparison, the enzyme was also immobilised on microcrystalline cellulose through its protein moiety. High retention of activity and a decrease in Km and Vmax were observed when beta-D-glucosidase was immobilised by these methods. An increase in the thermal stability of the immobilized beta-D-glucosidase preparations over the soluble enzyme was achieved if it was treated with glutaraldehyde before its adsorption on concanavalin A-Sepharose or if the enzyme immobilised on cyanogen bromide-activated Sepharose was subsequently treated with glutaraldehyde. Treatment of beta-D-glucosidase immobilised on microcrystalline cellulose with glutaraldehyde hardly increased its thermal stability over the soluble enzyme. (Refs. 24).

Adsorption of β-glucosidases in two commercial preparations onto pretreated biomass and lignin

DEFF Research Database (Denmark)

Haven, Mai Østergaard; Jørgensen, Henning

2013-01-01

adsorbed strongly to lignin.The extent of adsorption of β-glucosidase from Cellic® CTec2 was affected by both type of biomass and pretreatment method. With approximately 65% of the β-glucosidases from Cellic® CTec2 adsorbed onto lignin from pretreated wheat straw, the activity of the β......Background: Enzyme recycling is a method to reduce the production costs for advanced bioethanol by lowering the overall use of enzymes. Commercial cellulase preparations consist of many different enzymes that are important for efficient and complete cellulose (and hemicellulose) hydrolysis...... commercial preparations (Novozym 188 and Cellic® CTec2) to substrates mimicking the components in pretreated wheat straw revealed that the Aspergillus niger β-glucosidase in Novozym 188 did not adsorb significantly to any of the components in pretreated wheat straw, whereas the β-glucosidase in Cellic® CTec2...

Pomegranate ellagitannins inhibit α-glucosidase activity in vitro and reduce starch digestibility under simulated gastro-intestinal conditions.

Science.gov (United States)

Bellesia, Andrea; Verzelloni, Elena; Tagliazucchi, Davide

2015-02-01

Pomegranate extract was tested for its ability to inhibit α-amylase and α-glucosidase activity. Pomegranate extract strongly inhibited rat intestinal α-glucosidase in vitro whereas it was a weak inhibitor of porcine α-amylase. The inhibitory activity was recovered in an ellagitannins-enriched fraction and punicalagin, punicalin, and ellagic acid were identified as α-glucosidase inhibitors (IC(50) of 140.2, 191.4, and 380.9 μmol/L, respectively). Kinetic analysis suggested that the pomegranate extract and ellagitannins inhibited α-glucosidase activity in a mixed mode. The inhibitory activity was demonstrated using an in vitro digestion system, mimicking the physiological gastro-intestinal condition, and potatoes as food rich in starch. Pre-incubation between ellagitannins and α-glucosidase increased the inhibitory activity, suggesting that they acted by binding to α-glucosidase. During digestion punicalin and punicalagin concentration decreased. Despite this loss, the pomegranate extract retained high inhibitory activity. This study suggests that pomegranate ellagitannins may inhibit α-glucosidase activity in vitro possibly affecting in vivo starch digestion.

Extensive Lysine Methylation in Hyperthermophilic Crenarchaea: Potential Implications for Protein Stability and Recombinant Enzymes

Directory of Open Access Journals (Sweden)

Catherine H. Botting

2010-01-01

Full Text Available In eukarya and bacteria, lysine methylation is relatively rare and is catalysed by sequence-specific lysine methyltransferases that typically have only a single-protein target. Using RNA polymerase purified from the thermophilic crenarchaeum Sulfolobus solfataricus, we identified 21 methyllysines distributed across 9 subunits of the enzyme. The modified lysines were predominantly in α-helices and showed no conserved sequence context. A limited survey of the Thermoproteus tenax proteome revealed widespread modification with 52 methyllysines in 30 different proteins. These observations suggest the presence of an unusual lysine methyltransferase with relaxed specificity in the crenarchaea. Since lysine methylation is known to enhance protein thermostability, this may be an adaptation to a thermophilic lifestyle. The implications of this modification for studies and applications of recombinant crenarchaeal enzymes are discussed.

Drug: D03342 [KEGG MEDICUS

Lifescience Database Archive (English)

Full Text Available D03342 Drug Camiglibose (USAN) ... (C13H25NO9)2. 3H2O D03342.gif ... Antidiabetic agen...t ... DG01663 ... alpha-Glucosidase inhibitor ... DG01803 ... Antidiabetic, alpha-glucosidase inhibitor Unclassified ... D...G02044 ... Hypoglycemics ... DG01803 ... Antidiabetic, alpha-glucosidase inhibitor ... CAS: 132438-21-2 PubChem: 17397492 LigandBox: D03342 ...

Acquired thermotolerance and heat shock in the extremely thermophilic archaebacterium Sulfolobus sp. strain B12.

OpenAIRE

Trent, J D; Osipiuk, J; Pinkau, T

1990-01-01

The extreme thermophile Sulfolobus sp. strain B12 exhibits an acquired thermotolerance response. Thus, survival of cells from a 70 degrees C culture at the lethal temperature of 92 degrees C was enhanced by as much as 6 orders of magnitude over a 2-h period if the culture was preheated to 88 degrees C for 60 min or longer before being exposed to the lethal temperature. In eubacteria and eucaryotes, acquired thermotolerance correlates with the induced synthesis of a dozen or so proteins known ...

Crystallization and preliminary X-ray analysis of Streptococcus mutans dextran glucosidase

Energy Technology Data Exchange (ETDEWEB)

Saburi, Wataru; Hondoh, Hironori, E-mail: hondoh@abs.agr.hokudai.ac.jp [Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589 (Japan); Unno, Hideaki [Faculty of Engineering, Nagasaki University, Bunkyo-machi, Nagasaki 852-8521 (Japan); Okuyama, Masayuki; Mori, Haruhide [Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589 (Japan); Nakada, Toshitaka [Faculty of Science and Engineering, Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); Matsuura, Yoshiki [Institute for Protein Research, Osaka University, Suita, Osaka 565-0871 (Japan); Kimura, Atsuo [Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589 (Japan)

2007-09-01

Dextran glucosidase from S. mutans was crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.2 Å resolution. Dextran glucosidase from Streptococcus mutans is an exo-hydrolase that acts on the nonreducing terminal α-1,6-glucosidic linkage of oligosaccharides and dextran with a high degree of transglucosylation. Based on amino-acid sequence similarity, this enzyme is classified into glycoside hydrolase family 13. Recombinant dextran glucosidase was purified and crystallized by the hanging-drop vapour-diffusion technique using polyethylene glycol 6000 as a precipitant. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 72.72, b = 86.47, c = 104.30 Å. A native data set was collected to 2.2 Å resolution from a single crystal.

Crystallization and preliminary X-ray analysis of Streptococcus mutans dextran glucosidase

International Nuclear Information System (INIS)

Saburi, Wataru; Hondoh, Hironori; Unno, Hideaki; Okuyama, Masayuki; Mori, Haruhide; Nakada, Toshitaka; Matsuura, Yoshiki; Kimura, Atsuo

2007-01-01

Dextran glucosidase from S. mutans was crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.2 Å resolution. Dextran glucosidase from Streptococcus mutans is an exo-hydrolase that acts on the nonreducing terminal α-1,6-glucosidic linkage of oligosaccharides and dextran with a high degree of transglucosylation. Based on amino-acid sequence similarity, this enzyme is classified into glycoside hydrolase family 13. Recombinant dextran glucosidase was purified and crystallized by the hanging-drop vapour-diffusion technique using polyethylene glycol 6000 as a precipitant. The crystals belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 72.72, b = 86.47, c = 104.30 Å. A native data set was collected to 2.2 Å resolution from a single crystal

Maplexins, new α-glucosidase inhibitors from red maple (Acer rubrum) stems.

Science.gov (United States)

Wan, Chunpeng; Yuan, Tao; Li, Liya; Kandhi, Vamsikrishna; Cech, Nadja B; Xie, Mingyong; Seeram, Navindra P

2012-01-01

Thirteen gallic acid derivatives including five new gallotannins, named maplexins A-E, were isolated from red maple (Acer rubrum) stems. The compounds were identified by spectral analyses. The maplexins varied in number and location of galloyl groups attached to 1,5-anhydro-d-glucitol. The isolates were evaluated for α-glucosidase inhibitory and antioxidant activities. Maplexin E, the first compound identified with three galloyl groups linked to three different positions of 1,5-anhydro-d-glucitol, was 20 fold more potent than the α-glucosidase inhibitory drug, Acarbose (IC(50)=8 vs 160 μM). Structure-activity related studies suggested that both number and position of galloyls attached to 1,5-anhydro-d-glucitol were important for α-glucosidase inhibition. Copyright © 2011 Elsevier Ltd. All rights reserved.

Purification and characterization of recombinant high pI Barley α-Glucosidase

DEFF Research Database (Denmark)

Næsted, Henrik; Bojsen, Kirsten; Svensson, Birte

(MACGREGOR & sissons). recently expression and characterization of the recombinant full length and fully functional barley high pi α-glucosidase in pichia pastoris has been achieved. in order to facilitate protein yield in the mg range, a clone representing an n-terminal hexa histidine tagged recombinant...... form of the enzyme was grown under high cell-density fermentation conditions. this approach enabled a successful protein expression profile under the highly sensitive methanol utilization phase using a biotatb 5 l reactor for the fermentation procedure. the enzyme was purified from 3.5 liter α...... of the native enzyme indicates a possible post-translational glycosylation of the recombinant α-glucosidase. preliminary enzyme kinetic analysis has demonstrated that the purified α-glucosidase displayed high stability during the 5 day long fermenentation and exhibited a specific activity in the range...

Cloning and expression of N-glycosylation-related glucosidase from Glaciozyma antarctica

Science.gov (United States)

Yajit, Noor Liana Mat; Kamaruddin, Shazilah; Hashim, Noor Haza Fazlin; Bakar, Farah Diba Abu; Murad, Abd. Munir Abd.; Mahadi, Nor Muhammad; Mackeen, Mukram Mohamed

2016-11-01

The need for functional oligosaccharides in various field is ever growing. The enzymatic approach for synthesis of oligosaccharides is advantageous over traditional chemical synthesis because of the regio- and stereo- selectivity that can be achieved without the need for protection chemistry. In this study, the α-glucosidase I protein sequence from Saccharomyces cerevisiae (UniProt database) was compared using Basic Local Alignment Search Tool (BLAST) with Glaciozyma antarctica genome database. Results showed 33% identity and an E-value of 1 × 10-125 for α-glucosidase I. The gene was amplified, cloned into the pPICZα C vector and used to transform Pichia pastoris X-33 cells. Soluble expression of α-Glucosidase I (˜91 kDa) was achieved at 28 °C with 1.0 % of methanol.

Purification and enzymatic characterization of a novel β-1,6-glucosidase from Aspergillus oryzae.

Science.gov (United States)

Watanabe, Akira; Suzuki, Moe; Ujiie, Seiryu; Gomi, Katsuya

2016-03-01

In this study, among the 10 genes that encode putative β-glucosidases in the glycoside hydrolase family 3 (GH3) with a signal peptide in the Aspergillus oryzae genome, we found a novel gene (AO090038000425) encoding β-1,6-glucosidase with a substrate specificity for gentiobiose. The transformant harboring AO090038000425, which we named bglH, was overexpressed under the control of the improved glaA gene promoter to form a small clear zone around the colony in a plate assay using 4-methylumbelliferyl β-d-glucopyranoside as the fluorogenic substrate for β-glucosidase. We purified BglH to homogeneity and enzymatically characterize this enzyme. The thermal and pH stabilities of BglH were higher than those of other previously studied A. oryzae β-glucosidases, and BglH was stable over a wide temperature range (4°C-60°C). BglH was inhibited by Hg(2+), Zn(2+), glucono-δ-lactone, glucose, dimethyl sulfoxide, and ethanol, but not by ethylenediaminetetraacetic acid. Interestingly, BglH preferentially hydrolyzed gentiobiose rather than other oligosaccharides and aryl β-glucosides, thereby demonstrating that this enzyme is a β-1,6-glucosidase. To the best of our knowledge, this is the first report of the purification and characterization of β-1,6-glucosidase from Aspergillus fungi or from other eukaryotes. This study suggests that it may be possible to find a more suitable β-glucosidase such as BglH for reducing the bitter taste of gentiobiose, and thus for controlling the sweetness of starch hydrolysates in the food industry via genome mining. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Characterization of an extracellular β-glucosidase from Dekkera bruxellensis for resveratrol production

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Hsiao-Ping Kuo

2018-01-01

Full Text Available Polygonum cuspidatum is a widely grown crop with a rich source of polydatin (also called piceid for resveratrol production. Resveratrol is produced from piceid via enzymatic cleavage of the sugar moiety of piceid. In this study, Dekkera bruxellensis mutants were selected based on their high p-nitrophenyl-β-d-glucopyranoside and piceid conversion activities. The enzyme responsible for piceid conversion was a heterodimeric protein complex that was predominantly secreted to the extracellular medium and consisted of two subunits at an equal ratio with molecular masses of 30.5 kDa and 48.3 kDa. The two subunits were identified as SCW4p and glucan-β-glucosidase precursor in D. bruxellensis. Both proteins were individually expressed in Saccharomyces cerevisiae exg1Δ mutants, which lack extracellular β-glucosidase activity, to confirm each protein's enzymatic activities. Only the glucan-β-glucosidase precursor was shown to be a secretory protein with piceid deglycosylation activity. Our pilot experiments of piceid bioconversion demonstrate the possible industrial applications for this glucan-β-glucosidase precursor in the future.

In vitro inhibition activity of polyphenol-rich extracts from Syzygium aromaticum (L.) Merr. & Perry (Clove) buds against carbohydrate hydrolyzing enzymes linked to type 2 diabetes and Fe(2+)-induced lipid peroxidation in rat pancreas.

Science.gov (United States)

Adefegha, Stephen Adeniyi; Oboh, Ganiyu

2012-10-01

To investigate and compare the inhibitory properties of free and bound phenolic extracts of clove bud against carbohydrate hydrolyzing enzymes (alpha-amylase & alpha-glucosidase) and Fe(2+)-induced lipid peroxidation in rat pancreas in vitro. The free phenolics were extracted with 80% (v/v) acetone, while bound phenolics were extracted from the alkaline and acid hydrolyzed residue with ethyl acetate. Then, the interaction of the extracts with alpha-amylase and alpha-glucosidase was subsequently assessed. Thereafter, the total phenolic contents and antioxidant activities of the extracts were determined. The result revealed that both extracts inhibited alpha-amylase and alpha-glucosidase in a dose-dependent manner. However, the alpha-glucosidase inhibitory activity of the extracts were significantly (Ppancreas in vitro. This study provides a biochemical rationale by which clove elicits therapeutic effect on type 2 diabetes.

Chinese Medicine Amygdalin and β-Glucosidase Combined with Antibody Enzymatic Prodrug System As A Feasible Antitumor Therapy.

Science.gov (United States)

Li, Yun-Long; Li, Qiao-Xing; Liu, Rui-Jiang; Shen, Xiang-Qian

2018-03-01

Amarogentin is an efficacious Chinese herbal medicine and a component of the bitter apricot kernel. It is commonly used as an expectorant and supplementary anti-cancer drug. β-Glucosidase is an enzyme that hydrolyzes the glycosidic bond between aryl and saccharide groups to release glucose. Upon their interaction, β-glucosidase catalyzes amarogentin to produce considerable amounts of hydrocyanic acid, which inhibits cytochrome C oxidase, the terminal enzyme in the mitochondrial respiration chain, and suspends adenosine triphosphate synthesis, resulting in cell death. Hydrocyanic acid is a cell-cycle-stage-nonspecific agent that kills cancer cells. Thus, β-glucosidase can be coupled with a tumor-specific monoclonal antibody. β-Glucosidase can combine with cancer-cell-surface antigens and specifically convert amarogentin to an active drug that acts on cancer cells and the surrounding antibodies to achieve a killing effect. β-Glucosidase is injected intravenously and recognizes cancer-cell-surface antigens with the help of an antibody. The prodrug amarogentin is infused after β-glucosidase has reached the target position. Coupling of cell membrane peptides with β-glucosidase allows the enzyme to penetrate capillary endothelial cells and clear extracellular deep solid tumors to kill the cells therein. The Chinese medicine amarogentin and β-glucosidase will become an important treatment for various tumors when an appropriate monoclonal antibody is developed.

Determination of a-glucosidase inhibitory activity from selected Fabaceae plants.

Science.gov (United States)

Dej-Adisai, Sukanya; Pitakbut, Thanet

2015-09-01

Nineteen plants from Fabaceae family, which were used in Thai traditional medicine for treatment of diabetes, were determined of α-glucosidase inhibitory activity via enzymatic reaction. In this reaction, α-glucosidase was used as enzyme, which, reacted with the substrate, p-nitrophenol-D-glucopyranoside (pNPG). After that the product, p-nitro phenol (pNP) will be occurred and observed the yellow colour at 405 nm. In this study, acarbose was used as positive standard which, inhibited this enzyme with IC₅₀ as 331 ± 4.73 μg/ml. Caesalpinia pulcherrima leaves showed the highest activity with IC₅₀ as 436.97 ± 9.44 μg/ml. Furthermore, Bauhinia malabarica leaves presented moderately activity with IC₅₀ as 745.08 ± 11.15 μg/ml. However, the other plants showed mild to none activity of α-glucosidase inhibition. Accordingly, this study can support anti-diabetes of these plants in traditional medicine and it will be the database of the biological activity of Fabaceae plant.

Influence of C-terminal tail deletion on structure and stability of hyperthermophile Sulfolobus tokodaii RNase HI.

Science.gov (United States)

Chen, Lin; Zhang, Ji-Long; Zheng, Qing-Chuan; Chu, Wen-Ting; Xue, Qiao; Zhang, Hong-Xing; Sun, Chia-Chung

2013-06-01

The C-terminus tail (G144-T149) of the hyperthermophile Sulfolobus tokodaii (Sto-RNase HI) plays an important role in this protein's hyperstabilization and may therefore be a good protein stability tag. Detailed understanding of the structural and dynamic effects of C-terminus tail deletion is required for gaining insights into the thermal stability mechanism of Sto-RNase HI. Focused on Sulfolobus tokodaii RNase HI (Sto-RNase HI) and its derivative lacking the C-terminal tail (ΔC6 Sto-RNase HI) (PDB codes: 2EHG and 3ALY), we applied molecular dynamics (MD) simulations at four different temperatures (300, 375, 475, and 500 K) to examine the effect of the C-terminal tail on the hyperstabilization of Sto-RNase HI and to investigate the unfolding process of Sto-RNase HI and ΔC6 Sto-RNase HI. The simulations suggest that the C-terminal tail has significant impact in hyperstabilization of Sto-RNase HI and the unfolding of these two proteins evolves along dissimilar pathways. Essential dynamics analysis indicates that the essential subspaces of the two proteins at different temperatures are non-overlapping within the trajectories and they exhibit different directions of motion. Our work can give important information to understand the three-state folding mechanism of Sto-RNase HI and to offer alternative strategies to improve the protein stability.

α-Glucosidase inhibitory activities of fatty acids purified from the internal organ of sea cucumber Stichopus japonicas.

Science.gov (United States)

Nguyen, T H; Kim, S M

2015-04-01

α-Glucosidase inhibitory activities of the various solvent fractions (n-hexane, CHCl3 , EtOAc, BuOH, and water) of sea cucumber internal organ were investigated. 1,3-Dipalmitolein (1) and cis-9-octadecenoic acid (2) with potent α-glucosidase inhibitory activity were purified from the n-hexane fraction of sea cucumber internal organ. IC50 values of compounds 1 and 2 were 4.45 and 14.87 μM against Saccharomyces cerevisiae α-glucosidase. These compounds mildly inhibited rat-intestinal α-glucosidase. In addition, both compounds showed a mixed competitive inhibition against S. cerevisiae α-glucosidase and were very stable at pH 2 up to 60 min. The KI values of compounds 1 and 2 were 0.48 and 1.24 μM, respectively. Therefore, the internal organ of sea cucumber might be a potential new source of α-glucosidase inhibitors suitably used for prevention of obesity and diabetes mellitus. © 2015 Institute of Food Technologists®

Covalent Immobilization of β-Glucosidase on Magnetic Particles for Lignocellulose Hydrolysis

DEFF Research Database (Denmark)

Alftrén, Johan; Hobley, Timothy John

2013-01-01

β-Glucosidase hydrolyzes cellobiose to glucose and is an important enzyme in the consortium used for hydrolysis of cellulosic and lignocellulosic feedstocks. In the present work, β-glucosidase was covalently immobilized on non-porous magnetic particles to enable re-use of the enzyme. It was found...... that particles activated with cyanuric chloride and polyglutaraldehyde gave the highest bead-related immobilized enzyme activity when tested with p-nitrophenyl-β-D-glucopyranoside (104.7 and 82.2 U/g particles, respectively). Furthermore, the purified β-glucosidase preparation from Megazyme gave higher bead......-related enzyme activities compared to Novozym 188 (79.0 and 9.8 U/g particles, respectively). A significant improvement in thermal stability was observed for immobilized enzyme compared to free enzyme; after 5 h (at 65 °C), 36 % of activity remained for the former, while there was no activity in the latter...

Structure-activity relationships of lanostane-type triterpenoids from Ganoderma lingzhi as α-glucosidase inhibitors.

Science.gov (United States)

Fatmawati, Sri; Kondo, Ryuichiro; Shimizu, Kuniyoshi

2013-11-01

A series of lanostane-type triterpenoids, identified as ganoderma alcohols and ganoderma acids, were isolated from the fruiting body of Ganoderma lingzhi. Some of these compounds were confirmed as active inhibitors of the in vitro human recombinant aldose reductase. This paper aims to explain the structural requirement for α-glucosidase inhibition. Our structure-activity studies of ganoderma alcohols showed that the OH substituent at C-3 and the double-bond moiety at C-24 and C-25 are necessary to increase α-glucosidase inhibitory activity. The structure-activity relationships of ganoderma acids revealed that the OH substituent at C-11 is an important feature and that the carboxylic group in the side chain is essential for the recognition of α-glucosidase inhibitory activity. Moreover, the double-bond moiety at C-20 and C-22 in the side chain and the OH substituent at C-3 of ganoderma acids improve α-glucosidase inhibitory activity. These results provide an approach with which to consider the structural requirements of lanostane-type triterpenoids from G. lingzhi. An understanding of these requirements is considered necessary in order to improve a new type of α-glucosidase inhibitor. Copyright © 2013 Elsevier Ltd. All rights reserved.

Beta-glucosidases and nucleic acids encoding same

DEFF Research Database (Denmark)

2012-01-01

The present invention relates to the identification of a novel and improved beta-glucosidase producing strain of the fungus Aspergillus, namely Aspergillus saccharolyticus, which is efficient in the degradation of lignocellulosic biomasses into glucose for production of biofuels, biochemicals and...

Regulation of the cellulolytic system in Trichoderma reesei by sophorose: induction of cellulase and repression of beta-glucosidase.

OpenAIRE

Sternberg, D; Mandels, G R

1980-01-01

Sophorose has two regulatory roles in the production of cellulase enzymes in Trichoderma reesei: beta-glucosidase repression and cellulase induction. Sophorose also is hydrolyzed by the mycelial-associated beta-glucosidase. Repression of beta-glucosidase reduces sophorose hydrolysis and thus may increase cellulase induction.

Chemical Composition and α-Glucosidase Inhibitory Activity of Vietnamese Citrus Peels Essential Oils

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Nguyen Hai Dang

2016-01-01

Full Text Available Background. Inhibition of α-glucosidase is an important factor to control postprandial hyperglycemia in type 2 diabetes mellitus. Citrus essential oils (CEO are among the most widely used essential oils, and some of them exhibited promising antidiabetic effect. However, the α-glucosidase inhibition of CEO has not been investigated so far. The present work aims to evaluate the α-glucosidase inhibition of essential oils from six Vietnamese Citrus peels. Methods. The chemical composition of essential oils obtained by hydrodistillation from six Citrus peels was analyzed by GC-MS. All essential oils were tested for their inhibitory activity on α-glucosidase using p-nitrophenyl-α-D-glucopyranoside as substrate. Results. In Buddha’s hand and lime peels, the major components were limonene (59.0–61.31% and γ-terpinene (13.98–23.84% while limonene (90.95–95.74% was most abundant in pomelo, orange, tangerine, and calamondin peels. Among the essential oils, the Buddha’s hand oil showed the most significant α-glucosidase inhibitory effect with the IC50 value of 412.2 μg/mL. The combination of the Buddha’s hand essential oil and the antidiabetic drug acarbose increased the inhibitory effect. Conclusions. The results suggested the potential use of Buddha’s hand essential oil as an alternative in treatment of type 2 diabetes mellitus.

Transcription termination in the plasmid/virus hybrid pSSVx from Sulfolobus islandicus

DEFF Research Database (Denmark)

Contursi, Patrizia; Cannio, Raffaele; She, Qunxin

2010-01-01

The pSSVx from Sulfolobus islandicus, strain REY15/4, is a hybrid between a plasmid and a fusellovirus. A systematic study previously performed revealed the presence of nine major transcripts, the expression of which was differentially and temporally regulated over the growth cycle of S. islandicus....... In this study, two new transcripts were identified. Then, 3' termini of all the RNAs were mapped using adaptor RT-PCR and RNase protection assays, and termination/arrest positions were identified for each transcript. The majority of the identified ending positions were located in the close vicinity of a T...... and counter-transcripts might be responsible for the transcription termination at these T-track-minus loci in the closely spaced pSSVx genes....

A novel extracellular β-glucosidase from Trichosporon asahii: yield prediction, evaluation and application for aroma enhancement of Cabernet Sauvignon.

Science.gov (United States)

Wang, Yuxia; Xu, Yan; Li, Jiming

2012-08-01

The production and application of novel β-glucosidase from Trichosporon asahii were studied. The β-glucosidase yield was improved by response surface methodology, and the optimal media constituents were determined to be dextrin 4.67% (w/v), yeast extract 2.99% (w/v), MgSO(4) 0.01% (w/v), and K(2) HPO(4) 0.02% (w/v). As a result, β-glucosidase production was enhanced from 123.72 to 215.66 U/L. The effects of different enological factors on the activity of β-glucosidases from T. asahii were investigated in comparison to commercial enzymes. β-Glucosidase from T. asahii was activated in the presence of sugars in the range from 10% to 40% (w/v), with the exception of glucose (slight inhibition), and retained higher relative activities than commercial enzymes under the same conditions. In addition, ethanol, in concentrations between 5% and 20% (v/v), also increased the β-glucosidase activity. Although the β-glucosidase activity decreased with decreasing pH, the residual activity of T. asahii was still above 50% at the average wine pH (pH 3.5). Due to these properties, extracellular β-glucosidase from T. asahii exhibited a better ability than commercial enzymes in hydrolyzing aromatic precursors that remained in young finished wine. The excellent performs of this β-glucosidase in wine aroma enhancement and sensory evaluation indicated that the β-glucosidase has a potential application to individuate suitable preparations that can complement and optimize grape or wine quality during the winemaking process or in the final wine. The present study demonstrated the usefulness of response surface methodology based on the central composite design for yield enhancement of β-glucosidase from T. asahii. The investigation of the primary characteristics of the enzyme and its application in young red wine suggested that the β-glucosidase from T. asahii can provide more impetus for aroma improvement in the future. © 2012 Institute of Food Technologists®

Polynucleotides encoding polypeptides having beta-glucosidase activity

Science.gov (United States)

Harris, Paul; Golightly, Elizabeth

2010-03-02

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for antiviral defense (CASCADE).

Science.gov (United States)

Lintner, Nathanael G; Kerou, Melina; Brumfield, Susan K; Graham, Shirley; Liu, Huanting; Naismith, James H; Sdano, Matthew; Peng, Nan; She, Qunxin; Copié, Valérie; Young, Mark J; White, Malcolm F; Lawrence, C Martin

2011-06-17

In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA. The Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2-Cas5a complex is sufficient to bind crRNA and complementary ssDNA. The structure of Csa2 reveals a crescent-shaped structure unexpectedly composed of a modified RNA-recognition motif and two additional domains present as insertions in the RNA-recognition motif. Conserved residues indicate potential crRNA- and target DNA-binding sites, and the H160A variant shows significantly reduced affinity for crRNA. We propose a general subunit architecture for CASCADE in other bacteria and Archaea.

Formation and release of. beta. -glucosidase by Aspergillus niger ZIMET 43 746 in correlation to process operations

Energy Technology Data Exchange (ETDEWEB)

Kerns, G; Dalchow, E; Klappach, G; Meyer, D

1986-01-01

The total formation of ..beta..-glucosidase by the wild strain of Aspergillus niger ZIMET 43 746 is non-growth-associated. In discontinuous culture the total ..beta..-glucosidase activity related to the mycelium is increasing with the age of the mycelium. The complete release of the remaining mycelial-associated ..beta..-glucosidase is dependent on the structure of the mycelium. In the cases of the mycelium forms pellets throughout the growth phase than the release of ..beta..-glucosidase is accelerated compared to the release from loosly branched mycelium. Increasing shear stress caused by increasing of the impeller speed promotes the formation of pellets.

Cinnamon extract inhibits α-glucosidase activity and dampens postprandial glucose excursion in diabetic rats

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Thirumurugan Kavitha

2011-06-01

Full Text Available Abstract Background α-glucosidase inhibitors regulate postprandial hyperglycemia (PPHG by impeding the rate of carbohydrate digestion in the small intestine and thereby hampering the diet associated acute glucose excursion. PPHG is a major risk factor for diabetic vascular complications leading to disabilities and mortality in diabetics. Cinnamomum zeylanicum, a spice, has been used in traditional medicine for treating diabetes. In this study we have evaluated the α-glucosidase inhibitory potential of cinnamon extract to control postprandial blood glucose level in maltose, sucrose loaded STZ induced diabetic rats. Methods The methanol extract of cinnamon bark was prepared by Soxhlet extraction. Phytochemical analysis was performed to find the major class of compounds present in the extract. The inhibitory effect of cinnamon extract on yeast α-glucosidase and rat-intestinal α-glucosidase was determined in vitro and the kinetics of enzyme inhibition was studied. Dialysis experiment was performed to find the nature of the inhibition. Normal male Albino wistar rats and STZ induced diabetic rats were treated with cinnamon extract to find the effect of cinnamon on postprandial hyperglycemia after carbohydrate loading. Results Phytochemical analysis of the methanol extract displayed the presence of tannins, flavonoids, glycosides, terpenoids, coumarins and anthraquinones. In vitro studies had indicated dose-dependent inhibitory activity of cinnamon extract against yeast α-glucosidase with the IC 50 value of 5.83 μg/ml and mammalian α-glucosidase with IC 50 value of 670 μg/ml. Enzyme kinetics data fit to LB plot pointed out competitive mode of inhibition and the membrane dialysis experiment revealed reversible nature of inhibition. In vivo animal experiments are indicative of ameliorated postprandial hyperglycemia as the oral intake of the cinnamon extract (300 mg/kg body wt. significantly dampened the postprandial hyperglycemia by 78.2% and 52

Cinnamon extract inhibits α-glucosidase activity and dampens postprandial glucose excursion in diabetic rats

Science.gov (United States)

2011-01-01

Background α-glucosidase inhibitors regulate postprandial hyperglycemia (PPHG) by impeding the rate of carbohydrate digestion in the small intestine and thereby hampering the diet associated acute glucose excursion. PPHG is a major risk factor for diabetic vascular complications leading to disabilities and mortality in diabetics. Cinnamomum zeylanicum, a spice, has been used in traditional medicine for treating diabetes. In this study we have evaluated the α-glucosidase inhibitory potential of cinnamon extract to control postprandial blood glucose level in maltose, sucrose loaded STZ induced diabetic rats. Methods The methanol extract of cinnamon bark was prepared by Soxhlet extraction. Phytochemical analysis was performed to find the major class of compounds present in the extract. The inhibitory effect of cinnamon extract on yeast α-glucosidase and rat-intestinal α-glucosidase was determined in vitro and the kinetics of enzyme inhibition was studied. Dialysis experiment was performed to find the nature of the inhibition. Normal male Albino wistar rats and STZ induced diabetic rats were treated with cinnamon extract to find the effect of cinnamon on postprandial hyperglycemia after carbohydrate loading. Results Phytochemical analysis of the methanol extract displayed the presence of tannins, flavonoids, glycosides, terpenoids, coumarins and anthraquinones. In vitro studies had indicated dose-dependent inhibitory activity of cinnamon extract against yeast α-glucosidase with the IC 50 value of 5.83 μg/ml and mammalian α-glucosidase with IC 50 value of 670 μg/ml. Enzyme kinetics data fit to LB plot pointed out competitive mode of inhibition and the membrane dialysis experiment revealed reversible nature of inhibition. In vivo animal experiments are indicative of ameliorated postprandial hyperglycemia as the oral intake of the cinnamon extract (300 mg/kg body wt.) significantly dampened the postprandial hyperglycemia by 78.2% and 52.0% in maltose and sucrose

[Decontamination of organophosphorus compounds: Towards new alternatives].

Science.gov (United States)

Poirier, L; Jacquet, P; Elias, M; Daudé, D; Chabrière, E

2017-05-01

Organophosphorus coumpounds (OP) are toxic chemicals mainly used for agricultural purpose such as insecticides and were also developed and used as warfare nerve agents. OP are inhibitors of acetylcholinesterase, a key enzyme involved in the regulation of the central nervous system. Chemical, physical and biological approaches have been considered to decontaminate OP. This review summarizes the current and emerging strategies that are investigated to tackle this issue with a special emphasis on enzymatic remediation methods. During the last decade, many studies have been dedicated to the development of biocatalysts for OP removal. Among these, recent reports have pointed out the promising enzyme SsoPox isolated from the archaea Sulfolobus solfataricus. Considering both its intrinsic stability and activity, this hyperthermostable enzyme is highly appealing for the decontamination of OP. Copyright © 2017 Académie Nationale de Pharmacie. All rights reserved.

Current and emerging strategies for organophosphate decontamination: special focus on hyperstable enzymes.

Science.gov (United States)

Jacquet, Pauline; Daudé, David; Bzdrenga, Janek; Masson, Patrick; Elias, Mikael; Chabrière, Eric

2016-05-01

Organophosphorus chemicals are highly toxic molecules mainly used as pesticides. Some of them are banned warfare nerve agents. These compounds are covalent inhibitors of acetylcholinesterase, a key enzyme in central and peripheral nervous systems. Numerous approaches, including chemical, physical, and biological decontamination, have been considered for developing decontamination methods against organophosphates (OPs). This work is an overview of both validated and emerging strategies for the protection against OP pollution with special attention to the use of decontaminating enzymes. Considerable efforts have been dedicated during the past decades to the development of efficient OP degrading biocatalysts. Among these, the promising biocatalyst SsoPox isolated from the archaeon Sulfolobus solfataricus is emphasized in the light of recently published results. This hyperthermostable enzyme appears to be particularly attractive for external decontamination purposes with regard to both its catalytic and stability properties.

CrRNA-Protospacer Recognition during CRISPR- Directed DNA Interference Sulfolobus islandicus REY 15A and Structural Studies of CRISPR Binding Proteins (CBP) of Crenarchaeon Sulfolobus

DEFF Research Database (Denmark)

Mousaei, Marzieh

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and associated proteins) is one of the important known immune mechanisms in archaea and bacteria. This adaptive immune system degrades invading genetic elements and protects the cell. Amongst 3 main types I, II and III...... of CRISPR system, two types (I and III) are found in archaea. However, in Sulfolobus species, subtypes IA, I-D, and III-B, III-D and rarely III-A are found. The model organism used for interference and structural studies is S. islandicus REY15A which carries subtypes I-A and III-B (α and β). Besides CRISPR...... ribonucleoprotein complex which is involved directly in defense, there are some less- known parts of the system including CPBs (CRISPR repeat-binding proteins) which are suggested to play a role in transcription. In the first part of my thesis, I provide a brief introduction to archaea and viruses that infect...

[Chemical Constituents from Leaves of Hibiscus syriacus and Their α-Glucosidase Inhibitory Activities].

Science.gov (United States)

Wei, Qiang; Ji, Xiao-ying; Xu, Fei; Li, Qian-rong; Yin, Hao

2015-05-01

To study the chemical constituents from Hibiscus syriacus leaves and their α-glucosidase inhibitory activities. Column chromatography including macroporous resins, silica gel and Sephadex LH-20 were used for the isolation and purification of all compounds. Spectroscopic methods including physical and chemical properties, 1H-NMR and 13C-NMR were used for the identification of structures. Their α-glucosidase inhibitory activities were detected by a 96-well microplate. 15 compounds were isolated and identified as β-sitosterol(1), β-daucostero (2), β-amyrin (3), oleanolic acid (4), stigmast-4-en-3-one (5), friedelin (6), syriacusin A (7), kaempferol (8), isovitexin (9), vitexin (10), apigenin (11), apigenin-7-O-β-D-glucopyranoside (12), luteolin-7-O-β-D-glucopyranoside (13), vitexin-7-O-β-D-glucopyranoside (14) and rutin (15). All the compounds are isolated from the leaves of Hibiscus syriacus for the first time. Taking acarbose as positive control, the α-glucosidase inhibitory activities of 15 compounds were evaluated. Compounds 7 and 9 have shown strong α-glucosidase inhibitory activities with IC50 of 39.03 ± 0.38 and 32.12 ± 0.62 mg/L, inhibition ratio of 94.95% and 97.15%, respectively.

REGULATION OF EXPRESSION OF MULTIPLE BETA- GLUCOSIDASES OF ASPERGILLUS TERREUS AND THEIR PURIFICATION AND CHARACTERIZATION

Directory of Open Access Journals (Sweden)

Asiya Nazir

2009-02-01

Full Text Available This study reports the regulation and purification of -glucosidases from a thermotolerant Aspergillus terreus AN1 strain, previously reported for efficient deinking of composite paper waste. The differential expression of four -glucosidase isoforms, in response to carbon sources in production medium, was studied by electrophoretically resolving proteins by polyacrylamide gel electro-phoresis analysis (PAGE and developing zymograms using methylum-belliferyl -D glucoside as substrate. Three -glucosidases (GI, GII & GIII were purified using chromatographic techniques. SDS-PAGE revealed the respective molecular masses of GI, GII, and GIII, as 29, 43, and 98 KDa, and isoelectric point (pI to be 2.8, 3.7, and 3.0. The -glucosidases exhibited diverse pH and temperature optima as well as stability. -Glucosidase I (GI specifically recog-nized pNP--glucopyranoside (pNPG as a substrate, whereas, -glucosidase II (GII and III (GIII also showed activities against cellobiose and salicin. In contrast to GII and GIII, the activity of GI was positively influenced in the presence of hexoses/pentoses and alcohols. Km and Vmax for hydrolysis of pNPG by GI, GII, andGIII were found to be 14.2 mM and 166.9 µmol -1mg protein -1, 4.37 mM, and 34.7 µmol -1mg proteins -1, and 11.1 mM and 378.7µ mol -1 mg protein -1, respectively.

The architects of crenarchaeal chromatin : A biophysical characterization of chromatin proteins from Sulfolobus solfataricus

NARCIS (Netherlands)

Driessen, Rosalie Paula Catharina

2014-01-01

Understanding of chromatin organization and compaction in Archaea is currently limited. The genome of several megabasepairs long is folded by a set of small chromatin proteins to fit into the micron-sized cell. A first step in understanding archaeal chromatin organization is to study the action of

Heterocyclic Compounds: Effective α-Amylase and α-Glucosidase Inhibitors.

Science.gov (United States)

Saeedi, Mina; Hadjiakhondi, Abbas; Nabavi, Seyed Mohammad; Manayi, Azadeh

2017-01-01

Diabetes Mellitus (DM) is a metabolic disease characterized by high blood sugar levels. Recently, it has emerged as an important and global health problem with long-term complications and high economic burden. α-Amylase (α-Amy) and α-glucosidase (α-Gls) are two enzymes which are involved in the hydrolysis of starch into sugars and disaccharides leading to the increase of blood glucose level. Hence, inhibition of α-amylase and α-glucosidase plays key role in the treatment of type 2 diabetes. Heterocyclic compounds -both synthetic and naturally occurring derivatives- possess efficient biological properties. At this juncture, they have demonstrated potent inhibitory activity against α-Amy and α-Gls and were found to be versatile tools for the development of novel anti-diabetic agents.

pTC Plasmids from Sulfolobus Species in the Geothermal Area of Tengchong, China: Genomic Conservation and Naturally-Occurring Variations as a Result of Transposition by Mobile Genetic Elements.

Science.gov (United States)

Xiang, Xiaoyu; Huang, Xiaoxing; Wang, Haina; Huang, Li

2015-02-12

Plasmids occur frequently in Archaea. A novel plasmid (denoted pTC1) containing typical conjugation functions has been isolated from Sulfolobus tengchongensis RT8-4, a strain obtained from a hot spring in Tengchong, China, and characterized. The plasmid is a circular double-stranded DNA molecule of 20,417 bp. Among a total of 26 predicted pTC1 ORFs, 23 have homologues in other known Sulfolobus conjugative plasmids (CPs). pTC1 resembles other Sulfolobus CPs in genome architecture, and is most highly conserved in the genomic region encoding conjugation functions. However, attempts to demonstrate experimentally the capacity of the plasmid for conjugational transfer were unsuccessful. A survey revealed that pTC1 and its closely related plasmid variants were widespread in the geothermal area of Tengchong. Variations of the plasmids at the target sites for transposition by an insertion sequence (IS) and a miniature inverted-repeat transposable element (MITE) were readily detected. The IS was efficiently inserted into the pTC1 genome, and the inserted sequence was inactivated and degraded more frequently in an imprecise manner than in a precise manner. These results suggest that the host organism has evolved a strategy to maintain a balance between the insertion and elimination of mobile genetic elements to permit genomic plasticity while inhibiting their fast spreading.

pTC Plasmids from Sulfolobus Species in the Geothermal Area of Tengchong, China: Genomic Conservation and Naturally-Occurring Variations as a Result of Transposition by Mobile Genetic Elements

Directory of Open Access Journals (Sweden)

Xiaoyu Xiang

2015-02-01

Full Text Available Plasmids occur frequently in Archaea. A novel plasmid (denoted pTC1 containing typical conjugation functions has been isolated from Sulfolobus tengchongensis RT8-4, a strain obtained from a hot spring in Tengchong, China, and characterized. The plasmid is a circular double-stranded DNA molecule of 20,417 bp. Among a total of 26 predicted pTC1 ORFs, 23 have homologues in other known Sulfolobus conjugative plasmids (CPs. pTC1 resembles other Sulfolobus CPs in genome architecture, and is most highly conserved in the genomic region encoding conjugation functions. However, attempts to demonstrate experimentally the capacity of the plasmid for conjugational transfer were unsuccessful. A survey revealed that pTC1 and its closely related plasmid variants were widespread in the geothermal area of Tengchong. Variations of the plasmids at the target sites for transposition by an insertion sequence (IS and a miniature inverted-repeat transposable element (MITE were readily detected. The IS was efficiently inserted into the pTC1 genome, and the inserted sequence was inactivated and degraded more frequently in an imprecise manner than in a precise manner. These results suggest that the host organism has evolved a strategy to maintain a balance between the insertion and elimination of mobile genetic elements to permit genomic plasticity while inhibiting their fast spreading.

Strain improvement and optimization for β-glucosidase production in Aspergillus niger by low-energy N+ implantation

International Nuclear Information System (INIS)

Diao Jinshan; Wang Li; Chen Zhen; Liu Hui; Nie Guangjun; Zheng Zhiming

2010-01-01

Low-energy N + implantation was employed to mutate Aspergillus niger Au to enhance productivity of β-glucosidase. Effects of N + on strains, survival and mutation rate were studied. After several rounds of implantation, activity of β-glucosidase of the final mutant Au 0847 reached 13.75 U/mL, which is higher by 106.8% than that of original strain Au, and its heritability was stabilized. Activity of β-glucosidase of Au 0847 reached 30.53 U/mL after further fermentation condition optimization. (authors)

Antioxidant and α-glucosidase inhibitory ingredients identified from Jerusalem artichoke flowers.

