bioprospecting-extremophilic-microorganisms-to-address-environmental

Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution

Geothermal springs are rich in various metal ions due to the interaction between rock and water that takes place in the deep aquifer. Moreover, due to ...

University of Naples Federico II

University of Napoli Federico II

Bcp2 was identified as a putative peroxiredoxin (Prx) in the genome database of the aerobic hyperthermophilic archaeon Sulfolobus solfataricus. Its role in oxidative stress was investigated by transcriptional analysis of RNA isolated from cultures that had been stressed with various oxidant agents. Its specific involvement was confirmed by a considerable increase in the bcp2 transcript following induction with H2O2. The 5' end of the transcript was mapped by primer extension analysis and the promoter region was characterized. bcp2 was cloned and expressed in Escherichia coli, the recombinant enzyme was purified and the predicted molecular mass was confirmed. Using dithiothreitol as an electron donor, this enzyme acts as a catalyst in H2O2 reduction and protects plasmid DNA from nicking by the metal-catalysed oxidation system. Western blot analysis revealed that the Bpc2 expression was induced as a cellular adaptation in response to the addition of exogenous stressors. The results obtained indicate that Bcp2 plays an important role in the peroxide-scavaging system in S. solfataricus. Mutagenesis studies have shown that the only cysteine, Cys49, present in the Bcp2 sequence, is involved in the catalysis. Lastly, the presence of this Cys in the sequence confirms that Bcp2 is the first archaeal 1-Cysteine peroxiredoxin (1-Cys Prx) so far identified.

Identification and characterization of 1-Cys peroxiredoxin from Sulfolobus solfataricus and its involvement in the response to oxidative stress.

Sulfolobus solfataricus protein disulphide oxidoreductase (SsPDO) contains three disulphide bridges linking residues C(41)XXC(44), C(155)XXC(158), C(173)XXXXC(178). To get information on the role played by these cross-links in determining the structural and functional properties of the protein, we performed site-directed mutagenesis on Cys residues and investigated the changes in folding, stability and functional features of the mutants and analysed the results with computational analysis. The reductase activity of SsPDO and its mutants was evaluated by insulin and thioredoxin reductase assays also coupled with peroxiredoxin Bcp1 of S. solfataricus. The three-dimensional model of SsPDO was constructed and correlated with circular dichroism data and functional results. Biochemical analysis indicated a key function for the redox site constituted by Cys155 and Cys158. To discriminate between the role of the two cysteine residues, each cysteine was mutagenized and the behaviour of the single mutants was investigated elucidating the basis of the electron-shuffling mechanism for SsPDO. Finally, cysteine pK values were calculated and the accessible surface for the cysteine side chains in the reduced form was measured, showing higher reactivity and solvent exposure for Cys155.

Sulfolobus solfataricus protein disulphide oxidoreductase: insight into the roles of its redox sites.

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20mM and 15mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram+bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a kcat/KM value of 1.2×10(4)M(-1)s(-1). It also exhibited weak phosphatase activity with a kcat/KM value of 2.7×10(-4)M(-1)s(-1). The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural-functional characterization of a thermophilic arsenate reductase. 

A novel arsenate reductase from the bacterium Thermus thermophilus HB27: its role in arsenic detoxification.

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 K  and V  of DturCelB were determined for both glucomannan and CMC: they were 4.70 mg/ml and 473.1 μmol/min mg and 1.83 mg/ml and 1.349 μmol/min mg, respectively. Its optimal activity towards temperature and pH resulted to be 70 °C and pH 5.4, respectively. Further characterization highlighted good thermal stability (~ 50% of enzymatic activity after 2 h at 70 °C) and pH stability over a broad range (> 90% of activity after 1 h in buffer, ranging pH 5-9); resistance to chemicals was also observed.

Biochemical characterization of a thermostable endomannanase/endoglucanase from Dictyoglomus turgidum.

Dtur_0462 gene from the hypertermophilic bacterium Dictyoglomus turgidum, encoding a β-glucosidase, was synthetically produced and expressed in Escherichia coli BL21(DE3)-RIL. DturβGlu was purified to homogeneity by affinity chromatography and its homotetrameric structure was determined by gel filtration. The monomer is composed by 418 amino acidic residues and showed high sequence similarity with Glycoside Hydrolases (GHs) belonging to GH1 family. The maximum activity of DturβGlu was observed at 80°C and at pH5.4. DturβGlu was stable in the range of pH5-8 and retained 70% of its activity after 2h of incubation at 70°C. Metal ions and chemical reagents differently influenced the β-glucosidase activity; furthermore, DturβGlu displays a good ethanol and glucose tolerance (K 750mM). The enzyme is active on p-nitrophenyl-β-d-glucopyranoside (pNPGlu) (K 0.84mM) and p-nitrophenyl-β-d-galactopyranoside (pNPGal) (K 1.36mM) and shows a broad substrate specificity towards natural compounds as salicin, cellobiose and genistin. The ability to hydrolyze different substrates, the activation in the presence of surfactants, the good thermal resistance, and finally the high glucose and ethanol tolerance make this enzyme a good candidate for industrial applications.

