Papers by Gabriella Fiorentino
Antioxidants
Reactive species of oxygen (ROS), responsible for oxidative stress, accumulate in various tissues... more Reactive species of oxygen (ROS), responsible for oxidative stress, accumulate in various tissues damaged by burns, decubitus ulcers, and vascular lesions. Antioxidants play an important and well-documented role in healing of chronic and acute wounds. Rigenase ® , a specific extract of Triticum vulgare manufactured by Farmaceutici Damor, is employed in products used for the regeneration of tissue injuries. In this work, we show that Rigenase ® exhibits a scavenging effect toward free radicals, thus pointing to its relevant antioxidant activity.
International Journal of Molecular Sciences, 2022
The production of biochemicals requires the use of microbial strains with efficient substrate con... more The production of biochemicals requires the use of microbial strains with efficient substrate conversion and excellent environmental robustness, such as Weizmannia coagulans species. So far, the genomes of 47 strains have been sequenced. Herein, we report a comparative genomic analysis of nine strains on the full repertoire of Carbohydrate-Active enZymes (CAZymes), secretion systems, and resistance mechanisms to environmental challenges. Moreover, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) immune system along with CRISPR-associated (Cas) genes, was also analyzed. Overall, this study expands our understanding of the strain’s genomic diversity of W. coagulans to fully exploit its potential in biotechnological applications.
International Journal of Molecular Sciences, 2022
Arsenic (As) pollution is a widespread problem worldwide. In recent years, biosensors based on en... more Arsenic (As) pollution is a widespread problem worldwide. In recent years, biosensors based on enzymatic inhibition have been developed for arsenic detection, making the study of the effect of inhibitors on the selected enzymatic activity crucial for their setup. The arsenate reductase of Thermus thermophilus HB27, TtArsC, reduces As(V) into As(III), but is also endowed with phosphatase activity. This work investigates the inhibitory effects of As(V) and As(III) on phosphatase activity by taking advantage of a simple colorimetric assay; the results show that both of them are non-competitive inhibitors affecting the Vmax but not the KM of the reaction. However, their Ki values are different from each other (15.2 ± 1.6 μM for As(V) and 394.4 ± 40.3 µm with As(III)), indicating a higher inhibitory effect by As(V). Moreover, the inhibition-based biosystem results to be selective for As(V) since several other metal ions and salts do not affect TtArsC phosphatase activity; it exhibits a s...
The production of bio-chemicals requires the use of microbial strains with efficient substrate co... more The production of bio-chemicals requires the use of microbial strains with efficient substrate conversion and excellent environmental robustness, such as Bacillus coagulans spp. So far the genomes of about 50 strains have been sequenced. Herein, we report a comparative genomic analysis of nine strains on the full repertoire of CAZymes, secretion systems, and resistance mechanisms to environmental challenges. Moreover, B. coagulans Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) immune system along with CRISPR-associated Cas) genes, was also analysed. Overall, this study expands our understanding of the strains genomic diversity of B. coagulans to fully exploit its potential in biotechnological applications.
Microbial Cell Factories, 2021
Background The spore-forming lactic acid bacterium Bacillus coagulans MA-13 has been isolated fro... more 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 ...
Extremophiles, 2001
Abstract. A genomic region upstream of the alcohol dehydrogenase (Ssadh) gene was cloned and sequ... more Abstract. 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...
Frontiers in Microbiology, 2021
Extreme environments are excellent places to find microorganisms capable of tolerating extreme te... more Extreme environments are excellent places to find microorganisms capable of tolerating extreme temperature, pH, salinity pressure, and elevated concentration of heavy metals and other toxic compounds. In the last decades, extremophilic microorganisms have been extensively studied since they can be applied in several fields of biotechnology along with their enzymes. In this context, the characterization of heavy metal resistance determinants in thermophilic microorganisms is the starting point for the development of new biosystems and bioprocesses for environmental monitoring and remediation. This work focuses on the isolation and the genomic exploration of a new arsenic-tolerant microorganism, classified as Alicyclobacillus mali FL18. The bacterium was isolated from a hot mud pool of the solfataric terrains in Pisciarelli, a well-known hydrothermally active zone of the Campi Flegrei volcano near Naples in Italy. A. mali FL18 showed a good tolerance to arsenite (MIC value of 41 mM), ...
Antioxidants, 2020
To fight reactive oxygen species (ROS) produced by both the metabolism and strongly oxidative hab... more To fight reactive oxygen species (ROS) produced by both the metabolism and strongly oxidative habitats, hyperthermophilic archaea are equipped with an array of antioxidant enzymes whose role is to protect the biological macromolecules from oxidative damage. The most common ROS, such as superoxide radical (O2•−) and hydrogen peroxide (H2O2), are scavenged by superoxide dismutase, peroxiredoxins, and catalase. These enzymes, together with thioredoxin, protein disulfide oxidoreductase, and thioredoxin reductase, which are involved in redox homeostasis, represent the core of the antioxidant system. In this review, we offer a panorama of progression of knowledge on the antioxidative system in aerobic or microaerobic (hyper)thermophilic archaea and possible industrial applications of these enzymes.