Science.gov (United States)

Wang, Yan-Ming; Zhao, Jian-Qiang; Yang, Jun-Li; Idong, Pema Tsering; Mei, Li-Juan; Tao, Yan-Duo; Shi, Yan-Ping

2017-11-09

Jerusalem artichoke (JA, Helianthus tuberosus L.) has been researched extensively due to its wide range of uses, but there are limited studies on its flowers. In this study, we report the first detailed phytochemical study on JA flowers, which yielded 21 compounds. Compound 4 was identified as a major water-soluble yellow pigment of JA flowers. In addition, the methanol extract of JA flowers and the isolates were evaluated for their antioxidant and α-glucosidase inhibitory activities. Among the tested compounds, compound 13 showed the strongest ABTS + free radical scavenging activity with SC 50 value of 2.30 ± 0.13 μg/mL, and compound 6 showed most potent α-glucosidase inhibitory activity with inhibition rate of 60.0% ± 10.3% at a concentration of 250 μg/mL. Results showed that methanol extract of JA flowers exhibited antioxidant and α-glucosidase inhibitory activities which could be attributed to its phenolic ingredients including chlorogenic acid derivatives, flavonoids and phenols.

New α-Glucosidase inhibitors from Croton bonplandianum Croton ...

African Journals Online (AJOL)

26.7 μg/mL, relative to that of the positive control, acarbose (IC50, 38.2 µg/mL). Conclusion: The ... Chemicals, reagents and instrumentation α-Glucosidase ... measurements performed on the MAT 312 mass ... the extraction process, 20.2 g of.

Rapid Screening for α-Glucosidase Inhibitors from Gymnema sylvestre by Affinity Ultrafiltration–HPLC-MS

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Mingquan Guo

2017-04-01

Full Text Available Gymnema sylvestre R. Br. (Asclepiadaceae has been known to posses potential anti-diabetic activity, and the gymnemic acids were reported as the main bioactive components in this plant species. However, the specific components responsible for the hypoglycemic effect still remain unknown. In the present study, the in vitro study revealed that the extract of G. sylvestre exhibited significant inhibitory activity against α-glucosidase with IC50 at 68.70 ± 1.22 μg/mL compared to acarbose (positive control at 59.03 ± 2.30 μg/mL, which further indicated the potential anti-diabetic activity. To this end, a method based on affinity ultrafiltration coupled with liquid chromatography mass spectrometry (UF-HPLC-MS was established to rapidly screen and identify the α-glucosidase inhibitors from G. sylvestre. In this way, 9 compounds with higher enrichment factors (EFs were identified according to their MS/MS spectra. Finally, the structure-activity relationships revealed that glycosylation could decrease the potential antisweet activity of sapogenins, and other components except gymnemic acids in G. sylvestre could also be good α-glucosidase inhibitors due to their synergistic effects. Taken together, the proposed method combing α-glucosidase and UF-HPLC-MS presents high efficiency for rapidly screening and identifying potential inhibitors of α-glucosidase from complex natural products, and could be further explored as a valuable high-throughput screening (HTS platform in the early anti-diabetic drug discovery stage.

Dual role of imidazole as activator/inhibitor of sweet almond (Prunus dulcis β-glucosidase

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Sara Caramia

2017-07-01

Full Text Available The activity of Prunus dulcis (sweet almond β-glucosidase at the expense of p-nitrophenyl-β-d-glucopyranoside at pH 6 was determined, both under steady-state and pre-steady-state conditions. Using crude enzyme preparations, competitive inhibition by 1–5 mM imidazole was observed under both kinetic conditions tested. However, when imidazole was added to reaction mixtures at 0.125–0.250 mM, we detected a significant enzyme activation. To further inspect this effect exerted by imidazole, β-glucosidase was purified to homogeneity. Two enzyme isoforms were isolated, i.e. a full-length monomer, and a dimer containing a full-length and a truncated subunit. Dimeric β-glucosidase was found to perform much better than the monomeric enzyme, independently of the kinetic conditions used to assay enzyme activity. In addition, the sensitivity towards imidazole was found to differ between the two isoforms. While monomeric enzyme was indeed found to be relatively insensitive to imidazole, dimeric β-glucosidase was observed to be significantly activated by 0.125–0.250 mM imidazole under pre-steady-state conditions. Further, steady-state assays revealed that the addition of 0.125 mM imidazole to reaction mixtures increases the Km of dimeric enzyme from 2.3 to 6.7 mM. The activation of β-glucosidase dimer by imidazole is proposed to be exerted via a conformational transition poising the enzyme towards proficient catalysis.

Dual role of imidazole as activator/inhibitor of sweet almond (Prunus dulcis) β-glucosidase.

Science.gov (United States)

Caramia, Sara; Gatius, Angela Gala Morena; Dal Piaz, Fabrizio; Gaja, Denis; Hochkoeppler, Alejandro

2017-07-01

The activity of Prunus dulcis (sweet almond) β-glucosidase at the expense of p -nitrophenyl-β-d-glucopyranoside at pH 6 was determined, both under steady-state and pre-steady-state conditions. Using crude enzyme preparations, competitive inhibition by 1-5 mM imidazole was observed under both kinetic conditions tested. However, when imidazole was added to reaction mixtures at 0.125-0.250 mM, we detected a significant enzyme activation. To further inspect this effect exerted by imidazole, β-glucosidase was purified to homogeneity. Two enzyme isoforms were isolated, i.e. a full-length monomer, and a dimer containing a full-length and a truncated subunit. Dimeric β-glucosidase was found to perform much better than the monomeric enzyme, independently of the kinetic conditions used to assay enzyme activity. In addition, the sensitivity towards imidazole was found to differ between the two isoforms. While monomeric enzyme was indeed found to be relatively insensitive to imidazole, dimeric β-glucosidase was observed to be significantly activated by 0.125-0.250 mM imidazole under pre-steady-state conditions. Further, steady-state assays revealed that the addition of 0.125 mM imidazole to reaction mixtures increases the K m of dimeric enzyme from 2.3 to 6.7 mM. The activation of β-glucosidase dimer by imidazole is proposed to be exerted via a conformational transition poising the enzyme towards proficient catalysis.

A novel beta-glucosidase from the cell wall of maize (Zea mays L.): rapid purification and partial characterization

Science.gov (United States)

Nematollahi, W. P.; Roux, S. J.

1999-01-01

Plants have a variety of glycosidic conjugates of hormones, defense compounds, and other molecules that are hydrolyzed by beta-glucosidases (beta-D-glucoside glucohydrolases, E.C. 3.2.1.21). Workers have reported several beta-glucosidases from maize (Zea mays L.; Poaceae), but have localized them mostly by indirect means. We have purified and partly characterized a 58-Ku beta-glucosidase from maize, which we conclude from a partial sequence analysis, from kinetic data, and from its localization is not identical to any of those already reported. A monoclonal antibody, mWP 19, binds this enzyme, and localizes it in the cell walls of maize coleoptiles. An earlier report showed that mWP19 inhibits peroxidase activity in crude cell wall extracts and can immunoprecipitate peroxidase activity from these extracts, yet purified preparations of the 58 Ku protein had little or no peroxidase activity. The level of sequence similarity between beta-glucosidases and peroxidases makes it unlikely that these enzymes share epitopes in common. Contrary to a previous conclusion, these results suggest that the enzyme recognized by mWP19 is not a peroxidase, but there is a wall peroxidase closely associated with the 58 Ku beta-glucosidase in crude preparations. Other workers also have co-purified distinct proteins with beta-glucosidases. We found no significant charge in the level of immunodetectable beta-glucosidase in mesocotyls or coleoptiles that precedes the red light-induced changes in the growth rate of these tissues.

Drug: D00216 [KEGG MEDICUS

Lifescience Database Archive (English)

Full Text Available etic agent ... DG01663 ... alpha-Glucosidase inhibitor ... DG01803 ... Antidiabetic, alpha-glu...cosidase inhibitor Unclassified ... DG02044 ... Hypoglycemics ... DG01803 ... Antidiabetic, alpha-glucosidase inhibitor... D00216 Drug Acarbose (JAN/USAN/INN); Precose (TN) ... C25H43NO18 D00216.gif ... Actinoplanes [TAX:1865] Antidiab

Alpha-Glucosidase Enzyme Biosensor for the Electrochemical Measurement of Antidiabetic Potential of Medicinal Plants.

Science.gov (United States)

Mohiuddin, M; Arbain, D; Islam, A K M Shafiqul; Ahmad, M S; Ahmad, M N

2016-12-01

A biosensor for measuring the antidiabetic potential of medicinal plants was developed by covalent immobilization of α-glucosidase (AG) enzyme onto amine-functionalized multi-walled carbon nanotubes (MWCNTs-NH2). The immobilized enzyme was entrapped in freeze-thawed polyvinyl alcohol (PVA) together with p-nitrophenyl-α-D-glucopyranoside (PNPG) on the screen-printed carbon electrode at low pH to prevent the premature reaction between PNPG and AG enzyme. The enzymatic reaction within the biosensor is inhibited by bioactive compounds in the medicinal plant extracts. The capability of medicinal plants to inhibit the AG enzyme on the electrode correlates to the potential of the medicinal plants to inhibit the production of glucose from the carbohydrate in the human body. Thus, the inhibition indicates the antidiabetic potential of the medicinal plants. The performance of the biosensor was evaluated to measure the antidiabetic potential of three medicinal plants such as Tebengau (Ehretis laevis), Cemumar (Micromelum pubescens), and Kedondong (Spondias dulcis) and acarbose (commercial antidiabetic drug) via cyclic voltammetry, amperometry, and spectrophotometry. The cyclic voltammetry (CV) response for the inhibition of the AG enzyme activity by Tebengau plant extracts showed a linear relation in the range from 0.423-8.29 μA, and the inhibition detection limit was 0.253 μA. The biosensor exhibited good sensitivity (0.422 μA/mg Tebengau plant extracts) and rapid response (22 s). The biosensor retains approximately 82.16 % of its initial activity even after 30 days of storage at 4 °C.

Production and characterization of β-glucosidase from Gongronella ...

African Journals Online (AJOL)

sunny t

2016-04-20

Apr 20, 2016 ... Among the enzymes of the cellulolytic complex, β-glucosidases are noteworthy due to the possibility of their application in different industrial processes, such as production of biofuels, winemaking, and development of functional foods. This study aimed to evaluate the production and characterization of β-.

In-silico analysis of Aspergillus niger beta-glucosidases

Science.gov (United States)

Yeo S., L.; Shazilah, K.; Suhaila, S.; Abu Bakar F., D.; Murad A. M., A.

2014-09-01

Genomic data mining was carried out and revealed a total of seventeen β-glucosidases in filamentous fungi Aspergillus niger. Two of them belonged to glycoside hydrolase family 1 (GH1) while the rest belonged to genes in family 3 (GH3). These proteins were then named according to the nomenclature as proposed by the International Union of Biochemistry (IUB), starting from the lowest pI and glycoside hydrolase family. Their properties were predicted using various bionformatic tools showing the presence of domains for signal peptide and active sites. Interestingly, one particular domain, PA14 (protective antigen) was present in four of the enzymes, predicted to be involved in carbohydrate binding. A phylogenetic tree grouped the two glycoside hydrolase families with GH1 and GH3 related organisms. This study showed that the various domains present in these β-glucosidases are postulated to be crucial for the survival of this fungus, as supported by other analysis.

Light induced expression of β-glucosidase in Escherichia coli with autolysis of cell.

Science.gov (United States)

Chang, Fei; Zhang, Xianbing; Pan, Yu; Lu, Youxue; Fang, Wei; Fang, Zemin; Xiao, Yazhong

2017-11-07

β-Glucosidase has attracted substantial attention in the scientific community because of its pivotal role in cellulose degradation, glycoside transformation and many other industrial processes. However, the tedious and costly expression and purification procedures have severely thwarted the industrial applications of β-glucosidase. Thus development of new strategies to express β-glucosidases with cost-effective and simple procedure to meet the increasing demands on enzymes for biocatalysis is of paramount importance. Light activated cassette YF1/FixJ and the SRRz lysis system were successfully constructed to produce Bgl1A(A24S/F297Y), a mutant β-glucosidase tolerant to both glucose and ethanol. By optimizing the parameters for light induction, Bgl1A(A24S/F297Y) activity reached 33.22 ± 2.0 U/mL and 249.92 ± 12.25 U/mL in 250-mL flask and 3-L fermentation tank, respectively, comparable to the controls of 34.02 ± 1.96 U/mL and 322.21 ± 10.16 U/mL under similar culture conditions with IPTG induction. To further simplify the production of our target protein, the SRRz lysis gene cassette from bacteriophage Lambda was introduced to trigger cell autolysis. As high as 84.53 ± 6.79% and 77.21 ± 4.79% of the total β-glucosidase were released into the lysate after cell autolysis in 250 mL flasks and 3-L scale fermentation with lactose as inducer of SRRz. In order to reduce the cost of protein purification, a cellulose-binding module (CBM) from Clostridium thermocellum was fused into the C-terminal of Bgl1A(A24S/F297Y) and cellulose was used as an economic material to adsorb the fusion enzyme from the lysate. The yield of the fusion protein could reach 92.20 ± 2.27% after one-hour adsorption at 25 °C. We have developed an efficient and inexpensive way to produce β-glucosidase for potential industrial applications by using the combination of light induction, cell autolysis, and CBM purification strategy.

Digestive beta-glucosidases from the wood-feeding higher termite, Nasutitermes takasagoensis: intestinal distribution, molecular characterization, and alteration in sites of expression.

Science.gov (United States)

Tokuda, Gaku; Miyagi, Mio; Makiya, Hiromi; Watanabe, Hirofumi; Arakawa, Gaku

2009-12-01

beta-Glucosidase [EC 3.2.1.21] hydrolyzes cellobiose or cello-oligosaccharides into glucose during cellulose digestion in termites. SDS-PAGE and zymogram analyses of the digestive system in the higher termite Nasutitermes takasagoensis revealed that beta-glucosidase activity is localized in the salivary glands and midgut as dimeric glycoproteins. Degenerate PCR using primers based on the N-terminal amino acid sequences of the salivary beta-glucosidase resulted in cDNA fragments of 1.7 kb, encoding 489 amino acids with a sequence similar to glycosyl hydrolase family 1. Moreover, these primers amplified cDNA fragments from the midgut, and the deduced amino acid sequences are 87-91% identical to those of the salivary beta-glucosidases. Successful expression of the cDNAs in Escherichia coli implies that these sequences also encode functional beta-glucosidases. These results indicate that beta-glucosidases that primarily contribute to the digestive process of N. takasagoensis are produced in the midgut. Reverse transcription-PCR analysis indicated the site-specific expression of beta-glucosidase mRNAs in the salivary glands and midgut. These results suggest that termites have developed the ability to produce beta-glucosidases in the midgut, as is the case for endo-beta-1,4-glucanase, in which the site of expression has shifted from the salivary glands of lower termites to the midgut of higher termites. Copyright 2009 Elsevier Ltd. All rights reserved.

Dgroup: DG01663 [KEGG MEDICUS

Lifescience Database Archive (English)

Full Text Available DG01663 DGroup alpha-Glucosidase inhibitor -bose, -glustat ... DG01803 ... Antidiabetic,...at ... D09605 ... Duvoglustat (USAN/INN) ... D09606 ... Duvoglustat hydrochloride (USAN) Antidiabetic agent ... alpha-glucosidase [KO:K12316 K12317 K12047] ...

Evaluation of glucosidases of Aspergillus niger strain comparing with other glucosidases in transformation of ginsenoside Rb1 to ginsenosides Rg3

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Kyung Hoon Chang

2014-01-01

Full Text Available The transformation of ginsenoside Rb1 into a specific minor ginsenoside using Aspergillus niger KCCM 11239, as well as the identification of the transformed products and the pathway via thin layer chromatography and high performance liquid chromatography were evaluated to develop a new biologically active material. The conversion of ginsenoside Rb1 generated Rd, Rg3, Rh2, and compound K although the reaction rates were low due to the low concentration. In enzymatic conversion, all of the ginsenoside Rb1 was converted to ginsenoside Rd and ginsenoside Rg3 after 24 h of incubation. The crude enzyme (β-glucosidase from A. niger KCCM 11239 hydrolyzed the β-(1→6-glucosidic linkage at the C-20 of ginsenoside Rb1 to generate ginsenoside Rd and ginsenoside Rg3. Our experimental demonstration showing that A. niger KCCM 11239 produces the ginsenoside-hydrolyzing β-glucosidase reflects the feasibility of developing a specific bioconversion process to obtain active minor ginsenosides.

Direct ethanol production from cassava pulp using a surface-engineered yeast strain co-displaying two amylases, two cellulases, and {beta}-glucosidase

Energy Technology Data Exchange (ETDEWEB)

Apiwatanapiwat, Waraporn; Rugthaworn, Prapassorn [Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Ibaraki (Japan). Post-Harvest Science and Technology Div.; Kasetsart Univ., Bangkok (Thailand). Nanotechnology and Biotechnology Div.; Murata, Yoshinori; Kosugi, Akihiko; Arai, Takamitsu; Mori, Yutaka [Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Ibaraki (Japan). Post-Harvest Science and Technology Div.; Yamada, Ryosuke; Kondo, Akihiko [Kobe Univ. (Japan). Dept. of Chemical Science and Engineering

2011-04-15

In order to develop a method for producing fuel ethanol from cassava pulp using cell surface engineering (arming) technology, an arming yeast co-displaying {alpha}-amylase ({alpha}-AM), glucoamylase, endoglucanase, cellobiohydrase, and {beta}-glucosidase on the surface of the yeast cells was constructed. The novel yeast strain, possessing the activities of all enzymes, was able to produce ethanol directly from soluble starch, barley {beta}-glucan, and acid-treated Avicel. Cassava is a major crop in Southeast Asia and used mainly for starch production. In the starch manufacturing process, large amounts of solid wastes, called cassava pulp, are produced. The major components of cassava pulp are starch (approximately 60%) and cellulose fiber (approximately 30%). We attempted simultaneous saccharification and ethanol fermentation of cassava pulp with this arming yeast. During fermentation, ethanol concentration increased as the starch and cellulose fiber substrates contained in the cassava pulp decreased. The results clearly showed that the arming yeast was able to produce ethanol directly from cassava pulp without addition of any hydrolytic enzymes. (orig.)

Proteolysis in hyperthermophilic microorganisms

Directory of Open Access Journals (Sweden)

Donald E. Ward

2002-01-01

Full Text Available Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus, the crenarchaeote Sulfolobus solfataricus, and the bacterium Thermotoga maritima. An overview of the proteases in these organisms is given based on those proteases that have been characterized and on putative proteases that have been identified from genomic sequences, but have yet to be characterized. The analysis revealed both similarities and differences in the mechanisms utilized for proteolysis by each of these hyperthermophiles and indicated how these mechanisms relate to proteolysis in less thermophilic cells and organisms.

General misincorporation frequency: Re-evaluation of the fidelity of DNA polymerases.

Science.gov (United States)

Yang, Jie; Li, Bianbian; Liu, Xiaoying; Tang, Hong; Zhuang, Xiyao; Yang, Mingqi; Xu, Ying; Zhang, Huidong; Yang, Chun

2018-02-19

DNA replication in cells is performed in the presence of four dNTPs and four rNTPs. In this study, we re-evaluated the fidelity of DNA polymerases using the general misincorporation frequency consisting of three incorrect dNTPs and four rNTPs but not using the traditional special misincorporation frequency with only the three incorrect dNTPs. We analyzed both the general and special misincorporation frequencies of nucleotide incorporation opposite dG, rG, or 8-oxoG by Pseudomonas aeruginosa phage 1 (PaP1) DNA polymerase Gp90 or Sulfolobus solfataricus DNA polymerase Dpo4. Both misincorporation frequencies of other DNA polymerases published were also summarized and analyzed. The general misincorporation frequency is obviously higher than the special misincorporation frequency for many DNA polymerases, indicating the real fidelity of a DNA polymerase should be evaluated using the general misincorporation frequency. Copyright © 2018 Elsevier Inc. All rights reserved.

QSAR Studies on Andrographolide Derivatives as α-Glucosidase Inhibitors

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Shaohui Cai

2010-03-01

Full Text Available Andrographolide derivatives were shown to inhibit α-glucosidase. To investigate the relationship between activities and structures of andrographolide derivatives, a training set was chosen from 25 andrographolide derivatives by the principal component analysis (PCA method, and a quantitative structure-activity relationship (QSAR was established by 2D and 3D QSAR methods. The cross-validation r2 (0.731 and standard error (0.225 illustrated that the 2D-QSAR model was able to identify the important molecular fragments and the cross-validation r2 (0.794 and standard error (0.127 demonstrated that the 3D-QSAR model was capable of exploring the spatial distribution of important fragments. The obtained results suggested that proposed combination of 2D and 3D QSAR models could be useful in predicting the α-glucosidase inhibiting activity of andrographolide derivatives.

PRODUCTION OF RECOMBINANT HIGH pI-BARLEY α-GLUCOSIDASE

DEFF Research Database (Denmark)

Næsted, Henrik; Svensson, Birte

plantlet [1]. Recently, expression and characterization of the recombinant full length, fully functional barley high pI α-glucosidase in Pichia pastoris has been achieved. To enable production of recombinant protein in mg amounts, a transformant harbouring a clone encoding the N-terminally hexa histidine...... tagged recombinant form of the enzyme was propagated using a high cell-density fermentation procedure. This system resulted in successful expression under the highly sensitive methanol utilization phase conducting the fermentation process using a BiostatB 5 L reactor. The recombinant high pI α...... glycosylation of the recombinant α-glucosidase. The enzyme activity was highly stable during the 5 day long fermentation. Characterisation of the enzymatic properties confirmed the specific activity actually to be superior to that of the native enzyme purified from malt [2]. The kinetic parameters Km, Vmax...

Chemical Constituents of Malaysian U. cordata var. ferruginea and Their in Vitro α-Glucosidase Inhibitory Activities

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Nur Hakimah Abdullah

2016-04-01

Full Text Available Continuing our interest in the Uncaria genus, the phytochemistry and the in-vitro α-glucosidase inhibitory activities of Malaysian Uncaria cordata var. ferruginea were investigated. The phytochemical study of this plant, which employed various chromatographic techniques including recycling preparative HPLC, led to the isolation of ten compounds with diverse structures comprising three phenolic acids, two coumarins, three flavonoids, a terpene and an iridoid glycoside. These constituents were identified as 2-hydroxybenzoic acid or salicylic acid (1, 2,4-dihydroxybenzoic acid (2, 3,4-dihydroxybenzoic acid (3, scopoletin or 7-hydroxy-6-methoxy-coumarin (4, 3,4-dihydroxy-7-methoxycoumarin (5, quercetin (6, kaempferol (7, taxifolin (8, loganin (9 and β-sitosterol (10. Structure elucidation of the compounds was accomplished with the aid of 1D and 2D Nuclear Magnetic Resonance (NMR spectral data and Ultraviolet-Visible (UV-Vis, Fourier Transform Infrared (FTIR spectroscopy and mass spectrometry (MS. In the α-glucosidase inhibitory assay, the crude methanolic extract of the stems of the plant and its acetone fraction exhibited strong α-glucosidase inhibition activity of 87.7% and 89.2%, respectively, while its DCM fraction exhibited only moderate inhibition (75.3% at a concentration of 1 mg/mL. The IC50 values of both fractions were found to be significantly lower than the standard acarbose suggesting the presence of potential α-glucosidase inhibitors. Selected compounds isolated from the active fractions were then subjected to α-glucosidase assay in which 2,4-dihydroxybenzoic acid and quercetin showed strong inhibitory effects against the enzyme with IC50 values of 549 and 556 μg/mL compared to acarbose (IC50 580 μg/mL while loganin and scopoletin only showed weak α-glucosidase inhibition of 44.9% and 34.5%, respectively. This is the first report of the isolation of 2-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid and loganin from the genus

A proteomics strategy to discover beta-glucosidases from Aspergillus fumigatus with two-dimensional page in-gel activity assay and tandem mass spectrometry.

Science.gov (United States)

Kim, Kee-Hong; Brown, Kimberly M; Harris, Paul V; Langston, James A; Cherry, Joel R

2007-12-01

Economically competitive production of ethanol from lignocellulosic biomass by enzymatic hydrolysis and fermentation is currently limited, in part, by the relatively high cost and low efficiency of the enzymes required to hydrolyze cellulose to fermentable sugars. Discovery of novel cellulases with greater activity could be a critical step in overcoming this cost barrier. beta-Glucosidase catalyzes the final step in conversion of glucose polymers to glucose. Despite the importance, only a few beta-glucosidases are commercially available, and more efficient ones are clearly needed. We developed a proteomics strategy aiming to discover beta-glucosidases present in the secreted proteome of the cellulose-degrading fungus Aspergillus fumigatus. With the use of partial or complete protein denaturing conditions, the secretory proteome was fractionated in a 2DGE format and beta-glucosidase activity was detected in the gel after infusion with a substrate analogue that fluoresces upon hydrolysis. Fluorescing spots were subjected to tryptic-digestion, and identification as beta-glucosidases was confirmed by tandem mass spectrometry. Two novel beta-glucosidases of A. fumigatus were identified by this in situ activity staining method, and the gene coding for a novel beta-glucosidase ( EAL88289 ) was cloned and heterologously expressed. The expressed beta-glucosidase showed far superior heat stability to the previously characterized beta-glucosidases of Aspergillus niger and Aspergillus oryzae. Improved heat stability is important for development of the next generation of saccharifying enzymes capable of performing fast cellulose hydrolysis reactions at elevated temperatures, thereby lowering the cost of bioethanol production. The in situ activity staining approach described here would be a useful tool for cataloguing and assessing the efficiency of beta-glucosidases in a high throughput fashion.

Effects of Fruit Toxins on Intestinal and Microbial β-Glucosidase Activities of Seed-Predating and Seed-Dispersing Rodents (Acomys spp.).

Science.gov (United States)

Kohl, Kevin D; Samuni-Blank, Michal; Lymberakis, Petros; Kurnath, Patrice; Izhaki, Ido; Arad, Zeev; Karasov, William H; Dearing, M Denise

2016-01-01

Plant secondary compounds (PSCs) have profound influence on the ecological interaction between plants and their consumers. Glycosides, a class of PSC, are inert in their intact form and become toxic on activation by either plant β-glucosidase enzymes or endogenous β-glucosidases produced by the intestine of the plant-predator or its microbiota. Many insect herbivores decrease activities of endogenous β-glucosidases to limit toxin exposure. However, such an adaptation has never been investigated in nonmodel mammals. We studied three species of spiny mice (Acomys spp.) that vary in their feeding behavior of the glycoside-rich fruit of Ochradenus baccatus. Two species, the common (Acomys cahirinus) and Crete (Acomys minous) spiny mice, behaviorally avoid activating glycosides, while the golden spiny mouse (Acomys russatus) regularly consumes activated glycosides. We fed each species a nontoxic diet of inert glycosides or a toxic diet of activated fruit toxins and investigated the responses of intestinal and microbial β-glucosidase activities. We found that individuals feeding on activated toxins had lower intestinal β-glucosidase activity and that the species that behaviorally avoid activating glycosides also had lower intestinal β-glucosidase activity regardless of treatment. The microbiota represented a larger source of toxin liberation, and the toxin-adapted species (golden spiny mouse) exhibited almost a fivefold increase in microbial β-glucosidase when fed activated toxins, while other species showed slight decreases. These results are contrary to those in insects, where glycoside-adapted species have lower β-glucosidase activity. The glycoside-adapted golden spiny mouse may have evolved tolerance mechanisms such as enhanced detoxification rather than avoidance mechanisms.

Genetic Studies on CRISPR-Cas Functions in Invader Defense in Sulfolobus islandicus

DEFF Research Database (Denmark)

Peng, Wenfang

Archaea and bacteria contain CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) systems that protect themselves against invasion by viruses and plasmids. There are three major types of CRISPR-Cas systems, type I, II and III, that are further divided...... into at least 11 subtypes. I employed Sulfolobus islandicus Rey15A as the model to study CRISPR mechanisms. The model archaeon encodes one subtype I-A (Cascade) and two subtype III-B (Cmr-α and Cmr-β) interference systems with no apparent redundancy in cas genes or in CRISPR systems, which is ideal for genetic...... analysis of cas gene function. Furthermore, a range of genetic tools have been developed for S. islandicus Rey15A in our laboratory and a plasmid interference assay has been successfully developed for testing CRISPR-directed DNA targeting activity, which have provided a solid basis for studying...

Isolation and characterization of β-glucosidase producing bacteria ...

African Journals Online (AJOL)

glucosidase activity. This activity was tested by growth in medium supplemented with esculin and ferric ammonium citrate. The esculin positive strains from both the sources were characterized biochemically and checked for their ability to transform ginsenoside Rb1. The growth medium and the pH for maximum growth were ...

Kinetics, improved activity and thermostability of endoglucanase and beta glucosidase from a mutant-derivative of aspergillus niger ms82

International Nuclear Information System (INIS)

Sohail, M.; Ahmad, A.; Khan, S.A.; Uddin, F.

2013-01-01

A mutant MS301 of Aspergillus niger MS82 showed 1.5 to 2.5-fold improved endoglucanase and beta-glucosidase activity when grown on crude lignocellulosic substrates under solid-state and submerged conditions. Indicators of thermal stability of enzymes (Tm and T1/2) showed that the wild type and mutant endoglucanase was more heat-resistant compared to beta-glucosidase. However, mutant and parent enzymes shared almost the same values for melting temperatures and half-lives. Endoglucanase and beta-glucosidase from both the strains showed optimum activity under acidic pH. Energy of activation (Ea) of mutant beta-glucosidase was substantially lower than the parent enzyme while Ea of mutant endoglucanase was slightly less than the parent. The lowered Ea values can be attributed to the improved beta-glucosidase activity of the mutant strain. Moreover, the MS301 enzymes were better in hydrolyzing purified and crude cellulosic materials than the parent MS82. (author)

Differential Involvement of β-Glucosidases from Hypocrea jecorina in Rapid Induction of Cellulase Genes by Cellulose and Cellobiose

Science.gov (United States)

Zhou, Qingxin; Xu, Jintao; Kou, Yanbo; Lv, Xinxing; Zhang, Xi; Zhao, Guolei; Zhang, Weixin; Chen, Guanjun

2012-01-01

Appropriate perception of cellulose outside the cell by transforming it into an intracellular signal ensures the rapid production of cellulases by cellulolytic Hypocrea jecorina. The major extracellular β-glucosidase BglI (CEL3a) has been shown to contribute to the efficient induction of cellulase genes. Multiple β-glucosidases belonging to glycosyl hydrolase (GH) family 3 and 1, however, exist in H. jecorina. Here we demonstrated that CEL1b, like CEL1a, was an intracellular β-glucosidase displaying in vitro transglycosylation activity. We then found evidence that these two major intracellular β-glucosidases were involved in the rapid induction of cellulase genes by insoluble cellulose. Deletion of cel1a and cel1b significantly compromised the efficient gene expression of the major cellulase gene, cbh1. Simultaneous absence of BglI, CEL1a, and CEL1b caused the induction of the cellulase gene by cellulose to further deteriorate. The induction defect, however, was not observed with cellobiose. The absence of the three β-glucosidases, rather, facilitated the induced synthesis of cellulase on cellobiose. Furthermore, addition of cellobiose restored the productive induction on cellulose in the deletion strains. The results indicate that the three β-glucosidases may not participate in transforming cellobiose beyond hydrolysis to provoke cellulase formation in H. jecorina. They may otherwise contribute to the accumulation of cellobiose from cellulose as inducing signals. PMID:23002106

NCBI nr-aa BLAST: CBRC-DNOV-01-1573 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-DNOV-01-1573 ref|ZP_01446186.1| probable alpha-glucosidase protein [Roseovariu...s sp. HTCC2601] gb|EAU43606.1| probable alpha-glucosidase protein [Roseovarius sp. HTCC2601] ZP_01446186.1 0.59 38% ...

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...

Differential effects of sugars and the alpha-glucosidase inhibitor acarbose (Bay g 5421) on satiety in the Zucker obese rat.

Science.gov (United States)

Maggio, C A; Decarr, L B; Vasselli, J R

1987-01-01

To examine the satiety responses of Zucker obese and lean rats to simple sugars, adult male rats were given equicaloric intragastric infusions of fructose, glucose, and sucrose. All three sugars reduced the short-term intakes of both genotypes, although no reliable between-genotype differences in the satiety effects of the sugars were observed. Within each genotype, fructose had a larger satiety effect than sucrose. To examine a potential basis for the observed effects, rats were given sucrose infusions containing the intestinal glucosidase inhibitor acarbose (Bay g 5421). In obese rats, addition of a low dose of acarbose increased the satiety effect of sucrose infusion. Delaying carbohydrate absorption via acarbose administration may alter gastrointestinal and/or postabsorptive satiety processes, and may prove useful as a probe for investigating the nature of satiety signals.

LC-MS guided isolation of diterpenoids from Sapium insigne with α-glucosidase inhibitory activities.

Science.gov (United States)

Yan, De-Xiu; Geng, Chang-An; Yang, Tong-Hua; Huang, Xiao-Yan; Li, Tian-Ze; Gao, Zhen; Ma, Yun-Bao; Peng, Hua; Zhang, Xue-Mei; Chen, Ji-Jun

2018-04-08

Ten new (1-10) and ten known (11-20) diterpenoids involving ent-atisane, ent-seco-atisane, ent-kaurane and ent-seco-kaurane types were isolated from Sapium insigne under the guidance of LCMS-IT-TOF analyses. Their structures were characterized by extensive spectroscopic analyses (HRESIMS, UV, IR, 1D and 2D NMR). A putative biosynthetic pathway was proposed for ent-seco-atisane diterpenoids. Their inhibitory activities on α-glucosidase in vitro were tested for the first time. Compound 4 showed moderate inhibitory effect on α-glucosidase with an IC 50 value of 0.34 mM via a noncompetitive inhibition mechanism (K i  = 0.27 mM). The preliminary structure-activity relationships of the ent-atisane diterpenoids inhibiting α-glucosidase were discussed. Copyright © 2018 Elsevier B.V. All rights reserved.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

Science.gov (United States)

Harris, Paul; Golightly, Elizabeth

2012-11-27

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

α-Glucosidase inhibitory activities of isoflavanones, isoflavones, and pterocarpans from Mucuna pruriens.

Science.gov (United States)

Dendup, Tshewang; Prachyawarakorn, Vilailak; Pansanit, Acharavadee; Mahidol, Chulabhorn; Ruchirawat, Somsak; Kittakoop, Prasat

2014-05-01

Three new isoflavanones (1-3) and thirteen known compounds (4-16) were isolated from the roots of Mucuna pruriens. The absolute configurations of isoflavanones 1-3 and parvisoflavanone (4), lespedeol C (5), and uncinanone C (6) were addressed by a circular dichroism technique. Isoflavanones, isoflavones, and pterocarpans of M. pruriens were found to be α-glucosidase inhibitors. Medicarpin (7) and parvisoflavone B (9) were potent α-glucosidase inhibitors (twofold less active than the standard drug acarbose). The production of bioactive metabolites in M. pruriens seems to be season-dependent. Georg Thieme Verlag KG Stuttgart · New York.

Bio-assay guided isolation of α-glucosidase inhibitory constituents from Hibiscus mutabilis leaves.

Science.gov (United States)

Kumar, Deepak; Kumar, Hemanth; Vedasiromoni, J R; Pal, Bikas C

2012-01-01

The increasing demand for natural-product-based medicines and health-care products for the management of diabetes encouraged investigation of this commonly available Indian plant. To establish the anti-diabetic (α-glucosidase inhibitory) activity of H. mutabilis leaf extract, isolate and identify the constituents responsible for the activity, and validate a HPLC method for quantification of the active constituents for standardisation of the extract. The methanolic extract of leaves was partitioned between water, n-butanol and ethyl acetate. Bio-assay guided fractionation, based on inhibition of α-glucosidase, allowed isolation and identification of the active components. The active components were quantified using RP-HPLC-DAD validated for linearity, limit of detection, limit of quantification, precision, accuracy and robustness for this plant extract and the partitioned fractions. Ferulic acid and caffeic acid were identified as the α-glucosidase inhibitors present in H. mutabilis. They were partitioned into an ethyl acetate fraction. The HPLC-DAD calibration curve showed good linearity (r² > 0.99). For the recovery studies the %RSD was less than 2%. The interday and intraday variations were found to be less than 4% RSD for retention time and response. The identification of α-glucosidase inhibition activity in H. mutabilis supports further investigations into the possible use of the plant for the management of diabetes. The HPLC method validated for these extracts will be useful in future research with the plant. Copyright © 2011 John Wiley & Sons, Ltd.

Yeast α-Glucosidase Inhibitory Phenolic Compounds Isolated from Gynura medica Leaf

Directory of Open Access Journals (Sweden)

Chao Tan

2013-01-01

Full Text Available Gynura medica leaf extract contains significant amounts of flavonols and phenolic acids and exhibits powerful hypoglycemic activity against diabetic rats in vivo. However, the hypoglycemic active constituents that exist in the plant have not been fully elaborated. The purpose of this study is to isolate and elaborate the hypoglycemic activity compounds against inhibition the yeast α-glucosidase in vitro. Seven phenolic compounds including five flavonols and two phenolic acids were isolated from the leaf of G. medica. Their structures were identified by the extensive NMR and mass spectral analyses as: kaempferol (1, quercetin (2, kaempferol-3-O-β-D-glucopyranoside (3, kaempferol-3-O-rutinoside (4, rutin (5, chlorogenic acid (6 and 3,5-dicaffeoylquinic acid methyl ester (7. All of the compounds except 1 and 3 were isolated for the first time from G. medica. Compounds 1–7 were also assayed for their hypoglycemic activity against yeast α-glucosidase in vitro. All of the compounds except 1 and 6 showed good yeast α-glucosidase inhibitory activity with the IC50 values of 1.67 mg/mL, 1.46 mg/mL, 0.38 mg/mL, 0.10 mg/mL and 0.53 mg/mL, respectively.

Production and localization of cellulases and. beta. -glucosidase from the thermophilic fungus Thielavia terrestris

Energy Technology Data Exchange (ETDEWEB)

Breuil, C; Wojtczak, G; Saddler, J N

1986-01-01

The enzyme production and localization of Thielavia terrestris strains C464 and NRRL 8126 were compared to determine their optimum temperature and pH for cellulase activity. High levels of intracellular ..beta..-glucosidase activity were detected in the former strain. The intracellular ..beta..-glucosidase of both strains were more thermostable than the extra-cellular enzyme; the half life of T. terrestris (C464) endoglucanase activity at 60 degrees C was greater than 96 hours. 12 references.

Antioxidant, Iron-chelating and Anti-glucosidase Activities of Typha ...

African Journals Online (AJOL)

Iron chelating activity was assessed using a ferrozine-based assay. Anti- glucosidase activity was determined using 4-nitrophenyl ... flavonoid (TF) content was determined based an aluminum chloride colorimetric assay [6]. TF content was ..... Dietary iron restriction or iron chelation protects from diabetes and loss of β-cell.

Conditions for gene disruption by homologous recombination of exogenous DNA into the Sulfolobus solfataricus genome

NARCIS (Netherlands)

Albers, Sonja-Verena; Driessen, Arnold J.M.

2008-01-01

The construction of directed gene deletion mutants is an essential tool in molecular biology that allows functional studies on the role of genes in their natural environment. For hyperthermophilic archaea, it has been difficult to obtain a reliable system to construct such mutants. However, during

Life Cycle Characterization of Sulfolobus Monocaudavirus 1, an Extremophilic Spindle-Shaped Virus with Extracellular Tail Development.

Science.gov (United States)

Uldahl, Kristine B; Jensen, Signe B; Bhoobalan-Chitty, Yuvaraj; Martínez-Álvarez, Laura; Papathanasiou, Pavlos; Peng, Xu

2016-06-15

We provide here, for the first time, insights into the initial infection stages of a large spindle-shaped archaeal virus and explore the following life cycle events. Our observations suggest that Sulfolobus monocaudavirus 1 (SMV1) exhibits a high adsorption rate and that virions adsorb to the host cells via three distinct attachment modes: nosecone association, body association, and body/tail association. In the body/tail association mode, the entire virion, including the tail(s), aligns to the host cell surface and the main body is greatly flattened, suggesting a possible fusion entry mechanism. Upon infection, the intracellular replication cycle lasts about 8 h, at which point the virions are released as spindle-shaped tailless particles. Replication of the virus retarded host growth but did not cause lysis of the host cells. Once released from the host and at temperatures resembling that of its natural habitat, SMV1 starts developing one or two tails. This exceptional property of undergoing a major morphological development outside, and independently of, the host cell has been reported only once before for the related Acidianus two-tailed virus. Here, we show that SMV1 can develop tails of more than 900 nm in length, more than quadrupling the total virion length. Very little is known about the initial life cycle stages of viruses infecting hosts of the third domain of life, Archaea This work describes the first example of an archaeal virus employing three distinct association modes. The virus under study, Sulfolobus monocaudavirus 1, is a representative of the large spindle-shaped viruses that are frequently found in acidic hot springs. The results described here will add valuable knowledge about Archaea, the least studied domain in the virology field. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Synthesis and Biological Evaluation of 3-Benzylidene-4-chromanone Derivatives as Free Radical Scavengers and α-Glucosidase Inhibitors.

Science.gov (United States)

Takao, Koichi; Yamashita, Marimo; Yashiro, Aruki; Sugita, Yoshiaki

2016-01-01

A series of 3-benzylidene-4-chromanone derivatives (3-20) were synthesized and the structure-activity relationships for antioxidant and α-glucosidase inhibitory activities were evaluated. Among synthesized compounds, compounds 5, 13, 18, which contain catechol moiety, showed the potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity (5: EC50 13 µM; 13: EC50 14 µM; 18: EC50 13 µM). The compounds 12, 14, 18 showed higher α-glucosidase inhibitory activity (12: IC50 15 µM; 14: IC50 25 µM; 18: IC50 28 µM). The compound 18 showed both of potent DPPH radical scavenging and α-glucosidase inhibitory activities. These data suggest that 3-benzylidene-4-chromanone derivatives, such as compound 18, may serve as the lead compound for the development of novel α-glucosidase inhibitors with antioxidant activity.

Aqueous extracts of Roselle (Hibiscus sabdariffa Linn.) varieties inhibit α-amylase and α-glucosidase activities in vitro.

Science.gov (United States)

Ademiluyi, Adedayo O; Oboh, Ganiyu

2013-01-01

This study sought to investigate the inhibitory effect of aqueous extracts of two varieties (red and white) of Hibiscus sabdariffa (Roselle) calyces on carbohydrate hydrolyzing enzymes (α-amylase and α-glucosidase), with the aim of providing the possible mechanism for their antidiabetes properties. Aqueous extracts were prepared (1:100 w/v) and the supernatant used for the analysis. The extracts caused inhibition of α-amylase and α-glucosidase activities in vitro.The IC(50) revealed that the red variety (25.2 μg/mL) exhibited higher α-glucosidase inhibitory activity than the white variety (47.4 μg/mL), while the white variety (90.5 μg/mL) exhibited higher α-amylase inhibitory activity than the red variety (187.9 μg/mL). However, the α-glucosidase inhibitory activities of both calyces were higher than that of their α-amylase. In addition, the red variety possessed higher antioxidant capacity as exemplified by the (•)OH scavenging abilities, Fe(2+) chelating ability, and inhibition of Fe(2+)-induced pancreatic lipid peroxidation in vitro. The enzyme inhibitory activities and antioxidant properties of the roselle extracts agreed with their phenolic content. Hence, inhibition of α-amylase and α-glucosidase, coupled with strong antioxidant properties could be the possible underlying mechanism for the antidiabetes properties of H. sabdariffa calyces; however, the red variety appeared to be more potent.

