53 research outputs found
Ultra-rapid glutathionylation of chymotrypsinogen in its molten globule-like conformation: a comparison to archaeal proteins
Chymotrypsinogen, when reduced and taken to its molten globule-like conformation, displays a single cysteine with an unusual kinetic propensity toward oxidized glutathione (GSSG) and other organic thiol reagents. A single residue, identified by mass spectrometry like Cys1, reacts with GSSG about 1400 times faster than an unperturbed protein cysteine. A reversible protein-GSSG complex and a low pK(a) (8.1 +/- 0.1) make possible such astonishing kinetic property which is absent toward other natural disulfides like cystine, homocystine and cystamine. An evident hyper-reactivity toward 5,5 ' -dithiobis-(2-nitrobenzoic acid) (DTNB) and 1-chloro-2,4-dinitrobenzene (CDNB) was also found for this specific residue. The extraordinary reactivity toward GSSG is absent in two proteins of the thermophilic archaeon Sulfolobus solfataricus, an organism lacking glutathione: the Protein Disulphide Oxidoreductase (SsPDO) and the Bacterioferritin Comigratory Protein 1 (Bcp1) that displays Cys residues with an even lower pK(a) value (7.5 +/- 0.1) compared to chymotrypsinogen. This study, which also uses single mutants in Cys residues for Bcp1, proposes that this hyper-reactivity of a single cysteine, similar to that found in serum albumin, lysozyme, ribonuclease, may have relevance to drive the "incipit" of the oxidative folding of proteins from organisms where the glutathione/oxidized glutathione (GSH/GSSG) system is present
Prebiotic properties of Bacillus coagulans MA-13: production of galactoside hydrolyzing enzymes and characterization of the transglycosylation properties of a GH42 β-galactosidase
Background: The spore-forming lactic acid bacterium Bacillus coagulans MA-13 has been isolated from canned beans manufacturing and successfully employed for the sustainable production of lactic acid from lignocellulosic biomass. Among lactic acid bacteria, B. coagulans strains are generally recognized as safe (GRAS) for human consumption. Low-cost microbial production of industrially valuable products such as lactic acid and various enzymes devoted to the hydrolysis of oligosaccharides and lactose, is of great importance to the food industry. Specifically, α- and β-galactosidases are attractive for their ability to hydrolyze not-digestible galactosides present in the food matrix as well as in the human gastrointestinal tract. Results: In this work we have explored the potential of B. coagulans MA-13 as a source of metabolites and enzymes to improve the digestibility and the nutritional value of food. A combination of mass spectrometry analysis with conventional biochemical approaches has been employed to unveil the intra- and extra- cellular glycosyl hydrolase (GH) repertoire of B. coagulans MA-13 under diverse growth conditions. The highest enzymatic activity was detected on β-1,4 and α-1,6-glycosidic linkages and the enzymes responsible for these activities were unambiguously identified as β-galactosidase (GH42) and α-galactosidase (GH36), respectively. Whilst the former has been found only in the cytosol, the latter is localized also extracellularly. The export of this enzyme may occur through a not yet identified secretion mechanism, since a typical signal peptide is missing in the α-galactosidase sequence. A full biochemical characterization of the recombinant β-galactosidase has been carried out and the ability of this enzyme to perform homo- and hetero-condensation reactions to produce galacto-oligosaccharides, has been demonstrated. Conclusions: Probiotics which are safe for human use and are capable of producing high levels of both α-galactosidase and β-galactosidase are of great importance to the food industry. In this work we have proven the ability of B. coagulans MA-13 to over-produce these two enzymes thus paving the way for its potential use in treatment of gastrointestinal diseases. [Figure not available: see fulltext.
Genome Sequence of a Mesophilic Hydrogenotrophic Methanogen Methanocella paludicola, the First Cultivated Representative of the Order Methanocellales
We report complete genome sequence of a mesophilic hydrogenotrophic methanogen Methanocella paludicola, the first cultured representative of the order Methanocellales once recognized as an uncultured key archaeal group for methane emission in rice fields. The genome sequence of M. paludicola consists of a single circular chromosome of 2,957,635 bp containing 3004 protein-coding sequences (CDS). Genes for most of the functions known in the methanogenic archaea were identified, e.g. a full complement of hydrogenases and methanogenesis enzymes. The mixotrophic growth of M. paludicola was clarified by the genomic characterization and re-examined by the subsequent growth experiments. Comparative genome analysis with the previously reported genome sequence of RC-IMRE50, which was metagenomically reconstructed, demonstrated that about 70% of M. paludicola CDSs were genetically related with RC-IMRE50 CDSs. These CDSs included the genes involved in hydrogenotrophic methane production, incomplete TCA cycle, assimilatory sulfate reduction and so on. However, the genetic components for the carbon and nitrogen fixation and antioxidant system were different between the two Methanocellales genomes. The difference is likely associated with the physiological variability between M. paludicola and RC-IMRE50, further suggesting the genomic and physiological diversity of the Methanocellales methanogens. Comparative genome analysis among the previously determined methanogen genomes points to the genome-wide relatedness of the Methanocellales methanogens to the orders Methanosarcinales and Methanomicrobiales methanogens in terms of the genetic repertoire. Meanwhile, the unique evolutionary history of the Methanocellales methanogens is also traced in an aspect by the comparative genome analysis among the methanogens
Insight into the proteome of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis: the major cytosolic and membrane proteins
Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic Crenarchaeon, is the host of Nanoarchaeum equitans. Together, they form an intimate association, the first among Archaea. Membranes are of fundamental importance for the interaction of I. hospitalis and N. equitans, as they harbour the proteins necessary for the transport of macromolecules like lipids, amino acids, and cofactors between these organisms. Here, we investigated the protein inventory of I. hospitalis cells, and were able to identify 20 proteins in total. Experimental evidence and predictions let us conclude that 11 are soluble cytosolic proteins, eight membrane or membrane-associated proteins, and a single one extracellular. The quantitatively dominating proteins in the cytoplasm (peroxiredoxin; thermosome) antagonize oxidative and temperature stress which I. hospitalis cells are exposed to at optimal growth conditions. Three abundant membrane protein complexes are found: the major protein of the outer membrane, which might protect the cell against the hostile environment, forms oligomeric complexes with pores of unknown selectivity; two other complexes of the cytoplasmic membrane, the hydrogenase and the ATP synthase, play a key role in energy production and conversion
Diagnosis, treatment and prevention of pediatric obesity: consensus position statement of the Italian Society for Pediatric Endocrinology and Diabetology and the Italian Society of Pediatrics
The Italian Consensus Position Statement on Diagnosis, Treatment and Prevention of Obesity in Children and Adolescents integrates and updates the previous guidelines to deliver an evidence based approach to the disease. The following areas were reviewed: (1) obesity definition and causes of secondary obesity; (2) physical and psychosocial comorbidities; (3) treatment and care settings; (4) prevention.