International Journal of Environmental Research and Public Health, 2020
Microorganisms thriving in hot springs and hydrothermally active volcanic areas are dynamically i... more Microorganisms thriving in hot springs and hydrothermally active volcanic areas are dynamically involved in heavy-metal biogeochemical cycles; they have developed peculiar resistance systems to cope with such metals which nowadays can be considered among the most permanent and toxic pollutants for humans and the environment. For this reason, their exploitation is functional to unravel mechanisms of toxic-metal detoxification and to address bioremediation of heavy-metal pollution with eco-sustainable approaches. In this work, we isolated a novel strain of the thermophilic bacterium Geobacillus stearothermophilus from the solfataric mud pool in Pisciarelli, a well-known hydrothermally active zone of the Campi Flegrei volcano located near Naples in Italy, and characterized it by ribotyping, 16S rRNA sequencing and mass spectrometry analyses. The minimal inhibitory concentration (MIC) toward several heavy-metal ions indicated that the novel G. stearothermophilus isolate is particularly ...
The Open Biochemistry Journal, 2018
Extreme terrestrial and marine hot environments are excellent niches for specialized microorganis... more Extreme terrestrial and marine hot environments are excellent niches for specialized microorganisms belonging to the domains of Bacteria and Archaea; these microorganisms are considered extreme from an anthropocentric point of view because they are able to populate harsh habitats tolerating a variety of conditions, such as extreme temperature and/or pH, high metal concentration and/or salt; moreover, like all the microorganisms, they are also able to respond to sudden changes in the environmental conditions. Therefore, it is not surprising that they possess an extraordinary variety of dynamic and versatile mechanisms for facing different chemical and physical stresses. Such features have attracted scientists also considering an applicative point of view. In this review we will focus on the molecular mechanisms responsible for survival and adaptation of thermophiles to toxic metals, with particular emphasis on As(V), As(III), Cd(II), and on current biotechnologies for their detection, extraction and removal.
Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. I... more Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pull-down assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosyl-Lmethionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyze SAM- dependent arsenite methylation with formation of monomethylarsenite (MMAs) and dimet...
Scientific Reports
The correct immobilization and orientation of enzymes on nanosurfaces is a crucial step either fo... more The correct immobilization and orientation of enzymes on nanosurfaces is a crucial step either for the realization of biosensors, as well as to guarantee the efficacy of the developed biomaterials. In this work we produced two versions of a chimeric protein, namely ArsC-Vmh2 and Vmh2-ArsC, which combined the self-assembling properties of Vmh2, a hydrophobin from Pleurotus ostreatus, with that of TtArsC, a thermophilic arsenate reductase from Thermus thermophilus; both chimeras were heterologously expressed in Escherichia coli and purified from inclusion bodies. They were characterized for their enzymatic capability to reduce As(V) into As(III), as well as for their immobilization properties on polystyrene and gold in comparison to the native TtArsC. The chimeric proteins immobilized on polystyrene can be reused up to three times and stored for 15 days with 50% of activity loss. Immobilization on gold electrodes showed that both chimeras follow a classic Langmuir isotherm model towar...
International Journal of Environmental Research and Public Health
Extremophiles are microorganisms that populate habitats considered inhospitable from an anthropoc... more Extremophiles are microorganisms that populate habitats considered inhospitable from an anthropocentric point of view and are able to tolerate harsh conditions such as high temperatures, extreme pHs, high concentrations of salts, toxic organic substances, and/or heavy metals. These microorganisms have been broadly studied in the last 30 years and represent precious sources of biomolecules and bioprocesses for many biotechnological applications; in this context, scientific efforts have been focused on the employment of extremophilic microbes and their metabolic pathways to develop biomonitoring and bioremediation strategies to face environmental pollution, as well as to improve biorefineries for the conversion of biomasses into various chemical compounds. This review gives an overview on the peculiar metabolic features of certain extremophilic microorganisms, with a main focus on thermophiles, which make them attractive for biotechnological applications in the field of environmental ...
International Journal of Biological Macromolecules
Archaea
Peroxiredoxins (Prxs) are ubiquitous thiol peroxidases that are involved in the reduction of pero... more Peroxiredoxins (Prxs) are ubiquitous thiol peroxidases that are involved in the reduction of peroxides. It has been reported that prokaryotic Prxs generally show greater structural robustness than their eukaryotic counterparts, making them less prone to inactivation by overoxidation. This difference has inspired the search for new antioxidants from prokaryotic sources that can be used as possible therapeutic biodrugs. Bacterioferritin comigratory proteins (Bcps) of the hyperthermophilic archaeonSulfolobus solfataricusthat belong to the Prx family have recently been characterized. One of these proteins, Bcp1, was chosen to determine its antioxidant effects in H9c2 rat cardiomyoblast cells. Bcp1 activity was measuredin vitrounder physiological temperature and pH conditions that are typical of mammalian cells; the yeast thioredoxin reductase (yTrxR)/thioredoxin (yTrx) reducing system was used to evaluate enzyme activity. A TAT-Bcp1 fusion protein was constructed to allow its internaliz...