Inhibition of protein tyrosine phosphatase (PTP1B) and α-glucosidase by geranylated flavonoids from Paulownia tomentosa.

Science.gov (United States)

Song, Yeong Hun; Uddin, Zia; Jin, Young Min; Li, Zuopeng; Curtis-Long, Marcus John; Kim, Kwang Dong; Cho, Jung Keun; Park, Ki Hun

2017-12-01

Protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase are important targets to treat obesity and diabetes, due to their deep correlation with insulin and leptin signalling, and glucose regulation. The methanol extract of Paulownia tomentosa fruits showed potent inhibition against both enzymes. Purification of this extract led to eight geranylated flavonoids (1-8) displaying dual inhibition of PTP1B and α-glucosidase. The isolated compounds were identified as flavanones (1-5) and dihydroflavonols (6-8). Inhibitory potencies of these compounds varied accordingly, but most of the compounds were highly effective against PTP1B (IC 50  = 1.9-8.2 μM) than α-glucosidase (IC 50  = 2.2-78.9 μM). Mimulone (1) was the most effective against PTP1B with IC 50  = 1.9 μM, whereas 6-geranyl-3,3',5,5',7-pentahydroxy-4'-methoxyflavane (8) displayed potent inhibition against α-glucosidase (IC 50  = 2.2 μM). All inhibitors showed mixed type Ι inhibition toward PTP1B, and were noncompetitive inhibitors of α-glucosidase. This mixed type behavior against PTP1B was fully demonstrated by showing a decrease in V max , an increase of K m , and K ik /K iv ratio ranging between 2.66 and 3.69.

Structural basis for cyclophellitol inhibition of a β-glucosidase

DEFF Research Database (Denmark)

Gloster, Tracey M.; Madsen, Robert; Davies, Gideon J.

2007-01-01

The structural basis for b-glucosidase inhibition by cyclophellitol is demonstrated using X-ray crystallography, enzyme kinetics and mass spectrometry. The natural product was shown to bind by a covalent bond in the active site of the enzyme. This bond is formed by ring-opening of the epoxide...

Molecular cloning and characterization of the α-glucosidase II from Bombyx mori and Spodoptera frugiperda.

Science.gov (United States)

Watanabe, Satoko; Kakudo, Akemi; Ohta, Masato; Mita, Kazuei; Fujiyama, Kazuhito; Inumaru, Shigeki

2013-04-01

The α-glucosidase II (GII) is a heterodimer of α- and β-subunits and important for N-glycosylation processing and quality control of nascent glycoproteins. Although high concentration of α-glucosidase inhibitors from mulberry leaves accumulate in silkworms (Bombyx mori) by feeding, silkworm does not show any toxic symptom against these inhibitors and N-glycosylation of recombinant proteins is not affected. We, therefore, hypothesized that silkworm GII is not sensitive to the α-glucosidase inhibitors from mulberry leaves. However, the genes for B. mori GII subunits have not yet been identified, and the protein has not been characterized. Therefore, we isolated the B. mori GII α- and β-subunit genes and the GII α-subunit gene of Spodoptera frugiperda, which does not feed on mulberry leaves. We used a baculovirus expression system to produce the recombinant GII subunits and identified their enzyme characteristics. The recombinant GII α-subunits of B. mori and S. frugiperda hydrolyzed p-nitrophenyl α-d-glucopyranoside (pNP-αGlc) but were inactive toward N-glycan. Although the B. mori GII β-subunit was not required for the hydrolysis of pNP-αGlc, a B. mori GII complex of the α- and β-subunits was required for N-glycan cleavage. As hypothesized, the B. mori GII α-subunit protein was less sensitive to α-glucosidase inhibitors than was the S. frugiperda GII α-subunit protein. Our observations suggest that the low sensitivity of GII contributes to the ability of B. mori to evade the toxic effect of α-glucosidase inhibitors from mulberry leaves. Copyright © 2013 Elsevier Ltd. All rights reserved.

In Vitro α-Glucosidase Inhibitory Activity of Ethanol Extract of Buas-buas (Premna serratifolia Linn

Directory of Open Access Journals (Sweden)

Dini Hadiarti

2017-08-01

Full Text Available In 2008, diabetics in Indonesia has reached 8,5 million of 11,1 % prevalence in West Kalimantan. It was estimated to reach 14,1 million in 2035. The treatment of diabetes may occur adverse reactions such as hypoglycemia, lipoatrophy, lipohypertrophy, lactic acidosis, gastrointestinal disturbances, allergic reactions, and obesity. Therefore, it is necessary to find an alternative medicine to overcome this problem. Buas-buas (Premna serratifolia Linn could supposedly be an anti diabetic by inhibiting the α-glucosidase enzyme, due to the compositions of secondary metabolites, cardioprotective activity and it has a similar genus with Premna serratifolia Linn. The soxhlet extraction result from Premna serrtifolia Linn leaf powders produced of 34,1 % yield. Meanwhile, the activity of α-glucosidase in vitroinhibition test with amicroplate reader in various concentrations of samples of 0,125, 0,5, 1, 1,5, 2, dan 2,5 2% (b/v were obtained the absorptions of 37,95, 74,77, 86,15, 91,03 and 91,69 %, respectively. The extraction of Premna serratifolia Linn leaf revealed that the concentration of 2 % of sample inhibited in vitro α-glucosidase with a percentage of 91,03 %. The extraction of Premna serratifolia Linn leafinhibited α-glucosidase of 97 % from the percentage of Acarboseinhibition.

Australine, a pyrrolizidine alkaloid that inhibits amyloglucosidase and glycoprotein processing

Energy Technology Data Exchange (ETDEWEB)

Tropea, J.E.; Molyneux, R.J.; Kaushal, G.P.; Pan, Y.T.; Mitchell, M.; Elbein, A.D. (Univ. of Texas Health Science Center, San Antonio (USA))

1989-03-07

Australine is a polyhydroxylated pyrrolizidine alkaloid that was isolated from the seeds of the Australian tree Castanospermum australe and characterized by NMR and X-ray diffraction analysis. Since swainsonine and catanospermine are polyhydroxylated indolizidine alkaloids that inhibit specific glycosidases, the authors tested australine against a variety of exoglycosidases to determine whether it would inhibit any of these enzymes. This alkaloid proved to be a good inhibitor of the {alpha}-glucosidase amyloglucosidase (50% inhibition at 5.8 {mu}M), but it did not inhibit {beta}-glucosidase, {alpha}- or {beta}-mannosidase, or {alpha}- or {beta}-galactosidase. The inhibition of amyloglucosidase was of a competitive nature. Australine also inhibited the glycoprotein processing enzyme glucosidase I, but had only slight activity toward glucosidase II. When incubated with cultured cells, this alkaloid inhibited glycoprotein processing at the glucosidase I step and caused the accumulation of glycoproteins with Glc{sub 3}Man{sub 7-9}(GlcNAc){sub 2}-oligosaccharides.

Lanostane triterpenes from the mushroom Ganoderma resinaceum and their inhibitory activities against α-glucosidase.

Science.gov (United States)

Chen, Xian-Qiang; Zhao, Jing; Chen, Ling-Xiao; Wang, Shen-Fei; Wang, Ying; Li, Shao-Ping

2018-05-01

Eighteen previously undescribed lanostane triterpenes and thirty known analogues were obtained from the fruiting bodies of Ganoderma resinaceum. Resinacein C was isolated from a natural source for the first time. The structures of all the above compounds were elucidated by extensive spectroscopic analysis and comparisons of their spectroscopic data with those reported in the literature. Furthermore, in an in vitro assay, Resinacein C, ganoderic acid Y, lucialdehyde C, 7-oxo-ganoderic acid Z 3 , 7-oxo-ganoderic acid Z, and lucidadiol showed strong inhibitory effects against α-glucosidase compared with the positive control drug acarbose. The structure-activity relationships of ganoderma triterpenes on α-glucosidase inhibition showed that the C-24/C-25 double bond is necessary for α-glucosidase inhibitory activity. Moreover, the carboxylic acid group at C-26 and the hydroxy group at C-15 play important roles in enhancing inhibitory effects of these triterpenes. Copyright © 2018. Published by Elsevier Ltd.

Cellulolytic and xylanolytic potential of high β-glucosidase-producing Trichoderma from decaying biomass.

Science.gov (United States)

Okeke, Benedict C

2014-10-01

Availability, cost, and efficiency of microbial enzymes for lignocellulose bioconversion are central to sustainable biomass ethanol technology. Fungi enriched from decaying biomass and surface soil mixture displayed an array of strong cellulolytic and xylanolytic activities. Strains SG2 and SG4 produced a promising array of cellulolytic and xylanolytic enzymes including β-glucosidase, usually low in cultures of Trichoderma species. Nucleotide sequence analysis of internal transcribed spacer 2 (ITS2) region of rRNA gene revealed that strains SG2 and SG4 are closely related to Trichoderma inhamatum, Trichoderma piluliferum, and Trichoderma aureoviride. Trichoderma sp. SG2 crude culture supernatant correspondingly displayed as much as 9.84 ± 1.12, 48.02 ± 2.53, and 30.10 ± 1.11 units mL(-1) of cellulase, xylanase, and β-glucosidase in 30 min assay. Ten times dilution of culture supernatant of strain SG2 revealed that total activities were about 5.34, 8.45, and 2.05 orders of magnitude higher than observed in crude culture filtrate for cellulase, xylanase, and β-glucosidase, respectively, indicating that more enzymes are present to contact with substrates in biomass saccharification. In parallel experiments, Trichoderma species SG2 and SG4 produced more β-glucosidase than the industrial strain Trichoderma reesei RUT-C30. Results indicate that strains SG2 and SG4 have potential for low cost in-house production of primary lignocellulose-hydrolyzing enzymes for production of biomass saccharides and biofuel in the field.

High-resolution α-glucosidase inhibition profiling combined with HPLC-HRMS-SPE-NMR for identification of anti-diabetic compounds in Eremanthus crotonoides (Asteraceae)

DEFF Research Database (Denmark)

Lana e Silva, Eder; Felipe Revoredo Lobo, Jonathas; Vinther, Joachim Møllesøe

2016-01-01

with an inhibitory concentration (IC50) of 34.5 μg/mL towards α-glucosidase was investigated by high-resolution α-glucosidase inhibition profiling combined with HPLC-HRMS-SPE-NMR. This led to identification of six α-glucosidase inhibitors, namely quercetin (16), trans-tiliroside (17), luteolin (19), quercetin-3.......93 and 5.20 μM, respectively. This is the first report of the α-glucosidase inhibitory activity of compounds 20, 26, and 29, and the findings support the important role of Eremanthus species as novel sources of new drugs and/or herbal remedies for treatment of type 2 diabetes....

Photobiosynthesis of stable and functional silver/silver chloride nanoparticles with hydrolytic activity using hyperthermophilic β-glucosidases with industrial potential.

Science.gov (United States)

Araújo, Juscemácia N; Tofanello, Aryane; da Silva, Viviam M; Sato, Juliana A P; Squina, Fabio M; Nantes, Iseli L; Garcia, Wanius

2017-09-01

The β-glucosidases are important enzymes employed in a large number of processes and industrial applications, including biofuel production from biomass. Therefore, in this study, we reported for the first time the photobiosynthesis of stable and functional silver/silver chloride nanoparticles (Ag/AgCl-NPs) using two hyperthermostable bacterial β-glucosidases with industrial potential. The syntheses were straightforward and rapid processes carried out by mixing β-glucosidase and silver nitrate (in buffer 10mM Tris-HCl, pH 8) under irradiation with light (over a wavelength range of 450-600nm), therefore, compatible with the green chemistry procedure. Synthesized Ag/AgCl-NPs were characterized using a series of physical techniques. Absorption spectroscopy showed a strong absorption band centered at 460nm due to surface plasmon resonance of the Ag-NPs. X-ray diffraction analysis revealed that the Ag/AgCl-NPs were purely crystalline in nature. Under electron microscopy, Ag/AgCl-NPs of variable diameter ranging from 10 to 100nm can be visualized. Furthermore, electron microscopy, zeta potential and Fourier transform infrared spectroscopy results confirmed the presence of β-glucosidases coating and stabilizing the Ag/AgCl-NPs. Finally, the results showed that the enzymatic activities were maintained in the β-glucosidases assisted Ag/AgCl-NPs. The information described here should provide a useful basis for future studies of β-glucosidases assisted Ag/AgCl-NPs, including biotechnological applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Synthesis of Sulochrin-125I and Its Binding Affinity as α-Glucosidase Inhibitor using Radioligand Binding Assay (RBA Method

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W. Lestari

2014-04-01

Full Text Available Most of diabetics patients have type 2 diabetes mellitus or non insulin dependent diabetes mellitus. Treatment type 2 diabetes mellitus can be done by inhibiting α-glucosidase enzyme which converts carbohydrates into glucose. Sulochrin is one of the potential compounds which can inhibit the function of α-glucosidase enzyme. This study was carried out to obtain data of sulochrin binding with α-glucosidase enzyme as α-glucosidase inhibitor using Radioligand Binding Assay (RBA method. Primary reagent required in RBA method is labeled radioactive ligand (radioligand. In this study, the radioligand was sulochrin-125I and prior to sulochrin-125I synthesis, the sulochrin-I was synthesized. Sulochrin-I and sulochrin-125I were synthesized and their bindings were studied using Radioligand Binding Assay method. Sulochrin-I was synthesized with molecular formula C17H15O7I and molecular weight 457.9940. Sulochrin-125I was synthesized from sulochrin-I by isotope exchange method. From the RBA method, dissociation constant (Kd and maximum binding (Bmax were obtained 26.316 nM and Bmax 9.302 nM respectively. This low Kd indicated that sulochrin was can bind to α-glucosidase

Every OGT Is Illuminated … by Fluorescent and Synchrotron Lights

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Riccardo Miggiano

2017-12-01

Full Text Available O6-DNA-alkyl-guanine-DNA-alkyl-transferases (OGTs are evolutionarily conserved, unique proteins that repair alkylation lesions in DNA in a single step reaction. Alkylating agents are environmental pollutants as well as by-products of cellular reactions, but are also very effective chemotherapeutic drugs. OGTs are major players in counteracting the effects of such agents, thus their action in turn affects genome integrity, survival of organisms under challenging conditions and response to chemotherapy. Numerous studies on OGTs from eukaryotes, bacteria and archaea have been reported, highlighting amazing features that make OGTs unique proteins in their reaction mechanism as well as post-reaction fate. This review reports recent functional and structural data on two prokaryotic OGTs, from the pathogenic bacterium Mycobacterium tuberculosis and the hyperthermophilic archaeon Sulfolobus solfataricus, respectively. These studies provided insight in the role of OGTs in the biology of these microorganisms, but also important hints useful to understand the general properties of this class of proteins.

Analysis of the crystal structure of an active MCM hexamer.

Science.gov (United States)

Miller, Justin M; Arachea, Buenafe T; Epling, Leslie B; Enemark, Eric J

2014-09-29

In a previous Research article (Froelich et al., 2014), we suggested an MCM helicase activation mechanism, but were limited in discussing the ATPase domain because it was absent from the crystal structure. Here we present the crystal structure of a nearly full-length MCM hexamer that is helicase-active and thus has all features essential for unwinding DNA. The structure is a chimera of Sulfolobus solfataricus N-terminal domain and Pyrococcus furiosus ATPase domain. We discuss three major findings: 1) a novel conformation for the A-subdomain that could play a role in MCM regulation; 2) interaction of a universally conserved glutamine in the N-terminal Allosteric Communication Loop with the AAA+ domain helix-2-insert (h2i); and 3) a recessed binding pocket for the MCM ssDNA-binding motif influenced by the h2i. We suggest that during helicase activation, the h2i clamps down on the leading strand to facilitate strand retention and regulate ATP hydrolysis.

Monitoring Acidophilic Microbes with Real-Time Polymerase Chain Reaction (PCR) Assays

Energy Technology Data Exchange (ETDEWEB)

Frank F. Roberto

2008-08-01

Many techniques that are used to characterize and monitor microbial populations associated with sulfide mineral bioleaching require the cultivation of the organisms on solid or liquid media. Chemolithotrophic species, such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, or thermophilic chemolithotrophs, such as Acidianus brierleyi and Sulfolobus solfataricus can grow quite slowly, requiring weeks to complete efforts to identify and quantify these microbes associated with bioleach samples. Real-time PCR (polymerase chain reaction) assays in which DNA targets are amplified in the presence of fluorescent oligonucleotide primers, allowing the monitoring and quantification of the amplification reactions as they progress, provide a means of rapidly detecting the presence of microbial species of interest, and their relative abundance in a sample. This presentation will describe the design and use of such assays to monitor acidophilic microbes in the environment and in bioleaching operations. These assays provide results within 2-3 hours, and can detect less than 100 individual microbial cells.

Prespacer processing and specific integration in a Type I-A CRISPR system

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Rollie, Clare; Graham, Shirley; Rouillon, Christophe

2018-01-01

Abstract The CRISPR–Cas system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. Adaptation is dependent on the Cas1 and Cas2 proteins along with varying accessory proteins. Here we analyse the process in Sulfolobus solfataricus, showing that while Cas1 and Cas2 catalyze spacer integration in vitro, host factors are required for specificity. Specific integration also requires at least 400 bp of the leader sequence, and is dependent on the presence of hydrolysable ATP, suggestive of an active process that may involve DNA remodelling. Specific spacer integration is associated with processing of prespacer 3′ ends in a PAM-dependent manner. This is reflected in PAM-dependent processing of prespacer 3′ ends in vitro in the presence of cell lysate or the Cas4 nuclease, in a reaction consistent with PAM-directed binding and protection of prespacer DNA. These results highlight the diverse interplay between CRISPR–Cas elements and host proteins across CRISPR types. PMID:29228332

Magnetic ligand fishing as a targeting tool for HPLC-HRMS-SPE-NMR: α-glucosidase inhibitory ligands and alkylresorcinol glycosides from Eugenia catharinae

DEFF Research Database (Denmark)

Wubshet, Sileshi Gizachew; Brighente, Inês M. C.; Moaddel, Ruin

2015-01-01

A bioanalytical platform combining magnetic ligand fishing for α-glucosidase inhibition profiling and HPLC-HRMS-SPE-NMR for structural identification of α-glucosidase inhibitory ligands, both directly from crude plant extracts, is presented. Magnetic beads with N-terminus-coupled α-glucosidase we...

Polypeptides having beta-glucosidase activity and polynucleotides encoding the same

Science.gov (United States)

Brown, Kimberly; Harris, Paul

2013-12-17

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Combined use of high-resolution α-glucosidase inhibition profiling and HPLC-HRMS-SPE-NMR for investigation of antidiabetic principles in crude plant extracts

DEFF Research Database (Denmark)

Kongstad, Kenneth Thermann; Özdemir, Ceylan; Barzak, Asmah

2015-01-01

Type 2 diabetes is a metabolic disorder affecting millions of people worldwide, and new drug leads or functional foods containing selective α-glucosidase inhibitors are needed. Crude extract of 24 plants were assessed for α-glucosidase inhibitory activity. Methanol extracts of Cinnamomum zeylanicum...... bark, Rheum rhabarbarum peel, and Rheum palmatum root and ethyl acetate extracts of C. zeylanicum bark, Allium ascalonicum peel, and R. palmatum root showed IC50 values below 20 μg/mL. Subsequently, high-resolution α-glucosidase profiling was used in combination with high-performance liquid...... chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy for identification of metabolites responsible for the α-glucosidase inhibitory activity. Quercetin (1) and its dimer (2), trimer (3), and tetramer (4) were identified as main α-glucosidase inhibitors...

α-Glucosidase inhibition by flavonoids: an in vitro and in silico structure-activity relationship study.

Science.gov (United States)

Proença, Carina; Freitas, Marisa; Ribeiro, Daniela; Oliveira, Eduardo F T; Sousa, Joana L C; Tomé, Sara M; Ramos, Maria J; Silva, Artur M S; Fernandes, Pedro A; Fernandes, Eduarda

2017-12-01

α-Glucosidase inhibitors are described as the most effective in reducing post-prandial hyperglycaemia (PPHG) from all available anti-diabetic drugs used in the management of type 2 diabetes mellitus. As flavonoids are promising modulators of this enzyme's activity, a panel of 44 flavonoids, organised in five groups, was screened for their inhibitory activity of α-glucosidase, based on in vitro structure-activity relationship studies. Inhibitory kinetic analysis and molecular docking calculations were also applied for selected compounds. A flavonoid with two catechol groups in A- and B-rings, together with a 3-OH group at C-ring, was the most active, presenting an IC 50 much lower than the one found for the most widely prescribed α-glucosidase inhibitor, acarbose. The present work suggests that several of the studied flavonoids have the potential to be used as alternatives for the regulation of PPHG.

Effect of O-methylated and glucuronosylated flavonoids from Tamarix gallica on α-glucosidase inhibitory activity: structure-activity relationship and synergistic potential.

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Ben Hmidene, Asma; Smaoui, Abderrazak; Abdelly, Chedly; Isoda, Hiroko; Shigemori, Hideyuki

2017-03-01

O-Methylated and glucuronosylated flavonoids were isolated from Tamarix gallica as α-glucosidase inhibitors. Structure-activity relationship of these flavonoids suggests that catechol moiety and glucuronic acid at C-3 are factors in the increase in α-glucosidase inhibitory activity. Furthermore, rhamnetin, tamarixetin, rhamnazin, KGlcA, KGlcA-Me, QGlcA, and QGlcA-Me exhibit synergistic potential when applied with a very low concentration of acarbose to α-glucosidase from rat intestine.

Structural and functional insights of β-glucosidases identified from the genome of Aspergillus fumigatus

Science.gov (United States)

Dodda, Subba Reddy; Aich, Aparajita; Sarkar, Nibedita; Jain, Piyush; Jain, Sneha; Mondal, Sudipa; Aikat, Kaustav; Mukhopadhyay, Sudit S.

2018-03-01

Thermostable glucose tolerant β-glucosidase from Aspergillus species has attracted worldwide interest for their potentiality in industrial applications and bioethanol production. A strain of Aspergillus fumigatus (AfNITDGPKA3) identified by our laboratory from straw retting ground showed higher cellulase activity, specifically the β-glucosidase activity, compared to other contemporary strains. Though A. fumigatus has been known for high cellulase activity, detailed identification and characterization of the cellulase genes from their genome is yet to be done. In this work we have been analyzed the cellulase genes from the genome sequence database of Aspergillus fumigatus (Af293). Genome analysis suggests two cellobiohydrolase, eleven endoglucanase and seventeen β-glucosidase genes present. β-Glucosidase genes belong to either Glycohydro1 (GH1 or Bgl1) or Glycohydro3 (GH3 or Bgl3) family. The sequence similarity suggests that Bgl1 and Bgl3 of A. fumagatus are phylogenetically close to those of A. fisheri and A. oryzae. The modelled structure of the Bgl1 predicts the (β/α)8 barrel type structure with deep and narrow active site, whereas, Bgl3 shows the (α/β)8 barrel and (α/β)6 sandwich structure with shallow and open active site. Docking results suggest that amino acids Glu544, Glu466, Trp408,Trp567,Tyr44,Tyr222,Tyr770,Asp844,Asp537,Asn212,Asn217 of Bgl3 and Asp224,Asn242,Glu440, Glu445, Tyr367, Tyr365,Thr994,Trp435,Trp446 of Bgl1 are involved in the hydrolysis. Binding affinity analyses suggest that Bgl3 and Bgl1 enzymes are more active on the substrates like 4-methylumbelliferyl glycoside (MUG) and p-nitrophenyl-β-D-1, 4-glucopyranoside (pNPG) than on cellobiose. Further docking with glucose suggests that Bgl1 is more glucose tolerant than Bgl3. Analysis of the Aspergillus fumigatus genome may help to identify a β-glucosidase enzyme with better property and the structural information may help to develop an engineered recombinant enzyme.

Functional characterization of the origin of replication of pRN1 from Sulfolobus islandicus REN1H1.

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Chijioke J Joshua

Full Text Available Plasmid pRN1 from Sulfolobus islandicus REN1H1 is believed to replicate by a rolling circle mechanism but its origin and mechanism of replication are not well understood. We sought to create minimal expression vectors based on pRN1 that would be useful for heterologous gene expression in S. acidocaldarius, and in the process improve our understanding of the mechanism of replication. We constructed and transformed shuttle vectors that harbored different contiguous stretches of DNA from pRN1 into S. acidocaldarius E4-39, a uracil auxotroph. A 232-bp region 3' of orf904 was found to be critical for pRN1 replication and is therefore proposed to be the putative origin of replication. This 232-bp region contains a 100-bp stem-loop structure believed to be the double-strand origin of replication. The loop of the 100-bp structure contains a GTG tri-nucleotide motif, a feature that was previously reported to be important for the primase activity of Orf904. This putative origin and the associated orf56 and orf904 were identified as the minimal replicon of pRN1 because transformants of plasmids lacking any of these three features were not recovered. Plasmids lacking orf904 and orf56 but harboring the putative origin were transformable when orf904 and orf56 were provided in-trans; a 75-bp region 5' of the orf904 start codon was found to be essential for this complementation. Detailed knowledge of the pRN1 origin of replication will broaden the application of the plasmid as a genetic tool for Sulfolobus species.

Deletion of the topoisomerase III gene in the hyperthermophilic archaeon Sulfolobus islandicus results in slow growth and defects in cell cycle control

DEFF Research Database (Denmark)

Li, Xiyang; Guo, Li; Deng, Ling

2011-01-01

Topoisomerase III (topo III), a type IA topoisomerase, is widespread in hyperthermophilic archaea. In order to interrogate the in vivo role of archaeal topo III, we constructed and characterized a topo III gene deletion mutant of Sulfolobus islandicus. The mutant was viable but grew more slowly...... results suggest that the enzyme may serve roles in chromosomal segregation and control of the level of supercoiling in the cell....

Heat inactivation kinetics of Hypocrea orientalis β-glucosidase with enhanced thermal stability by glucose.

Science.gov (United States)

Xu, Xin-Qi; Shi, Yan; Wu, Xiao-Bing; Zhan, Xi-Lan; Zhou, Han-Tao; Chen, Qing-Xi

2015-11-01

Thermal inactivation kinetics of Hypocrea orientalis β-glucosidase and effect of glucose on thermostability of the enzyme have been determined in this paper. Kinetic studies showed that the thermal inactivation was irreversible and first-order reaction. The microscopic rate constants for inactivation of free enzyme and substrate-enzyme complex were both determined, which suggested that substrates can protect β-glucosidase against thermal deactivation effectively. On the other hand, glucose was found to protect β-glucosidase from heat inactivation to remain almost whole activity below 70°C at 20mM concentration, whereas the apparent inactivation rate of BG decreased to be 0.3×10(-3)s(-1) in the presence of 5mM glucose, smaller than that of sugar-free enzyme (1.91×10(-3)s(-1)). The intrinsic fluorescence spectra results showed that glucose also had stabilizing effect on the conformation of BG against thermal denaturation. Docking simulation depicted the interaction mode between glucose and active residues of the enzyme to produce stabilizing effect. Copyright © 2015 Elsevier B.V. All rights reserved.

β-d-Glucosidase as "key enzyme" for sorghum cyanogenic glucoside (dhurrin) removal and beer bioflavouring.

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Tokpohozin, Sedjro Emile; Fischer, Susann; Sacher, Bertram; Becker, Thomas

2016-11-01

Sorghum malt used during African beer processing contains a high level of cyanogenic glucoside (dhurrin), up to 1375 ppm. In traditional sorghum malting and mashing, dhurrin is not sufficiently hydrolyzed due to uncontrolled germination and a high gelatinization temperature. The cyanide content of traditional African beers (11 ppm) is higher than the minimum dose (1 ppm) required to form carcinogenic ethyl carbamate during alcoholic fermentation. In the detoxification process, aryl-β-d-glucosidase (dhurrinase) is the "key component". For significant dhurrin hydrolysis during mashing, optimizing dhurrinase synthesis during malting is a good solution to reduce dhurrin completely to below the harmful dose in the sorghum wort. Lactic acid bacteria which exhibit aryl-β-d-glucosidase prior to alcoholic fermentation may help to reduce ethyl carbamate content in alcoholic beverages. Moreover, some specific β-d-glucosidases have a dual property, being able to cleave and synthesize glucosides bonds and thereby generating good precursors for beer bioflavouring. Copyright © 2016 Elsevier Ltd. All rights reserved.

Transglycosylation properties of maltodextrin glucosidase (MalZ) from Escherichia coli and its application for synthesis of a nigerose-containing oligosaccharide

Energy Technology Data Exchange (ETDEWEB)

Song, Kyung-Mo [Center for Agricultural Biomaterials and Department of Food Science and Biotechnology, Seoul National University, Seoul 151-921 (Korea, Republic of); Shim, Jae-Hoon [Department of Biology, University of Incheon, Incheon 406-772 (Korea, Republic of); Park, Jong-Tae; Kim, Sung-Hee [Center for Agricultural Biomaterials and Department of Food Science and Biotechnology, Seoul National University, Seoul 151-921 (Korea, Republic of); Kim, Young-Wan [Department of Food and Biotechnology, Korea University, Jochiwon 339-700 (Korea, Republic of); Boos, Winfried [Department of Biology, University of Konstanz, Konstanz 78457 (Germany); Park, Kwan-Hwa, E-mail: parkkh@incheon.ac.kr [Department of Biology, University of Incheon, Incheon 406-772 (Korea, Republic of)

2010-06-18

The transglycosylation reaction of maltodextrin glucosidase (MalZ) cloned and purified from Escherichia coli K12 was characterized and applied to the synthesis of branched oligosaccharides. Purified MalZ preferentially catalyzed the hydrolysis of maltodextrin, {gamma}-cyclodextrin (CD), and cycloamylose (CA). In addition, when the enzyme was incubated with 5% maltotriose (G3), a series of transfer products were produced. The resulting major transfer products, annotated as T1, T2, and T3, were purified and their structures were determined by TLC, MALDI-TOF/MS, {sup 13}C NMR, and enzymatic analysis. T1 was identified as a novel compound, maltosyl {alpha}-1,3-maltose, whereas T2 and T3 were determined to be isopanose and maltosyl-{alpha}-1,6-maltose, respectively. These results indicated that MalZ transferred sugar moiety mainly to C-3 or C-6-OH of glucose of the acceptor molecule. To obtain highly concentrated transfer products, the enzyme was reacted with 10% liquefied cornstarch, and then glucose and maltose were removed by immobilized yeast. The T1 content of the resulting reaction mixture reached 9.0%. The mixture of T1 containing a nigerose moiety can have an immunopotentiating effect on the human body and may be a potential functional sugar stuff.

Screening of β-Glucosidase and β-Xylosidase Activities in Four Non-Saccharomyces Yeast Isolates.

Science.gov (United States)

López, María Consuelo; Mateo, José Juan; Maicas, Sergi

2015-08-01

The finding of new isolates of non-Saccharomyces yeasts, showing beneficial enzymes (such as β-glucosidase and β-xylosidase), can contribute to the production of quality wines. In a selection and characterization program, we have studied 114 isolates of non-Saccharomyces yeasts. Four isolates were selected because of their both high β-glucosidase and β-xylosidase activities. The ribosomal D1/D2 regions were sequenced to identify them as Pichia membranifaciens Pm7, Hanseniaspora vineae Hv3, H. uvarum Hu8, and Wickerhamomyces anomalus Wa1. The induction process was optimized to be carried on YNB-medium supplemented with 4% xylan, inoculated with 106 cfu/mL and incubated 48 h at 28 °C without agitation. Most of the strains had a pH optimum of 5.0 to 6.0 for both the β-glucosidase and β-xylosidase activities. The effect of sugars was different for each isolate and activity. Each isolate showed a characteristic set of inhibition, enhancement or null effect for β-glucosidase and β-xylosidase. The volatile compounds liberated from wine incubated with each of the 4 yeasts were also studied, showing an overall terpene increase (1.1 to 1.3-folds) when wines were treated with non-Saccharomyces isolates. In detail, terpineol, 4-vinyl-phenol and 2-methoxy-4-vinylphenol increased after the addition of Hanseniaspora isolates. Wines treated with Hanseniaspora, Wickerhamomyces, or Pichia produced more 2-phenyl ethanol than those inoculated with other yeasts. © 2015 Institute of Food Technologists®

Purification and characterization of a beta-glucosidase from the root parasitic plant Orobanche minor Sm.

Science.gov (United States)

Sasanuma, Izumi; Hirakawa, Go

2010-01-01

The beta-glucosidase of a root parasitic angiosperm, Orobanche minor Sm., was purified and characterized. The optimum pH and temperature for activity of the enzyme were 5.0 and 50 degrees C. The beta-glucosidase was stable at up to 50 degrees C at pH 4.0-10.0. The M(r) was estimated to be 33 kD by SDS-PAGE. The enzyme hydrolyzed p-nitrophenyl-beta-D-glucopyranoside and salicin, but not the cell wall of O. minor or cellohexaose.

Modulation of CRISPR locus transcription by the repeat-binding protein Cbp1 in Sulfolobus

DEFF Research Database (Denmark)

Deng, Ling; Kenchappa, Chandra Shekar; Peng, Xu

2012-01-01

CRISPR loci are essential components of the adaptive immune system of archaea and bacteria. They consist of long arrays of repeats separated by DNA spacers encoding guide RNAs (crRNA), which target foreign genetic elements. Cbp1 (CRISPR DNA repeat binding protein) binds specifically to the multiple...... direct repeats of CRISPR loci of members of the acidothermophilic, crenarchaeal order Sulfolobales. cbp1 gene deletion from Sulfolobus islandicus REY15A produced a strong reduction in pre-crRNA yields from CRISPR loci but did not inhibit the foreign DNA targeting capacity of the CRISPR/Cas system....... Conversely, overexpression of Cbp1 in S. islandicus generated an increase in pre-crRNA yields while the level of reverse strand transcripts from CRISPR loci remained unchanged. It is proposed that Cbp1 modulates production of longer pre-crRNA transcripts from CRISPR loci. A possible mechanism...

BGL6 beta-glucosidase and nucleic acids encoding the same

Science.gov (United States)

Dunn-Coleman, Nigel [Los Gatos, CA; Ward, Michael [San Francisco, CA

2009-09-01

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

A defence-related Olea europaea β-glucosidase hydrolyses and activates oleuropein into a potent protein cross-linking agent.

Science.gov (United States)

Koudounas, Konstantinos; Banilas, Georgios; Michaelidis, Christos; Demoliou, Catherine; Rigas, Stamatis; Hatzopoulos, Polydefkis

2015-04-01

Oleuropein, the major secoiridoid compound in olive, is involved in a sophisticated two-component defence system comprising a β-glucosidase enzyme that activates oleuropein into a toxic glutaraldehyde-like structure. Although oleuropein deglycosylation studies have been monitored extensively, an oleuropein β-glucosidase gene has not been characterized as yet. Here, we report the isolation of OeGLU cDNA from olive encoding a β-glucosidase belonging to the defence-related group of terpenoid-specific glucosidases. In planta recombinant protein expression assays showed that OeGLU deglycosylated and activated oleuropein into a strong protein cross-linker. Homology and docking modelling predicted that OeGLU has a characteristic (β/α)8 TIM barrel conformation and a typical construction of a pocket-shaped substrate recognition domain composed of conserved amino acids supporting the β-glucosidase activity and non-conserved residues associated with aglycon specificity. Transcriptional analysis in various olive organs revealed that the gene was developmentally regulated, with its transcript levels coinciding well with the spatiotemporal patterns of oleuropein degradation and aglycon accumulation in drupes. OeGLU upregulation in young organs reflects its prominent role in oleuropein-mediated defence system. High gene expression during drupe maturation implies an additional role in olive secondary metabolism, through the degradation of oleuropein and reutilization of hydrolysis products. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Overexpression of an exotic thermotolerant β-glucosidase in trichoderma reesei and its significant increase in cellulolytic activity and saccharification of barley straw

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Dashtban Mehdi

2012-05-01

Full Text Available Abstract Background Trichoderma reesei is a widely used industrial strain for cellulase production, but its low yield of β-glucosidase has prevented its industrial value. In the hydrolysis process of cellulolytic residues by T. reesei, a disaccharide known as cellobiose is produced and accumulates, which inhibits further cellulases production. This problem can be solved by adding β-glucosidase, which hydrolyzes cellobiose to glucose for fermentation. It is, therefore, of high vvalue to construct T. reesei strains which can produce sufficient β-glucosidase and other hydrolytic enzymes, especially when those enzymes are capable of tolerating extreme conditions such as high temperature and acidic or alkali pH. Results We successfully engineered a thermostable β-glucosidase gene from the fungus Periconia sp. into the genome of T. reesei QM9414 strain. The engineered T. reesei strain showed about 10.5-fold (23.9 IU/mg higher β-glucosidase activity compared to the parent strain (2.2 IU/mg after 24 h of incubation. The transformants also showed very high total cellulase activity (about 39.0 FPU/mg at 24 h of incubation whereas the parent strain almost did not show any total cellulase activity at 24 h of incubation. The recombinant β-glucosidase showed to be thermotolerant and remains fully active after two-hour incubation at temperatures as high as 60°C. Additionally, it showed to be active at a wide pH range and maintains about 88% of its maximal activity after four-hour incubation at 25°C in a pH range from 3.0 to 9.0. Enzymatic hydrolysis assay using untreated, NaOH, or Organosolv pretreated barley straw as well as microcrystalline cellulose showed that the transformed T. reesei strains released more reducing sugars compared to the parental strain. Conclusions The recombinant T. reesei overexpressing Periconia sp. β-glucosidase in this study showed higher β-glucosidase and total cellulase activities within a shorter incubation

α-Glucosidase inhibitory hydrolyzable tannins from Eugenia jambolana seeds.

Science.gov (United States)

Omar, Raed; Li, Liya; Yuan, Tao; Seeram, Navindra P

2012-08-24

Three new hydrolyzable tannins including two gallotannins, jamutannins A (1) and B (2), and an ellagitannin, iso-oenothein C (3), along with eight known phenolic compounds were isolated from the seeds of Eugenia jambolana fruit. The structures were elucidated on the basis of spectroscopic data analysis. All compounds isolated were evaluated for α-glucosidase inhibitory effects compared to the clinical drug acarbose.

CRISPR associated diversity within a population of Sulfolobus islandicus.

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Nicole L Held

2010-09-01

Full Text Available Predator-prey models for virus-host interactions predict that viruses will cause oscillations of microbial host densities due to an arms race between resistance and virulence. A new form of microbial resistance, CRISPRs (clustered regularly interspaced short palindromic repeats are a rapidly evolving, sequence-specific immunity mechanism in which a short piece of invading viral DNA is inserted into the host's chromosome, thereby rendering the host resistant to further infection. Few studies have linked this form of resistance to population dynamics in natural microbial populations.We examined sequence diversity in 39 strains of the archeaon Sulfolobus islandicus from a single, isolated hot spring from Kamchatka, Russia to determine the effects of CRISPR immunity on microbial population dynamics. First, multiple housekeeping genetic markers identify a large clonal group of identical genotypes coexisting with a diverse set of rare genotypes. Second, the sequence-specific CRISPR spacer arrays split the large group of isolates into two very different groups and reveal extensive diversity and no evidence for dominance of a single clone within the population.The evenness of resistance genotypes found within this population of S. islandicus is indicative of a lack of strain dominance, in contrast to the prediction for a resistant strain in a simple predator-prey interaction. Based on evidence for the independent acquisition of resistant sequences, we hypothesize that CRISPR mediated clonal interference between resistant strains promotes and maintains diversity in this natural population.

Inhibitory effect of rhubarb on intestinal α-glucosidase activity in type ...

African Journals Online (AJOL)

insulin, and intestinal α-glucosidase were also determined. Results: ... As a serious and chronic metabolic disorder, diabetes ... increased levels of hemoglobin, fasting blood glucose ... Diabetic patients manifest impaired glucose ... improving insulin sensitivity and decreasing ... Guide, is the first-line therapy of postprandial.

Dgroup: DG01803 [KEGG MEDICUS

Lifescience Database Archive (English)

Full Text Available DG01803 DGroup Antidiabetic, alpha-glucosidase inhibitor -bose ... D00216 ... Acarbose (...e (USAN) D09779 ... Emiglitate (JAN/INN) Antidiabetic agent ... DG01663 ... alpha-Glucosidase inhibitor Unclassified ... DG02044 ... Hypoglycemics ATC code: A10BF Antidiabetics GAA [HSA:2548] [KO:K12316] GANC [HSA:2595] [KO:K12317] MGAM [HSA:8972] [KO:K12047] ...

Identification of newly isolated Talaromyces pinophilus and statistical optimization of β-glucosidase production under solid-state fermentation.

Science.gov (United States)

El-Naggar, Noura El-Ahmady; Haroun, S A; Oweis, Eman A; Sherief, A A

2015-01-01

Fungi able to degrade agriculture wastes were isolated from different soil samples, rice straw, and compost; these isolates were screened for their ability to produce β-glucosidase. The most active fungal isolate was identified as Talaromyces pinophilus strain EMOO 13-3. The Plackett-Burman design is used for identifying the significant variables that influence β-glucosidase production under solid-state fermentation. Fifteen variables were examined for their significances on the production of β-glucosidase in 20 experimental runs. Among the variables screened, moisture content, Tween 80, and (NH4)2SO4 had significant effects on β-glucosidase production with confidence levels above 90% (p fermentation conditions: substrate amount 0.5 (g/250 mL flask), NaNO3 0.5 (%), KH2PO4 0.3 (%), KCl 0.02 (%), MgSO4 · 7H2O 0.01 (%), CaCl2 0.01 (%), yeast extract 0.07 (%), FeSO4 · 7H2O 0.0002 (%), Tween 80 0.02 (%), (NH4)2SO4 0.3 (%), pH 6.5, temperature 25°C, moisture content 1 (mL/g dry substrate), inoculum size 0.5 (mL/g dry substrate), and incubation period 5 days.

Transcriptome changes in STSV2-infected Sulfolobus islandicus REY15A undergoing continuous CRISPR spacer acquisition

DEFF Research Database (Denmark)

León-Sobrino, Carlos; Kot, Witold P; Garrett, Roger A

2016-01-01

A transcriptome study was performed on Sulfolobus islandicus REY15A actively undergoing CRISPR spacer acquisition from the crenarchaeal monocaudavirus STSV2 in rich and basal media over a 6 day period. Spacer acquisition preceded strong host growth retardation, altered transcriptional activity...... of four different CRISPR-Cas modules and changes in viral copy numbers, and with significant differences in the two media. Transcript levels of proteins involved in the cell cycle were reduced, while those of DNA replication, DNA repair, transcriptional regulation, and some antitoxin-toxin pairs...... and transposases were unchanged or enhanced. Antisense RNAs were implicated in the transcriptional regulation of adaptation and interference modules of the type I-A CRISPR-Cas system and evidence was found for the occurrence of functional coordination between the single CRISPR-Cas adaptation module...

Total phenolic compounds, antioxidant potential and α-glucosidase inhibition by Tunisian Euphorbia paralias L.