The main novelties deriving from the Italian experience lie in the definition, screening of the cardiometabolic and hepatic risk factors and the endorsement of a staged approach to treatment. The evidence based efficacy of behavioral intervention versus pharmacological or surgical treatments is reported. Lastly, the prevention by promoting healthful diet, physical activity, sleep pattern, and environment is strongly recommended since the intrauterine phase
Antioxidant system of peroxiredoxins in Sulfolobus solfataricus.
The maintenance of the proper intracellular redox environment in aerobic microorganisms is guaranteed by redox systems and antioxidants that safeguard cells from the attack of reactive oxgen species (ROS) such as superoxide anions (O∙-), hydrogen peroxide (H2O2), hydroxyl radicals (OH∙). The increase of ROS concentration inside the cell damage biomolecules, membranes and essential metabolic functions. To kept low the intracellular level of ROS the cells are equipped of an array of antioxidant systems that have in the first line the superoxide dismutase (SODs) that catalyze the dismutation of O∙- to form H2O2 and oxygen. The H2O2 is reduced by different systems represented mainly by catalases and peroxidases.
The peroxiredoxins (Prx) are thiol-peroxidase that can scavange the peroxides utilizing generally Thioredoxin Reductase (TR) / Thioredoxin (Trx) system as electron donors that allow the recycling of the enzymes (1). Prxs are ubiquitous enzymes that share the same basic catalytic mechanisms, in which an activated cysteine (the peroxidative cysteine) is oxidized to sulphenic acid by peroxide substrate. Reductive regeneration of the oxidized catalytic thiol generally depends on glutathione, thioredoxin, glutaredoxin, cyclophilin and tryparedoxin.
The aim of this project is to expand the knowledge on antioxidant system in Sulfolobus solfataricus. For this purpose we have expressed in E. coli and characterized the four Prxs, Bcp1, Bcp2 (2), Bcp3, Bcp4. The purified recombinant proteins are able to remove both H2O2 and tert-butyl hydroperoxide and show high thermostability. Furthermore Bcp1, Bcp3 and Bcp4 can use as electron donor in the reycling of enzymes, the redox system Protein Disulfide Oxidoreductase (SsPDO) / TR previously characterized in S.solfataricus (3) while Bcp2 can use only DTT.
Comparative functional and transcriptional analysis suggest different role of Bcps during oxidative stress: Bcp2 and Bcp3 could act as inducible enzyme when the cell are attacked by exogenous peroxides, while Bcp1 and Bcp4 could support a protective role against endogenous peroxide formed during metabolism
A thermophilic Bacillus coagulans strain isolated from beans-waste is promising for cellulosic biomass saccharification
Comparative activity of Ceftizoxime and four other Cephalosporins against Gram negative bacteria and their sensitivity to Beta Lactamases. Microbiologica:
Identification and molecular characterisation of an endoglucanase gene, celS, from the extremely thermophilic archaeon Sulfolobus solfataricus.
A genomic region upstream of the alcohol dehydrogenase
(
Ssadh
) gene was cloned and sequenced from a
library of
Sulfolobus solfataricus
MT4 strain. The isolated
4,040-bp DNA fragment revealed an open reading frame
(
celS
), lying in the opposite direction to
Ssadh
, which
showed significant similarity to
endo
-
β
-1,4-glucanases from
Pyrococcus furiosus
,
Thermotoga maritima
, and
Thermotoga
neapolitana
.
celS
was shown to be a functional gene in
vivo: a specific
celS
mRNA was detected by primer extension
analysis showing a unique initiation transcription site
coinciding with the ATG translation initiation codon. The
specific gene product was detected as an extracellular cellulase
after enzyme staining by carboxymethyl cellulose
(CMC) SDS-PAGE, showing a molecular weight in agreement
with that deduced from the open reading frame.
Depending on growth conditions, different levels of cellulase
activity and specific
celS
transcript were detected,
revealing an inductive effect of CMC and suggesting a
repressive role of glucose
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