International Journal of Biological Macromolecules
World Journal of Microbiology and Biotechnology
Extremophilic microorganisms are valuable sources of enzymes for various industrial applications.... more Extremophilic microorganisms are valuable sources of enzymes for various industrial applications. In fact, given their optimal catalytic activity and operational stability under harsh physical and chemical conditions, they represent a suitable alternative to their mesophilic counterparts. For instance, extremophilic enzymes are important to foster the switch from fossil-based to lignocellulose-based industrial processes. Indeed, more stable enzymes are needed, because the conversion of the lignocellulosic biomass to a wide palette of value-added products requires extreme chemo-physical pre-treatments. Galactomannans are part of the hemicellulose fraction in lignocellulosic biomass. They are heteropolymers constituted by a β-1,4-linked mannan backbone substituted with side chains of α-1,6-linked galactose residues. Therefore, the joint action of different hydrolytic enzymes (i.e. β-mannanase, β-mannosidase and α-galactosidase) is needed to accomplish their complete hydrolysis. So far, numerous galactomannan-degrading enzymes have been isolated and characterized from extremophilic microorganisms. Besides applications in biorefinery, these biocatalysts are also useful to improve the quality (i.e. digestibility and prebiotic properties) of food and feed as well as in paper industries to aid the pulp bleaching process. In this review, an overview about the structure, function and applications of galactomannans is provided. Moreover, a survey of (hyper)-thermophilic galactomannans-degrading enzymes, mainly characterized in the last decade, has been carried out. These extremozymes are described in the light of their biotechnological application in industrial processes requiring harsh conditions.
Microbial cell factories, Jan 18, 2018
The characterization of the molecular determinants of metal resistance has potential biotechnolog... more The characterization of the molecular determinants of metal resistance has potential biotechnological application in biosensing and bioremediation. In this context, the bacterium Thermus thermophilus HB27 is a metal tolerant thermophile containing a set of genes involved in arsenic resistance which, differently from other microbes, are not organized into a single operon. They encode the proteins: arsenate reductase, TtArsC, arsenic efflux membrane transporter, TtArsX, and transcriptional repressor, TtSmtB. In this work we show that the arsenic efflux protein TtArsX and the arsenic responsive transcriptional repressor TtSmtB are required to provide resistance to cadmium. We analyzed the sensitivity to Cd(II) of mutants lacking TtArsX, finding that they are more sensitive to this metal than the wild type strain. In addition, using promoter probe reporter plasmids, we show that the transcription of TtarsX is also stimulated by the presence of Cd(II) in a TtSmtB-dependent way. Actually,...
Enzyme and microbial technology, 2018
The full utilization of hemicellulose sugars (pentose and exose) present in lignocellulosic mater... more The full utilization of hemicellulose sugars (pentose and exose) present in lignocellulosic material, is required for an efficient bio-based fuels and chemicals production. Two recombinant thermophilic enzymes, an endo-1,4-β-mannanase from Dictyoglomus turgidum (DturCelB) and an α-galactosidase from Thermus thermophilus (TtGalA), were assayed at 80 °C, to assess their heterosynergystic association on galactomannans degradation, particularly abundant in hemicellulose. The enzymes were tested under various combinations simultaneously and sequentially, in order to estimate the optimal conditions for the release of reducing sugars. The results showed that the most efficient degree of synergy was obtained in simultaneous assay with a protein ratio of 25% of DturCelB and 75% of TtGalA, using Locust bean gum as substrate. On the other hand, the mechanism of action was demonstrated through the sequential assays, i.e. when TtGalA acting as first to enhance the subsequent hydrolysis performed...
Extremophiles : life under extreme conditions, 2018
Dictyoglomus turgidum is a hyperthermophilic, anaerobic, gram-negative bacterium that shows an ar... more Dictyoglomus turgidum is a hyperthermophilic, anaerobic, gram-negative bacterium that shows an array of putative glycoside hydrolases (GHs) encoded by its genome, a feature that makes this microorganism very interesting for biotechnological applications. The aim of this work is the characterization of a hyperthermophilic GH5, Dtur_0671, of D. turgidum, annotated as endoglucanase and herein named DturCelB in agreement to DturCelA, which was previously characterized. The synthetic gene was expressed in Escherichia coli. The purified recombinant enzyme is active as a monomer (40 kDa) and CD structural studies showed a conserved α/β structure at different temperatures (25 and 70 °C) and high thermoresistance (Tm of 88 °C). Interestingly, the enzyme showed high endo-β-1,4-mannanase activity vs various mannans, but low endo-β-1,4 glucanase activity towards carboxymethylcellulose. The Kand Vof DturCelB were determined for both glucomannan and CMC: they were 4.70 mg/ml and 473.1 μmol/min mg...
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Papers by Gabriella Fiorentino