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Malek Besbes Hlila

2016-08-01

Full Text Available Objective: To examine the potential antioxidant and anti-α-glucosidase inhibitory activities of Tunisian Euphorbia paralias L. leaves and stems extracts and their composition of total polyphenol and flavonoids. Methods: The different samples were tested for their antiradical activities by using 2, 2’- azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS and 1,1-diphenyl-2-picrylhydrazyl (DPPH assays. In α-glucosidase activity, α-glucosidase (0.3 IU/mL and substrate, 2500 µmol/ L p-nitrophenyl α-D-glucopyranoside were used; absorbance was registered at 405 nm. Results: The leaves acetonic extract exhibited the strongest α-glucosidase inhibition [IC50 = (0.0035 ± 0.001 µg/mL], which was 20-fold more active than the standard product (acarbose [IC50 = (0.07 ± 0.01 µg/mL]. Acetonic extract of the leaves exhibited the highest quantity of total phenolic [(95.54 ± 0.04 µg gallic acid equivalent/mg] and flavonoid [(55.16 ± 0.25 µg quercetin equivalent/mg]. The obtained findings presented also that this extract was detected with best antioxidant capacity [IC50 = (0.015 ± 0.01 µg/mL] against DPPH and a value of IC50 equal to (0.02 ± 0.01 µg/mL against ABTS. Positive relationship between polyphenolic content of the tested Euphorbia paralias L. leaves and stems extracts and its antioxidant activity (DPPH and ABTS was detected. Elevated positive linear correlation was got between ABTS and total phenolic (R2 = 0.751. Conclusions: The findings clearly demonstrate that the use of a polar solvent enables extraction of significant quantities of phenol compounds and flavonoids.

Inhibition of β-galactosidase and α-glucosidase synthesis in petroleum refinery effluent bacteria by phenolic compounds

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Gideon C. Okpokwasili

2011-04-01

Full Text Available Inhibition of α-glucosidase (EC 3.2.1.20 and β-galactosidase (EC 3.2.1.23 biosynthesis by phenolic compounds (phenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol in Escherichia coli, Bacillus and Pseudomonas species isolated from petroleum refinery wastewater was assessed. At sufficient concentrations, phenols inhibited the induction of α-glucosidase and β-galactosidase. The patterns of these toxic effects can be mathematically described with logistic and sigmoid dose-response models. The median inhibitory concentrations (IC50 varied among the phenols, the bacteria and enzymes. Quantitative structure–activity relationship (QSAR models based on the logarithm of the octanol–water partition coefficient (log10Kow were developed for each bacterium. The correlation coefficients varied between 0.84and 0.99 for the enzymes. The test results indicated α-glucosidase and β-galactosidase biosynthesis as important microbial indices for evaluation of toxicity of phenolic compounds.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of Thermotoga neapolitana β-glucosidase B

Energy Technology Data Exchange (ETDEWEB)

Turner, Pernilla [Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund (Sweden); Pramhed, Anna [Department of Molecular Biophysics, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund (Sweden); Kanders, Erik; Hedström, Martin; Karlsson, Eva Nordberg, E-mail: eva.nordberg-karlsson@biotek.lu.se [Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund (Sweden); Logan, Derek T., E-mail: eva.nordberg-karlsson@biotek.lu.se [Department of Molecular Biophysics, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund (Sweden); Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund (Sweden)

2007-09-01

Here, the expression, purification, crystallization and X-ray diffraction data of a family 3 β-glucosidase from the hyperthermophilic bacterium Thermotoga neapolitana are reported. β-Glucosidases belong to families 1, 3 and 9 of the glycoside hydrolases and act on cello-oligosaccharides. Family 1 and 3 enzymes are retaining and are reported to have transglycosylation activity, which can be used to produce oligosaccharides and glycoconjugates. Family 3 enzymes are less well characterized than their family 1 homologues and to date only two crystal structures have been solved. Here, the expression, purification, crystallization and X-ray diffraction data of a family 3 β-glucosidase from the hyperthermophilic bacterium Thermotoga neapolitana are reported. Crystals of selenomethionine-substituted protein have also been grown. The crystals belong to space group C222{sub 1}, with unit-cell parameters a = 74.9, b = 127.0, c = 175.2 Å. Native data have been collected to 2.4 Å resolution and the structure has been solved to 2.7 Å using the selenomethionine MAD method. Model building and refinement of the structure are under way.

Combining rational and random strategies in β-glucosidase Zm-p60.1 protein library construction.

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Dušan Turek

Full Text Available Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques for assessing effects of varying residues at selected positions on proteins' structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations' effects. Thus, in the presented study we applied the technique to randomize position W373 in β-glucosidase Zm-p60.1, which is highly conserved among β-glucosidases. Unexpectedly, β-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.

Combining rational and random strategies in β-glucosidase Zm-p60.1 protein library construction.

Science.gov (United States)

Turek, Dušan; Klimeš, Pavel; Mazura, Pavel; Brzobohatý, Břetislav

2014-01-01

Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques) for assessing effects of varying residues at selected positions on proteins' structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations' effects. Thus, in the presented study we applied the technique to randomize position W373 in β-glucosidase Zm-p60.1, which is highly conserved among β-glucosidases. Unexpectedly, β-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.

Immobilization of β-glucosidase onto mesoporous silica support: Physical adsorption and covalent binding of enzyme

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Ivetić Darjana Ž.

2014-01-01

Full Text Available This paper investigates β-glucosidase immobilization onto mesoporous silica support by physical adsorption and covalent binding. The immobilization was carried out onto micro-size silica aggregates with the average pore size of 29 nm. During physical adsorption the highest yield of immobilized β-glucosidase was obtained at initial protein concentration of 0.9 mg ml-1. Addition of NaCl increased 1.7-fold, while Triton X-100 addition decreased 6-fold yield of adsorption in comparison to the one obtained without any addition. Covalently bonded β-glucosidase, via glutaraldehyde previously bonded to silanized silica, had higher yield of immobilized enzyme as well as higher activity and substrate affinity in comparison to the one physically adsorbed. Covalent binding did not considerably changed pH and temperature stability of obtained biocatalyst in range of values that are commonly used in reactions in comparison to unbounded enzyme. Furthermore, covalent binding provided biocatalyst which retained over 70% of its activity after 10 cycles of reuse. [Projekat Ministarstva nauke Republike Srbije, br. III 45021

Fermentation of purple Jerusalem artichoke extract to improve the α-glucosidase inhibitory effect in vitro and ameliorate blood glucose in db/db mice.

Science.gov (United States)

Wang, Zhiqiang; Hwang, Seung Hwan; Lee, Sun Youb; Lim, Soon Sung

2016-06-01

Jerusalem artichoke has inhibitory activity against α-glucosidase and decreases fasting serum glucose levels, which may be related to its fructan content. The biological activity of fructan can be influenced by the degree of polymerization. Thus, in this study, the inhibitory effects of original and fermented purple Jerusalem artichoke (PJA) on α-glucosidase were compared in vitro. Additionally, the anti-diabetes effect of Lactobacillus plantarum-fermented PJA (LJA) was studied in a non-insulin-dependent diabetes mellitus animal model (C57BIKsJ db/db). The water extract of PJA was fermented by L. plantarum, and two strains of Bacillus subtilis to compare their anti-α-glucosidase activities in vitro by α-glucosidase assays. The anti-diabetes effect of LJA was studied in a non-insulin-dependent diabetes mellitus animal model (C57BIKsJ db/db) for seven weeks. During the experiment, food intake, body weight, and fasting blood glucose were measured every week. At the end of the treatment period, several diabetic parameters and the intestinal α-glucosidase activity were measured. The LJA showed the highest α-glucosidase inhibitory activity in vitro. In the in vivo study, it resulted in a significantly lower blood glucose concentration than the control. Serum insulin and HDL cholesterol levels were significantly higher and the concentrations of triglycerides, non-esterified fatty acids, and total cholesterol were significant lower in mice treated with LJA after seven weeks. In addition, the intestinal α-glucosidase activity was partially inhibited. These results suggested that LJA regulates blood glucose and has potential use as a dietary supplement.

New Biflavonoids with α-Glucosidase and Pancreatic Lipase Inhibitory Activities from Boesenbergia rotunda

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Nutputsorn Chatsumpun

2017-10-01

Full Text Available Roots of Boesenbergia rotunda (L. Mansf. are prominent ingredients in the cuisine of several Asian countries, including Thailand, Malaysia, Indonesia, India, and China. An extract prepared from the roots of this plant showed strong inhibitory activity against enzymes α-glucosidase and pancreatic lipase and was subjected to chromatographic separation to identify the active components. Three new biflavonoids of the flavanone-chalcone type (9, 12, and 13 were isolated, along with 12 known compounds. Among the 15 isolates, the three new compounds showed stronger inhibitory activity against α-glucosidase than the drug acarbose but displayed lower pancreatic lipase inhibitory effect than the drug orlistat. The results indicated the potential of B. rotunda roots as a functional food for controlling after-meal blood glucose levels.

ORF Alignment: NC_004350 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_004350 gi|24379337 >1g5aA 90 627 3 535 7e-83 ... gb|AAN58598.1| dextran glucosidas...e DexB [Streptococcus mutans UA159] ... ref|NP_721292.1| dextran glucosidase DexB [Streptococcus ... ... ... (Exo-1,6-alpha-glucosidase) (Glucodextranase) ... Length = 533 ... Que

Chemo-enzymatic synthesis route to poly(glucosyl-acrylates) using glucosidase from almonds

NARCIS (Netherlands)

Kloosterman, Wouter M. J.; Roest, Steven; Priatna, Siti R.; Stavila, Erythrina; Loos, Katja

2014-01-01

Novel types of glucosyl-acrylate monomers are obtained by beta-glucosidase from almond catalyzed glycosidation reaction. The saccharide-acrylate monomers were synthesized by reaction of D-glucose with hydroxyl functional acrylates: 2-hydroxyethyl acrylate (2-HEA), 2-hydroxyethyl methacrylate

Lactucaxanthin - a potential anti-diabetic carotenoid from lettuce (Lactuca sativa) inhibits α-amylase and α-glucosidase activity in vitro and in diabetic rats.

Science.gov (United States)

Gopal, Sowmya Shree; Lakshmi, Magisetty Jhansi; Sharavana, Gurunathan; Sathaiah, Gunaseelan; Sreerama, Yadahally N; Baskaran, Vallikannan

2017-03-22

Intestinal and pancreatic α-amylase and α-glucosidase inhibitors offer an approach to lower the levels of post-prandial hyperglycemia through the control of dietary starch breakdown in digestion. This study hypothesized that lactucaxanthin (Lxn) in lettuce (Lactuca sativa) inhibits the activity of α-amylase and α-glucosidase. In this study, the interaction of Lxn with α-amylase and α-glucosidase in silico and its inhibitory effect on these enzymes were studied using in vitro and STZ-induced diabetic rat models. Lxn was isolated from lettuce with 96% purity confirmed by HPLC and LCMS. The in silico analysis showed that Lxn has a lower binding energy (-6.05 and -6.34 kcal mol -1 ) with α-amylase and α-glucosidase compared to their synthetic inhibitors, acarbose (-0.21 kcal mol -1 ) and miglitol (-2.78 kcal mol -1 ), respectively. In vitro α-amylase and α-glucosidase inhibition assays revealed that Lxn had IC 50 values of 435.5 μg mL -1 and 1.84 mg mL -1 , but acarbose has values of 2.5 and 16.19 μg mL -1 . The in vivo results showed an increased activity for α-amylase and α-glucosidase in the intestine (4.7 and 1.30 fold, p < 0.05) and pancreas (1.3 and 1.48 fold, p < 0.05) of STZ induced diabetic rats compared to normal rats. Whereas the activity decreased (p < 0.05) in the Lxn fed diabetic rats, except for the intestinal α-glucosidase activity (1.69 ± 0.12 PNP per min per mg protein). This was confirmed by the low blood glucose level (239.4 ± 18.2 mg dL -1 ) in diabetic rats fed Lxn compared to the diabetic group (572.2 ± 30.5 mg dL -1 , p < 0.05). Lxn significantly inhibited (p < 0.05) the activity of α-amylase and α-glucosidase and could be of medical and nutritional relevance in the treatment of diabetes.

Molecular Architecture of Strictosidine Glucosidase: The Gateway to the Biosynthesis of the Monoterpenoid Indole Alkaloid Family[W

Science.gov (United States)

Barleben, Leif; Panjikar, Santosh; Ruppert, Martin; Koepke, Juergen; Stöckigt, Joachim

2007-01-01

Strictosidine β-d-glucosidase (SG) follows strictosidine synthase (STR1) in the production of the reactive intermediate required for the formation of the large family of monoterpenoid indole alkaloids in plants. This family is composed of ∼2000 structurally diverse compounds. SG plays an important role in the plant cell by activating the glucoside strictosidine and allowing it to enter the multiple indole alkaloid pathways. Here, we report detailed three-dimensional information describing both native SG and the complex of its inactive mutant Glu207Gln with the substrate strictosidine, thus providing a structural characterization of substrate binding and identifying the amino acids that occupy the active site surface of the enzyme. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-207, Glu-416, His-161, and Trp-388 in catalysis. Comparison of the catalytic pocket of SG with that of other plant glucosidases demonstrates the structural importance of Trp-388. Compared with all other glucosidases of plant, bacterial, and archaeal origin, SG's residue Trp-388 is present in a unique structural conformation that is specific to the SG enzyme. In addition to STR1 and vinorine synthase, SG represents the third structural example of enzymes participating in the biosynthetic pathway of the Rauvolfia alkaloid ajmaline. The data presented here will contribute to deciphering the structure and reaction mechanism of other higher plant glucosidases. PMID:17890378

The enhanced inhibition of water extract of black tea under baking treatment on α-amylase and α-glucosidase.

Science.gov (United States)

Tong, Da-Peng; Zhu, Ke-Xue; Guo, Xiao-Na; Peng, Wei; Zhou, Hui-Ming

2018-02-01

This paper studied the inhibition of water extract of natural or baked black tea on the activity of α-amylase and α- glucosidase. Baking treatment was found to be one effective way to enhance the inhibition of black tea on both α-amylase and α- glucosidase, and IC 50 of water extract of baked black tea (BBTWE) were 1.213mg/mL and 4.190mg/mL, respectively, while IC 50 of water extract of black tea (BTWE) were 1.723mg/mL and 6.056mg/mL, respectively. This study further studied the mechanism of the effect of water extract on α-amylase and α- glucosidase using HPLC, circular dichroism, and synchronous fluorescence. HPLC analysis of tea polyphenols showed that the content of tea polyphenols with low polarity increased after baking. In addition, BBTWE had higer abilty on decreasing the hydrophobicity of tryptophan residues than BTWE for both α-amylase and α- glucosidase.The increase of α-helix proportion of α-amylase when treated with BBTWE was more obvious than that when treated with BTWE. In a word, thermal process of baked foods may be beneficial for tea polyphenols to reduce the rate of starch digestion. Copyright © 2017 Elsevier B.V. All rights reserved.

α-Glucosidase and pancreatic lipase inhibitory activities and glucose uptake stimulatory effect of phenolic compounds from Dendrobium formosum

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Prachyaporn Inthongkaew

Full Text Available ABSTRACT A methanol extract from the whole plant of Dendrobium formosum Roxb. ex Lindl., Orchidaceae, showed inhibitory potential against α-glucosidase and pancreatic lipase enzymes. Chromatographic separation of the extract resulted in the isolation of twelve phenolic compounds. The structures of these compounds were determined through analysis of NMR and HR-ESI-MS data. All of the isolates were evaluated for their α-glucosidase and pancreatic lipase inhibitory activities, as well as glucose uptake stimulatory effect. Among the isolates, 5-methoxy-7-hydroxy-9,10-dihydro-1,4-phenanthrenequinone (12 showed the highest α-glucosidase and pancreatic lipase inhibitory effects with an IC50 values of 126.88 ± 0.66 µM and 69.45 ± 10.14 µM, respectively. An enzyme kinetics study was conducted by the Lineweaver-Burk plot method. The kinetics studies revealed that compound 12 was a non-competitive inhibitor of α-glucosidase and pancreatic lipase enzymes. Moreover, lusianthridin at 1 and 10 µg/ml and moscatilin at 100 µg/ml showed glucose uptake stimulatory effect without toxicity on L6 myotubes. This study is the first report on the phytochemical constituents and anti-diabetic and anti-obesity activities of D. formosum.

Synthesis of disaccharides using β-glucosidases from Aspergillus niger, A. awamori and Prunus dulcis.

Science.gov (United States)

da Silva, Ayla Sant'Ana; Molina, Javier Freddy; Teixeira, Ricardo Sposina Sobral; Valdivieso Gelves, Luis G; Bon, Elba P S; Ferreira-Leitão, Viridiana S

2017-11-01

Glucose conversion into disaccharides was performed with β-glucosidases from Prunus dulcis (β-Pd), Aspergillus niger (β-An) and A. awamori (β-Aa), in reactions containing initial glucose of 700 and 900 g l -1 . The reactions' time courses were followed regarding glucose and product concentrations. In all cases, there was a predominant formation of gentiobiose over cellobiose and also of oligosaccharides with a higher molecular mass. For reactions containing 700 g glucose l -1 , the final substrate conversions were 33, 38, and 23.5% for β-An, β-Aa, and β-Pd, respectively. The use of β-An yielded 103 g gentiobiose l -1 (15.5% yield), which is the highest reported for a fungal β-glucosidase. The increase in glucose concentration to 900 g l -1 resulted in a significant increase in disaccharide synthesis by β-Pd, reaching 128 g gentiobiose l -1 (15% yield), while for β-An and β-Aa, there was a shift toward the synthesis of higher oligosaccharides. β-Pd and the fungal β-An and β-Aa β-glucosidases present quite dissimilar kinetics and selective properties regarding the synthesis of disaccharides; while β-Pd showed the highest productivity for gentiobiose synthesis, β-An presented the highest specificity.

Immobilization of Aspergillus awamori β-glucosidase on commercial gelatin: An inexpensive and efficient process.

Science.gov (United States)

Nishida, Verônica S; de Oliveira, Roselene F; Brugnari, Tatiane; Correa, Rúbia Carvalho G; Peralta, Rosely A; Castoldi, Rafael; de Souza, Cristina G M; Bracht, Adelar; Peralta, Rosane M

2018-05-01

In this work, a β-glucosidase of Aspergillus awamori with a molecular weight of 180 kDa was produced in solid-state cultures using a mixture of pineapple crown leaves and wheat bran. Maximum production of the enzyme (820 ± 30 U/g substrate) was obtained after 8 days of culture at 28 °C and initial moisture of 80%. The crude enzyme was efficiently immobilized on glutaraldehyde cross-linked commercial gelatin. Immobilization changed the kinetics of the enzyme, whose behavior could no longer be described by a saturation function of the Michaelis-Menten type. Comparative evaluation of the free and immobilized enzyme showed that the immobilized enzyme was more thermostable and less inhibited by glucose than the free form. In consequence of these properties, the immobilized enzyme was able to hydrolyze cellobiose more extensively. In association with Trichoderma reesei cellulase, the free and immobilized β-glucosidase increased the liberation of glucose from cellulose 3- and 5-fold, respectively. Immobilization of the A. awamori β-glucosidase on glutaraldehyde cross-linked commercial gelatin is an efficient and cheap method allowing the reuse of the enzyme by at least 10 times. Copyright © 2018 Elsevier B.V. All rights reserved.

Honokiol trimers and dimers via biotransformation catalyzed by Momordica charantia peroxidase: novel and potent α-glucosidase inhibitors.

Science.gov (United States)

He, Ye; Wang, Xiao-Bing; Fan, Bo-Yi; Kong, Ling-Yi

2014-01-15

Ten honokiol oligomers (1-10), including four novel trimers (1-4) and four novel dimers (5-8), were obtained by means of biotransformation of honokiol catalyzed by Momordica charantia peroxidase (MCP) for the first time. Their structures were established on the basis of spectroscopic methods. The biological results demonstrated that most of the oligomers were capable of inhibiting α-glucosidase with significant abilities, which were one to two orders of magnitude more potent than the substrate, honokiol. In particular, compound 2, the honokiol trimer, displayed the greatest inhibitory activity against α-glucosidase with an IC50 value of 1.38μM. Kinetic and CD studies indicated that 2 inhibited α-glucosidase in a reversible, mixed-type manner and caused conformational changes in the secondary structure of the enzyme protein. These findings suggested that 2 might be exploited as a promising drug candidate for the treatment of diabetes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Assessment of constituents in Allium by multivariate data analysis, high-resolution α-glucosidase inhibition assay and HPLC-SPE-NMR

DEFF Research Database (Denmark)

Schmidt, Jeppe Secher; Nyberg, Nils; Stærk, Dan

2014-01-01

Bulbs and leaves of 35 Allium species and cultivars bought or collected in 2010–2012 were investigated with multivariate data analysis, high-resolution α-glucosidase inhibition assays and HPLC-HRMS-SPE-NMR with the aim of exploring the potential of Allium as a future functional food for management...... of type 2 diabetes. It was found that 30 out of 106 crude extracts showed more than 80% inhibition of the α-glucosidase enzyme at a concentration of 40 mg/mL (dry sample) or 0.4 g/mL (fresh sample). High-resolution α-glucosidase biochromatograms of these extracts allowed fast identification of three...

Identification of an Unfolding Intermediate for a DNA Lesion Bypass Polymerase

Science.gov (United States)

Sherrer, Shanen M.; Maxwell, Brian A.; Pack, Lindsey R.; Fiala, Kevin A.; Fowler, Jason D.; Zhang, Jun; Suo, Zucai

2012-01-01

Sulfolobus solfataricusDNA Polymerase IV (Dpo4), a prototype Y-family DNA polymerase, has been well characterized biochemically and biophysically at 37 °C or lower temperatures. However, the physiological temperature of the hyperthermophile S. solfataricus is approximately 80 °C. With such a large discrepancy in temperature, the in vivo relevance of these in vitro studies of Dpo4 has been questioned. Here, we employed circular dichroism spectroscopy and fluorescence-based thermal scanning to investigate the secondary structural changes of Dpo4 over a temperature range from 26 to 119 °C. Dpo4 was shown to display a high melting temperature characteristic of hyperthermophiles. Unexpectedly, the Little Finger domain of Dpo4, which is only found in the Y-family DNA polymerases, was shown to be more thermostable than the polymerase core. More interestingly, Dpo4 exhibited a three-state cooperative unfolding profile with an unfolding intermediate. The linker region between the Little Finger and Thumb domains of Dpo4 was found to be a source of structural instability. Through site-directed mutagenesis, the interactions between the residues in the linker region and the Palm domain were identified to play a critical role in the formation of the unfolding intermediate. Notably, the secondary structure of Dpo4 was not altered when the temperature was increased from 26 to 87.5 °C. Thus, in addition to providing structural insights into the thermal stability and an unfolding intermediate of Dpo4, our work also validated the relevance of the in vitro studies of Dpo4 performed at temperatures significantly lower than 80 °C. PMID:22667759

Selected Tea and Tea Pomace Extracts Inhibit Intestinal α-Glucosidase Activity in Vitro and Postprandial Hyperglycemia in Vivo

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Jungbae Oh

2015-04-01

Full Text Available Type 2 diabetes mellitus (T2DM is a metabolic disorder characterized by postprandial hyperglycemia, which is an early defect of T2DM and thus a primary target for anti-diabetic drugs. A therapeutic approach is to inhibit intestinal α-glucosidase, the key enzyme for dietary carbohydrate digestion, resulting in delayed rate of glucose absorption. Although tea extracts have been reported to have anti-diabetic effects, the potential bioactivity of tea pomace, the main bio waste of tea beverage processing, is largely unknown. We evaluated the anti-diabetic effects of three selected tea water extracts (TWE and tea pomace extracts (TPE by determining the relative potency of extracts on rat intestinal α-glucosidase activity in vitro as well as hypoglycemic effects in vivo. Green, oolong, and black tea bags were extracted in hot water and the remaining tea pomace were dried and further extracted in 70% ethanol. The extracts were determined for intestinal rat α-glucosidases activity, radical scavenging activity, and total phenolic content. The postprandial glucose-lowering effects of TWE and TPE of green and black tea were assessed in male Sprague-Dawley (SD rats and compared to acarbose, a known pharmacological α-glucosidase inhibitor. The IC50 values of all three tea extracts against mammalian α-glucosidase were lower or similar in TPE groups than those of TWE groups. TWE and TPE of green tea exhibited the highest inhibitory effects against α-glucosidase activity with the IC50 of 2.04 ± 0.31 and 1.95 ± 0.37 mg/mL respectively. Among the specific enzymes tested, the IC50 values for TWE (0.16 ± 0.01 mg/mL and TPE (0.13 ± 0.01 mg/mL of green tea against sucrase activity were the lowest compared to those on maltase and glucoamylase activities. In the animal study, the blood glucose level at 30 min after oral intake (0.5 g/kg body wt of TPE and TWE of both green and black tea was significantly reduced compared to the control in sucrose-loaded SD

In vivo biotinylation of recombinant beta-glucosidase enables simultaneous purification and immobilization on streptavidin coated magnetic particles

DEFF Research Database (Denmark)

Alftrén, Johan; Ottow, Kim Ekelund; Hobley, Timothy John

2013-01-01

Beta-glucosidase from Bacillus licheniformis was in vivo biotinylated in Escherichia coli and subsequently immobilized directly from cell lysate on streptavidin coated magnetic particles. In vivo biotinylation was mediated by fusing the Biotin Acceptor Peptide to the C-terminal of beta......-glucosidase and co-expressing the BirA biotin ligase. The approach enabled simultaneous purification and immobilization of the enzyme from crude cell lysate on magnetic particles because of the high affinity and strong interaction between biotin and streptavidin. After immobilization of the biotinylated beta...

Expression, purification and preliminary crystallographic analysis of the recombinant β-glucosidase (BglA) from the halothermophile Halothermothrix orenii

International Nuclear Information System (INIS)

Kori, Lokesh D.; Hofmann, Andreas; Patel, Bharat K. C.

2010-01-01

A β-glucosidase A (BglA) from the thermophile Halothermothrix orenii has been cloned, purified and crystallized in an orthorhombic space group. X-ray diffraction data have been collected to 3.5 Å resolution, and the structure was solved by molecular replacement, revealing the presence of two molecules in the asymmetric unit. The β-glucosidase A gene (bglA) has been cloned from the halothermophilic bacterium Halothermothrix orenii and the recombinant enzyme (BglA; EC 3.2.1.21) was bacterially expressed, purified using metal ion-affinity chromatography and subsequently crystallized. Orthorhombic crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystal structure with two molecules in the asymmetric unit was solved by molecular replacement using a library of known glucosidase structures. Attempts to collect higher resolution diffraction data from crystals grown under different conditions and structure refinement are currently in progress

Aqueous Extract of Annona macroprophyllata: A Potential α-Glucosidase Inhibitor

Science.gov (United States)

Brindis, F.; González-Trujano, M. E.; González-Andrade, M.; Aguirre-Hernández, E.; Villalobos-Molina, R.

2013-01-01

Annona genus contains plants used in folk medicine for the treatment of diabetes. In the present study, an aqueous extract prepared from Annona macroprophyllata (Annonaceae, also known as A. diversifolia) leaves was evaluated on both the activity of yeast α-glucosidase (an in vitro assay) and sucrose tolerance in Wistar rats. The results have shown that the aqueous extract from A. macroprophyllata inhibits the yeast α-glucosidase with an IC50 = 1.18 mg/mL, in a competitive manner with a K i = 0.97 mg/mL, a similar value to that of acarbose (K i = 0.79 mg/mL). The inhibitory activity of A. macroprophyllata was reinforced by its antihyperglycemic effect, at doses of 100, 300, and 500 mg/kg in rats. Chromatographic analysis identified the flavonoids rutin and isoquercitrin in the most polar fractions of A. macroprophyllata crude extract, suggesting that these flavonoids are part of the active constituents in the plant. Our results support the use of A. macroprophyllata in Mexican folk medicine to control postprandial glycemia in people with diabetes mellitus, involving active constituents of flavonoid nature. PMID:24298552

α-Glucosidase and Protein Tyrosine Phosphatase 1B Inhibitory Activity of Plastoquinones from Marine Brown Alga Sargassum serratifolium

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Md. Yousof Ali

2017-12-01

Full Text Available Sargassum serratifolium C. Agardh (Phaeophyceae, Fucales is a marine brown alga that belongs to the family Sargassaceae. It is widely distributed throughout coastal areas of Korea and Japan. S. serratifolium has been found to contain high concentrations of plastoquinones, which have strong anti-cancer, anti-inflammatory, antioxidant, and neuroprotective activity. This study aims to investigate the anti-diabetic activity of S. serratifolium and its major constituents through inhibition of protein tyrosine phosphatase 1B (PTP1B, α-glucosidase, and ONOO−-mediated albumin nitration. S. serratifolium ethanolic extract and fractions exhibited broad PTP1B and α-glucosidase inhibitory activity (IC50, 1.83~7.04 and 3.16~24.16 µg/mL for PTP1B and α-glucosidase, respectively. In an attempt to identify bioactive compounds, three plastoquinones (sargahydroquinoic acid, sargachromenol and sargaquinoic acid were isolated from the active n-hexane fraction of S. serratifolium. All three plastoquinones exhibited dose-dependent inhibitory activity against PTP1B in the IC50 range of 5.14–14.15 µM, while sargachromenol and sargaquinoic acid showed dose-dependent inhibitory activity against α-glucosidase (IC50 42.41 ± 3.09 and 96.17 ± 3.48 µM, respectively. In the kinetic study of PTP1B enzyme inhibition, sargahydroquinoic acid and sargaquinoic acid led to mixed-type inhibition, whereas sargachromenol displayed noncompetitive-type inhibition. Moreover, plastoquinones dose-dependently inhibited ONOO−-mediated albumin nitration. Docking simulations of these plastoquinones demonstrated negative binding energies and close proximity to residues in the binding pocket of PTP1B and α-glucosidase, indicating that these plastoquinones have high affinity and tight binding capacity towards the active site of the enzymes. These results demonstrate that S. serratifolium and its major plastoquinones may have the potential as functional food ingredients for the

Inhibition of α-glucosidase by polysaccharides from the fruit hull of Camellia oleifera Abel.

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Zhang, Sheng; Li, Xiang-Zhou

2015-01-22

We isolated and purified polysaccharides from the Camellia oleifera Abel. fruit hull and studied its hypoglycemic potential. Our results revealed six polysaccharides (CFPA-1-5 & CFPB) from the aqueous extract from the defatted C. oleifera fruit hull. Purified polysaccharides (purity >90%) were investigated for the inhibition of α-glucosidase activity in vitro. Two polysaccharides, CFPB and CFPA-3 were present in high concentration in the fruit hull and showed a dose-dependent inhibition of α-glucosidase activity, with IC50 concentrations of 11.80 and 10.95 μg/mL, respectively. This result suggests that polysaccharides (CFP) extracted from the fruit hull of C. oleifera may have potential as functional foods with featuring a hypoglycemic effect. Copyright © 2014 Elsevier Ltd. All rights reserved.

Differential active site loop conformations mediate promiscuous activities in the lactonase SsoPox.

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Julien Hiblot

Full Text Available Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263 that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability.

Biochemical and structural characterization of Cren7, a novel chromatin protein conserved among Crenarchaea.

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Guo, Li; Feng, Yingang; Zhang, Zhenfeng; Yao, Hongwei; Luo, Yuanming; Wang, Jinfeng; Huang, Li

2008-03-01

Archaea contain a variety of chromatin proteins consistent with the evolution of different genome packaging mechanisms. Among the two main kingdoms in the Archaea, Euryarchaeota synthesize histone homologs, whereas Crenarchaeota have not been shown to possess a chromatin protein conserved at the kingdom level. We report the identification of Cren7, a novel family of chromatin proteins highly conserved in the Crenarchaeota. A small, basic, methylated and abundant protein, Cren7 displays a higher affinity for double-stranded DNA than for single-stranded DNA, constrains negative DNA supercoils and is associated with genomic DNA in vivo. The solution structure and DNA-binding surface of Cren7 from the hyperthermophilic crenarchaeon Sulfolobus solfataricus were determined by NMR. The protein adopts an SH3-like fold. It interacts with duplex DNA through a beta-sheet and a long flexible loop, presumably resulting in DNA distortions through intercalation of conserved hydrophobic residues into the DNA structure. These data suggest that the crenarchaeal kingdom in the Archaea shares a common strategy in chromatin organization.

Inhibitory effect of rhubarb on intestinal α-glucosidase activity in type ...

African Journals Online (AJOL)

Purpose: To investigate the inhibitory effect of rhubarb on α-glucosidase activity in the small intestine of rats with type 1 diabetes. Methods: Type 1 diabetic rat model was established by intraperitoneally injecting 30 male SD rats with 1 % streptozocin (STZ). Rats with fasting blood glucose > 11 mmol/L (24) were used for the ...

Kinetics of α-amylase and α-glucosidase inhibitory potential of Zea mays Linnaeus (Poaceae), Stigma maydis aqueous extract: An in vitro assessment.

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Sabiu, S; O'Neill, F H; Ashafa, A O T

2016-05-13

Corn silk (Zea mays L., Stigma maydis) is an important herb used traditionally in many parts of the world to treat array of diseases including diabetes mellitus. Inhibitors of α-amylase and α-glucosidase offer an effective strategy to modulate levels of post prandial hyperglycaemia via control of starch metabolism. This study evaluated α-amylase and α-glucosidase inhibitory potentials of corn silk aqueous extract. Active principles and antioxidant attributes of the extract were also analysed. The α-amylase inhibitory potential of the extract was investigated by reacting its different concentrations with α-amylase and starch solution, while α-glucosidase inhibition was determined by pre-incubating α-glucosidase with different concentrations of the extract followed by addition of p-nitrophenylglucopyranoside. The mode(s) of inhibition of the enzymes were determined using Lineweaver-Burke plot. In vitro analysis of the extract showed that it exhibited potent and moderate inhibitory potential against α-amylase and α-glucosidase, respectively. The inhibition was concentration-dependent with respective half-maximal inhibitory concentration (IC50) values of 5.89 and 0.93mg/mL. Phytochemical analyses revealed the presence of alkaloids, flavonoids, phenols, saponins, tannins and phytosterols as probable inhibitory constituents. Furthermore, the extract remarkably scavenges reactive oxygen species like DPPH and nitric oxide radicals, elicited good reducing power and a significant metal chelating attributes. Overall, the non-competitive and uncompetitive mechanism of action of corn silk extract is due to its inhibitory effects on α-amylase and α-glucosidase, respectively. Consequently, this will reduce the rate of starch hydrolysis, enhance palliated glucose levels, and thus, lending credence to hypoglycaemic candidature of corn silk. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Synthesis, α-glucosidase inhibition and molecular docking study of coumarin based derivatives.

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Taha, Muhammad; Shah, Syed Adnan Ali; Afifi, Muhammad; Imran, Syahrul; Sultan, Sadia; Rahim, Fazal; Khan, Khalid Mohammed

2018-04-01

We have synthesized seventeen Coumarin based derivatives (1-17), characterized by 1 HNMR, 13 CNMR and EI-MS and evaluated for α-glucosidase inhibitory potential. Among the series, all derivatives exhibited outstanding α-glucosidase inhibition with IC 50 values ranging between 1.10 ± 0.01 and 36.46 ± 0.70 μM when compared with the standard inhibitor acarbose having IC 50 value 39.45 ± 0.10 μM. The most potent derivative among the series is derivative 3 having IC 50 value 1.10 ± 0.01 μM, which are many folds better than the standard acarbose. The structure activity relationship (SAR) was mainly based upon by bring about difference of substituent's on phenyl part. Molecular docking studies were carried out to understand the binding interaction of the most active compounds. Copyright © 2018 Elsevier Inc. All rights reserved.

Release Profile of Andrographis paniculata Leaf Extract Nanocapsule as α-Glucosidase Inhibitors

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Zahrani, K.; Imansari, F.; Utami, T. S.; Arbianti, R.

2017-07-01

Andrographis paniculata is one of 13 leading commodities Indonesian medicinal plants through the Ditjen POM. Andrographolide as main active compound has been shown to have many pharmacological activities, one of which is as α-glucosidase enzyme inhibitors which has clinical potential as an antitumor, antiviral, antidiabetic, and immunoregulator agents. This study aims to do nanoencapsulation of Andrographis paniculatar leaf extract to increase its active compound bioavailability and get a release profile through synthetic fluids media simulation. Nanoencapsulation with ionic gelation method result the encapsulation efficiency and loading capacity values of 73.47% and 46.29% at 2%: 1% of chitosan: STPP ratio. The maximum α-glucosidase inhibition of 37.17% was obtained at 16% concentration. Burst release at gastric pH conditions indicate that most of the drug (in this study is an Andrographis paniculata leaf extract) adsorbed on the surface of the nanoparticles an indicates that the kind of nanoparticle formed is nanosphere.

High-resolution bioactivity profiling combined with HPLC-HRMS-SPE-NMR: α-glucosidase inhibitors and acetylated ellagic acid rhamnosides from Myrcia palustris DC. (Myrtaceae)

DEFF Research Database (Denmark)

Wubshet, Sileshi Gizachew; Moresco, Henrique H.; Tahtah, Yousof

2015-01-01

, and therefore improved drug leads or functional foods containing α-glucosidase inhibitors are needed for management of blood glucose. In this study, leaves of Myrcia palustris were investigated by high-resolution α-glucosidase inhibition profiling combined with HPLC–HRMS–SPE–NMR. This led to identification...

Crystal Structures of Two Isozymes of Citrate Synthase from Sulfolobus tokodaii Strain 7

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Midori Murakami

2016-01-01

Full Text Available Thermoacidophilic archaeon Sulfolobus tokodaii strain 7 has two citrate synthase genes (ST1805-CS and ST0587-CS in the genome with 45% sequence identity. Because they exhibit similar optimal temperatures of catalytic activity and thermal inactivation profiles, we performed structural comparisons between these isozymes to elucidate adaptation mechanisms to high temperatures in thermophilic CSs. The crystal structures of ST1805-CS and ST0587-CS were determined at 2.0 Å and 2.7 Å resolutions, respectively. Structural comparison reveals that both of them are dimeric enzymes composed of two identical subunits, and these dimeric structures are quite similar to those of citrate synthases from archaea and eubacteria. ST0587-CS has, however, 55 ion pairs within whole dimer structure, while having only 36 in ST1805-CS. Although the number and distributions of ion pairs are distinct from each other, intersubunit ion pairs between two domains of each isozyme are identical especially in interterminal region. Because the location and number of ion pairs are in a trend with other CSs from thermophilic microorganisms, the factors responsible for thermal adaptation of ST-CS isozymes are characterized by ion pairs in interterminal region.

In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag.

Science.gov (United States)

Visone, Valeria; Han, Wenyuan; Perugino, Giuseppe; Del Monaco, Giovanni; She, Qunxin; Rossi, Mosè; Valenti, Anna; Ciaramella, Maria

2017-01-01

Protein imaging, allowing a wide variety of biological studies both in vitro and in vivo, is of great importance in modern biology. Protein and peptide tags fused to proteins of interest provide the opportunity to elucidate protein location and functions, detect protein-protein interactions, and measure protein activity and kinetics in living cells. Whereas several tags are suitable for protein imaging in mesophilic organisms, the application of this approach to microorganisms living at high temperature has lagged behind. Archaea provide an excellent and unique model for understanding basic cell biology mechanisms. Here, we present the development of a toolkit for protein imaging in the hyperthermophilic archaeon Sulfolobus islandicus. The system relies on a thermostable protein tag (H5) constructed by engineering the alkylguanine-DNA-alkyl-transferase protein of Sulfolobus solfataricus, which can be covalently labeled using a wide range of small molecules. As a suitable host, we constructed, by CRISPR-based genome-editing technology, a S. islandicus mutant strain deleted for the alkylguanine-DNA-alkyl-transferase gene (Δogt). Introduction of a plasmid-borne H5 gene in this strain led to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms.

A diterpenoid sugiol from Metasequoia glyptostroboides with α-glucosidase and tyrosinase inhibitory potential

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Vivek K. Bajpai

2014-08-01

Full Text Available Nowadays use of plant derived natural compounds have become a topic of increasing interest in food and medicine industries due to their multitude of biological and therapeutic properties. In this study, a diterpenoid compound sugiol, isolated from Metasequoia glyptostroboides was evaluated for α–glucosidase and tyrosinase inhibitory efficacy in terms of its potent anti-diabetic and anti-melanogenesis potential, respectively. As a result, sugiol at the concentration range of (100-10,000 µg/mL and (20-500 µg/mL showed potent efficacy on inhibiting α-glucosidase and tyrosinase enzymes in vitro ranging from 12.34-63.47% and 28.22-67.43%, respectively. These findings confirm the therapeutic potential of diterpenoid compound sugiol from M. glyptostroboides as a novel candidate for using in food and medicine industry which may have practical potential to cure skin and diabetes mellitus type-2 related disorders.

Antidiabetic potential of Conocarpus lancifolius

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Malik Saadullah

2014-06-01

Full Text Available The antidiabetic activity of Conocarpus lancifolius extract was investigated in vitro, as alpha glucosidase inhibition and in vivo as alloxan induced diabetic rabbits with other biochemical parameters (LDL, HDL, SGPT, SGOT, cretinine, urea and triglyceride. Alpha-glucosidase inhibition activity was performed by using acorbose as standred. Methanolic extract show alpha-glucosidase inhibition activity. The dose of 200 mg/kg body weight significantly (p<0.05 decreases the blood glucose level, plasma total cholesterol, triglycerides and LDL in treated rabbits as compared to diabetic rabbits. This dose significantly increased the level of HDL in treated group. The activity of SGOT and SGPT also significantly (p<0.05 decreased in treated diabetic rabbits. Phytochemical studies show the presence of glycosides, tannins, saponins and terpenoids. The antidiabetic potential is may be due to its saponin contents.

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

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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.

New α-Glucosidase Inhibitory Triterpenic Acid from Marine Macro Green Alga Codium dwarkense Boergs

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Liaqat Ali

2015-07-01

Full Text Available The marine ecosystem has been a key resource for secondary metabolites with promising biological roles. In the current study, bioassay-guided phytochemical investigations were carried out to assess the presence of enzyme inhibitory chemical constituents from the methanolic extract of marine green alga—Codium dwarkense. The bioactive fractions were further subjected to chromatographic separations, which resulted in the isolation of a new triterpenic acid; dwarkenoic acid (1 and the known sterols; androst-5-en-3β-ol (2, stigmasta-5,25-dien-3β,7α-diol (3, ergosta-5,25-dien-3β-ol (4, 7-hydroxystigmasta-4,25-dien-3-one-7-O-β-d-fucopyranoside (5, 7-hydroxystigmasta-4,25-dien-3-one (6, and stigmasta-5,25-dien-3β-ol (7. The structure elucidation of the new compound was carried out by combined mass spectrometry and 1D (1H and 13C and 2D (HSQC, HMBC, COSY, and NOESY NMR spectroscopic data. The sub-fractions and pure constituents were assayed for enzymatic inhibition of alpha-glucosidase. Compound 1 showed significant inhibition at all concentrations. Compounds 2, 3, 5, and 7 exhibited a dose-dependent response, whereas compounds 4–6 showed moderate inhibition. Utilizing such marine-derived biological resources could lead to drug discoveries related to anti-diabetics.

Identification of a β-glucosidase from the Mucor circinelloides genome by peptide pattern recognition.

Science.gov (United States)

Huang, Yuhong; Busk, Peter Kamp; Grell, Morten Nedergaard; Zhao, Hai; Lange, Lene

2014-12-01

Mucor circinelloides produces plant cell wall degrading enzymes that allow it to grow on complex polysaccharides. Although the genome of M. circinelloides has been sequenced, only few plant cell wall degrading enzymes are annotated in this species. We applied peptide pattern recognition, which is a non-alignment based method for sequence analysis to map conserved sequences in glycoside hydrolase families. The conserved sequences were used to identify similar genes in the M. circinelloides genome. We found 12 different novel genes encoding members of the GH3, GH5, GH9, GH16, GH38, GH47 and GH125 families in M. circinelloides. One of the two GH3-encoding genes was predicted to encode a β-glucosidase (EC 3.2.1.21). We expressed this gene in Pichia pastoris KM71H and found that the purified recombinant protein had relative high β-glucosidase activity (1.73U/mg) at pH5 and 50°C. The Km and Vmax with p-nitrophenyl-β-d-glucopyranoside as substrate was 0.20mM and 2.41U/mg, respectively. The enzyme was not inhibited by glucose and retained 84% activity at glucose concentrations up to 140mM. Although zygomycetes are not considered to be important degraders of lignocellulosic biomass in nature, the present finding of an active β-glucosidase in M. circinelloides demonstrates that enzymes from this group of fungi have a potential for cellulose degradation. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of Total Flavonoids, Myricetin, and Quercetin from Hovenia dulcis Thunb. As Inhibitors of α-Amylase and α-Glucosidase.

Science.gov (United States)

Meng, Yonghong; Su, Anping; Yuan, Shuang; Zhao, Huaguo; Tan, Siyuan; Hu, Chingyuan; Deng, Hong; Guo, Yurong

2016-12-01

This study was designed to investigate the inhibition effect and mechanism of total flavonoids, myricetin and quercetin extracted from Hovenia dulcis Thunb. on α-amylase and α-glucosidase in order to explore the potential use of Hovenia flavonoids in alleviating postprandial hyperglycemia. The results demonstrate that total flavonoids, myricetin, and quercetin were effective inhibitors of α-amylase with IC 50 values of 32.8, 662 and 770 μg ml -1 , respectively. And all three were effective inhibitors of α-glucosidase with IC 50 values of 8, 3 and 32 μg ml -1 , respectively. Enzyme kinetics tests and Lineweaver-Burk results showed the inhibition effects of total flavonoids, myricetin and quercrtin on α-amylase were all reversible and competitive, and the effects on α-glucosidase were all reversible but non-competitive. This study revealed that Hovenia flavonoids, especially myricetin, are effective and promising functional foods in alleviating type 2 diabetes mellitus.

Separation of antioxidant and α-glucosidase inhibitory flavonoids from the aerial parts of Asterothamnus centrali-asiaticus.

Science.gov (United States)

Wang, Yan-Ming; Zhao, Jian-Qiang; Yang, Jun-Li; Tao, Yan-Duo; Mei, Li-Juan; Shi, Yan-Ping

2017-06-01

A new flavonoid, along with 16 known ones, was separated from the aerial parts of Asterothamnus centrali-asiaticus. Their structures were elucidated by extensive spectroscopic methods, including 1D and 2D NMR techniques and HRESIMS. To confirm the structure of the new compound, computational prediction of its 13 C chemical shifts was performed. All of the 17 flavonoids were reported from A. centrali-asiaticus for the first time. In addition, all flavonoids were evaluated for their antioxidant and α-glucosidase inhibitory activities. The results showed that 10 of them exhibited antioxidant activity. Meanwhile, four flavonoids displayed α-glucosidase inhibitory effect with IC 50 values ranging from 38.9 to 299.7 μM.

The α-amylase and α-glucosidase inhibitory effects of Irish seaweed extracts.

Science.gov (United States)

Lordan, Sinéad; Smyth, Thomas J; Soler-Vila, Anna; Stanton, Catherine; Ross, R Paul

2013-12-01

To date, numerous studies have reported on the antidiabetic properties of various plant extracts through inhibition of carbohydrate-hydrolysing enzymes. The objective of this research was to evaluate extracts of seaweeds for α-amylase and α-glucosidase inhibitory effects. Cold water and ethanol extracts of 15 seaweeds were initially screened and from this, five brown seaweed species were chosen. The cold water and ethanol extracts of Ascophyllum nodosum had the strongest α-amylase inhibitory effect with IC50 values of 53.6 and 44.7 μg/ml, respectively. Moreover, the extracts of Fucus vesiculosus Linnaeus were found to be potent inhibitors of α-glucosidase with IC50 values of 0.32 and 0.49 μg/ml. The observed effects were associated with the phenolic content and antioxidant activity of the extracts, and the concentrations used were below cytotoxic levels. Overall, our findings suggest that brown seaweed extracts may limit the release of simple sugars from the gut and thereby alleviate postprandial hyperglycaemia. Copyright © 2013. Published by Elsevier Ltd.

Carbon dots for fluorescent detection of α-glucosidase activity using enzyme activated inner filter effect and its application to anti-diabetic drug discovery

Energy Technology Data Exchange (ETDEWEB)

Kong, Weiheng [Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu 273165 (China); Wu, Di [School of Life Sciences, Xiamen University, Xiamen 361005 (China); Xia, Lian [Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu 273165 (China); Chen, Xuefeng [School of Food and Biological Engineering, Shaanxi University of Science & Technology, Xian 710021 (China); Li, Guoliang, E-mail: 61254368@163.com [School of Food and Biological Engineering, Shaanxi University of Science & Technology, Xian 710021 (China); Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu 273165 (China); Key Laboratory of Food Safety Risk Assessment, Ministry of Health, China National Centre for Food Safety Risk Assessment, Beijing 100021 (China); Qiu, Nannan [Key Laboratory of Food Safety Risk Assessment, Ministry of Health, China National Centre for Food Safety Risk Assessment, Beijing 100021 (China); Chen, Guang; Sun, Zhiwei; You, Jinmao [Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu 273165 (China); Wu, Yongning, E-mail: wuyongning@cfsa.net.cn [Key Laboratory of Food Safety Risk Assessment, Ministry of Health, China National Centre for Food Safety Risk Assessment, Beijing 100021 (China)

2017-06-22

Recently, α-glucosidase inhibitor has been widely used in clinic for diabetic therapy. In the present study, a facile and sensitive fluorescent assay based on enzyme activated inner filter effect (IFE) on nitrogen-doped carbon dots (CDs) was first developed for the detection of α-glucosidase. The N-doped CDs with green emission were prepared by a one-step hydrothermal synthesis and gave the fluorescence quantum yield of 30%, which were used as the signal output. Through α-glucosidase catalysis, 4-nitrophenol was released from 4-nitrophenyl-α-D-glucopyranoside (NGP). Interestingly, the absorption of 4-nitrophenol and the excitation of CDs were completely overlapping. Due to its great molar absorptivity, 4-nitrophenol was capable of acting as a powerful absorber to affect the fluorescent signal of CDs (i.e. IFE). By converting the absorption signals into fluorescence signals, the facile fluorescence assay strategy could be realized for α-glucosidase activity sensing, which effectively avoided the complex modification of the surface of CDs or construction of the nanoprobes. The established IFE-based sensing platform offered a low detection limit of 0.01 U/mL (S/N = 3). This proposed sensing approach has also been expanded to the inhibitor screening and showed excellent applicability. As a typical α-glucosidase inhibitor, acarbose was investigated with a low detection limit of 10{sup −8} M. This developed method enjoyed many merits including simplicity, lost cost, high sensitivity, good reproducibility and excellent selectivity, which also provided a new insight on the application of CDs to develop the facile and sensitive biosensor. - Highlights: • Green N-doped CDs were first prepared by a facile synthesis process. • IFE-based sensor without covalent linking or surface modifications was developed. • The method was successfully applied to α-glucosidase detection. • The method can be employed for sensitive screening of anti-diabetes drugs.

Mammalian mucosal α-glucosidases coordinate with α-amylase in the initial starch hydrolysis stage to have a role in starch digestion beyond glucogenesis.

Science.gov (United States)

Dhital, Sushil; Lin, Amy Hui-Mei; Hamaker, Bruce R; Gidley, Michael J; Muniandy, Anbuhkani

2013-01-01

Starch digestion in the human body is typically viewed in a sequential manner beginning with α-amylase and followed by α-glucosidase to produce glucose. This report indicates that the two enzyme types can act synergistically to digest granular starch structure. The aim of this study was to investigate how the mucosal α-glucosidases act with α-amylase to digest granular starch. Two types of enzyme extracts, pancreatic and intestinal extracts, were applied. The pancreatic extract containing predominantly α-amylase, and intestinal extract containing a combination of α-amylase and mucosal α-glucosidase activities, were applied to three granular maize starches with different amylose contents in an in vitro system. Relative glucogenesis, released maltooligosaccharide amounts, and structural changes of degraded residues were examined. Pancreatic extract-treated starches showed a hydrolysis limit over the 12 h incubation period with residues having a higher gelatinization temperature than the native starch. α-Amylase combined with the mucosal α-glucosidases in the intestinal extract showed higher glucogenesis as expected, but also higher maltooligosaccharide amounts indicating an overall greater degree of granular starch breakdown. Starch residues after intestinal extract digestion showed more starch fragmentation, higher gelatinization temperature, higher crystallinity (without any change in polymorph), and an increase of intermediate-sized or small-sized fractions of starch molecules, but did not show preferential hydrolysis of either amylose or amylopectin. Direct digestion of granular starch by mammalian recombinant mucosal α-glucosidases was observed which shows that these enzymes may work either independently or together with α-amylase to digest starch. Thus, mucosal α-glucosidases can have a synergistic effect with α-amylase on granular starch digestion, consistent with a role in overall starch digestion beyond their primary glucogenesis function.

Carbon dots for fluorescent detection of α-glucosidase activity using enzyme activated inner filter effect and its application to anti-diabetic drug discovery

International Nuclear Information System (INIS)

Kong, Weiheng; Wu, Di; Xia, Lian; Chen, Xuefeng; Li, Guoliang; Qiu, Nannan; Chen, Guang; Sun, Zhiwei; You, Jinmao; Wu, Yongning

2017-01-01

Recently, α-glucosidase inhibitor has been widely used in clinic for diabetic therapy. In the present study, a facile and sensitive fluorescent assay based on enzyme activated inner filter effect (IFE) on nitrogen-doped carbon dots (CDs) was first developed for the detection of α-glucosidase. The N-doped CDs with green emission were prepared by a one-step hydrothermal synthesis and gave the fluorescence quantum yield of 30%, which were used as the signal output. Through α-glucosidase catalysis, 4-nitrophenol was released from 4-nitrophenyl-α-D-glucopyranoside (NGP). Interestingly, the absorption of 4-nitrophenol and the excitation of CDs were completely overlapping. Due to its great molar absorptivity, 4-nitrophenol was capable of acting as a powerful absorber to affect the fluorescent signal of CDs (i.e. IFE). By converting the absorption signals into fluorescence signals, the facile fluorescence assay strategy could be realized for α-glucosidase activity sensing, which effectively avoided the complex modification of the surface of CDs or construction of the nanoprobes. The established IFE-based sensing platform offered a low detection limit of 0.01 U/mL (S/N = 3). This proposed sensing approach has also been expanded to the inhibitor screening and showed excellent applicability. As a typical α-glucosidase inhibitor, acarbose was investigated with a low detection limit of 10"−"8 M. This developed method enjoyed many merits including simplicity, lost cost, high sensitivity, good reproducibility and excellent selectivity, which also provided a new insight on the application of CDs to develop the facile and sensitive biosensor. - Highlights: • Green N-doped CDs were first prepared by a facile synthesis process. • IFE-based sensor without covalent linking or surface modifications was developed. • The method was successfully applied to α-glucosidase detection. • The method can be employed for sensitive screening of anti-diabetes drugs.

Mammalian mucosal α-glucosidases coordinate with α-amylase in the initial starch hydrolysis stage to have a role in starch digestion beyond glucogenesis.

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Sushil Dhital

Full Text Available Starch digestion in the human body is typically viewed in a sequential manner beginning with α-amylase and followed by α-glucosidase to produce glucose. This report indicates that the two enzyme types can act synergistically to digest granular starch structure. The aim of this study was to investigate how the mucosal α-glucosidases act with α-amylase to digest granular starch. Two types of enzyme extracts, pancreatic and intestinal extracts, were applied. The pancreatic extract containing predominantly α-amylase, and intestinal extract containing a combination of α-amylase and mucosal α-glucosidase activities, were applied to three granular maize starches with different amylose contents in an in vitro system. Relative glucogenesis, released maltooligosaccharide amounts, and structural changes of degraded residues were examined. Pancreatic extract-treated starches showed a hydrolysis limit over the 12 h incubation period with residues having a higher gelatinization temperature than the native starch. α-Amylase combined with the mucosal α-glucosidases in the intestinal extract showed higher glucogenesis as expected, but also higher maltooligosaccharide amounts indicating an overall greater degree of granular starch breakdown. Starch residues after intestinal extract digestion showed more starch fragmentation, higher gelatinization temperature, higher crystallinity (without any change in polymorph, and an increase of intermediate-sized or small-sized fractions of starch molecules, but did not show preferential hydrolysis of either amylose or amylopectin. Direct digestion of granular starch by mammalian recombinant mucosal α-glucosidases was observed which shows that these enzymes may work either independently or together with α-amylase to digest starch. Thus, mucosal α-glucosidases can have a synergistic effect with α-amylase on granular starch digestion, consistent with a role in overall starch digestion beyond their primary

Mammalian Mucosal α-Glucosidases Coordinate with α-Amylase in the Initial Starch Hydrolysis Stage to Have a Role in Starch Digestion beyond Glucogenesis

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Dhital, Sushil; Lin, Amy Hui-Mei; Hamaker, Bruce R.; Gidley, Michael J.; Muniandy, Anbuhkani

2013-01-01

Starch digestion in the human body is typically viewed in a sequential manner beginning with α-amylase and followed by α-glucosidase to produce glucose. This report indicates that the two enzyme types can act synergistically to digest granular starch structure. The aim of this study was to investigate how the mucosal α-glucosidases act with α-amylase to digest granular starch. Two types of enzyme extracts, pancreatic and intestinal extracts, were applied. The pancreatic extract containing predominantly α-amylase, and intestinal extract containing a combination of α-amylase and mucosal α-glucosidase activities, were applied to three granular maize starches with different amylose contents in an in vitro system. Relative glucogenesis, released maltooligosaccharide amounts, and structural changes of degraded residues were examined. Pancreatic extract-treated starches showed a hydrolysis limit over the 12 h incubation period with residues having a higher gelatinization temperature than the native starch. α-Amylase combined with the mucosal α-glucosidases in the intestinal extract showed higher glucogenesis as expected, but also higher maltooligosaccharide amounts indicating an overall greater degree of granular starch breakdown. Starch residues after intestinal extract digestion showed more starch fragmentation, higher gelatinization temperature, higher crystallinity (without any change in polymorph), and an increase of intermediate-sized or small-sized fractions of starch molecules, but did not show preferential hydrolysis of either amylose or amylopectin. Direct digestion of granular starch by mammalian recombinant mucosal α-glucosidases was observed which shows that these enzymes may work either independently or together with α-amylase to digest starch. Thus, mucosal α-glucosidases can have a synergistic effect with α-amylase on granular starch digestion, consistent with a role in overall starch digestion beyond their primary glucogenesis function. PMID

Enzymatic Synthesis of the Flavone Glucosides, Prunin and Isoquercetin, and the Aglycones, Naringenin and Quercetin, with Selective α-L-Rhamnosidase and β-D-Glucosidase Activities of Naringinase

Directory of Open Access Journals (Sweden)

Hélder Vila-Real

2011-01-01

Full Text Available The production of flavonoid glycosides by removing rhamnose from rutinosides can be accomplished through enzymatic catalysis. Naringinase is an enzyme complex, expressing both α-L-rhamnosidase and β-D-glucosidase activities, with application in glycosides hydrolysis. To produce monoglycosylated flavonoids with naringinase, the expression of β-D-glucosidase activity is not desirable leading to the need of expensive methods for α-L-rhamnosidase purification. Therefore, the main purpose of this study was the inactivation of β-D-glucosidase activity expressed by naringinase keeping α-L-rhamnosidase with a high retention activity. Response surface methodology (RSM was used to evaluate the effects of temperature and pH on β-D-glucosidase inactivation. A selective inactivation of β-D-glucosidase activity of naringinase was achieved at 81.5∘C and pH 3.9, keeping a very high residual activity of α-L-rhamnosidase (78%. This was a crucial achievement towards an easy and cheap production method of very expensive flavonoids, like prunin and isoquercetin starting from naringin and rutin, respectively.

Direct ethanol production from barley beta-glucan by sake yeast displaying Aspergillus oryzae beta-glucosidase and endoglucanase.

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Kotaka, Atsushi; Bando, Hiroki; Kaya, Masahiko; Kato-Murai, Michiko; Kuroda, Kouichi; Sahara, Hiroshi; Hata, Yoji; Kondo, Akihiko; Ueda, Mitsuyoshi

2008-06-01

Three beta-glucosidase- and two endoglucanase-encoding genes were cloned from Aspergillus oryzae, and their gene products were displayed on the cell surface of the sake yeast, Saccharomyces cerevisiae GRI-117-UK. GRI-117-UK/pUDB7 displaying beta-glucosidase AO090009000356 showed the highest activity against various substrates and efficiently produced ethanol from cellobiose. On the other hand, GRI-117-UK/pUDCB displaying endoglucanase AO090010000314 efficiently degraded barley beta-glucan to glucose and smaller cellooligosaccharides. GRI-117-UK/pUDB7CB codisplaying both beta-glucosidase AO090009000356 and endoglucanase AO090010000314 was constructed. When direct ethanol fermentation from 20 g/l barley beta-glucan as a model substrate was performed with the codisplaying strain, the ethanol concentration reached 7.94 g/l after 24 h of fermentation. The conversion ratio of ethanol from beta-glucan was 69.6% of the theoretical ethanol concentration produced from 20 g/l barley beta-glucan. These results showed that sake yeast displaying A. oryzae cellulolytic enzymes can be used to produce ethanol from cellulosic materials. Our constructs have higher ethanol production potential than the laboratory constructs previously reported.

The catalytic potency of ß-glucosidase from Pyroccus furiosus in the direct glucosylation reaction

NARCIS (Netherlands)

Roode, de B.M.; Meer, van der T.D.; Kaper, T.; Franssen, M.C.R.; Padt, van der A.; Oost, van der J.; Boom, R.M.

2001-01-01

Enzymes from extremophiles operate at conditions that are different from their `normal' counterparts, and are therefore a useful extension of the enzyme toolbox. In this paper, the direct glucosylation reaction mediated by a hyperthermophilic -glucosidase from Pyrocuccus furiosus was investigated.

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.

Glucosidase inhibitory activity and antioxidant activity of flavonoid compound and triterpenoid compound from Agrimonia Pilosa Ledeb.

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Liu, Xi; Zhu, Liancai; Tan, Jun; Zhou, Xuemei; Xiao, Ling; Yang, Xian; Wang, Bochu

2014-01-10

In Chinese traditional medicine, Agrimonia pilosa Ledeb (APL) exhibits great effect on treatment of type 2 diabetes mellitus (T2DM), however its mechanism is still unknown. Considering that T2DM are correlated with postprandial hyperglycemia and oxidative stress, we investigated the α-glucosidase inhibitory activity and the antioxidant activity of flavonoid compound (FC) and triterpenoid compound (TC) from APL. Entire plants of APL were extracted using 95% ethanol and 50% ethanol successively. The resulting extracts were partitioned and isolated by applying liquid chromatography using silica gel column and Sephadex LH 20 column to give FC and TC. The content of total flavonoids in FC and the content of total triterpenoids in TC were determined by using UV spectrophotometry. HPLC analysis was used to identify and quantify the monomeric compound in FC and TC. The α-glucosidase inhibitory activities were determined using the chromogenic method with p-nitrophenyl-α-D-glucopyranoside as substrate. Antioxidant activities were assessed through three kinds of radical scavenging assays (DPPH radical, ABTS radical and hydroxyl radical) & β-carotene-linoleic acid assay. The results indicate FC is abundant of quercitrin, and hyperoside, and TC is abundant of 1β, 2β, 3β, 19α-tetrahydroxy-12-en-28-oic acid (265.2 mg/g) and corosolic acid (100.9 mg/g). The FC & the TC have strong α-glucosidase inhibitory activities with IC50 of 8.72 μg/mL and 3.67 μg/mL, respectively. We find that FC show competitive inhibition against α-glucosidase, while the TC exhibits noncompetitive inhibition. Furthermore, The FC exhibits significant radical scavenging activity with the EC50 values of 7.73 μg/mL, 3.64 μg/mL and 5.90 μg/mL on DPPH radical, hydroxyl radical and ABTS radical, respectively. The FC also shows moderate anti-lipid peroxidation activity with the IC50 values of 41.77 μg/mL on inhibiting β-carotene bleaching. These results imply that the FC and the TC could be

Magnetic Ligand Fishing as a Targeting Tool for HPLC-HRMS-SPE-NMR: α-Glucosidase Inhibitory Ligands and Alkylresorcinol Glycosides from Eugenia catharinae.

Science.gov (United States)

Wubshet, Sileshi G; Brighente, Inês M C; Moaddel, Ruin; Staerk, Dan

2015-11-25

A bioanalytical platform combining magnetic ligand fishing for α-glucosidase inhibition profiling and HPLC-HRMS-SPE-NMR for structural identification of α-glucosidase inhibitory ligands, both directly from crude plant extracts, is presented. Magnetic beads with N-terminus-coupled α-glucosidase were synthesized and characterized for their inherent catalytic activity. Ligand fishing with the immobilized enzyme was optimized using an artificial test mixture consisting of caffeine, ferulic acid, and luteolin before proof-of-concept with the crude extract of Eugenia catharinae. The combination of ligand fishing and HPLC-HRMS-SPE-NMR identified myricetin 3-O-α-L-rhamnopyranoside, myricetin, quercetin, and kaempferol as α-glucosidase inhibitory ligands in E. catharinae. Furthermore, HPLC-HRMS-SPE-NMR analysis led to identification of six new alkylresorcinol glycosides, i.e., 5-(2-oxopentyl)resorcinol 4-O-β-D-glucopyranoside, 5-propylresorcinol 4-O-β-D-glucopyranoside, 5-pentylresorcinol 4-O-[α-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside, 5-pentylresorcinol 4-O-β-D-glucopyranoside, 4-hydroxy-3-O-methyl-5-pentylresorcinol 1-O-β-D-glucopyranoside, and 3-O-methyl-5-pentylresorcinol 1-O-[β-D-glucopyranosyl-(1→6)]-β-D-glucopyranoside.

Induction and catabolite repression of α-glucosidase synthesis in protoplasts of Saccharomyces carlsbergensis

NARCIS (Netherlands)

Wijk, R. van; Ouwehand, J.; Bos, T. van den; Koningsberger, V.V.

1969-01-01

1. 1. Kinetic data on the repression, the derepression and the induction of α-glucosidase synthesis in protoplasts of Saccharomyces carlsbergensis suggested that some site other than the stereospecific site for the induction by maltose was involved in the repression by glucose. 2. 2. A study of the

A β-glucosidase hyper-production Trichoderma reesei mutant reveals a potential role of cel3D in cellulase production.

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Li, Chengcheng; Lin, Fengming; Li, Yizhen; Wei, Wei; Wang, Hongyin; Qin, Lei; Zhou, Zhihua; Li, Bingzhi; Wu, Fugen; Chen, Zhan

2016-09-01

The conversion of cellulose by cellulase to fermentable sugars for biomass-based products such as cellulosic biofuels, biobased fine chemicals and medicines is an environment-friendly and sustainable process, making wastes profitable and bringing economic benefits. Trichoderma reesei is the well-known major workhorse for cellulase production in industry, but the low β-glucosidase activity in T. reesei cellulase leads to inefficiency in biomass degradation and limits its industrial application. Thus, there are ongoing interests in research to develop methods to overcome this insufficiency. Moreover, although β-glucosidases have been demonstrated to influence cellulase production and participate in the regulation of cellulase production, the underlying mechanism remains unclear. The T. reesei recombinant strain TRB1 was constructed from T. reesei RUT-C30 by the T-DNA-based mutagenesis. Compared to RUT-C30, TRB1 displays a significant enhancement of extracellular β-glucosidase (BGL1) activity with 17-fold increase, a moderate increase of both the endoglucanase (EG) activity and the exoglucanase (CBH) activity, a minor improvement of the total filter paper activity, and a faster cellulase induction. This superiority of TRB1 over RUT-C30 is independent on carbon sources and improves the saccharification ability of TRB1 cellulase on pretreated corn stover. Furthermore, TRB1 shows better resistance to carbon catabolite repression than RUT-C30. Secretome characterization of TRB1 shows that the amount of CBH, EG and BGL in the supernatant of T. reesei TRB1 was indeed increased along with the enhanced activities of these three enzymes. Surprisingly, qRT-PCR and gene cloning showed that in TRB1 β-glucosidase cel3D was mutated through the random insertion by AMT and was not expressed. The T. reesei recombinant strain TRB1 constructed in this study is more desirable for industrial application than the parental strain RUT-C30, showing extracellular β-glucosidase hyper

Quickly Screening for Potential α-Glucosidase Inhibitors from Guava Leaves Tea by Bioaffinity Ultrafiltration Coupled with HPLC-ESI-TOF/MS Method.

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Wang, Lu; Liu, Yufeng; Luo, You; Huang, Kuiying; Wu, Zhenqiang

2018-02-14

Guava leaves tea (GLT) has a potential antihyperglycemic effect. Nevertheless, it is unclear which compound plays a key role in reducing blood sugar. In this study, GLT extract (IC 50 = 19.37 ± 0.21 μg/mL) exhibited a stronger inhibitory potency against α-glucosidase than did acarbose (positive control) at IC 50 = 178.52 ± 1.37 μg/mL. To rapidly identify the specific α-glucosidase inhibitor components from GLT, an approach based on bioaffinity ultrafiltration combined with high performance liquid chromatography coupled to electrospray ionization-time-of-flight-mass spectrometry (BAUF-HPLC-ESI-TOF/MS) was developed. Under the optimal bioaffinity ultrafiltration conditions, 11 corresponding potential α-glucosidase inhibitors with high affinity degrees (ADs) were screened and identified from the GLT extract. Quercetin (IC 50 = 4.51 ± 0.71 μg/mL) and procyanidin B3 (IC 50 = 28.67 ± 5.81 μg/mL) were determined to be primarily responsible for the antihyperglycemic effect, which further verified the established screening method. Moreover, structure-activity relationships were discussed. In conclusion, the BAUF-HPLC-ESI-TOF/MS method could be applied to determine the potential α-glucosidase inhibitors from complex natural products quickly.

Drug treatment of type 2 diabetes mellitus in patients for whom metformin is contraindicated [Corrigendum

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Irons BK

2017-10-01

Full Text Available Irons BK, Minze MG. Drug treatment of type 2 diabetes mellitus in patients for whom metformin is contraindicated. Diabetes Metab Syndr Obes. 2014;7:15–24.There is an error in the text on page 19:“Alpha-glucosidase inhibitors are infrequently used in the treatment of T2DM.3 These agents competitively inhibit the absorption of alpha-glucosidase along the brush border of the small intestine, which slows gut absorption of carbohydrates and ultimately reduces post-prandial blood glucose.34–36” should read as “Alpha-glucosidase inhibitors are infrequently used in the treatment of T2DM.3 These agents competitively inhibit the degradation of complex carbohydrates along the brush border of the small intestine, which slows gut absorption of metabolized carbohydrates and ultimately reduces post-prandial blood glucose.34–36”Read the original article

Improved oligosaccharide synthesis by protein engineering of b-glucosidase from hyperthermophilic Pyrococcus furiosus

NARCIS (Netherlands)

Hanson, T.; Kaper, T.; Oost, van der J.; Vos, de W.M.

2001-01-01

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type -glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type -mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme

Effect of Senna alata (L) Roxb (Fabaceae) Leaf Extracts on alpha ...

African Journals Online (AJOL)

Purpose: To investigate the inhibitory effect of S. alata leaf extracts on α-amylase and α-glucosidase, and its potential for reducing postprandial blood glucose level of rats. Methods: The α-amylase and α-glucosidase inhibitory potentials of acetone, ethylacetate and hexane extracts of S. alata were investigated by reacting ...

Comprehensive enzymatic analysis of the amylolytic system in the digestive fluid of the sea hare, Aplysia kurodai: Unique properties of two α-amylases and two α-glucosidases

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Akihiko Tsuji

2014-01-01

Full Text Available Sea lettuce (Ulva pertusa is a nuisance species of green algae that is found all over the world. East-Asian species of the marine gastropod, the sea hare Aplysia kurodai, shows a clear feeding preference for sea lettuce. Compared with cellulose, sea lettuce contains a higher amount of starch as a storage polysaccharide. However, the entire amylolytic system in the digestive fluid of A. kurodai has not been studied in detail. We purified α-amylases and α-glucosidases from the digestive fluid of A. kurodai and investigated the synergistic action of these enzymes on sea lettuce. A. kurodai contain two α-amylases (59 and 80 kDa and two α-glucosidases (74 and 86 kDa. The 59-kDa α-amylase, but not the 80-kDa α-amylase, was markedly activated by Ca2+ or Cl−. Both α-amylases degraded starch and maltoheptaose, producing maltotriose, maltose, and glucose. Glucose production from starch was higher with 80-kDa α-amylase than with 59-kDa α-amylase. Kinetic analysis indicated that 74-kDa α-glucosidase prefers short α-1,4-linked oligosaccharide, whereas 86-kDa α-glucosidase prefers large α-1,6 and α-1,4-linked polysaccharides such as glycogen. When sea lettuce was used as a substrate, a 2-fold greater amount of glucose was released by treatment with 59-kDa α-amylase and 74-kDa α-glucosidase than by treatment with 45-kDa cellulase and 210-kDa β-glucosidase of A. kurodai. Unlike mammals, sea hares efficiently digest sea lettuce to glucose by a combination of two α-amylases and two α-glucosidases in the digestive fluids without membrane-bound maltase–glucoamylase and sucrase–isomaltase complexes.

Characterization and kinetic analysis of a thermostable GH3 ß-glucosidase from Penicillium brasilianum

DEFF Research Database (Denmark)

Krogh, Kristian Bertel Rømer; Harris, P.V.; Olsen, C.L.

2010-01-01

A GH3 beta-glucosidase (BGL) from Penicillium brasilianum was purified to homogeneity after cultivation on a cellulose and xylan rich medium. The BGL was identified in a genomic library, and it was successfully expressed in Aspergillus oryzae. The BGL had excellent stability at elevated...

Functional diversity of family 3 β-glucosidases from thermophilic cellulolytic fungus Humicola insolens Y1.

Science.gov (United States)

Xia, Wei; Bai, Yingguo; Cui, Ying; Xu, Xinxin; Qian, Lichun; Shi, Pengjun; Zhang, Wei; Luo, Huiying; Zhan, Xiuan; Yao, Bin

2016-06-08

The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose. However, studies on the β-glucosidases from this fungus remain insufficient, especially on glycosyl hydrolase family 3 enzymes. In the present study, we analyzed the functional diversity of three distant family 3 β-glucosidases from Humicola insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastoris strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50-60 °C and pH 5.5-6.0) and high glucose tolerance, but differed in substrate specificities and kinetics. HiBgl3B was solely active towards aryl β-glucosides while HiBgl3A and HiBgl3C showed broad substrate specificities including both disaccharides and aryl β-glucosides. Of the three enzymes, HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Substitutions of three key residues Ile48, Ile278 and Thr484 of HiBgl3B to the corresponding residues of HiBgl3A conferred the enzyme activity towards sophorose, and vice versa. This study reveals the functional diversity of GH3 β-glucosidases as well as the key residues in recognizing +1 subsite of different substrates.

Mammalian Mucosal ?-Glucosidases Coordinate with ?-Amylase in the Initial Starch Hydrolysis Stage to Have a Role in Starch Digestion beyond Glucogenesis

OpenAIRE

Dhital, Sushil; Lin, Amy Hui-Mei; Hamaker, Bruce R.; Gidley, Michael J.; Muniandy, Anbuhkani

2013-01-01

Starch digestion in the human body is typically viewed in a sequential manner beginning with α-amylase and followed by α-glucosidase to produce glucose. This report indicates that the two enzyme types can act synergistically to digest granular starch structure. The aim of this study was to investigate how the mucosal α-glucosidases act with α-amylase to digest granular starch. Two types of enzyme extracts, pancreatic and intestinal extracts, were applied. The pancreatic extract containing pre...

Production and biochemical characterization of α-glucosidase from Aspergillus niger ITV-01 isolated from sugar cane bagasse.

Science.gov (United States)

Del Moral, S; Barradas-Dermitz, D M; Aguilar-Uscanga, M G

2018-01-01

Aspergillus niger ITV-01 presents amylolytic activity, identified as α-glucosidase, an enzyme that only produces α-d-glucose from soluble starch and that presents transglucosylase activity on α-d-glucopyranosyl-(1-4)-α-d-glucopyranose (maltose) (200 gL -1 ). Biochemical characterization was performed on A. niger ITV-01 α-glucosidase; its optimum parameters were pH 4.3, temperature 80 °C but stable at 40 °C, with an energy of activation (Ea) 176.25 kJ mol -1 . Using soluble starch as the substrate, K m and V max were 5 mg mL -1 and 1000 U mg -1 , respectively. As α-glucosidase is not a metalloenzyme, calcium and EDTA did not have any effect on its activity. The molecular weight was estimated by SDS-PAGE to be about 75 kDa. It was also active in methanol and ethanol. When ammonium sulfate (AS) and yeast extract (YE) nitrogen sources and calcium effect were evaluated, the greatest activity occurred using YE and calcium, as opposed to AS media where no activity was detected. The results obtained showed that this enzyme has industrial application potential in the processes to produce either ethanol or malto-oligosaccharides from α-d-glucopyranosyl-(1-4)-α-d-glucopyranose (maltose).

Physiochemical and Thermodynamic Characterization of Highly Active Mutated Aspergillus niger β-glucosidase for Lignocellulose Hydrolysis.

Science.gov (United States)

Javed, Muhammad Rizwan; Rashid, Muhammad Hamid; Riaz, Muhammad; Nadeem, Habibullah; Qasim, Muhammad; Ashiq, Nourin

2018-01-01

Cellulose represents a major source of fermentable sugars in lignocellulosic biomass and a combined action of hydrolytic enzymes (exoglucanases , endoglucanases and β-glucosidases) is required to effectively convert cellulose to glucose that can be fermented to bio-ethanol. However, in-order to make the production of bio-ethanol an economically feasible process, the costs of the enzymes to be used for hydrolysis of the raw material need to be reduced and an increase in specific activity or production efficiency of cellulases is required. Among the cellulases, β-glucosidase not only hydrolyzes cellobiose to glucose but it also reduces the cellobiose inhibition, resulting in efficient functioning of endo- and exo-glucanases. Therefore, in the current study kinetic and thermodynamic characteristics of highly active β-glucosidase from randomly mutated Aspergillus niger NIBGE-06 have been evaluated for its industrial applications. The main objective of this study was the identification of mutations and determination of their effect on the physiochemical, kinetic and thermodynamic characteristics of β-glucosidase activity and stability. Pure cultures of Aspergillus niger NIBGE and its 2-Deoxy-D-glucose resistant γ-rays mutant Aspergillus niger NIBGE-06 were grown on Vogel's medium containing wheat bran (3% w/v), at 30±1 °C for 96-108 h. Crude enzymes from both strains were subjected to ammonium sulfate precipitation and column chromatography on Fast Protein Liquid Chromatography (FPLC) system. The purified β-glucosidases from both fungal sources were characterized for their native and subunit molecular mass through FPLC and SDS-PAGE, respectively. The purified enzymes were then comparatively characterized for their optimum temperature, activation energy (Ea), temperature quotient (Q10), Optimum pH, Heat of ionization (ΔHI) of active site residues , Michaelis-Menten constants (Vmax, Km, kcat and kcat/Km) and thermodynamics of irreversible inactivation through

Soluble and Membrane-Bound β-Glucosidases Are Involved in Trimming the Xyloglucan Backbone.

Science.gov (United States)

Sampedro, Javier; Valdivia, Elene R; Fraga, Patricia; Iglesias, Natalia; Revilla, Gloria; Zarra, Ignacio

2017-02-01

In many flowering plants, xyloglucan is a major component of primary cell walls, where it plays an important role in growth regulation. Xyloglucan can be degraded by a suite of exoglycosidases that remove specific sugars. In this work, we show that the xyloglucan backbone, formed by (1→4)-linked β-d-glucopyranosyl residues, can be attacked by two different Arabidopsis (Arabidopsis thaliana) β-glucosidases from glycoside hydrolase family 3. While BGLC1 (At5g20950; for β-glucosidase active against xyloglucan 1) is responsible for all or most of the soluble activity, BGLC3 (At5g04885) is usually a membrane-anchored protein. Mutations in these two genes, whether on their own or combined with mutations in other exoglycosidase genes, resulted in the accumulation of partially digested xyloglucan subunits, such as GXXG, GXLG, or GXFG. While a mutation in BGLC1 had significant effects on its own, lack of BGLC3 had only minor effects. On the other hand, double bglc1 bglc3 mutants revealed a synergistic interaction that supports a role for membrane-bound BGLC3 in xyloglucan metabolism. In addition, bglc1 bglc3 was complemented by overexpression of either BGLC1 or BGLC3 In overexpression lines, BGLC3 activity was concentrated in a microsome-enriched fraction but also was present in soluble form. Finally, both genes were generally expressed in the same cell types, although, in some cases, BGLC3 was expressed at earlier stages than BGLC1 We propose that functional specialization could explain the separate localization of both enzymes, as a membrane-bound β-glucosidase could specifically digest soluble xyloglucan without affecting the wall-bound polymer. © 2017 American Society of Plant Biologists. All Rights Reserved.

Chemical constituents of gold-red apple and their α-glucosidase inhibitory activities.

Science.gov (United States)

He, Qian-Qian; Yang, Liu; Zhang, Jia-Yu; Ma, Jian-Nan; Ma, Chao-Mei

2014-10-01

Ten compounds were isolated and purified from the peels of gold-red apple (Malus domestica) for the 1st time. The identified compounds are 3β, 20β-dihydroxyursan-28-oic acid (1), 2α-hydroxyoleanolic acid (2), euscaphic acid (3), 3-O-p-coumaroyl tormentic acid (4), ursolic acid (5), 2α-hydroxyursolic acid (6), oleanolic acid (7), betulinic acid (8), linolic acid (9), and α-linolenic acid (10). Their structures were determined by interpreting their nuclear magnetic resonance and mass spectrometry (MS) spectra, and by comparison with literature data. Compound 1 is new, and compound 2 is herein reported for the 1st time for the genus Malus. α-Glucosidase inhibition assay revealed 6 of the triterpenoid isolates as remarkable α-glucosidase inhibitors, with betulinic acid showing the strongest inhibition (IC50 = 15.19 μM). Ultra-performance liquid chromatography-electrospray ionization MS analysis of the fruit peels, pomace, flesh, and juice revealed that the peels and pomace contained high levels of triterpenes, suggesting that wastes from the fruit juice industry could serve as rich sources of bioactive triterpenes. © 2014 Institute of Food Technologists®

Purification, crystallization and preliminary X-ray analysis of a hexameric β-glucosidase from wheat

International Nuclear Information System (INIS)

Sue, Masayuki; Yamazaki, Kana; Kouyama, Jun-ichi; Sasaki, Yasuyuki; Ohsawa, Kanju; Miyamoto, Toru; Iwamura, Hajime; Yajima, Shunsuke

2005-01-01

Recombinant β-glucosidase from wheat seedlings complexed with a substrate aglycone has been crystallized in a hexameric active form. A diffraction data set has been collected at 1.7 Å. The wheat β-glucosidase TaGlu1b, which is only active in a hexameric form, was tagged with 6×His at the N-terminus, overexpressed in Escherichia coli and purified in two steps. The protein complexed with a substrate aglycone was crystallized at 293 K from a solution containing 10 mM HEPES pH 7.2, 1 M LiSO 4 and 150 mM NaCl using the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.7 Å at the Photon Factory. The crystal belongs to space group P4 1 32, with unit-cell parameters a = b = c = 194.65 Å, α = β = γ = 90°. The asymmetric unit was confirmed by molecular-replacement solution to contain one monomer, giving a solvent content of 72.1%

Small molecule inhibitors of ER α-glucosidases are active against multiple hemorrhagic fever viruses.

Science.gov (United States)

Chang, Jinhong; Warren, Travis K; Zhao, Xuesen; Gill, Tina; Guo, Fang; Wang, Lijuan; Comunale, Mary Ann; Du, Yanming; Alonzi, Dominic S; Yu, Wenquan; Ye, Hong; Liu, Fei; Guo, Ju-Tao; Mehta, Anand; Cuconati, Andrea; Butters, Terry D; Bavari, Sina; Xu, Xiaodong; Block, Timothy M

2013-06-01

Host cellular endoplasmic reticulum α-glucosidases I and II are essential for the maturation of viral glycosylated envelope proteins that use the calnexin mediated folding pathway. Inhibition of these glycan processing enzymes leads to the misfolding and degradation of these viral glycoproteins and subsequent reduction in virion secretion. We previously reported that, CM-10-18, an imino sugar α-glucosidase inhibitor, efficiently protected the lethality of dengue virus infection of mice. In the current study, through an extensive structure-activity relationship study, we have identified three CM-10-18 derivatives that demonstrated superior in vitro antiviral activity against representative viruses from four viral families causing hemorrhagic fever. Moreover, the three novel imino sugars significantly reduced the mortality of two of the most pathogenic hemorrhagic fever viruses, Marburg virus and Ebola virus, in mice. Our study thus proves the concept that imino sugars are promising drug candidates for the management of viral hemorrhagic fever caused by variety of viruses. Copyright © 2013 Elsevier B.V. All rights reserved.

Inhibitory effects of chickpea and Tribulus terrestris on lipase, α-amylase and α-glucosidase.

Science.gov (United States)

Ercan, Pınar; El, Sedef Nehir

2016-08-15

The total saponin content and its in vitro bioaccessibilities in Tribulus terrestris and chickpea were determined by a static in vitro digestion method (COST FA1005 Action INFOGEST). Also, in vitro inhibitory effects of the chosen food samples on lipid and starch digestive enzymes were determined by evaluating the lipase, α-amylase and α-glucosidase activities. The tested T. terrestris and chickpea showed inhibitory activity against α-glucosidase (IC50 6967 ± 343 and 2885 ± 85.4 μg/ml, respectively) and α-amylase (IC50 343 ± 26.2 and 167 ± 6.12 μg/ml, respectively). The inhibitory activities of T. terrestris and chickpea against lipase were 15.3 ± 2.03 and 9.74 ± 1.09 μg/ml, respectively. The present study provides the first evidence that these food samples (T. terrestris, chickpea) are potent inhibitors of key enzymes in digestion of carbohydrates and lipids in vitro. Copyright © 2016 Elsevier Ltd. All rights reserved.

Edible seaweed as future functional food: Identification of α-glucosidase inhibitors by combined use of high-resolution α-glucosidase inhibition profiling and HPLC-HRMS-SPE-NMR

DEFF Research Database (Denmark)

Liu, Bingrui; Kongstad, Kenneth Thermann; Wiese, Stefanie

2016-01-01

-glucosidase inhibition profiling combined with high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy (HR-bioassay/HPLC–HRMS–SPE–NMR). The results showed Ascophyllum nodosum and Fucus vesicolosus to be rich in antioxidants, equaling...... as fatty acids – with oleic acid, linoleic acid and eicosapentaenoic acid being the most potent with IC50 values of 0.069, 0.075 and 0.10 mM, respectively, and showing a mixed-type inhibition mode....

Intestinal α-glucosidase and some pancreatic enzymes inhibitory effect of hydroalcholic extract of Moringa stenopetala leaves.

Science.gov (United States)

Toma, Alemayehu; Makonnen, Eyasu; Mekonnen, Yelamtsehay; Debella, Asfaw; Addisakwattana, Sirichai

2014-06-03

Moringa stenopetala has been used in traditional health systems to treat diabetes mellitus. One of the successful methods to prevent of the onset of diabetes is to control postprandial hyperglycemia by the inhibition of α-glucosidase and pancreatic α-amylase activities, resulting in the aggressive delay of the carbohydrate digestion of absorbable monosaccharides. The aim of the present study is to investigate the effect of the extract of the leaves of Moringa stenopetala on α-glucosidase, pancreatic α-amylase, pancreatic lipase, and pancreatic cholesterol esterase activities, and, therefore find out the relevance of the plant in controlling blood sugar and lipid levels. The dried leaves of Moringa stenopetala were extracted with hydroalcoholic solvent and dried using rotary vapor under reduced pressure. The dried extracts were determined for the total phenolic compounds, flavonoid content and condensed tannins content by using Folin-Ciocateu's reagent, AlCl3 and vanillin assay, respectively. The dried extract of plant-based food was further quantified with respect to intestinal α-glucosidase (maltase and sucrase) inhibition and pancreatic α-amylase inhibition by glucose oxidase method and dinitrosalicylic (DNS) reagent, respectively. The phytochemical analysis indicated that flavonoid, total phenolic, and condensed tannin contents in the extract were 71.73 ± 2.48 mg quercetin equivalent/g of crude extract, 79.81 ± 2.85 mg of gallic acid equivalent/g of crude extract, 8.82 ± 0.77 mg catechin equivalent/g of crude extract, respectively. The extract inhibited intestinal sucrase more than intestinal maltase with IC50 value of 1.47 ± 0.19 mg/ml. It also slightly inhibited pancreatic α-amylase, pancreatic lipase and pancreatic cholesterol esterase. The result demonstrated the beneficial biochemical effects of Moringa stenopetala by inhibiting intestinal α-glucosidase, pancreatic cholesterol esterase and pancreatic lipase activities. A

Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control.

Science.gov (United States)

Satoh, Tadashi; Toshimori, Takayasu; Noda, Masanori; Uchiyama, Susumu; Kato, Koichi

2016-11-01

The glycoside hydrolase family 31 (GH31) α-glucosidases play vital roles in catabolic and regulated degradation, including the α-subunit of glucosidase II (GIIα), which catalyzes trimming of the terminal glucose residues of N-glycan in glycoprotein processing coupled with quality control in the endoplasmic reticulum (ER). Among the known GH31 enzymes, only GIIα functions with its binding partner, regulatory β-subunit (GIIβ), which harbors a lectin domain for substrate recognition. Although the structural data have been reported for GIIα and the GIIβ lectin domain, the interaction mode between GIIα and GIIβ remains unknown. Here, we determined the structure of a complex formed between GIIα and the GIIα-binding domain of GIIβ, thereby providing a structural basis underlying the functional extension of this unique GH31 enzyme. © 2016 The Protein Society.

Silver(I) complexes of 2,4-dihydroxybenzaldehyde-amino acid Schiff bases-Novel noncompetitive α-glucosidase inhibitors.

Science.gov (United States)

Zheng, Jingwei; Ma, Lin

2015-01-01

A series of silver(I) complexes of 2,4-dihydroxybenzaldehyde-amino acid Schiff bases were designed and tested for α-glucosidase inhibition. Our results indicate that all the silver complexes (4a-18a) possessed strong inhibitory activity at μmolL(-1) level, especially glutamine (12a) and histidine (18a) Schiff base silver(I) complexes exhibited an IC50 value of less than 0.01μmolL(-1). This series of compounds exhibited noncompetitive inhibition characteristics in kinetic studies. In addition, we investigated the mechanism of inhibition and the structure-activity relationships of the amino acid Schiff base silver complexes. Our results reveal that Schiff base silver complexes may be explored for their therapeutic potential as alternatives of α-glucosidase inhibitors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Preparation of lactose-free pasteurized milk with a recombinant thermostable β-glucosidase from Pyrococcus furiosus

Science.gov (United States)

2013-01-01

Background Lactose intolerance is a common health concern causing gastrointestinal symptoms and avoidance of dairy products by afflicted individuals. Since milk is a primary source of calcium and vitamin D, lactose intolerant individuals often obtain insufficient amounts of these nutrients which may lead to adverse health outcomes. Production of lactose-free milk can provide a solution to this problem, although it requires use of lactase from microbial sources and increases potential for contamination. Use of thermostable lactase enzymes can overcome this issue by functioning under pasteurization conditions. Results A thermostable β-glucosidase gene from Pyrococcus furiosus was cloned in frame with the Saccharomyces cerecisiae a-factor secretory signal and expressed in Pichia pastoris strain X-33. The recombinant enzyme was purified by a one-step method of weak anion exchange chromatography. The optimum temperature and pH for this β-glucosidase activity was 100°C and pH 6.0, respectively. The enzyme activity was not significantly inhibited by Ca2+. We tested the additive amount, hydrolysis time, and the influence of glucose on the enzyme during pasteurization and found that the enzyme possessed a high level of lactose hydrolysis in milk that was not obviously influenced by glucose. Conclusions The thermostablity of this recombinant β-glucosidase, combined with its neutral pH activity and favorable temperature activity optima, suggest that this enzyme is an ideal candidate for the hydrolysis of lactose in milk, and it would be suitable for application in low-lactose milk production during pasteurization. PMID:24053641

The chaperonin of the archaeon Sulfolobus solfataricus is an RNA-binding protein that participates in ribosomal RNA processing.

OpenAIRE

Ruggero, D; Ciammaruconi, A; Londei, P

1998-01-01

The 60 kDa molecular chaperones (chaperonins) are high molecular weight protein complexes having a characteristic double-ring toroidal shape; they are thought to aid the folding of denatured or newly synthesized polypeptides. These proteins exist as two functionally similar, but distantly related families, one comprising the bacterial and organellar chaperonins and another (the so-called CCT-TRiC family) including the chaperonins of the archaea and the eukaryotes. Although some evidence exist...

A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats.

Science.gov (United States)

Beloglazova, Natalia; Brown, Greg; Zimmerman, Matthew D; Proudfoot, Michael; Makarova, Kira S; Kudritska, Marina; Kochinyan, Samvel; Wang, Shuren; Chruszcz, Maksymilian; Minor, Wladek; Koonin, Eugene V; Edwards, Aled M; Savchenko, Alexei; Yakunin, Alexander F

2008-07-18

Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6A resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.

Mechanisms of mutagenesis: DNA replication in the presence of DNA damage.

Science.gov (United States)

Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F Peter; Zhang, Huidong

2016-01-01

Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, Escherichia coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural insight into dynamic bypass of the major cisplatin-DNA adduct by Y-family polymerase Dpo4

Energy Technology Data Exchange (ETDEWEB)

Wong, Jimson H.Y.; Brown, Jessica A.; Suo, Zucai; Blum, Paul; Nohmi, Takehiko; Ling, Hong (OSU); (NINA-Japan); (UNL); (UWO)

2010-08-23

Y-family DNA polymerases bypass Pt-GG, the cisplatin-DNA double-base lesion, contributing to the cisplatin resistance in tumour cells. To reveal the mechanism, we determined three structures of the Y-family DNA polymerase, Dpo4, in complex with Pt-GG DNA. The crystallographic snapshots show three stages of lesion bypass: the nucleotide insertions opposite the 3{prime}G (first insertion) and 5{prime}G (second insertion) of Pt-GG, and the primer extension beyond the lesion site. We observed a dynamic process, in which the lesion was converted from an open and angular conformation at the first insertion to a depressed and nearly parallel conformation at the subsequent reaction stages to fit into the active site of Dpo4. The DNA translocation-coupled conformational change may account for additional inhibition on the second insertion reaction. The structures illustrate that Pt-GG disturbs the replicating base pair in the active site, which reduces the catalytic efficiency and fidelity. The in vivo relevance of Dpo4-mediated Pt-GG bypass was addressed by a dpo-4 knockout strain of Sulfolobus solfataricus, which exhibits enhanced sensitivity to cisplatin and proteomic alterations consistent with genomic stress.

Smectite clays as solid supports for immobilization of beta-glucosidase : Synthesis, characterization, and biochemical properties

NARCIS (Netherlands)

Serefoglou, Evangelia; Litina, Kiriaki; Gournis, Dimitrios; Kalogeris, Emmanuel; Tzialla, Aikaterini A.; Pavlidis, Ioannis V.; Stamatis, Haralambos; Maccallini, Enrico; Lubomska, Monika; Rudolf, Petra

2008-01-01

Nanomaterials as solid supports can improve the efficiency of immobilized enzymes by reducing diffusional limitation as well as by increasing the surface area per mass unit and therefore improving enzyme loading. In this work, beta-glucosidase from almonds was immobilized on two smectite nanoclays.

ORF Alignment: NC_001263 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_001263 gi|15805432 >1bplA 2 169 309 525 3e-10 ... gb|AAF09983.1| alpha-dextran end...o-1,6-alpha-glucosidase [Deinococcus radiodurans] ... pir||D75524 alpha-dextran endo-1,6-alpha-glucos...idase - ... Deinococcus radiodurans (strain R1) ref|NP_294128.1| ... alpha-dextran endo-1,6-al

Characterization of β-glucosidase from Aspergillus terreus and its application in the hydrolysis of soybean isoflavones* #

Science.gov (United States)

Yan, Feng-ying; Xia, Wei; Zhang, Xiao-xu; Chen, Sha; Nie, Xin-zheng; Qian, Li-chun

2016-01-01

An extracellular β-glucosidase produced by Aspergillus terreus was identified, purified, characterized and was tested for the hydrolysis of soybean isoflavone. Matrix-assisted laser desorption/ionization with tandem time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) revealed the protein to be a member of the glycosyl hydrolase family 3 with an apparent molecular mass of about 120 kDa. The purified β-glucosidase showed optimal activity at pH 5.0 and 65 °C and was very stable at 50 °C. Moreover, the enzyme exhibited good stability over pH 3.0–8.0 and possessed high tolerance towards pepsin and trypsin. The kinetic parameters K m (apparent Michaelis-Menten constant) and V max (maximal reaction velocity) for p-nitrophenyl-β-D-glucopyranoside (pNPG) were 1.73 mmol/L and 42.37 U/mg, respectively. The K m and V max for cellobiose were 4.11 mmol/L and 5.7 U/mg, respectively. The enzyme efficiently converted isoflavone glycosides to aglycones, with a hydrolysis rate of 95.8% for daidzin, 86.7% for genistin, and 72.1% for glycitin. Meanwhile, the productivities were 1.14 mmol/(L·h) for daidzein, 0.72 mmol/(L·h) for genistein, and 0.19 mmol/(L·h) for glycitein. This is the first report on the application of A. terreus β-glucosidase for converting isoflavone glycosides to their aglycones in soybean products. PMID:27256679

Characterization of β-glucosidase from Aspergillus terreus and its application in the hydrolysis of soybean isoflavones.

Science.gov (United States)

Yan, Feng-Ying; Xia, Wei; Zhang, Xiao-Xu; Chen, Sha; Nie, Xin-Zheng; Qian, Li-Chun

2016-06-01

An extracellular β-glucosidase produced by Aspergillus terreus was identified, purified, characterized and was tested for the hydrolysis of soybean isoflavone. Matrix-assisted laser desorption/ionization with tandem time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) revealed the protein to be a member of the glycosyl hydrolase family 3 with an apparent molecular mass of about 120 kDa. The purified β-glucosidase showed optimal activity at pH 5.0 and 65 °C and was very stable at 50 °C. Moreover, the enzyme exhibited good stability over pH 3.0-8.0 and possessed high tolerance towards pepsin and trypsin. The kinetic parameters Km (apparent Michaelis-Menten constant) and Vmax (maximal reaction velocity) for p-nitrophenyl-β-D-glucopyranoside (pNPG) were 1.73 mmol/L and 42.37 U/mg, respectively. The Km and Vmax for cellobiose were 4.11 mmol/L and 5.7 U/mg, respectively. The enzyme efficiently converted isoflavone glycosides to aglycones, with a hydrolysis rate of 95.8% for daidzin, 86.7% for genistin, and 72.1% for glycitin. Meanwhile, the productivities were 1.14 mmol/(L·h) for daidzein, 0.72 mmol/(L·h) for genistein, and 0.19 mmol/(L·h) for glycitein. This is the first report on the application of A. terreus β-glucosidase for converting isoflavone glycosides to their aglycones in soybean products.

In silico design of fragment-based drug targeting host processing α-glucosidase i for dengue fever

Science.gov (United States)

Toepak, E. P.; Tambunan, U. S. F.

2017-02-01

Dengue is a major health problem in the tropical and sub-tropical regions. The development of antiviral that targeting dengue’s host enzyme can be more effective and efficient treatment than the viral enzyme. Host enzyme processing α-glucosidase I has an important role in the maturation process of dengue virus envelope glycoprotein. The inhibition of processing α-glucosidase I can become a promising target for dengue fever treatment. The antiviral approach using in silico fragment-based drug design can generate drug candidates with high binding affinity. In this research, 198.621 compounds were obtained from ZINC15 Biogenic Database. These compounds were screened to find the favorable fragments according to Rules of Three and pharmacological properties. The screening fragments were docked into the active site of processing α-glucosidase I. The potential fragment candidates from the molecular docking simulation were linked with castanospermine (CAST) to generate ligands with a better binding affinity. The Analysis of ligand - enzyme interaction showed ligands with code LRS 22, 28, and 47 have the better binding free energy than the standard ligand. Ligand LRS 28 (N-2-4-methyl-5-((1S,3S,6S,7R,8R,8aR)-1,6,7,8-tetrahydroxyoctahydroindolizin-3-yl) pentyl) indolin-1-yl) propionamide) itself among the other ligands has the lowest binding free energy. Pharmacological properties prediction also showed the ligands LRS 22, 28, and 47 can be promising as the dengue fever drug candidates.

Uji Penghambatan Aktivitas Alfa-glukosidase Ekstrak Dan Fraksi Daun Antidesma Montanum Blume

OpenAIRE

Nofiantini,; Elya, Berna; Azizahwati,

2012-01-01

Alpha-Glucosidase inhibitor has known to be a therapeutic agent for diabetes mellitus (DM) treatment, especially type 2 DM. Based on previous studies. There are various plants that have the effect of inhibiting the activity of a-glucosidase, one of which is garu leaves (Antidesma montanum Blume). This research aimed to get the fraction which had the highest Il-glucosidase inhibiting activity from ethanol extract of garu leaves and identify the chemical compounds from the most active fraction....

Effects of Hibiscus sabdariffa Linn. fruit extracts on α-glucosidase enzyme, glucose diffusion and wound healing activities

Directory of Open Access Journals (Sweden)

Raheem Mohssin Shadhan

2017-05-01

Conclusions: It is established that methanolic extract and fractions from H. sabdariffa Linn. fruit can inhibit the α-glucosidase enzyme and glucose movement as well as influence the wound healing activity positively.

Effect of steeping temperature on antioxidant and inhibitory activities of green tea extracts against α-amylase, α-glucosidase and intestinal glucose uptake.

Science.gov (United States)

Liu, Shuyuan; Ai, Zeyi; Qu, Fengfeng; Chen, Yuqiong; Ni, Dejiang

2017-11-01

The objective of the present study was to evaluate the effect of steeping temperature on the biological activities of green tea, including the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity, α-glucosidase and α-amylase inhibitory activities, and glucose uptake inhibitory activity in Caco-2 cells. Results showed that, with increasing extraction temperature, the polyphenol content increased, which contributed to enhance antioxidant activity and inhibitory effects on α-glucosidase and α-amylase. Green tea steeped at 100°C showed the highest DPPH radical-scavenging activity and inhibitory effects on α-glucosidase and α-amylase activities with EC 50 or IC 50 values of 6.15μg/mL, 0.09mg/mL, and 6.31mg/mL, respectively. However, the inhibitory potential on glucose uptake did not show an upward trend with increasing extraction temperature. Green tea steeped at 60°C had significantly stronger glucose uptake inhibitory activity (ptea. Copyright © 2017 Elsevier Ltd. All rights reserved.

Functional and structural characterization of a β-glucosidase involved in saponin metabolism from intestinal bacteria.

Science.gov (United States)

Yan, Shan; Wei, Peng-Cheng; Chen, Qiao; Chen, Xin; Wang, Shi-Cheng; Li, Jia-Ru; Gao, Chuan

2018-02-19

Saponins are natural glycosides widely used in medicine and the food industry. Although saponin metabolism in human is dependent on intestinal microbes, few involving bacteria enzymes have been identified. We cloned BlBG3, a GH3 β-glucosidase from Bifidobacterium longum, from human stool. We found that BlBG3 catalyzes the hydrolysis of glycoside furostanol and ginsenoside Rb1 at higher efficiency than other microbial β-glucosidases. Structural analysis of BlBG3 in complex with d-glucose revealed its three unique loops, which form a deep pocket and participate in substrate binding. To understand how substrate is bound to the pocket, molecular docking was performed and the binding interactions of protobioside with BlBG3 were revealed. Mutational study suggested that R484 and H642 are critical for enzymatic activity. Our study presents the first structural and functional analysis of a saponin-processing enzyme from human microbiota. Copyright © 2018 Elsevier Inc. All rights reserved.

α-Glucosidase and tyrosinase inhibitory effects of an abietane type diterpenoid taxoquinone from Metasequoia glyptostroboides.

Science.gov (United States)

Bajpai, Vivek K; Park, Yong-Ha; Na, MinKyun; Kang, Sun Chul

2015-03-26

Nowadays plant derived natural compounds have gained huge amount of research attention especially in food and medicine industries due to their multitude of biological and therapeutic properties as alternative medicines. In this study, a diterpenoid compound taxoquinone, isolated from Metasequoia glyptostroboides was evaluated for its α-glucosidase and tyrosinase inhibitory efficacy in terms of its potent anti-diabetic and depigmentation potential, respectively. As a result, taxoquinone at the concentration range of 100-3,000 μg/mL and 200-1,000 μg/mL showed potent efficacy on inhibiting α-glucosidase and tyrosinase enzymes by 9.24-51.32% and 11.14-52.32%, respectively. The findings of this study clearly evident potent therapeutic efficacy of an abietane diterpenoid taxoquinone isolated from M. glyptostroboides with a possibility for using it as a novel candidate in food and medicine industry as a natural alternative medicine to prevent diabetes mellitus type-2 related disorders and as a depigmentation agent.

In vitro and in vivo α-amylase and α-glucosidase inhibiting activities of the protein extracts from two varieties of bitter gourd (Momordica charantia L.).

Science.gov (United States)

Poovitha, Sundar; Parani, Madasamy

2016-07-18

α-amylase and α-glucosidase digest the carbohydrates and increase the postprandial glucose level in diabetic patients. Inhibiting the activity of these two enzymes can control postprandial hyperglycemia, and reduce the risk of developing diabetes. Bitter gourd or balsam pear is one of the important medicinal plants used for controlling postprandial hyperglycemia in diabetes patients. However, there is limited information available on the presence of α-amylase and α-glucosidase inhibiting compounds. In the current study, the protein extracts from the fruits of M. charantia var. charantia (MCC) and M. charantia var. muricata (MCM) were tested for α-amylase and α-glucosidase inhibiting activities in vitro, and glucose lowering activity after oral administration in vivo. The protein extract from both MCC and MCM inhibited the activity of α-amylase and α-glucosidase through competitive inhibition, which was on par with Acarbose as indicated by in vitro percentage of inhibition (66 to 69 %) and IC50 (0.26 to 0.29 mg/ml). Both the protein extracts significantly reduced peak blood glucose and area under the curve in Streptozotocin-induced diabetic rats, which were orally challenged with starch and sucrose. Protein extracts from the fruits of the two varieties of bitter gourd inhibited α-amylase and α-glucosidase in vitro and lowered the blood glucose level in vivo on par with Acarbose when orally administrated to Streptozotocin-induced diabetic rats. Further studies on mechanism of action and methods of safe and biologically active delivery will help to develop an anti-diabetic oral protein drug from these plants.

Radial-velocity variations in Alpha Ori, Alpha Sco, and Alpha Her

International Nuclear Information System (INIS)

Smith, M.A.; Patten, B.M.; Goldberg, L.

1989-01-01

Radial-velocity observations of Alpha Ori, Alpha Sco A, and Alpha Her A are used to study radial-velocity periodicities in M supergiants. The data refer to several metallic lines in the H-alpha region and to H-alpha itself. It is shown that Alpha Ori and Alpha Sco A have cycle lengths of about 1 yr and semiamplitudes of 2 km/s. It is suggested that many semiregular red supergiant varibles such as Alpha Ori may be heading toward chaos. All three stars show short-term stochastic flucutations with an amplitude of 1-2 km/s. It is found that the long-term variability of H-alpha velocities may be a consequence of intermittent failed ejections. 58 refs

Low concentration of sodium bicarbonate improves the bioactive compound levels and antioxidant and α-glucosidase inhibitory activities of tartary buckwheat sprouts.

Science.gov (United States)

Qin, Peiyou; Wei, Aichun; Zhao, Degang; Yao, Yang; Yang, Xiushi; Dun, Baoqing; Ren, Guixing

2017-06-01

This study aimed to investigate the effects of different concentrations of sodium bicarbonate (NaHCO 3 ) on the accumulation of flavonoids, total phenolics and d-chiro-inositol (DCI), as well as the antioxidant and α-glucosidase inhibitory activities, in tartary buckwheat sprouts. Treatment with low concentrations of NaHCO 3 (0.05, 0.1, and 0.2%) resulted in an increase in flavonoids, total phenolic compounds and DCI concentrations, and improved DPPH radical-scavenging and α-glucosidase inhibition activities compared with the control (0%). The highest levels of total flavonoids (26.69mg/g DW), individual flavonoids (rutin, isoquercitrin, quercetin, and kaempferol), total phenolic compounds (29.31mg/g DW), DCI (12.56mg/g DW), as well as antioxidant and α-glucosidase inhibition activities, were observed in tartary buckwheat sprouts treated with 0.05% NaHCO 3 for 96h. These results indicated that appropriate treatment with NaHCO 3 could improve the healthy benefits of tartary buckwheat sprouts. Copyright © 2016 Elsevier Ltd. All rights reserved.

Unexpected High Digestion Rate of Cooked Starch by the Ct-Maltase-Glucoamylase Small Intestine Mucosal α-Glucosidase Subunit

Science.gov (United States)

Lin, Amy Hui-Mei; Nichols, Buford L.; Quezada-Calvillo, Roberto; Avery, Stephen E.; Sim, Lyann; Rose, David R.; Naim, Hassan Y.; Hamaker, Bruce R.

2012-01-01

For starch digestion to glucose, two luminal α-amylases and four gut mucosal α-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal α-glucosidases on cooked (gelatinized) starch. Gelatinized normal maize starch was digested with N- and C-terminal subunits of recombinant mammalian maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) of varying amounts and digestion periods. Without the aid of α-amylase, Ct-MGAM demonstrated an unexpected rapid and high digestion degree near 80%, while other subunits showed 20 to 30% digestion. These findings suggest that Ct-MGAM assists α-amylase in digesting starch molecules and potentially may compensate for developmental or pathological amylase deficiencies. PMID:22563462

Effects of the ultra-high pressure on structure and α-glucosidase inhibition of polysaccharide from Astragalus.

Science.gov (United States)

Zhu, Zhen-Yuan; Luo, You; Dong, Guo-Ling; Ren, Yuan-Yuan; Chen, Li-Jing; Guo, Ming-Zhu; Wang, Xiao-Ting; Yang, Xue-Ying; Zhang, Yongmin

2016-06-01

A novel homogeneous polysaccharide fraction (APS) was extracted from Astragalus by hot water and purified by Sephadex G-100 and G-75 column. Its molecular weight was 693kDa. APS and APS with ultra-high pressure treatment exhibited significant inhibitory abilities on a-glucosidase, inhibition rate from high to low in order was 400MPa-APS, 300MPa-APS, 500MPa-APS and APS. The inhibition ​percentage of 400MPa-APS (1.5mg/mL) was 49% (max.). This suggested that the inhibitory activity of APS on a-glucosidase was improved by ultra-high pressure treatment. FT-IR, SEM, CD spectra, atomic force microscope and Congo red test analysis of APS and 400MPa-APS showed ultra-high pressure treatment didn't change the preliminary structure but had an effect on its advanced structure. Copyright © 2016 Elsevier B.V. All rights reserved.

Optimization of a fermented pumpkin-based beverage to improve Lactobacillus mali survival and α-glucosidase inhibitory activity: A response surface methodology approach

Directory of Open Access Journals (Sweden)

W.Y. Koh

2018-03-01

Full Text Available The aim of this research was to develop an optimum fermentation and composition model for a new fermented pumpkin-based beverage with high probiotic survival and α-glucosidase inhibitory activity. Relationship between fermentation temperature, inoculum and ingredient concentration with response variables (fermentation time at the fermentation endpoint pH 4.5, survival rate of Lactobacillus mali K8 in pumpkin-based beverage treated with simulated gastrointestinal tract enzyme fluids, α-glucosidase inhibitory activity and sensory overall acceptability after 4 weeks of refrigerated storage was investigated using response surface methodology. Optimal formulation was obtained at an approximation of 40% pumpkin puree concentration, 8 Log CFU/mL inoculum and at 35 °C. The product derived from this optimum formula reached the fermentation endpoint after 28.34 ± 0.10 h and the quality change during 4 weeks storage was studied. The product achieved 88.56 ± 0.67% of L. mali survival after treatment with simulated gastric and intestinal juices; demonstrated 95.89 ± 0.30% α-glucosidase inhibitory activity, as well as scored 6.99 ± 0.40 on sensory overall acceptability after 4 weeks of storage. These findings illustrated that the model is effective in improving probiotic survival and α-glucosidase inhibitory activity with excellent sensory acceptability, thus may offer a dietary means for the management of hyperglycaemia. Keywords: Probiotics, Response surface methodology, Box-Behnken, Hyperglycaemia, Functional food

Inhibition of protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase by xanthones from Cratoxylum cochinchinense, and their kinetic characterization.

Science.gov (United States)

Li, Zuo Peng; Song, Yeong Hun; Uddin, Zia; Wang, Yan; Park, Ki Hun

2018-02-01

Cratoxylum cochinchinense displayed significant inhibition against protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase, both of which are key target enzymes to attenuate diabetes and obesity. The compounds responsible for both enzymes inhibition were identified as twelve xanthones (1-12) among which compounds 1 and 2 were found to be new ones. All of them simultaneously inhibited PTP1B with IC 50 s of (2.4-52.5 µM), and α-glucosidase with IC 50 values of (1.7-72.7 µM), respectively. Cratoxanthone A (3) and γ-mangostin (7) were estimated to be most active inhibitors against both PTP1B (IC 50  = 2.4 µM for 3, 2.8 µM for 7) and α-glucosidase (IC 50  = 4.8 µM for 3, 1.7 µM for 7). In kinetic studies, all isolated xanthones emerged to be mixed inhibitors of α-glucosidase, whereas they behaved as competitive inhibitors of PTP1B. In time dependent experiments, compound 3 showed isomerization inhibitory behavior with following kinetic parameters: K i app  = 2.4 µM; k 5  = 0.05001 µM -1  S -1 and k 6  = 0.02076 µM -1  S -1 . Copyright © 2017 Elsevier Ltd. All rights reserved.

Biochemical and kinetic characterization of the multifunctional β-glucosidase/β-xylosidase/α-arabinosidase, Bgxa1.

Science.gov (United States)

Gruninger, R J; Gong, X; Forster, R J; McAllister, T A

2014-04-01

Functional screening of a metagenomic library constructed with DNA extracted from the rumen contents of a grass/hay-fed dairy cow identified a protein, β-glucosidase/β-xylosidase/α-arabinosidase gene (Bgxa1), with high levels of β-glucosidase activity. Purified Bgxa1 was highly active against p-nitrophenyl-β-D-glucopyranoside (pNPG), cellobiose, p-nitrophenyl-β-D-xylopyranoside (pNPX) and p-nitrophenyl-α-D-arabinofuranoside (pNPAf), suggesting it is a multifunctional β-glucosidase/β-xylosidase/α-arabinosidase. Kinetic analysis of the protein indicated that Bgxa1 has the greatest catalytic activity against pNPG followed by pNPAf and pNPX, respectively. The catalytic efficiency of β-glucosidase activity was 100× greater than β-xylosidase or α-arabinosidase. The pH and temperature optima for the hydrolysis of selected substrates also differed considerably with optima of pH 6.0/45 °C and pH 8.5/40 °C for pNPG and pNPX, respectively. The pH dependence of pNPAf hydrolysis displayed a bimodal distribution with maxima at both pH 6.5 and pH 8.5. The enzyme exhibited substrate-dependent responses to changes in ionic strength. Bgxa1 was highly stable over a broad pH range retaining at least 70 % of its relative catalytic activity from pH 5.0-10.0 with pNPG as a substrate. Homology modelling was employed to probe the structural basis of the unique specificity of Bgxa1 and revealed the deletion of the PA14 domain and insertions in loops adjacent to the active site. This domain has been found to be an important determinant in the substrate specificity of proteins related to Bgxa1. It is postulated that these indels are, in part, responsible for the multifunctional activity of Bgxa1. Bgxa1 acted synergistically with endoxylanase (Xyn10N18) when incubated with birchwood xylan, increasing the release of reducing sugars by 168 % as compared to Xyn10N18 alone. Examination of Bgxa1 and Xyn10N18 synergy with a cellulase for the saccharification of alkali-treated straw

Functional analysis of the active site of the maize beta-glucosidase Zm-p60.1

Czech Academy of Sciences Publication Activity Database

Fohlerová, Radka; Mazura, Pavel; Janda, L.; Chaloupková, R.; Jeřábek, P.; Damborský, J.; Brzobohatý, Břetislav

2005-01-01

Roč. 409, - (2005), S3 [2nd International Symposium Auxins and Cytokinins in Plant Development, 07.07.2005-12.07.2005] R&D Projects: GA ČR(CZ) GA203/02/0865 Institutional research plan: CEZ:AV0Z50040507 Keywords : beta-glucosidase * active site Subject RIV: BO - Biophysics

Expression, purification, crystallization and preliminary X-ray analysis of rice (Oryza sativa L.) Os4BGlu12 β-glucosidase

International Nuclear Information System (INIS)

Sansenya, Sompong; Ketudat Cairns, James R.; Opassiri, Rodjana

2010-01-01

Recombinant rice Os4BGlu12 β-glucosidase purified from E. coli was crystallized with and without 2,4-dinitrophenyl-2-deoxy-2-fluoro-β-d-glucopyranoside. Rice (Oryza sativa L.) Os4BGlu12, a glycoside hydrolase family 1 β-glucosidase (EC 3.2.1.21), was expressed as a fusion protein with an N-terminal thioredoxin/His 6 tag in Escherichia coli strain Origami B (DE3) and purified with subsequent removal of the N-terminal tag. Native Os4BGlu12 and its complex with 2,4-dinitrophenyl-2-deoxy-2-fluoro-β-d-glucopyranoside (DNP2FG) were crystallized using 19% polyethylene glycol (3350 or 2000, respectively) in 0.1 M Tris–HCl pH 8.5, 0.16 M NaCl at 288 K. Diffraction data sets for the apo and inhibitor-bound forms were collected to 2.50 and 2.45 Å resolution, respectively. The space group and the unit-cell parameters of the crystal indicated the presence of two molecules per asymmetric unit, with a solvent content of 50%. The structure of Os4BGlu12 was successfully solved in space group P4 3 2 1 2 by molecular replacement using the white clover cyanogenic β-glucosidase structure as a search model

Production of beta-glucosidase and hydrolysis of isoflavone phytoestrogens by Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus casei in soymilk.

Science.gov (United States)

Donkor, O N; Shah, N P

2008-01-01

The study determined beta-glucosidase activity of commercial probiotic organisms for hydrolysis of isoflavone to aglycones in fermenting soymilk. Soymilk made with soy protein isolate (SPI) was fermented with Lactobacillus acidophilus LAFTI L10, Bifidobacterium lactis LAFTI B94, and Lactobacillus casei LAFTI L26 at 37 degrees C for 48 h and the fermented soymilk was stored for 28 d at 4 degrees C. beta-Glucosidase activity of organisms was determined using rho-nitrophenyl beta-D-glucopyranoside as a substrate and the hydrolysis of isoflavone glycosides to aglycones by these organisms was carried out. The highest level of growth occurred at 12 h for L. casei L26, 24 h for B. lactis B94, and 36 h for L. acidophilus L10 during fermentation in soymilk. Survival after storage at 4 degrees C for 28 d was 20%, 15%, and 11% greater (P < 0.05) than initial cell counts, respectively. All the bacteria produced beta-glucosidase, which hydrolyzed isoflavone beta-glycosides to isoflavone aglycones. The decrease in the concentration of beta-glycosides and the increase in the concentration of aglycones were significant (P < 0.05) in the fermented soymilk. Increased isoflavone aglycone content in fermented soymilk is likely to improve the biological functionality of soymilk.

Uracil phosphoribosyltransferase from the extreme thermoacidophilic archaebacterium Sulfolobus shibatae is an allosteric enzyme, activated by GTP and inhibited by CTP

DEFF Research Database (Denmark)

Linde, Lise; Jensen, Kaj Frank

1996-01-01

Uracil phosphoribosyltransferase, which catalyses the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl a-1-pyrophosphate (PRPP), was partly purified from the extreme thermophilic archaebacterium Sulfolobus shibatae. The enzyme required divalent metal ions for activity...... and it showed the highest activity at pH 6.4. The specific activity of the enzyme was 50-times higher at 95°C than at 37°C, but the functional half-life was short at 95°C. The activity of uracil phosphoribosyltransferase was strongly activated by GTP, which increased Vmax of the reaction by approximately 20......-fold without much effect on Km for the substrates. The concentration of GTP required for half-maximal activation was about 80 µM. CTP was a strong inhibitor and acted by raising the concentration of GTP needed for half-maximal activation of the enzyme. We conclude that uracil phosphoribosyltransferase...

Xanthium strumarium as an Inhibitor of α-Glucosidase, Protein Tyrosine Phosphatase 1β, Protein Glycation and ABTS+ for Diabetic and Its Complication

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Seung Hwan Hwang

2016-09-01

Full Text Available Phytochemical investigation of the natural products from Xanthium strumarium led to the isolation of fourteen compounds including seven caffeoylquinic acid (CQA derivatives. The individual compounds were screened for inhibition of α-glucosidase, protein tyrosine phosphatase 1β (PTP1β, advanced glycation end products (AGEs, and ABTS+ radical scavenging activity using in vitro assays. Among the isolated compounds, methyl-3,5-di-caffeoyquinic acid exhibited significant inhibitory activity against α-glucosidase (18.42 μM, PTP1β (1.88 μM, AGEs (82.79 μM, and ABTS+ (6.03 μM. This effect was marked compared to that of the positive controls (acarbose 584.79 μM, sumarin 5.51 μM, aminoguanidine 1410.00 μM, and trolox 29.72 μM respectively. In addition, 3,5-di-O-CQA (88.14 μM and protocatechuic acid (32.93 μM had a considerable inhibitory effect against α-glucosidase and ABTS+. Based on these findings, methyl-3,5-di-caffeoyquinic acid was assumed to be potentially responsible for the anti-diabetic actions of X. strumarium.

Reclamation of Marine Chitinous Materials for the Production of α-Glucosidase Inhibitors via Microbial Conversion

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Van Bon Nguyen

2017-11-01

Full Text Available Six kinds of chitinous materials have been used as sole carbon/nitrogen (C/N sources for producing α-glucosidase inhibitors (aGI by Paenibacillus sp. TKU042. The aGI productivity was found to be highest in the culture supernatants using demineralized crab shell powder (deCSP and demineralized shrimp shell powder (deSSP as the C/N source. The half maximal inhibitory concentration (IC50 and maximum aGI activity of fermented deCSP (38 µg/mL, 98%, deSSP (108 µg/mL, 89%, squid pen powder (SPP (422 µg/mL, 98%, and shrimp head powder (SHP (455 µg/mL, 92% were compared with those of fermented nutrient broth (FNB (81 µg/mL, 93% and acarbose (1095 µg/mL, 74%, a commercial antidiabetic drug. The result of the protein/chitin ratio on aGI production showed that the optimal ratio was 0.2/1. Fermented deCSP showed lower IC50 and higher maximum inhibitory activity than those of acarbose against rat intestinal α-glucosidase.

Synthesis and evaluation of a 5-Membered Isoiminosugar as Glycosidase Inhibitor

DEFF Research Database (Denmark)

Lundt, Inge; Godskesen, Michael Anders

1998-01-01

(3R, 4R)-4-hydroxy-3-hydroxymethylpyrrolidine (6) was prepared from D-xylose and found to be a weak inhibitor of alpha-D-, beta-D-glucosidase and alpha-L-fucosidase. (C) 1998 Published by Elsevier Science Ltd. All rights reserved....

Focused directed evolution of beta-glucosidases: theoretical versus real effectiveness of a minimal working setup and simple robust screening

Czech Academy of Sciences Publication Activity Database

Mazura, P.; Filipi, T.; Souček, P.; Brzobohatý, Břetislav

2011-01-01

Roč. 346, č. 2 (2011), s. 238-242 ISSN 0008-6215 Institutional support: RVO:68081707 Keywords : Directed evolution * beta-Glucosidase * Mutagenesis Subject RIV: BO - Biophysics Impact factor: 2.332, year: 2011

Enhanced cellulase recovery without β-glucosidase supplementation for cellulosic ethanol production using an engineered strain and surfactant.

Science.gov (United States)

Huang, Renliang; Guo, Hong; Su, Rongxin; Qi, Wei; He, Zhimin

2017-03-01

Recycling cellulases by substrate adsorption is a promising strategy for reducing the enzyme cost of cellulosic ethanol production. However, β-glucosidase has no carbohydrate-binding module (CBM). Thus, additional enzymes are required in each cycle to achieve a high ethanol yield. In this study, we report a new method of recycling cellulases without β-glucosidase supplementation using lignocellulosic substrate, an engineered strain expressing β-glucosidase and Tween 80. The cellulases and Tween 80 were added to an aqueous suspension of diluted sulfuric acid/ammonia-treated corncobs in a simultaneous saccharification and fermentation (SSF) process for ethanol production. Subsequently, the addition of fresh pretreated corncobs to the fermentation liquor and remaining solid residue provided substrates with absorbed cellulases for the next SSF cycle. This method provided excellent ethanol production in three successive SSF cycles without requiring the addition of new cellulases. For a 10% (w/v) solid loading, a cellulase dosage of 30 filter paper units (FPU)/g cellulose, 0.5% Tween 80, and 2 g/L of the engineered strain, approximately 90% of the initial ethanol concentration from the first SSF process was obtained in the next two SSF processes, with a total ethanol production of 306.27 g/kg corncobs and an enzyme productivity of 0.044 g/FPU. Tween 80 played an important role in enhancing cellulase recovery. This new enzyme recycling method is more efficient and practical than other reported methods. Biotechnol. Bioeng. 2017;114: 543-551. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Combination of α-glucosidase inhibitor and ribavirin for the treatment of dengue virus infection in vitro and in vivo.

Science.gov (United States)

Chang, Jinhong; Schul, Wouter; Butters, Terry D; Yip, Andy; Liu, Boping; Goh, Anne; Lakshminarayana, Suresh B; Alonzi, Dominic; Reinkensmeier, Gabriele; Pan, Xiaoben; Qu, Xiaowang; Weidner, Jessica M; Wang, Lijuan; Yu, Wenquan; Borune, Nigel; Kinch, Mark A; Rayahin, Jamie E; Moriarty, Robert; Xu, Xiaodong; Shi, Pei-Yong; Guo, Ju-Tao; Block, Timothy M

2011-01-01

Cellular α-glucosidases I and II are enzymes that sequentially trim the three terminal glucoses in the N-linked oligosaccharides of viral envelope glycoproteins. This process is essential for the proper folding of viral glycoproteins and subsequent assembly of many enveloped viruses, including dengue virus (DENV). Imino sugars are substrate mimics of α-glucosidases I and II. In this report, we show that two oxygenated alkyl imino sugar derivatives, CM-9-78 and CM-10-18, are potent inhibitors of both α-glucosidases I and II in vitro and in treated animals, and efficiently inhibit DENV infection of cultured human cells. Pharmacokinetic studies reveal that both compounds are well tolerated at doses up to 100mg/kg in rats and have favorable pharmacokinetic properties and bioavailability in mice. Moreover, we showed that oral administration of either CM-9-78 or CM-10-18 reduces the peak viremia of DENV in mice. Interestingly, while treatment of DENV infected mice with ribavirin alone did not reduce the viremia, combination therapy of ribavirin with sub-effective dose of CM-10-18 demonstrated a significantly enhanced antiviral activity, as indicated by a profound reduction of the viremia. Our findings thus suggest that combination therapy of two broad-spectrum antiviral agents may provide a practically useful approach for the treatment of DENV infection. Copyright © 2010 Elsevier B.V. All rights reserved.

Development of a highly efficient indigo dyeing method using indican with an immobilized beta-glucosidase from Aspergillus niger.

Science.gov (United States)

Song, Jingyuan; Imanaka, Hiroyuki; Imamura, Koreyoshi; Kajitani, Kouichi; Nakanishi, Kazuhiro

2010-09-01

A highly efficient method for dyeing textiles with indigo is described. In this method, the substrate, indican is first hydrolyzed at an acidic pH of 3 using an immobilized beta-glucosidase to produce indoxyl, under which conditions indigo formation is substantially repressed. The textile sample is then dipped in the prepared indoxyl solution and the textile is finally exposed to ammonia vapor for a short time, resulting in rapid indigo dyeing. As an enzyme, we selected a beta-glucosidase from Aspergillus niger, which shows a high hydrolytic activity towards indican and was thermally stable at temperatures up to 50-60 degrees C, in an acidic pH region. The A. niger beta-glucosidase, when immobilized on Chitopearl BCW-3001 by treatment with glutaraldehyde, showed an optimum reaction pH similar to that of the free enzyme with a slightly higher thermal stability. The kinetics for the hydrolysis of indican at pH 3, using the purified free and immobilized enzymes was found to follow Michaelis-Menten type kinetics with weak competitive inhibition by glucose. Using the immobilized enzyme, we successfully carried out repeated-batch and continuous hydrolyses of indican at pH 3 when nitrogen gas was continuously supplied to the substrate solution. Various types of model textiles were dyed using the proposed method although the color yield varied, depending on the type of textile used. Copyright 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Xanthium strumarium as an Inhibitor of α-Glucosidase, Protein Tyrosine Phosphatase 1β, Protein Glycation and ABTS⁺ for Diabetic and Its Complication.

Science.gov (United States)

Hwang, Seung Hwan; Wang, Zhiqiang; Yoon, Ha Na; Lim, Soon Sung

2016-09-16

Phytochemical investigation of the natural products from Xanthium strumarium led to the isolation of fourteen compounds including seven caffeoylquinic acid (CQA) derivatives. The individual compounds were screened for inhibition of α-glucosidase, protein tyrosine phosphatase 1β (PTP1β), advanced glycation end products (AGEs), and ABTS⁺ radical scavenging activity using in vitro assays. Among the isolated compounds, methyl-3,5-di-caffeoyquinic acid exhibited significant inhibitory activity against α-glucosidase (18.42 μM), PTP1β (1.88 μM), AGEs (82.79 μM), and ABTS⁺ (6.03 μM). This effect was marked compared to that of the positive controls (acarbose 584.79 μM, sumarin 5.51 μM, aminoguanidine 1410.00 μM, and trolox 29.72 μM respectively). In addition, 3,5-di-O-CQA (88.14 μM) and protocatechuic acid (32.93 μM) had a considerable inhibitory effect against α-glucosidase and ABTS⁺. Based on these findings, methyl-3,5-di-caffeoyquinic acid was assumed to be potentially responsible for the anti-diabetic actions of X. strumarium.

Glycosidases in Brachionus plicatilis (Rotifera).

Science.gov (United States)

Kühle, K; Kleinow, W

1990-01-01

1. Tests for glycosidases were performed in homogenates of Brachionus plicatilis. 2. Hydrolytic activity was detected with the following substrates: (a) with synthetic substrates (NP = 4-nitrophenyl): NP-alpha- and NP-beta-D-glucopyranoside, NP-alpha- and NP-beta-D-galactopyranoside, NP-N-acetyl-beta-D-glucosaminide, NP-N-acetyl-beta-D-galactosaminide, NP-alpha- and NP-beta-D-mannopyranoside and NP-alpha-L-fucopyranoside; (b) with disaccharides: sucrose, maltose, trehalose, isomaltose, cellobiose, gentiobiose and lactose; (c) with polysaccharides: laminarine, carboxymethyl-cellulose, avicel, Micrococcus luteus (for lysozyme) and 4-nitrophenyl-alpha-D-maltoheptaoside (for amylase). 3. The pH dependence of the glycosidase activities was determined. 4. The distribution of enzyme activities within fractions from the homogenate was studied in order to localize them within the cell. 5. Proteins from Brachionus homogenate were separated by SDS-gel electrophoresis and the positions of the following glycosidase activities were detected by assays performed on the gels (estimated molecular weights in parentheses): alpha-glucosidase (250,000); beta-glucosidase (200,000); beta-galactosidase (70,000); N-acetyl-beta-glucosaminidase (60,000).

Identification of a feedback loop involving β-glucosidase 2 and its product sphingosine sheds light on the molecular mechanisms in Gaucher disease.

Science.gov (United States)

Schonauer, Sophie; Körschen, Heinz G; Penno, Anke; Rennhack, Andreas; Breiden, Bernadette; Sandhoff, Konrad; Gutbrod, Katharina; Dörmann, Peter; Raju, Diana N; Haberkant, Per; Gerl, Mathias J; Brügger, Britta; Zigdon, Hila; Vardi, Ayelet; Futerman, Anthony H; Thiele, Christoph; Wachten, Dagmar

2017-04-14

The lysosomal acid β-glucosidase GBA1 and the non-lysosomal β-glucosidase GBA2 degrade glucosylceramide (GlcCer) to glucose and ceramide in different cellular compartments. Loss of GBA2 activity and the resulting accumulation of GlcCer results in male infertility, whereas mutations in the GBA1 gene and loss of GBA1 activity cause the lipid-storage disorder Gaucher disease. However, the role of GBA2 in Gaucher disease pathology and its relationship to GBA1 is not well understood. Here, we report a GBA1-dependent down-regulation of GBA2 activity in patients with Gaucher disease. Using an experimental approach combining cell biology, biochemistry, and mass spectrometry, we show that sphingosine, the cytotoxic metabolite accumulating in Gaucher cells through the action of GBA2, directly binds to GBA2 and inhibits its activity. We propose a negative feedback loop, in which sphingosine inhibits GBA2 activity in Gaucher cells, preventing further sphingosine accumulation and, thereby, cytotoxicity. Our findings add a new chapter to the understanding of the complex molecular mechanism underlying Gaucher disease and the regulation of β-glucosidase activity in general. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Insight into glucosidase II from the red marine microalga Porphyridium sp. (Rhodophyta).

Science.gov (United States)

Levy-Ontman, Oshrat; Fisher, Merav; Shotland, Yoram; Tekoah, Yoram; Malis Arad, Shoshana

2015-12-01

N-glycosylation of proteins is one of the most important post-translational modifications that occur in various organisms, and is of utmost importance for protein function, stability, secretion, and loca-lization. Although the N-linked glycosylation pathway of proteins has been extensively characterized in mammals and plants, not much information is available regarding the N-glycosylation pathway in algae. We studied the α 1,3-glucosidase glucosidase II (GANAB) glycoenzyme in a red marine microalga Porphyridium sp. (Rhodophyta) using bioinformatic and biochemical approaches. The GANAB-gene was found to be highly conserved evolutionarily (compo-sed of all the common features of α and β subunits) and to exhibit similar motifs consistent with that of homolog eukaryotes GANAB genes. Phylogenetic analysis revealed its wide distribution across an evolutionarily vast range of organisms; while the α subunit is highly conserved and its phylogenic tree is similar to the taxon evolutionary tree, the β subunit is less conserved and its pattern somewhat differs from the taxon tree. In addition, the activity of the red microalgal GANAB enzyme was studied, including functional and biochemical characterization using a bioassay, indicating that the enzyme is similar to other eukaryotes ortholog GANAB enzymes. A correlation between polysaccharide production and GANAB activity, indicating its involvement in polysaccharide biosynthesis, is also demonstrated. This study represents a valuable contribution toward understanding the N-glycosylation and polysaccharide biosynthesis pathways in red microalgae. © 2015 Phycological Society of America.

Release Profile and Inhibition Test of The Nanoparticles A. Paniculata Extract as Inhibitor of α-Glucosidase in The Process of Carbohydrates Breakdown Into Glucose Diabetes Mellitus

Science.gov (United States)

Imansari, Farisa; Sahlan, Muhammad; Arbianti, Rita

2017-07-01

Andrographis paniculata (A.paniculata) contain the main active substances Andrographolide which helps lower glucose levels in diabetics by inhibiting the enzyme α-glucosidase. The ability of the extract A.paniculata in lowering glucose levels will increase with the technique encapsulation with a coating of composition Chitosan-STPP as a drug delivery to the target organ. This study aimed to get an overview of A.paniculata release profile of nanoparticles in a synthetic fluid media with various concentrations of coating and inhibition testing nasty shard extract in inhibiting the enzyme α-glucosidase. This research resulted in nanoparticles by coating efficiency and loading capacity of chitosan greatest variation of 2% and 1% STPP 60% and 46.29%. chitosan greatest variation of 2% and 1% STPP 60% and 46.29%. The ability of A.paniculata extracts as α-glucosidase enzyme inhibitors has been demonstrated in this study, the percent inhibition of 33.17%.

Molecular Characterization of Copper and Cadmium Resistance Determinants in the Biomining Thermoacidophilic Archaeon Sulfolobus metallicus

Directory of Open Access Journals (Sweden)

Alvaro Orell

2013-01-01

Full Text Available Sulfolobus metallicus is a thermoacidophilic crenarchaeon used in high-temperature bioleaching processes that is able to grow under stressing conditions such as high concentrations of heavy metals. Nevertheless, the genetic and biochemical mechanisms responsible for heavy metal resistance in S. metallicus remain uncharacterized. Proteomic analysis of S. metallicus cells exposed to 100 mM Cu revealed that 18 out of 30 upregulated proteins are related to the production and conversion of energy, amino acids biosynthesis, and stress responses. Ten of these last proteins were also up-regulated in S. metallicus treated in the presence of 1 mM Cd suggesting that at least in part, a common general response to these two heavy metals. The S. metallicus genome contained two complete cop gene clusters, each encoding a metallochaperone (CopM, a Cu-exporting ATPase (CopA, and a transcriptional regulator (CopT. Transcriptional expression analysis revealed that copM and copA from each cop gene cluster were cotranscribed and their transcript levels increased when S. metallicus was grown either in the presence of Cu or using chalcopyrite (CuFeS2 as oxidizable substrate. This study shows for the first time the presence of a duplicated version of the cop gene cluster in Archaea and characterizes some of the Cu and Cd resistance determinants in a thermophilic archaeon employed for industrial biomining.

Biochemical and Molecular Characterization of a Barley Seed ß-Glucosidase

DEFF Research Database (Denmark)

Leah, R.; Kigel, J.; Svendsen, I.

1995-01-01

blot analysis with the cDNA as probe indicated that BGQ60 is encoded by a single gene, and that BGQ60 mRNA only accumulates in the starchy endosperm tissue of late developing seeds. The bgq60 structural gene of approximately 5 kilobases contains an open reading frame encoding 485 amino acids...... during barley seed development and germination are discussed.......A 60-kDa ß-glucosidase (BGQ60) was purified and characterized from seeds of barley (Hordeum vulgare L.). BGQ60 catalytic activity was restricted to the cleavage of short-chain oligosaccharides composed of(1, 2) -,(1, 2, 3) -, and/or(1, 2, 3, 4) -ß-linked glucose or mannose units...

Probing the aglycon binding site of a b-glucosidase: a collection of C-1-modified 2,5-dideoxy-2,5-imino-D-mannitol derivatives and their structure-activity relationships as competitive inhibitors

DEFF Research Database (Denmark)

Wrodnigg, Tanja; Diness, Frederik; Gruber, Christoph

2004-01-01

A range of new C-1 modified derivatives of the powerful glucosidase inhibitor 2,5-dideoxy-2,5-imino-D-mannitol has been synthesised and their biological activities probed with the b-glucosidase from Agrobacterium sp. Ki values are compared with those of previously prepared close relatives. Findings...

Effect of pH, Temperature, and Chemicals on the Endoglucanases and β-Glucosidases from the Thermophilic Fungus Myceliophthora heterothallica F.2.1.4. Obtained by Solid-State and Submerged Cultivation

Directory of Open Access Journals (Sweden)

Vanessa de Cássia Teixeira da Silva

2016-01-01

Full Text Available This work reports endoglucanase and beta-glucosidase production by the thermophilic fungus Myceliophthora heterothallica in solid-state (SSC and submerged (SmC cultivation. Wheat bran and sugarcane bagasse were used for SSC and cardboard for SmC. Highest endoglucanase production in SSC occurred after 192 hours: 1,170.6 ± 0.8 U/g, and in SmC after 168 hours: 2,642 ± 561 U/g. The endoglucanases and beta-glucosidases produced by both cultivation systems showed slight differences concerning their optimal pH and temperature. The number of endoglucanases was also different: six isoforms in SSC and ten in SmC. Endoglucanase activity remained above 50% after incubation between pH 3.0 and 9.0 for 24 h for both cultivation systems. The effect of several chemicals displayed variation between SSC and SmC isoenzymes. Manganese activated the enzymes from SmC but inhibited those from SSC. For β-glucosidases, maximum production on SmC was 244 ± 48 U/g after 168 hours using cardboard as carbon source. In SSC maximum production reached 10.9 ± 0.3 U/g after 240 h with 1 : 1 wheat bran and sugarcane bagasse. Manganese exerted a significant activation on SSC β-glucosidases, and glucose inhibited the enzymes from both cultivation systems. FeCl3 exerted the strongest inhibition for endoglucanases and β-glucosidases.

Effects of Undaria pinnatifida, Himanthalia elongata and Porphyra umbilicalis extracts on in vitro α-glucosidase activity and glucose diffusion.

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Schultz Moreira, Adriana R; Garcimartín, Alba; Bastida, Sara; Jiménez-Escrig, Antonio; Rupérez, Pilar; Green, Brian D; Rafferty, Eamon; Sánchez-Muniz, Francisco J; Benedí, Juana

2014-06-01

Seaweeds are good sources of dietary fibre, which can influence glucose uptake and glycemic control. To investigate and compare the in vitro inhibitory activity of different extracts from Undaria pinnatifida (Wakame), Himanthalia elongata (Sea spaghetti) and Porphyra umbilicalis (Nori) on α-glucosidase activity and glucose diffusion. The in vitro effects Chloroform-, ethanol- and water-soluble extracts of the three algae were assayed on α- glucosidase activity and glucose diffusion through membrane. Principal Components Analysis (PCA) was applied to identify patterns in the data and to discriminate which extract will show the most proper effect. Only water extracts of Sea spaghetti possessed significant in vitro inhibitory effects on α-glucosidase activity (26.2% less mmol/L glucose production than control, p < 0.05) at 75 min. PCA distinguished Sea spaghetti effects, supporting that soluble fibre and polyphenols were involved. After 6 h, Ethanol-Sea spaghetti and water-Wakame extracts exerted the highest inhibitory effects on glucose diffusion (65.0% and 60.2% vs control, respectively). This extracts displayed the lowest slopes for glucose diffusion-time lineal adjustments (68.2% and 62.8% vs control, respectively). The seaweed hypoglycemic effects appear multi-faceted and not necessarily concatenated. According to present results, ethanol and water extracts of Sea spaghetti, and water extracts of Wakame could be useful for the development of functional foods with specific hypoglycemic properties. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

Comprehensive enzymatic analysis of the cellulolytic system in digestive fluid of the Sea Hare Aplysia kurodai. Efficient glucose release from sea lettuce by synergistic action of 45 kDa endoglucanase and 210 kDa ß-glucosidase.

Directory of Open Access Journals (Sweden)

Akihiko Tsuji

Full Text Available Although many endo-ß-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and seaweeds. In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the sea hare (Aplysia kurodai were screened and characterized to determine how the sea hare obtains glucose from sea lettuce (Ulva pertusa. Four endo-ß-1,4-glucanases (21K, 45K, 65K, and 95K cellulase and 2 ß-glucosidases (110K and 210K were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21K cellulase, whereas 45K and 65K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210K ß-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU-ß-glucoside, 4MU-ß-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210K ß-glucosidase possesses lactase activity. Although ß-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210K ß-glucosidase. Kinetic analysis of the inhibition of 210K ß-glucosidase by D-glucono-1,5-lactone suggested the presence of 2 active sites similar to those of mammalian lactase-phlorizin hydrolase. Saccharification of sea lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K ß-glucosidase. Our results indicate that 45K cellulase and 210K ß-glucosidase

Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment.

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Brouns, Stan J J; Walther, Jasper; Snijders, Ambrosius P L; van de Werken, Harmen J G; Willemen, Hanneke L D M; Worm, Petra; de Vos, Marjon G J; Andersson, Anders; Lundgren, Magnus; Mazon, Hortense F M; van den Heuvel, Robert H H; Nilsson, Peter; Salmon, Laurent; de Vos, Willem M; Wright, Phillip C; Bernander, Rolf; van der Oost, John

2006-09-15

The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for D-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to be differentially expressed compared with growth on D-glucose. These genes were heterologously overexpressed in Escherichia coli, and the recombinant proteins were purified and biochemically studied. This showed that D-arabinose is oxidized to 2-oxoglutarate by the consecutive action of a number of previously uncharacterized enzymes, including a D-arabinose dehydrogenase, a D-arabinonate dehydratase, a novel 2-keto-3-deoxy-D-arabinonate dehydratase, and a 2,5-dioxopentanoate dehydrogenase. Promoter analysis of these genes revealed a palindromic sequence upstream of the TATA box, which is likely to be involved in their concerted transcriptional control. Integration of the obtained biochemical data with genomic context analysis strongly suggests the occurrence of pentose oxidation pathways in both Archaea and Bacteria, and predicts the involvement of additional enzyme components. Moreover, it revealed striking genetic similarities between the catabolic pathways for pentoses, hexaric acids, and hydroxyproline degradation, which support the theory of metabolic pathway genesis by enzyme recruitment.

High-resolution bioactivity profiling combined with HPLC-HRMS-SPE-NMR: α-Glucosidase inhibitors and acetylated ellagic acid rhamnosides from Myrcia palustris DC. (Myrtaceae).

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Wubshet, Sileshi G; Moresco, Henrique H; Tahtah, Yousof; Brighente, Inês M C; Staerk, Dan

2015-08-01

Type 2 diabetes (T2D) is an endocrine metabolic disease with a worldwide prevalence of more than 8%, and an expected increase close to 50% in the next 15-20years. T2D is associated with severe and life-threatening complications like retinopathy, neuropathy, nephropathy, and cardiovascular diseases, and therefore improved drug leads or functional foods containing α-glucosidase inhibitors are needed for management of blood glucose. In this study, leaves of Myrcia palustris were investigated by high-resolution α-glucosidase inhibition profiling combined with HPLC-HRMS-SPE-NMR. This led to identification of casuarinin, myricetin 3-O-β-d-(6″-galloyl)galactopyranoside, kaempferol 3-O-β-d-galactopyranoside, myricetin, and quercetin as α-glucosidase inhibitors. In addition, four acetylated ellagic acid rhamnosides, i.e., 4-O-(2″,4″-O-diacetyl-α-l-rhamnopyranosyl)ellagic acid, 4-O-(2″,3″-O-diacetyl-α-l-rhamnopyranosyl)ellagic acid, 4-O-(3″,4″-O-diacetyl-α-l-rhamnopyranosyl)ellagic acid, and 4-O-(2″,3″,4″-O-triacetyl-α-l-rhamnopyranosyl)ellagic acid were identified. Copyright © 2015 Elsevier Ltd. All rights reserved.

Simultaneous quantification of ten constituents of Xanthoceras sorbifolia Bunge using UHPLC-MS methods and evaluation of their radical scavenging, DNA scission protective, and α-glucosidase inhibitory activities.

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Zhang, Yu; Ma, Jian-Nan; Ma, Chun-Li; Qi, Zhi; Ma, Chao-Mei

2015-11-01

The present study was designed to investigate the bioactive constituents of Xanthoceras sorbifolia in terms of amounts and their antioxidant, DNA scission protection, and α-glucosidase inhibitory activities. Simultaneous quantification of 10 X. sorbifolia constituents was carried out by a newly established ultra-high performance liquid chromatography-quadrupole mass spectrometry method (UHPLC-MS). The antioxidant activities were evaluated by measuring DPPH radical scavenging and DNA scission protective activities. The α-glucosidase inhibitory activities were investigated by using an assay with α-glucosidase from Bacillus Stearothermophilus and disaccharidases from mouse intestine. We found that the wood of X. sorbifolia was rich in phenolic compounds with the contents of catechin, epicatechin, myricetin, and dihydromyricetin being 0.12-0.19, 1.94-2.16, 0.77-0.91, and 6.76-7.89 mg·g(-1), respectively. The four constituents strongly scavenged DPPH radicals (with EC50 being 4.2, 3.8 and 5.7 μg·mL(-1), respectively) and remarkably protected peroxyl radical-induced DNA strand scission (92.10%, 94.66%, 75.44% and 89.95% of protection, respectively, at a concentration of 10 μmol·L(-1)). A dimeric flavan 3-ol, epigallocatechin-(4β→8, 2β→O-7)-epicatechin potently inhibited α-glucosidase with an IC50 value being as low as 1.2 μg·mL(-1). The established UHPLC-MS method could serve as a quality control tool for X. sorbifolia. In conclusion, the high contents of antioxidant and α-glucosidase inhibitory constituents in X. sorbifolia support its use as complementation of other therapeutic agents for metabolic disorders, such as diabetes and hypertension. Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

α-Glucosidase inhibitors and phytotoxins from Streptomyces xanthophaeus.

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Wei, Jing; Zhang, Xiu-Yun; Deng, Shan; Cao, Lin; Xue, Quan-Hong; Gao, Jin-Ming

2017-09-01

Twenty-four metabolites 1-24 were isolated from the fermentation broth of Streptomyces xanthophaeus. Their structures were elucidated on the basis of spectroscopic analysis and by comparison of their NMR data with literature data reported. Daidzein (1), genistein (2) and gliricidin (3) inhibited α-glucosidase in vitro with IC 50 values of 174.2, 36.1 and 47.4 μM, respectively, more potent than the positive control, acarbose. Docking study revealed that the amino acid residue Thr 215 is the essential binding site for active ligands 2. In addition, the phytotoxic effects of all compounds were assayed on radish seedlings, five of which, 3, 8, 13, 15 and 18, inhibited the growth of radish (Raphanus sativus) seedlings with inhibitory rates of >60% at a concentration of 100 ppm, which was comparable or superior to the positive control glyphosate. This is the first report of the phytotoxicity of the compounds.

Revisiting overexpression of a heterologous β-glucosidase in Trichoderma reesei: fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities.

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Xue, Xianli; Wu, Yilan; Qin, Xing; Ma, Rui; Luo, Huiying; Su, Xiaoyun; Yao, Bin

2016-07-11

The filamentous fungus Trichoderma reesei has the capacity to secret large amounts of cellulase and is widely used in a variety of industries. However, the T. reesei cellulase is weak in β-glucosidase activity, which results in accumulation of cellobiose inhibiting the endo- and exo-cellulases. By expressing an exogenous β-glucosidase gene, the recombinant T. reesei cellulase is expected to degrade cellulose into glucose more efficiently. The thermophilic β-glucosidase NfBgl3A from Neosartorya fischeri is chosen for overexpression in T. reesei due to its robust activity. In vitro, the Pichia pastoris-expressed NfBgl3A aided the T. reesei cellulase in releasing much more glucose with significantly lower amounts of cellobiose from crystalline cellulose. The NfBgl3A gene was hence fused to the cbh1 structural gene and assembled between the strong cbh1 promoter and cbh1 terminator to obtain pRS-NfBgl3A by using the DNA assembler method. pRS-NfBgl3A was transformed into the T. reesei uridine auxotroph strain TU-6. Six positive transformants showed β-glucosidase activities of 2.3-69.7 U/mL (up to 175-fold higher than that of wild-type). The largely different β-glucosidase activities in the transformants may be ascribed to the gene copy numbers of NfBgl3A or its integration loci. The T. reesei-expressed NfBgl3A showed highly similar biochemical properties to that expressed in P. pastoris. As expected, overexpression of NfBgl3A enhanced the overall cellulase activity of T. reesei. The CBHI activity in all transformants increased, possibly due to the extra copies of cbh1 gene introduced, while the endoglucanase activity in three transformants also largely increased, which was not observed in any other studies overexpressing a β-glucosidase. NfBgl3A had significant transglycosylation activity, generating sophorose, a potent cellulase inducer, and other oligosaccharides from glucose and cellobiose. We report herein the successful overexpression of a thermophilic N

Structural analysis of β-glucosidase mutants derived from a hyperthermophilic tetrameric structure

International Nuclear Information System (INIS)

Nakabayashi, Makoto; Kataoka, Misumi; Mishima, Yumiko; Maeno, Yuka; Ishikawa, Kazuhiko

2014-01-01

Substitutive mutations that convert a tetrameric β-glucosidase into a dimeric state lead to improvement of its crystal quality. β-Glucosidase from Pyrococcus furiosus (BGLPf) is a hyperthermophilic tetrameric enzyme which can degrade cellooligosaccharides to glucose under hyperthermophilic conditions and thus holds promise for the saccharification of lignocellulosic biomass at high temperature. Prior to the production of large amounts of this enzyme, detailed information regarding the oligomeric structure of the enzyme is required. Several crystals of BGLPf have been prepared over the past ten years, but its crystal structure had not been solved until recently. In 2011, the first crystal structure of BGLPf was solved and a model was constructed at somewhat low resolution (2.35 Å). In order to obtain more detailed structural data on BGLPf, the relationship between its tetrameric structure and the quality of the crystal was re-examined. A dimeric form of BGLPf was constructed and its crystal structure was solved at a resolution of 1.70 Å using protein-engineering methods. Furthermore, using the high-resolution crystal structural data for the dimeric form, a monomeric form of BGLPf was constructed which retained the intrinsic activity of the tetrameric form. The thermostability of BGLPf is affected by its oligomeric structure. Here, the biophysical and biochemical properties of engineered dimeric and monomeric BGLPfs are reported, which are promising prototype models to apply to the saccharification reaction. Furthermore, details regarding the oligomeric structures of BGLPf and the reasons why the mutations yielded improved crystal structures are discussed

Engineering the cytokinin-glucoside specificity of the maize beta-D-glucosidase Zm-p60.1 using site-directed random mutagenesis

Czech Academy of Sciences Publication Activity Database

Filipi, T.; Mazura, P.; Janda, L.; Kiran, N.S.; Brzobohatý, Břetislav

2012-01-01

Roč. 74, FEB2012 (2012), s. 40-48 ISSN 0031-9422 Institutional support: RVO:68081707 Keywords : beta-Glucosidase * cis-Zeatin-O-beta-D-glucopyranoside * Cytokinin metabolism Subject RIV: BO - Biophysics Impact factor: 3.050, year: 2012

Efficient production of lignocellulolytic enzymes xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1

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Leda Maria Fortes Gottschalk

2013-01-01

Full Text Available The production of xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by Aspergillus awamori 2B.361 U2/1, a hyper producer of glucoamylase and pectinase, was evaluated using selected conditions regarding nitrogen nutrition. Submerged cultivations were carried out at 30 ºC and 200 rpm in growth media containing 30 g wheat bran/L as main carbon source and either yeast extract, ammonium sulfate, sodium nitrate or urea, as nitrogen sources; in all cases it was used a fixed molar carbon to molar nitrogen concentration of 10.3. The use of poor nitrogen sources favored the accumulation of xylanase, β-xylosidase and ferulic acid esterase to a peak concentrations of 44,880; 640 and 118 U/L, respectively, for sodium nitrate and of 34,580, 685 and 170 U/L, respectively, for urea. However, the highest β-glucosidase accumulation of 10,470 U/L was observed when the rich organic nitrogen source yeast extract was used. The maxima accumulation of filter paper activity, xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of β-glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and β-xylosidase were over 2-fold higher. This strain also produced ferulic acid esterase (170 U/L, which was not detected in the T. reesei culture.

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African Journals Online (AJOL)

Effects of aqueous extract of Allium cepa (red onion) on ovalbumininduced allergic asthma in wistar rats. Abstract PDF · Vol 10, No 1 (2017) - Articles HPLC profile, in vitro alpha-amylase, alpha-glucosidase inhibitory and antioxidant activities of Gymnema sylvestre ethyl acetate leaf extract. Abstract PDF. ISSN: 2006-6996.

Intracellular β-Glucosidases CEL1a and CEL1b Are Essential for Cellulase Induction on Lactose in Trichoderma reesei

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Xu, Jintao; Zhao, Guolei; Kou, Yanbo; Zhang, Weixin; Zhou, Qingxin; Chen, Guanjun

2014-01-01

Lactose (1,4-O-β-d-galacto-pyranosyl-d-glucose) induces cellulolytic enzymes in Trichoderma reesei and is in fact one of the most important soluble carbon sources used to produce cellulases on an industrial level. The mechanism underlying the induction is, however, not fully understood. In this study, we investigated the cellular functions of the intracellular β-glucosidases CEL1a and CEL1b in the induction of cellulase genes by lactose in T. reesei. We demonstrated that while CEL1a and CEL1b were functionally equivalent in mediating the induction, the simultaneous absence of these intracellular β-glucosidases abolished cbh1 gene expression on lactose. d-Galactose restored the efficient cellulase gene induction in the Δcel1a strain independently of its reductive metabolism, but not in the Δcel1a Δcel1b strain. A further comparison of the transcriptional responses of the Δcel1a Δcel1b strain complemented with wild-type CEL1a or a catalytically inactive CEL1a version and the Δcel1a strain constitutively expressing CEL1a or the Kluyveromyces lactis β-galactosidase LAC4 showed that both the CEL1a protein and its glycoside hydrolytic activity were indispensable for cellulase induction by lactose. We also present evidence that intracellular β-glucosidase-mediated lactose induction is further conveyed to XYR1 to ensure the efficiently induced expression of cellulase genes. PMID:24879125

Expression of a codon-optimized β-glucosidase from Cellulomonas flavigena PR-22 in Saccharomyces cerevisiae for bioethanol production from cellobiose.

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Ríos-Fránquez, Francisco Javier; González-Bautista, Enrique; Ponce-Noyola, Teresa; Ramos-Valdivia, Ana Carmela; Poggi-Varaldo, Héctor Mario; García-Mena, Jaime; Martinez, Alfredo

2017-05-01

Bioethanol is one of the main biofuels produced from the fermentation of saccharified agricultural waste; however, this technology needs to be optimized for profitability. Because the commonly used ethanologenic yeast strains are unable to assimilate cellobiose, several efforts have been made to express cellulose hydrolytic enzymes in these yeasts to produce ethanol from lignocellulose. The C. flavigenabglA gene encoding β-glucosidase catalytic subunit was optimized for preferential codon usage in S. cerevisiae. The optimized gene, cloned into the episomal vector pRGP-1, was expressed, which led to the secretion of an active β-glucosidase in transformants of the S. cerevisiae diploid strain 2-24D. The volumetric and specific extracellular enzymatic activities using pNPG as substrate were 155 IU L -1 and 222 IU g -1 , respectively, as detected in the supernatant of the cultures of the S. cerevisiae RP2-BGL transformant strain growing in cellobiose (20 g L -1 ) as the sole carbon source for 48 h. Ethanol production was 5 g L -1 after 96 h of culture, which represented a yield of 0.41 g g -1 of substrate consumed (12 g L -1 ), equivalent to 76% of the theoretical yield. The S. cerevisiae RP2-BGL strain expressed the β-glucosidase extracellularly and produced ethanol from cellobiose, which makes this microorganism suitable for application in ethanol production processes with saccharified lignocellulose.

A novel class of α-glucosidase and HMG-CoA reductase inhibitors from Ganoderma leucocontextum and the anti-diabetic properties of ganomycin I in KK-Ay mice.

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Wang, Kai; Bao, Li; Ma, Ke; Zhang, Jinjin; Chen, Baosong; Han, Junjie; Ren, Jinwei; Luo, Huajun; Liu, Hongwei

2017-02-15

Three new meroterpenoids, ganoleucin A-C (1-3), together with five known meroterpenoids (4-8), were isolated from the fruiting bodies of Ganoderma leucocontextum. The structures of the new compounds were elucidated by extensive spectroscopic analysis, circular dichroism (CD) spectroscopy, and chemical transformation. The inhibitory effects of 1-8 on HMG-CoA reductase and α-glucosidase were tested in vitro. Ganomycin I (4), 5, and 8 showed stronger inhibitory activity against HMG-CoA reductase than the positive control atorvastatin. Compounds 1, and 3-8 presented potent noncompetitive inhibitory activity against α-glucosidase from both yeast and rat small intestinal mucosa. Ganomycin I (4), the most potent inhibitor against both α-glucosidase and HMG-CoA reductase, was synthesized and evaluated for its in vivo bioactivity. Pharmacological results showed that ganomycin I (4) exerted potent and efficacious hypoglycemic, hypolipidemic, and insulin-sensitizing effects in KK-A y mice. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

In vitro antioxidant and, α-glucosidase inhibitory activities and comprehensive metabolite profiling of methanol extract and its fractions from Clinacanthus nutans.

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Alam, Md Ariful; Zaidul, I S M; Ghafoor, Kashif; Sahena, F; Hakim, M A; Rafii, M Y; Abir, H M; Bostanudin, M F; Perumal, V; Khatib, A

2017-03-31

This study was aimed to evaluate antioxidant and α-glucosidase inhibitory activity, with a subsequent analysis of total phenolic and total flavonoid content of methanol extract and its derived fractions from Clinacanthus nutans accompanied by comprehensive phytochemical profiling. Liquid-liquid partition chromatography was used to separate methanolic extract to get hexane, ethyl acetate, butanol and residual aqueous fractions. The total antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazy (DPPH) radical scavenging and ferric reducing antioxidant power assay (FRAP). The antidiabetic activity of methanol extract and its consequent fractions were examined by α-glucosidase inhibitory bioassay. The chemical profiling was carried out by gas chromatography coupled with quadrupole time-of-flight mass spectrometry (GC Q-TOF MS). The total yield for methanol extraction was (12.63 ± 0.98) % (w/w) and highest fractionated value found for residual aqueous (52.25 ± 1.01) % (w/w) as compared to the other fractions. Significant DPPH free radical scavenging activity was found for methanolic extract (63.07 ± 0.11) % and (79.98 ± 0.31) % for ethyl acetate fraction among all the fractions evaluated. Methanol extract was the most prominent in case of FRAP (141.89 ± 0.87 μg AAE/g) whereas most effective reducing power observed in ethyl acetate fraction (133.6 ± 0.2987 μg AAE/g). The results also indicated a substantial α-glucosidase inhibitory activity for butanol fraction (72.16 ± 1.0) % and ethyl acetate fraction (70.76 ± 0.49) %. The statistical analysis revealed that total phenolic and total flavonoid content of the samples had the significant (p < 0.05) impact on DPPH free radical scavenging and α-glucosidase inhibitory activity. Current results proposed the therapeutic potential of Clinacanthus nutans, especially ethyl acetate and butanol fraction as chemotherapeutic agent against oxidative related cellular damages and control the

Human acid β-glucosidase: isolation and amino acid sequence of a peptide containing the catalytic site

International Nuclear Information System (INIS)

Dinur, T.; Osiecki, K.M.; Legler, G.; Gatt, S.; Desnick, R.J.; Grabowski, G.A.

1986-01-01

Human acid β-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) cleaves the glucosidic bonds of glucosylceramide and synthetic β-glucosides. The deficient activity of this hydrolase is the enzymatic defect in the subtypes and variants of Gaucher disease, the most prevalent lysosomal storage disease. To isolate and characterize the catalytic site of the normal enzyme, brominated 3 H-labeled conduritol B epoxide ( 3 H-Br-CBE), which inhibits the enzyme by binding covalently to this site, was used as an affinity label. Under optimal conditions 1 mol of 3 H-Br-CBE bound to 1 mol of pure enzyme protein, indicating the presence of a single catalytic site per enzyme subunit. After V 8 protease digestion of the 3 H-Br-CBE-labeled homogeneous enzyme, three radiolabeled peptides, designated peptide A, B, or C, were resolved by reverse-phase HPLC. The partial amino acid sequence (37 residues) of peptide A (M/sub r/, 5000) was determined. The sequence of this peptide, which contained the catalytic site, had exact homology to the sequence near the carboxyl terminus of the protein, as predicted from the nucleotide sequence of the full-length cDNA encoding acid β-glucosidase

Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

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Shoji Suzuki

2017-01-01

Full Text Available Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER. In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr- and arginine decarboxylase- (argD- deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

α-Glucosidase inhibition and antioxidant activity of an oenological commercial tannin. Extraction, fractionation and analysis by HPLC/ESI-MS/MS and (1)H NMR.

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Muccilli, Vera; Cardullo, Nunzio; Spatafora, Carmela; Cunsolo, Vincenzo; Tringali, Corrado

2017-01-15

Two batches of the oenological tannin Tan'Activ R, (toasted oak wood - Quercus robur), were extracted with ethanol. A fractionation on XAD-16 afforded four fractions for each extract. Extracts and fractions were evaluated for antioxidant activity (DPPH), polyphenol content (GAE) and yeast α-glucosidase inhibitory activity. Comparable results were obtained for both columns, fractions X1B and X2B showing the highest antioxidant activity. Fractions X1C and X2C notably inhibited α-glucosidase, with IC50=9.89 and 8.05μg/mL, respectively. Fractions were subjected to HPLC/ESI-MS/MS and (1)H NMR analysis. The main phenolic constituents of both X1B and X2B were a monogalloylglucose isomer (1), a HHDP-glucose isomer (2), castalin (3) gallic acid (4), vescalagin (5), and grandinin (or its isomer roburin E, 6). X1C and X2C showed a complex composition, including non-phenolic constituents. Fractionation of X2C gave a subfraction, with enhanced α-glucosidase inhibitory activity (IC50=6.15μg/mL), with castalagin (7) as the main constituent. Copyright © 2016 Elsevier Ltd. All rights reserved.

The S-Layer Glycoprotein of the Crenarchaeote Sulfolobus acidocaldarius Is Glycosylated at Multiple Sites with Chitobiose-Linked N-Glycans

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Elham Peyfoon

2010-01-01

Full Text Available Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L1004-Q1395. Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30–40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Glc1Man2GlcNAc2 plus 6-sulfoquinovose (QuiS, consistent with the tribranched hexasaccharide previously reported in the cytochrome b558/566 of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya.

Distribution of alpha3, alpha5 and alpha(v) integrin subunits in mature and immature human oocytes.

Science.gov (United States)

Capmany, G; Mart, M; Santaló, J; Bolton, V N

1998-10-01

The distribution of three integrin subunits, alpha3, alpha5 and alpha(v), in immature and mature human oocytes has been examined using immunofluorescence and confocal microscopy. The results demonstrate that both alpha5 and alpha(v) are present at the germinal vesicle stage, while alpha3 was only detected in oocytes after germinal vesicle breakdown, in metaphase I and II stage oocytes. The cortical concentration of integrin subunits alpha3 and alpha5 is consistent with their localization in the oolemma. In contrast, the homogeneous distribution of alpha(v) throughout the oocyte suggests the existence of cytoplasmic reservoirs of this protein in the oocyte.

Screening for potential α-glucosidase and α-amylase inhibitory constituents from selected Vietnamese plants used to treat type 2 diabetes

DEFF Research Database (Denmark)

Trinh, Thi Dieu Binh; Stærk, Dan; Jäger, Anna K

2016-01-01

Ethnopharmacological relevance The 18 plant species investigated in this study have been used as herbal antidiabetic remedies in Vietnamese traditional medicines. This study aimed to evaluate their ability to inhibit α-glucosidase and α-amylase, two key enzymes involved in serum glucose regulation...

Enzymatic and structural characterization of hydrolysis of gibberellin A4 glucosyl ester by a rice β-D-glucosidase.

Science.gov (United States)

Hua, Yanling; Sansenya, Sompong; Saetang, Chiraporn; Wakuta, Shinji; Ketudat Cairns, James R

2013-09-01

In order to identify a rice gibberellin ester β-D-glucosidase, gibberellin A4 β-D-glucosyl ester (GA4-GE) was synthesized and used to screen rice β-glucosidases. Os3BGlu6 was found to have the highest hydrolysis activity to GA4-GE among five recombinantly expressed rice glycoside hydrolase family GH1 enzymes from different phylogenic clusters. The kinetic parameters of Os3BGlu6 and its mutants E178Q, E178A, E394D, E394Q and M251N for hydrolysis of p-nitrophenyl β-D-glucopyranoside (pNPGlc) and GA4-GE confirmed the roles of the catalytic acid/base and nucleophile for hydrolysis of both substrates and suggested M251 contributes to binding hydrophobic aglycones. The activities of the Os3BGlu6 E178Q and E178A acid/base mutants were rescued by azide, which they transglucosylate to produce β-D-glucopyranosyl azide, in a pH-dependent manner, while acetate also rescued Os3BGlu6 E178A at low pH. High concentrations of sodium azide (200-400 mM) inhibited Os3BGlu6 E178Q but not Os3BGlu6 E178A. The structures of Os3BGlu6 E178Q crystallized with either GA4-GE or pNPGlc had a native α-D-glucosyl moiety covalently linked to the catalytic nucleophile, E394, which showed the hydrogen bonding to the 2-hydroxyl in the covalent intermediate. These data suggest that a GH1 β-glucosidase uses the same retaining catalytic mechanism to hydrolyze 1-O-acyl glucose ester and glucoside. Copyright © 2013 Elsevier Inc. All rights reserved.

High performance liquid chromatography profiling of health-promoting phytochemicals and evaluation of antioxidant, anti-lipoxygenase, iron chelating and anti-glucosidase activities of wetland macrophytes.

Science.gov (United States)

Ooh, Keng-Fei; Ong, Hean-Chooi; Wong, Fai-Chu; Sit, Nam-Weng; Chai, Tsun-Thai

2014-08-01

The phytochemistry and bioactivity of wetland macrophytes are underexplored. Plants are known as the natural sources of phytochemical beneficial to health. The objective of this study is to analyze the phytochemical profiles and bioactivities of 10 extracts prepared from different plant parts of wetland macrophytes Hanguana malayana, Ludwigia adscendens and Monochoria hastata. High performance liquid chromatography (HPLC) was used to analyze the phytochemical profile of the extracts. Antioxidant assay such as 2,2-diphenyl-1-picrylhydrazyl, nitric oxide (NO) radical scavenging activity and ferric reducing antioxidant power were performed. Bioactivity assays carried out were anti-lipoxygenase, anti-glucosidase, and iron chelating. Leaf extract of L. adscendens had the highest 2,2-diphenyl-1-picrylhydrazyl (half of maximal effective concentration [EC50] =0.97 mg/mL) and NO (EC50 = 0.31 mg/mL) scavenging activities. The extract also exhibited the highest iron chelating (EC50 = 3.24 mg/mL) and anti-glucosidase (EC50 = 27.5 μg/mL) activities. The anti-glucosidase activity of L. adscendens leaf extract was comparable or superior to those of acarbose, myricetin and quercetin. Correlation between iron chelating and radical scavenging activities among the extracts implies the presence of dual-function phytoconstituents with concurrent iron chelating and radical scavenging activities. HPLC analysis revealed the presence of p-coumaric acid (p-CA), gallic acid (GA) and myricetin in all or most extracts. M. hastata fruit and leaf extracts had the highest p-hydroxybenzoic acid content. Antioxidant and anti-glucosidase activities of the extracts were correlated with p-CA, GA, and myricetin contents. Our study demonstrated that wetland macrophytes H. malayana, L. adscendens and M. hastata are potential sources of health-promoting phytochemicals with potent therapeutically-relevant bioactivities.

High performance liquid chromatography profiling of health-promoting phytochemicals and evaluation of antioxidant, anti-lipoxygenase, iron chelating and anti-glucosidase activities of wetland macrophytes

Science.gov (United States)

Ooh, Keng-Fei; Ong, Hean-Chooi; Wong, Fai-Chu; Sit, Nam-Weng; Chai, Tsun-Thai

2014-01-01

Background: The phytochemistry and bioactivity of wetland macrophytes are underexplored. Plants are known as the natural sources of phytochemical beneficial to health. Objective: The objective of this study is to analyze the phytochemical profiles and bioactivities of 10 extracts prepared from different plant parts of wetland macrophytes Hanguana malayana, Ludwigia adscendens and Monochoria hastata. Materials and Methods: High performance liquid chromatography (HPLC) was used to analyze the phytochemical profile of the extracts. Antioxidant assay such as 2,2-diphenyl-1-picrylhydrazyl, nitric oxide (NO) radical scavenging activity and ferric reducing antioxidant power were performed. Bioactivity assays carried out were anti-lipoxygenase, anti-glucosidase, and iron chelating. Results: Leaf extract of L. adscendens had the highest 2,2-diphenyl-1-picrylhydrazyl (half of maximal effective concentration [EC50] =0.97 mg/mL) and NO (EC50 = 0.31 mg/mL) scavenging activities. The extract also exhibited the highest iron chelating (EC50 = 3.24 mg/mL) and anti-glucosidase (EC50 = 27.5 μg/mL) activities. The anti-glucosidase activity of L. adscendens leaf extract was comparable or superior to those of acarbose, myricetin and quercetin. Correlation between iron chelating and radical scavenging activities among the extracts implies the presence of dual-function phytoconstituents with concurrent iron chelating and radical scavenging activities. HPLC analysis revealed the presence of p-coumaric acid (p-CA), gallic acid (GA) and myricetin in all or most extracts. M. hastata fruit and leaf extracts had the highest p-hydroxybenzoic acid content. Antioxidant and anti-glucosidase activities of the extracts were correlated with p-CA, GA, and myricetin contents. Conclusion: Our study demonstrated that wetland macrophytes H. malayana, L. adscendens and M. hastata are potential sources of health-promoting phytochemicals with potent therapeutically-relevant bioactivities. PMID:25298659

Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids.

Science.gov (United States)

Gerasimenko, Irina; Sheludko, Yuri; Ma, Xueyan; Stöckigt, Joachim

2002-04-01

Strictosidine glucosidase (SG) is an enzyme that catalyses the second step in the biosynthesis of various classes of monoterpenoid indole alkaloids. Based on the comparison of cDNA sequences of SG from Catharanthus roseus and raucaffricine glucosidase (RG) from Rauvolfia serpentina, primers for RT-PCR were designed and the cDNA encoding SG was cloned from R. serpentina cell suspension cultures. The active enzyme was expressed in Escherichia coli and purified to homogeneity. Analysis of its deduced amino-acid sequence assigned the SG from R. serpentina to family 1 of glycosyl hydrolases. In contrast to the SG from C. roseus, the enzyme from R. serpentina is predicted to lack an uncleavable N-terminal signal sequence, which is believed to direct proteins to the endoplasmic reticulum. The temperature and pH optimum, enzyme kinetic parameters and substrate specificity of the heterologously expressed SG were studied and compared to those of the C. roseus enzyme, revealing some differences between the two glucosidases. In vitro deglucosylation of strictosidine by R. serpentina SG proceeds by the same mechanism as has been shown for the C. roseus enzyme preparation. The reaction gives rise to the end product cathenamine and involves 4,21-dehydrocorynantheine aldehyde as an intermediate. The enzymatic hydrolysis of dolichantoside (Nbeta-methylstrictosidine) leads to several products. One of them was identified as a new compound, 3-isocorreantine A. From the data it can be concluded that the divergence of the biosynthetic pathways leading to different classes of indole alkaloids formed in R. serpentina and C. roseus cell suspension cultures occurs at a later stage than strictosidine deglucosylation.

A novel low-temperature-active β-glucosidase from symbiotic Serratia sp. TN49 reveals four essential positions for substrate accommodation.

Science.gov (United States)

Zhou, Junpei; Zhang, Rui; Shi, Pengjun; Huang, Huoqing; Meng, Kun; Yuan, Tiezheng; Yang, Peilong; Yao, Bin

2011-10-01

A 2,373-bp full-length gene (bglA49) encoding a 790-residue polypeptide (BglA49) with a calculated mass of 87.8 kDa was cloned from Serratia sp. TN49, a symbiotic bacterium isolated from the gut of longhorned beetle (Batocera horsfieldi) larvae. The deduced amino acid sequence of BglA49 showed the highest identities of 80.1% with a conceptually translated protein from Pantoea sp. At-9b (EEW02556), 38.3% with the identified glycoside hydrolase (GH) family 3 β-glucosidase from Clostridium stercorarium NCBI 11754 (CAB08072), and sp. G5 (ABL09836) and Paenibacillus sp. C7 (AAX35883). The recombinant enzyme (r-BglA49) was expressed in Escherichia coli and displayed the typical characteristics of low-temperature-active enzymes, such as low temperature optimum (showing apparent optimal activity at 35°C), activity at low temperatures (retaining approximately 60% of its maximum activity at 20°C and approximately 25% at 10°C). Compared with the thermophilic GH 3 β-glucosidase, r-BglA49 had fewer hydrogen bonds and salt bridges and less proline residues. These features might relate to the increased structure flexibility and higher catalytic activity at low temperatures of r-BglA49. The molecular docking study of four GH 3 β-glucosidases revealed five conserved positions contributing to substrate accommodation, among which four positions of r-BglA49 (R192, Y228, D260, and E449) were identified to be essential based on site-directed mutagenesis analysis.

Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis

Energy Technology Data Exchange (ETDEWEB)

Ruppert, Martin [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Panjikar, Santosh [European Molecular Biology Laboratory Hamburg, Outstation Deutsches Elektronen-Synchrotron, Notkestrasse 85, D-22603 Hamburg (Germany); Barleben, Leif [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Stöckigt, Joachim [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); College of Pharmaceutical Sciences, Zhejiang University, 353 Yan An Road, 310031 Hangzhou (China)

2006-03-01

Raucaffricine glucosidase, an enzyme involved in the biosynthesis of monoterpenoid indole alkaloids in the plant Rauvolfia serpentina, was crystallized by the hanging-drop vapour-diffusion method using PEG4000 as precipitant. The crystals diffract to 2.3 Å resolution and belong to space group I222. Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 Å, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 Å.

Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis

International Nuclear Information System (INIS)

Ruppert, Martin; Panjikar, Santosh; Barleben, Leif; Stöckigt, Joachim

2006-01-01

Raucaffricine glucosidase, an enzyme involved in the biosynthesis of monoterpenoid indole alkaloids in the plant Rauvolfia serpentina, was crystallized by the hanging-drop vapour-diffusion method using PEG4000 as precipitant. The crystals diffract to 2.3 Å resolution and belong to space group I222. Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 Å, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 Å

Polyhydroxylated alkaloids isolated from mulberry trees (Morusalba L.) and silkworms (Bombyx mori L.).

Science.gov (United States)

Asano, N; Yamashita, T; Yasuda, K; Ikeda, K; Kizu, H; Kameda, Y; Kato, A; Nash, R J; Lee, H S; Ryu, K S

2001-09-01

New polyhydroxylated alkaloids, (2R,3R,4R)-2-hydroxymethyl-3,4-dihydroxypyrrolidine-N-propionamide from the root bark of Morus alba L., and 4-O-alpha-D-galactopyranosyl-calystegine B(2) and 3 beta,6 beta-dihydroxynortropane from the fruits, were isolated by column chromatography using a variety of ion-exchange resins. Fifteen other polyhydroxylated alkaloids were also isolated. 1-Deoxynojirimycin, a potent alpha-glucosidase inhibitor, was concentrated 2.7-fold by silkworms feeding on mulberry leaves. Some alkaloids contained in mulberry leaves were potent inhibitors of mammalian digestive glycosidases but not inhibitors of silkworm midgut glycosidases, suggesting that the silkworm has enzymes specially adapted to enable it to feed on mulberry leaves. The possibility of preventing the onset of diabetes and obesity using natural dietary supplements containing 1-deoxynojirimycin and other alpha-glucosidase inhibitors in high concentration is of great potential interest.

The determination of $\\alpha_s$ by the ALPHA collaboration

CERN Document Server

Bruno, Mattia

2016-01-01

We review the ALPHA collaboration strategy for obtaining the QCD coupling at high scale. In the three-flavor effective theory it avoids the use of perturbation theory at $\\alpha > 0.2$ and at the same time has the physical scales small compared to the cutoff $1/a$ in all stages of the computation. The result $\\Lambda_\\overline{MS}^{(3)}=332(14)$~MeV is translated to $\\alpha_\\overline{MS}(m_Z)=0.1179(10)(2)$ by use of (high order) perturbative relations between the effective theory couplings at the charm and beauty quark "thresholds". The error of this perturbative step is discussed and estimated as $0.0002$.

Influence of season, environment and feeding habits on the enzymatic activity of peptidase and β-glucosidase in the gastrointestinal tract of two Siluriformes fishes (Teleostei

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Silvana Duarte

2013-06-01

Full Text Available The enzymatic activities involved in the digestion of proteins and carbohydrates were compared among three organs of the digestive track of two Siluriformes fish species, and between two areas: a reservoir, and an area downriver of it. Our aim was to test the hypothesis that the digestive organs of species with varied feeding habits have different enzymatic activities, and that the enzymatic activity differs among seasons and environmental conditions. The iliophagous/herbivorous species Hypostomus auroguttatus Kner, 1854 had higher trypsin-like, chymotrypsin-like peptidase and β-glucosidase activity in the intestine when compared with the omnivorous species Pimelodus maculatus Lacepède, 1803, whereas the latter had more hepatic trypsin-like activity than the former. The peak of activity of the three enzymes in H. auroguttatus was recorded in the winter and spring. On the other hand, P. maculatus tended to have the more prominent peptidase and β-glucosidase activity in the summer, and the smallest in the winter. The intestine of H. auroguttatus had higher enzymatic (trypsin, chymotrypsin and β-glucosidase activity than the stomach and the liver. For P. maculatus, the highest β-glucosidase activity was found in the liver. The enzymatic activity of H. aurogutattus did not differ between lotic and lentic systems, whereas P. maculatus had comparatively higher stomach and hepatic trypsin levels and hepatic chymotrypsin-like activities in the reservoir than down in the river. These findings indicate that, in H. auroguttatus, most digestive activity occurs in the intestine, which is long and adapted for the digestion of bottom-river vegetable matter and detritus. The seasons and the type of the system (lentic vs. lotic seem to affect the enzymatic activity for these two species differently, a likely consequence of their different lifestyles.

Gene cloning and characterization of a cold-adapted β-glucosidase belonging to glycosyl hydrolase family 1 from a psychrotolerant bacterium Micrococcus antarcticus.

Science.gov (United States)

Fan, Hong-Xia; Miao, Li-Li; Liu, Ying; Liu, Hong-Can; Liu, Zhi-Pei

2011-06-10

The gene bglU encoding a cold-adapted β-glucosidase (BglU) was cloned from Micrococcus antarcticus. Sequence analysis revealed that the bglU contained an open reading frame of 1419 bp and encoded a protein of 472 amino acid residues. Based on its putative catalytic domains, BglU was classified as a member of the glycosyl hydrolase family 1 (GH1). BglU possessed lower arginine content and Arg/(Arg+Lys) ratio than mesophilic GH1 β-glucosidases. Recombinant BglU was purified with Ni2+ affinity chromatography and subjected to enzymatic characterization. SDS-PAGE and native staining showed that it was a monomeric protein with an apparent molecular mass of 48 kDa. BglU was particularly thermolabile since its half-life time was only 30 min at 30°C and it exhibited maximal activity at 25°C and pH 6.5. Recombinant BglU could hydrolyze a wide range of aryl-β-glucosides and β-linked oligosaccharides with highest activity towards cellobiose and then p-nitrophenyl-β-d-glucopyranoside (pNPG). Under the optimal conditions with pNPG as substrate, the K(m) and k(cat) were 7 mmol/L and 7.85 × 103/s, respectively. This is the first report of cloning and characterization of a cold-adapted β-glucosidase belonging to GH1 from a psychrotolerant bacterium. Copyright © 2011 Elsevier Inc. All rights reserved.

Contribution of Musa paradisiaca in the inhibition of α-amylase, α-glucosidase and Angiotensin-I converting enzyme in streptozotocin induced rats.

Science.gov (United States)

Shodehinde, Sidiqat A; Ademiluyi, Adedayo O; Oboh, Ganiyu; Akindahunsi, Afolabi A

2015-07-15

Unripe plantain based-diets are part of folklore remedy for the management of diabetes in tropical Africa; however, with the dearth of information on the rationale behind this practice; this study therefore, sought to investigate the antihyperglycemic effect of traditional unripe plantain products (Amala and Booli) in high fat fed/low dose streptozotocin-induced diabetic rats and to provide a possible rationale for their antidiabetic properties. Diabetes was induced experimentally by high fat fed/low dose streptozotocin-diabetic rats (25mg/kg body wt.) and the diabetic rats were fed diets supplemented with 20-40% Amala and Booli for 14 days. The effect of the diets on the blood glucose level, pancreatic α-amylase, intestinal α-glucosidase and Angiotensin-I converting enzyme (ACE) activities and plasma antioxidant status as well as amylose/amylopectin content of the unripe plantain products were determined. A marked increase in the blood glucose, α-amylase, α-glucosidase and ACE activities with a corresponding decrease in plasma antioxidant status was recorded in diabetic rats. However, these indices were significantly (P < 0.05) reversed after unripe plantain product supplemented diet treatments for 14 days. Also, the amylose/amylopectin ratio of the products is 1:3. This study revealed that unripe plantain products exert antihyperglycemic effects which could be attributed to the inhibition of α-amylase and α-glucosidase activities by their constituent phytochemicals as well as their amylose/amylopectin contents in the diabetic rats, hence, providing the possible rationale behind their antidiabetic properties. Copyright © 2015 Elsevier Inc. All rights reserved.

Differentiation of the mRNA transcripts originating from the alpha 1- and alpha 2-globin loci in normals and alpha-thalassemics.

OpenAIRE

Liebhaber, S A; Kan, Y W

1981-01-01

The alpha-globin polypeptide is encoded by two adjacent genes, alpha 1 and alpha 2. In the normal diploid state (alpha alpha/alpha alpha) all four alpha-globin genes are expressed. Loss or dysfunction of one or more of these genes leads to deficient alpha-globin production and results in alpha-thalassemia. We present a technique to differentially assess the steady-state levels of the alpha 1- and alpha-2-globin messenger RNA (mRNA) transcripts and thus delineate the relative level of expressi...

A proprietary alpha-amylase inhibitor from white bean (Phaseolus vulgaris): a review of clinical studies on weight loss and glycemic control.

Science.gov (United States)

Barrett, Marilyn L; Udani, Jay K

2011-03-17

Obesity, and resultant health hazards which include diabetes, cardiovascular disease and metabolic syndrome, are worldwide medical problems. Control of diet and exercise are cornerstones of the management of excess weight. Foods with a low glycemic index may reduce the risk of diabetes and heart disease as well as their complications. As an alternative to a low glycemic index diet, there is a growing body of research into products that slow the absorption of carbohydrates through the inhibition of enzymes responsible for their digestion. These products include alpha-amylase and glucosidase inhibitors. The common white bean (Phaseolus vulgaris) produces an alpha-amylase inhibitor, which has been characterized and tested in numerous clinical studies. A specific and proprietary product named Phase 2® Carb Controller (Pharmachem Laboratories, Kearny, NJ) has demonstrated the ability to cause weight loss with doses of 500 to 3000 mg per day, in either a single dose or in divided doses. Clinical studies also show that Phase 2 has the ability to reduce the post-prandial spike in blood glucose levels. Experiments conducted incorporating Phase 2 into food and beverage products have found that it can be integrated into various products without losing activity or altering the appearance, texture or taste of the food. There have been no serious side effects reported following consumption of Phase 2. Gastro-intestinal side effects are rare and diminish upon extended use of the product. In summary, Phase 2 has the potential to induce weight loss and reduce spikes in blood sugar caused by carbohydrates through its alpha-amylase inhibiting activity.

A proprietary alpha-amylase inhibitor from white bean (Phaseolus vulgaris: A review of clinical studies on weight loss and glycemic control

Directory of Open Access Journals (Sweden)

Barrett Marilyn L

2011-03-01

Full Text Available Abstract Obesity, and resultant health hazards which include diabetes, cardiovascular disease and metabolic syndrome, are worldwide medical problems. Control of diet and exercise are cornerstones of the management of excess weight. Foods with a low glycemic index may reduce the risk of diabetes and heart disease as well as their complications. As an alternative to a low glycemic index diet, there is a growing body of research into products that slow the absorption of carbohydrates through the inhibition of enzymes responsible for their digestion. These products include alpha-amylase and glucosidase inhibitors. The common white bean (Phaseolus vulgaris produces an alpha-amylase inhibitor, which has been characterized and tested in numerous clinical studies. A specific and proprietary product named Phase 2® Carb Controller (Pharmachem Laboratories, Kearny, NJ has demonstrated the ability to cause weight loss with doses of 500 to 3000 mg per day, in either a single dose or in divided doses. Clinical studies also show that Phase 2 has the ability to reduce the post-prandial spike in blood glucose levels. Experiments conducted incorporating Phase 2 into food and beverage products have found that it can be integrated into various products without losing activity or altering the appearance, texture or taste of the food. There have been no serious side effects reported following consumption of Phase 2. Gastro-intestinal side effects are rare and diminish upon extended use of the product. In summary, Phase 2 has the potential to induce weight loss and reduce spikes in blood sugar caused by carbohydrates through its alpha-amylase inhibiting activity.

Production of Ginsenoside F2 by Using Lactococcus lactis with Enhanced Expression of β-Glucosidase Gene from Paenibacillus mucilaginosus.

Science.gov (United States)

Li, Ling; Shin, So-Yeon; Lee, Soo Jin; Moon, Jin Seok; Im, Wan Taek; Han, Nam Soo

2016-03-30

This study aimed to produce a pharmacologically active minor ginsenoside F2 from the major ginsenosides Rb1 and Rd by using a recombinant Lactococcus lactis strain expressing a heterologous β-glucosidase gene. The nucleotide sequence of the gene (BglPm) was derived from Paenibacillus mucilaginosus and synthesized after codon optimization, and the two genes (unoptimized and optimized) were expressed in L. lactis NZ9000. Codon optimization resulted in reduction of unfavorable codons by 50% and a considerable increase in the expression levels (total activities) of β-glucosidases (0.002 unit/mL, unoptimized; 0.022 unit/mL, optimized). The molecular weight of the enzyme was 52 kDa, and the purified forms of the enzymes could successfully convert Rb1 and Rd into F2. The permeabilized L. lactis expressing BglPm resulted in a high conversion yield (74%) of F2 from the ginseng extract. Utilization of this microbial cell to produce F2 may provide an alternative method to increase the health benefits of Panax ginseng.

Stability of commercial glucanase and β-glucosidase preparations under hydrolysis conditions

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Oscar Rosales-Calderon

2014-06-01

Full Text Available The cost of enzymes makes enzymatic hydrolysis one of the most expensive steps in the production of lignocellulosic ethanol. Diverse studies have used commercial enzyme cocktails assuming that change in total protein concentration during hydrolysis was solely due to adsorption of endo- and exoglucanases onto the substrate. Given the sensitivity of enzymes and proteins to media conditions this assumption was tested by evaluating and modeling the protein concentration of commercial cocktails at hydrolysis conditions. In the absence of solid substrate, the total protein concentration of a mixture of Celluclast 1.5 L and Novozyme 188 decreased by as much as 45% at 50 °C after 4 days. The individual cocktails as well as a mixture of both were stable at 20 °C. At 50 °C, the protein concentration of Celluclast 1.5 was relatively constant but Novozyme 188 decreased by as much as 77%. It was hypothesized that Novozyme 188 proteins suffer a structural change at 50 °C which leads to protein aggregation and precipitation. Lyophilized β-glucosidase (P-β-glucosidase at 50 °C exhibited an aggregation rate which was successfully modeled using first order kinetics (R2 = 0.97. By incorporating the possible presence of chaperone proteins in Novozyme 188, the protein aggregation observed for this cocktail was successfully modeled (R2 = 0.96. To accurately model the increasing protein stability observed at high cocktail loadings, the model was modified to include the presence of additives in the cocktail (R2 = 0.98. By combining the measurement of total protein concentration with the proposed Novozyme 188 protein aggregation model, the endo- and exoglucanases concentration in the solid and liquid phases during hydrolysis can be more accurately determined. This methodology can be applied to various systems leading to optimization of enzyme loading by minimizing the excess of endo- and exoglucanases. In addition, the monitoring of endo- and exoglucanases

In Vitro Studies on the Antioxidant Property and Inhibition of α-Amylase, α-Glucosidase, and Angiotensin I-Converting Enzyme by Polyphenol-Rich Extracts from Cocoa (Theobroma cacao) Bean.

Science.gov (United States)

Oboh, Ganiyu; Ademosun, Ayokunle O; Ademiluyi, Adedayo O; Omojokun, Olasunkanmi S; Nwanna, Esther E; Longe, Kuburat O

2014-01-01

Background. This study sought to investigate the antidiabetic and antihypertensive mechanisms of cocoa (Theobroma cacao) bean through inhibition of α-amylase, α-glucosidase, angiotensin-1 converting enzyme, and oxidative stress. Methodology. The total phenol and flavonoid contents of the water extractable phytochemicals from the powdered cocoa bean were determined and the effects of the extract on α-amylase, α-glucosidase, and angiotensin-1 converting enzyme activities were investigated in vitro. Furthermore, the radicals [1,1-diphenyl-2 picrylhydrazyl (DPPH), 2,2..-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), hydroxyl (OH), and nitric oxide (NO)] scavenging ability and ferric reducing antioxidant property of the extract were assessed. Results. The results revealed that the extract inhibited α-amylase (1.81 ± 0.22 mg/mL), α-glucosidase (1.84 ± 0.17 mg/mL), and angiotensin-1 converting enzyme (0.674 ± 0.06 mg/mL [lungs], 1.006 ± 0.08 mg/mL [heart]) activities in a dose-dependent manner and also showed dose-dependent radicals [DPPH (16.94 ± 1.34 mg/mL), NO (6.98 ± 0.886 mg/mL), OH (3.72 ± 0.26 mg/mL), and ABTS (15.7 ± 1.06 mmol/TEAC·g] scavenging ability. Conclusion. The inhibition of α-amylase, α-glucosidase, and angiotensin-1 converting enzyme activities by the cocoa bean extract could be part of the possible mechanism by which the extract could manage and/or prevent type-2 diabetes and hypertension.

In Vitro Studies on the Antioxidant Property and Inhibition of α-Amylase, α-Glucosidase, and Angiotensin I-Converting Enzyme by Polyphenol-Rich Extracts from Cocoa (Theobroma cacao Bean

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Ganiyu Oboh

2014-01-01

Full Text Available Background. This study sought to investigate the antidiabetic and antihypertensive mechanisms of cocoa (Theobroma cacao bean through inhibition of α-amylase, α-glucosidase, angiotensin-1 converting enzyme, and oxidative stress. Methodology. The total phenol and flavonoid contents of the water extractable phytochemicals from the powdered cocoa bean were determined and the effects of the extract on α-amylase, α-glucosidase, and angiotensin-1 converting enzyme activities were investigated in vitro. Furthermore, the radicals [1,1-diphenyl-2 picrylhydrazyl (DPPH, 2,2..-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS, hydroxyl (OH, and nitric oxide (NO] scavenging ability and ferric reducing antioxidant property of the extract were assessed. Results. The results revealed that the extract inhibited α-amylase (1.81 ± 0.22 mg/mL, α-glucosidase (1.84 ± 0.17 mg/mL, and angiotensin-1 converting enzyme (0.674 ± 0.06 mg/mL [lungs], 1.006 ± 0.08 mg/mL [heart] activities in a dose-dependent manner and also showed dose-dependent radicals [DPPH (16.94 ± 1.34 mg/mL, NO (6.98 ± 0.886 mg/mL, OH (3.72 ± 0.26 mg/mL, and ABTS (15.7 ± 1.06 mmol/TEAC·g] scavenging ability. Conclusion. The inhibition of α-amylase, α-glucosidase, and angiotensin-1 converting enzyme activities by the cocoa bean extract could be part of the possible mechanism by which the extract could manage and/or prevent type-2 diabetes and hypertension.

In Vitro Studies on the Antioxidant Property and Inhibition of α-Amylase, α-Glucosidase, and Angiotensin I-Converting Enzyme by Polyphenol-Rich Extracts from Cocoa (Theobroma cacao) Bean

Science.gov (United States)

Ademosun, Ayokunle O.; Ademiluyi, Adedayo O.; Omojokun, Olasunkanmi S.; Nwanna, Esther E.; Longe, Kuburat O.

2014-01-01

Background. This study sought to investigate the antidiabetic and antihypertensive mechanisms of cocoa (Theobroma cacao) bean through inhibition of α-amylase, α-glucosidase, angiotensin-1 converting enzyme, and oxidative stress. Methodology. The total phenol and flavonoid contents of the water extractable phytochemicals from the powdered cocoa bean were determined and the effects of the extract on α-amylase, α-glucosidase, and angiotensin-1 converting enzyme activities were investigated in vitro. Furthermore, the radicals [1,1-diphenyl-2 picrylhydrazyl (DPPH), 2,2..-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), hydroxyl (OH), and nitric oxide (NO)] scavenging ability and ferric reducing antioxidant property of the extract were assessed. Results. The results revealed that the extract inhibited α-amylase (1.81 ± 0.22 mg/mL), α-glucosidase (1.84 ± 0.17 mg/mL), and angiotensin-1 converting enzyme (0.674 ± 0.06 mg/mL [lungs], 1.006 ± 0.08 mg/mL [heart]) activities in a dose-dependent manner and also showed dose-dependent radicals [DPPH (16.94 ± 1.34 mg/mL), NO (6.98 ± 0.886 mg/mL), OH (3.72 ± 0.26 mg/mL), and ABTS (15.7 ± 1.06 mmol/TEAC·g] scavenging ability. Conclusion. The inhibition of α-amylase, α-glucosidase, and angiotensin-1 converting enzyme activities by the cocoa bean extract could be part of the possible mechanism by which the extract could manage and/or prevent type-2 diabetes and hypertension. PMID:25295218

In vitro antidiabetic activity of various crude extracts of Boletus variipes

Science.gov (United States)

Muniandy, Sutha; Fazry, Shazrul; Daud, Fauzi; Senafi, Sahidan

2015-09-01

Diabetes mellitus is a complex metabolic disease that progressively spread worldwide and difficult to treat due to various physical and metabolic complications. Current treatment using synthetic drugs has lead to various undesirable side effects. Here we determined the effect of Boletus variipes extracts on diabetes related enzymes. In this study, hot water, cold water and methanol extracts of B. variipes were utilized in order to assess their in vitro antidiabetic activity by measuring the effect on α-amylase and α-glucosidase enzyme. Hot water extract possessed the highest inhibition activity of α-amylase and α-glucosidase in a concentration dependent manner with the IC50 value 87 mg/mL and 89 mg/mL respectively. The methanol extract also showed inhibition activity of α-amylase and α-glucosidase but significantly lower than the hot water extract. Whereas cold water extract did not show any inhibition activity towards both the enzymes. Therefore, it is hypothesized that the hot water extract of Boletus variipes contains bioactive compound that can inhibit alpha-amylase and alpha-glucosidase enzyme activity. At the request of all authors of the paper an updated version was published on 11 May 2016. The original version identified the species of mushroom as Boletus variipes, but new findings have proved the species of mushroom to be Boletus qriseipurpureus. The species name has been updated throughout the revised version of this paper.

The role of certain oxidative enzymes, catalase, and beta-glucosidase on virulence of Cephalosporium maydis.

Science.gov (United States)

Abd-Elrazik, A; Darweish, F A; Rushdi, M H

1978-01-01

Isolates of Cephalosporium maydis varied in their pathogenicity to D.C. 67 maize cultivar from highly to weakly pathogenic. Highly pathogenic isolates showed lower activity of polyphenol oxidase, peroxidase, cytochrome oxidase, and beta-glucosidase enzymes and higher activity of catalase and dehydrogenase than weakly pathogenic isolates. Enzymes production by the tested isolates increased as the culture age increased; except in case of catalase enzyme, the reverse action was detected. The role of these enzymes in the virulence of C. maydis is suggested and discussed.

Alpha-Glucosidase Inhibitory and Antioxidant Activity of Solvent ...

African Journals Online (AJOL)

regression analysis. Phytochemical contents and correlation with bioactivities. Total phenolic (TP), total proanthocyanidin. (TPro), and total hydroxycinnamic acid ..... An advantage of competitive inhibitors is that their inhibitory action is reversible, thus allowing undesirable effects to be readily mitigated by decreasing the ...

Alternative splicing of T cell receptor (TCR) alpha chain transcripts containing V alpha 1 or V alpha 14 elements.

Science.gov (United States)

Mahotka, C; Hansen-Hagge, T E; Bartram, C R

1995-10-01

Human acute lymphoblastic leukemia cell lines represent valuable tools to investigate distinct steps of the complex regulatory pathways underlying T cell receptor recombination and expression. A case in point are V delta 2D delta 3 and subsequent V delta 2D delta 3J alpha rearrangements observed in human leukemic pre-B cells as well as in normal lymphopoiesis. The functional expression of these unusual (VD) delta (JC) alpha hybrids is almost exclusively prevented by alternative splicing events. In this report we show that alternative splicing at cryptic splice donor sites within V elements is not a unique feature of hybrid TCR delta/alpha transcripts. Among seven V alpha families analyzed by RT-PCR, alternatively spliced products were observed in TCR alpha recombinations containing V alpha 1 or V alpha 14 elements. In contrast to normal peripheral blood cells and thymocytes, the leukemia cell line JM expressing functional V alpha 1J alpha 3C alpha transcripts lacked evidence of aberrant TCR alpha RNA species.

Four New Flavonoids with α-Glucosidase Inhibitory Activities from Morus alba var. tatarica.

Science.gov (United States)

Zhang, Ya-Long; Luo, Jian-Guang; Wan, Chuan-Xing; Zhou, Zhong-Bo; Kong, Ling-Yi

2015-11-01

Four new flavonoids, mortatarins A-D (1-4, resp.), along with eight known flavonoids (5-12) were isolated from the root bark of Morus alba var. tatarica. Their structures were established on the basis of spectroscopic data analysis, and the absolute configuration of 4 was determined by analysis of its CD spectrum. All isolates were tested for inhibitory activities against α-glucosidase. Compounds 4, 7, and 8 exhibited a significant degree of inhibition with IC50 values of 5.0 ± 0.3, 7.5 ± 0.5, and 5.9 ± 0.2 μM, respectively. Copyright © 2015 Verlag Helvetica Chimica Acta AG, Zürich.

Stability of the 'L12 stalk' in ribosomes from mesophilic and (hyper)thermophilic Archaea and Bacteria.

Science.gov (United States)

Shcherbakov, D; Dontsova, M; Tribus, M; Garber, M; Piendl, W

2006-01-01

The ribosomal stalk complex, consisting of one molecule of L10 and four or six molecules of L12, is attached to 23S rRNA via protein L10. This complex forms the so-called 'L12 stalk' on the 50S ribosomal subunit. Ribosomal protein L11 binds to the same region of 23S rRNA and is located at the base of the 'L12 stalk'. The 'L12 stalk' plays a key role in the interaction of the ribosome with translation factors. In this study stalk complexes from mesophilic and (hyper)thermophilic species of the archaeal genus Methanococcus and from the Archaeon Sulfolobus solfataricus, as well as from the Bacteria Escherichia coli, Geobacillus stearothermophilus and Thermus thermophilus, were overproduced in E.coli and purified under non-denaturing conditions. Using filter-binding assays the affinities of the archaeal and bacterial complexes to their specific 23S rRNA target site were analyzed at different pH, ionic strength and temperature. Affinities of both archaeal and bacterial complexes for 23S rRNA vary by more than two orders of magnitude, correlating very well with the growth temperatures of the organisms. A cooperative effect of binding to 23S rRNA of protein L11 and the L10/L12(4) complex from mesophilic and thermophilic Archaea was shown to be temperature-dependent.

Hyperthermophilic endoglucanase for in planta lignocellulose conversion

Directory of Open Access Journals (Sweden)

Klose Holger

2012-08-01

Full Text Available Abstract Background The enzymatic conversion of lignocellulosic plant biomass into fermentable sugars is a crucial step in the sustainable and environmentally friendly production of biofuels. However, a major drawback of enzymes from mesophilic sources is their suboptimal activity under established pretreatment conditions, e.g. high temperatures, extreme pH values and high salt concentrations. Enzymes from extremophiles are better adapted to these conditions and could be produced by heterologous expression in microbes, or even directly in the plant biomass. Results Here we show that a cellulase gene (sso1354 isolated from the hyperthermophilic archaeon Sulfolobus solfataricus can be expressed in plants, and that the recombinant enzyme is biologically active and exhibits the same properties as the wild type form. Since the enzyme is inactive under normal plant growth conditions, this potentially allows its expression in plants without negative effects on growth and development, and subsequent heat-inducible activation. Furthermore we demonstrate that the recombinant enzyme acts in high concentrations of ionic liquids and can therefore degrade α-cellulose or even complex cell wall preparations under those pretreatment conditions. Conclusion The hyperthermophilic endoglucanase SSO1354 with its unique features is an excellent tool for advanced biomass conversion. Here we demonstrate its expression in planta and the possibility for post harvest activation. Moreover the enzyme is suitable for combined pretreatment and hydrolysis applications.

Novel Bioengineered Cassava Expressing an Archaeal Starch Degradation System and a Bacterial ADP-Glucose Pyrophosphorylase for Starch Self-Digestibility and Yield Increase

Directory of Open Access Journals (Sweden)

Ayalew Ligaba-Osena

2018-02-01

Full Text Available To address national and global low-carbon fuel targets, there is great interest in alternative plant species such as cassava (Manihot esculenta, which are high-yielding, resilient, and are easily converted to fuels using the existing technology. In this study the genes encoding hyperthermophilic archaeal starch-hydrolyzing enzymes, α-amylase and amylopullulanase from Pyrococcus furiosus and glucoamylase from Sulfolobus solfataricus, together with the gene encoding a modified ADP-glucose pyrophosphorylase (glgC from Escherichia coli, were simultaneously expressed in cassava roots to enhance starch accumulation and its subsequent hydrolysis to sugar. A total of 13 multigene expressing transgenic lines were generated and characterized phenotypically and genotypically. Gene expression analysis using quantitative RT-PCR showed that the microbial genes are expressed in the transgenic roots. Multigene-expressing transgenic lines produced up to 60% more storage root yield than the non-transgenic control, likely due to glgC expression. Total protein extracted from the transgenic roots showed up to 10-fold higher starch-degrading activity in vitro than the protein extracted from the non-transgenic control. Interestingly, transgenic tubers released threefold more glucose than the non-transgenic control when incubated at 85°C for 21-h without exogenous application of thermostable enzymes, suggesting that the archaeal enzymes produced in planta maintain their activity and thermostability.

Investigation of sliding DNA clamp dynamics by single-molecule fluorescence, mass spectrometry and structure-based modeling

Science.gov (United States)

Gadkari, Varun V; Harvey, Sophie R; Raper, Austin T; Chu, Wen-Ting; Wang, Jin; Wysocki, Vicki H; Suo, Zucai

2018-01-01

Abstract Proliferating cell nuclear antigen (PCNA) is a trimeric ring-shaped clamp protein that encircles DNA and interacts with many proteins involved in DNA replication and repair. Despite extensive structural work to characterize the monomeric, dimeric, and trimeric forms of PCNA alone and in complex with interacting proteins, no structure of PCNA in a ring-open conformation has been published. Here, we use a multidisciplinary approach, including single-molecule Förster resonance energy transfer (smFRET), native ion mobility-mass spectrometry (IM-MS), and structure-based computational modeling, to explore the conformational dynamics of a model PCNA from Sulfolobus solfataricus (Sso), an archaeon. We found that Sso PCNA samples ring-open and ring-closed conformations even in the absence of its clamp loader complex, replication factor C, and transition to the ring-open conformation is modulated by the ionic strength of the solution. The IM-MS results corroborate the smFRET findings suggesting that PCNA dynamics are maintained in the gas phase and further establishing IM-MS as a reliable strategy to investigate macromolecular motions. Our molecular dynamic simulations agree with the experimental data and reveal that ring-open PCNA often adopts an out-of-plane left-hand geometry. Collectively, these results implore future studies to define the roles of PCNA dynamics in DNA loading and other PCNA-mediated interactions. PMID:29529283

ALPHA/AMPU, Radionuclide Radioactivity from Alpha Spectrometer Measurements

International Nuclear Information System (INIS)

Sill, D.S.

1990-01-01

1 - Description of program or function: The two computer programs, ALPHA and AMPU, take raw data obtained from alpha spectrometry and from these calculate activities and uncertainties of the radionuclides present in the sample. ALPHA determines activities of any alpha emitter in a sample that has been directly precipitated with NdF 3 . AMPU determines the Pu-239, Pu-238,and Am-241 activities using Pu-236 and Am-243 tracers. 2 - Method of solution: These programs propagate all random and systematic uncertainties, found anywhere in the experimental process, to the final result. The result is rounded and is in decimal agreement with the uncertainty. 3 - Restrictions on the complexity of the problem: In ALPHA, a chemical yield of 98% is assumed

Resting alpha activity predicts learning ability in alpha neurofeedback

Directory of Open Access Journals (Sweden)

Wenya eNan

2014-07-01

Full Text Available Individuals differ in their ability to learn how to regulate the alpha activity by neurofeedback. This study aimed to investigate whether the resting alpha activity is related to the learning ability of alpha enhancement in neurofeedback and could be used as a predictor. A total of 25 subjects performed 20 sessions of individualized alpha neurofeedback in order to learn how to enhance activity in the alpha frequency band. The learning ability was assessed by three indices respectively: the training parameter changes between two periods, within a short period and across the whole training time. It was found that the resting alpha amplitude measured before training had significant positive correlations with all learning indices and could be used as a predictor for the learning ability prediction. This finding would help the researchers in not only predicting the training efficacy in individuals but also gaining further insight into the mechanisms of alpha neurofeedback.

ORF Alignment: NC_004307 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_004307 gi|23465117 >1g5aA 80 628 2 589 5e-59 ... gb|AAL05573.1| alpha-glucosidase [Bifidobacterium adolesce...ntis] ... Length = 588 ... Query: 1 ... MTANNLNDDWWKQAVVYQIYPRSFKDVNGDGLGDIAGVTEK

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

Directory of Open Access Journals (Sweden)

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.

Extracts of Coreopsis tinctoria Nutt. Flower Exhibit Antidiabetic Effects via the Inhibition of α-Glucosidase Activity

Directory of Open Access Journals (Sweden)

Wujie Cai

2016-01-01

Full Text Available The aim of this study was to assay the effects of Coreopsis tinctoria Nutt. flower extracts on hyperglycemia of diet-induced obese mice and the underlying mechanisms. Coreopsis tinctoria flower was extracted with ethanol and water, respectively. The total phenol, flavonoid levels, and the constituents of the extracts were measured. For the animal experiments, C57BL/6 mice were fed with a chow diet, high-fat diet, or high-fat diet mixed with 0.4% (w/w water and ethanol extracts of Coreopsis tinctoria flower for 8 weeks. The inhibitory effects of the extracts on α-glucosidase activity and the antioxidant properties were assayed in vitro. We found that the extracts blocked the increase of fasting blood glucose, serum triglyceride (TG, insulin, leptin, and liver lipid levels and prevented the development of glucose tolerance impairment and insulin resistance in the C57BL/6 mice induced by a high-fat diet. The extracts inhibited α-glycosidase activity and increased oxidant activity in vitro. In conclusion, Coreopsis tinctoria flower extracts may ameliorate high-fat diet-induced hyperglycemia and insulin resistance. The underling mechanism may be via the inhibition of α-glucosidase activity. Our data indicate that Coreopsis tinctoria flower could be used as a beverage supplement and a potential source of drugs for treatment of diabetics.

[Evaluate drug interaction of multi-components in Morus alba leaves based on α-glucosidase inhibitory activity].

Science.gov (United States)

Ji, Tao; Su, Shu-Lan; Guo, Sheng; Qian, Da-Wei; Ouyang, Zhen; Duan, Jin-Ao

2016-06-01

Column chromatography was used for enrichment and separation of flavonoids, alkaloids and polysaccharides from the extracts of Morus alba leaves; glucose oxidase method was used with sucrose as the substrate to evaluate the multi-components of M. alba leaves in α-glucosidase inhibitory models; isobole method, Chou-Talalay combination index analysis and isobolographic analysis were used to evaluate the interaction effects and dose-effect characteristics of two components, providing scientific basis for revealing the hpyerglycemic mechanism of M. alba leaves. The components analysis showed that flavonoid content was 5.3%; organic phenolic acids content was 10.8%; DNJ content was 39.4%; and polysaccharide content was 18.9%. Activity evaluation results demonstrated that flavonoids, alkaloids and polysaccharides of M. alba leaves had significant inhibitory effects on α-glucosidase, and the inhibitory rate was increased with the increasing concentration. Alkaloids showed most significant inhibitory effects among these three components. Both compatibility of alkaloids and flavonoids, and the compatibility of alkaloids and polysaccharides demonstrated synergistic effects, but the compatibility of flavonoids and polysaccharides showed no obvious synergistic effects. The results have confirmed the interaction of multi-components from M. alba leaves to regulate blood sugar, and provided scientific basis for revealing hpyerglycemic effectiveness and mechanism of the multi-components from M. alba leaves. Copyright© by the Chinese Pharmaceutical Association.

Daidzin decreases blood glucose and lipid in streptozotocin ...

African Journals Online (AJOL)

hyperglycemic mice and improved oral glucose tolerance. The serum and ... Inhibition of α-glucosidase and stimulation of glucose consumption by muscles may account for ..... induced production of tumor necrosis factor-alpha and fibrinolysis ...

Transcriptome analysis of Sulfolobus solfataricus infected with two related fuselloviruses reveals novel insights into the regulation of CRISPR-Cas system

DEFF Research Database (Denmark)

Fusco, Salvatore; Liguori, Rossana; Limauro, Danila

2015-01-01

in a double-infected strain to explore both virus-host and virus-virus interactions. Whereas SSV1 did not induce major changes of the host gene expression, SSV2 elicited a strong host response, which includes the transcriptional activation of CRISPR loci and cas genes. As a consequence, a significant decrease...... of the SSV2 copy number has been observed, which in turn led to provirus-capture into the host chromosome. Results of this study have revealed novel aspects of the host-viral interaction in the frame of the CRISPR-response....

Structural and kinetic studies of the allosteric transition in Sulfolobus solfataricus uracil phosphoribosyltransferase: Permanent activation by engineering of the C-terminus

DEFF Research Database (Denmark)

Christoffersen, Stig; Kadziola, Anders; Johansson, Eva

2009-01-01

and PPi, in the other sites. Combined with three existing structures of uracil phosphoribosyltransferase in complex with UMP and the allosteric inhibitor cytidine triphosphate (CTP), these structures provide valuable insight into the mechanism of allosteric transition from inhibited to active enzyme...

Molecular basis for nondeletion alpha-thalassemia in American blacks. Alpha 2(116GAG----UAG).

OpenAIRE

Liebhaber, S A; Coleman, M B; Adams, J G; Cash, F E; Steinberg, M H

1987-01-01

An American black woman was found to have the phenotype of moderately severe alpha-thalassemia normally associated with the loss of two to three alpha-globin genes despite an alpha-globin gene map that demonstrated the loss of only a single alpha-globin gene (-alpha/alpha alpha). Several individuals in her kindred with normal alpha-globin gene mapping studies (alpha alpha/alpha alpha) had mild alpha-thalassemia hematologic values consistent with the loss of one to two alpha-globin genes. Thes...

[superscript 1]H NMR Spectroscopy-Based Configurational Analysis of Mono- and Disaccharides and Detection of ß-Glucosidase Activity: An Undergraduate Biochemistry Laboratory

Science.gov (United States)

Periyannan, Gopal R.; Lawrence, Barbara A.; Egan, Annie E.

2015-01-01

A [superscript 1]H NMR spectroscopy-based laboratory experiment explores mono- and disaccharide structural chemistry, and the enzyme-substrate specificity of glycosidic bond cleavage by ß-glucosidase towards cellobiose (ß-linked gluco-disaccharide) and maltose (a-linked gluco-disaccharide). Structural differences between cellobiose, maltose, and…

Seasonal variation and distribution of total and active microbial community of beta-glucosidase encoding genes in coniferous forest soil

Czech Academy of Sciences Publication Activity Database

Pathan, S.I.; Žifčáková, Lucia; Ceccherini, M.T.; Pantani, O.L.; Větrovský, Tomáš; Baldrian, Petr

2017-01-01

Roč. 105, February (2017), s. 71-80 ISSN 0038-0717 R&D Projects: GA ČR(CZ) GA16-08916S Grant - others:Transbiodiverse(CZ) 7. RP Marie Curie ITN FP7/2007e2013 project 289949 Institutional support: RVO:61388971 Keywords : Beta-Glucosidases * Forest soil * Bacteria Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 4.857, year: 2016

A ?-glucosidase hyper-production Trichoderma reesei mutant reveals a potential role of cel3D in cellulase production

OpenAIRE

Li, Chengcheng; Lin, Fengming; Li, Yizhen; Wei, Wei; Wang, Hongyin; Qin, Lei; Zhou, Zhihua; Li, Bingzhi; Wu, Fugen; Chen, Zhan

2016-01-01

Background The conversion of cellulose by cellulase to fermentable sugars for biomass-based products such as cellulosic biofuels, biobased fine chemicals and medicines is an environment-friendly and sustainable process, making wastes profitable and bringing economic benefits. Trichoderma reesei is the well-known major workhorse for cellulase production in industry, but the low ?-glucosidase activity in T. reesei cellulase leads to inefficiency in biomass degradation and limits its industrial ...

Anti-pp,. cap alpha cap alpha. and p. cap alpha. elastic scattering at high energies and Chou-Yang conjecture

Energy Technology Data Exchange (ETDEWEB)

Saleem, M.; Fazal-e-Aleem; Rifique, M.

1987-03-01

The recent experimental measurements for anti-pp and ..cap alpha cap alpha.. elastic scattering at high energies have shown that the Chou-Yang conjecture regarding the relationship between the electromagnetic and the hadronic form factor of a particle is only an approximation. A new ansatz has been proposed to obtain hadronic form factors of proton and the ..cap alpha..-particle. These form factors have been used to explain the various characteristics of anti-pp, ..cap alpha cap alpha.. and p..cap alpha.. elastic scattering at high energies.