Biochemical characterization of a novel thermostable β-glucosidase from Dictyoglomus turgidum.

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  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.

Antioxidant Capacity of Rigenase, a Specific Aqueous Extract of .

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.

Galactomannan degradation by thermophilic enzymes: a hot topic for biotechnological applications.

The transcription factors of the ArsR/SmtB family are widespread within the bacterial and archaeal kingdoms. They are transcriptional repressors able to sense a variety of metals and undergo allosteric conformational changes upon metal binding, resulting in derepression of genes involved in detoxification. So far, the molecular determinants of specificity, selectivity, and metal binding mechanism have been scarcely investigated in thermophilic microorganisms. TtSmtB, the only ArsR/SmtB member present in the genome of Thermus thermophilus HB27, was chosen as a model to shed light into such molecular mechanisms at high temperature. In the present study, using a multidisciplinary approach, a structural and functional characterization of the protein was performed focusing on its metal interaction and chemical-physical stability. Our data demonstrate that TtSmtB has two distinct metal binding sites per monomer and interacts with di-tri-penta-valent ions with different affinity. Detailed knowledge at molecular level of protein-metal interaction is remarkable to design metal binding domains as scaffolds in metal-based therapies as well as in metal biorecovery or biosensing in the environment.

A physicochemical investigation on the metal binding properties of TtSmtB, a thermophilic member of the ArsR/SmtB transcription factor family.

To fight oxidative damage due to reactive oxygen species (ROS), cells are equipped of different enzymes, among which Peroxiredoxins (Prxs) (EC 1.11.1.15) play a key role. Prxs are thiol-based enzymes containing one (1-Cys Prx) or two (2-Cys Prx) catalytic cysteine residues. In 2-Cys Prxs the cysteine residues form a disulfide bridge following reduction of peroxide which is in turn reduced by Thioredoxin reductase (Tr) /Thioredoxin (Trx) disulfide reducing system to regenerate the enzyme. In this paper we investigated on Prxs of Thermus thermophilus whose genome contains an ORF TT_C0933 encoding a putative Prx, belonging to the subfamily of Bacterioferritin comigratory protein (Bcp): the synthetic gene was produced and expressed in E. coli and the recombinant protein, TtBcp, was biochemically characterized. TtBcp was active on both organic and inorganic peroxides and showed stability at high temperatures. To get insight into disulfide reducing system involved in the recycling of the enzyme we showed that TtBcp catalically eliminates hydrogen peroxide using an unusual partner, the Protein Disulfide Oxidoreductase (TtPDO) that could replace regeneration of the enzyme. Altogether these results highlight not only a new anti-oxidative pathway but also a promising molecule for possible future biotechnological applications.

A peroxiredoxin of Thermus thermophilus HB27: Biochemical characterization of a new player in the antioxidant defence.

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.

Prebiotic properties of Bacillus coagulans MA-13: production of galactoside hydrolyzing enzymes and characterization of the transglycosylation properties of a GH42 β-galactosidase.

Article	Year
Identification and characterization of 1-Cys peroxiredoxin from Sulfolobus solfataricus and its involvement in the response to oxidative stress. The FEBS journal\| PubMed ID: 16441659	2006
Sulfolobus solfataricus protein disulphide oxidoreductase: insight into the roles of its redox sites. Protein engineering, design & selection : PEDS\| PubMed ID: 18988690	2009
A novel arsenate reductase from the bacterium Thermus thermophilus HB27: its role in arsenic detoxification. Biochimica et biophysica acta\| PubMed ID: 23800470	2013
Biochemical characterization of a thermostable endomannanase/endoglucanase from Dictyoglomus turgidum. Extremophiles : life under extreme conditions\| PubMed ID: 29177717	2018
Biochemical characterization of a novel thermostable β-glucosidase from Dictyoglomus turgidum. International journal of biological macromolecules\| PubMed ID: 29518444	2018
Antioxidant Capacity of Rigenase, a Specific Aqueous Extract of . Antioxidants (Basel, Switzerland)\| PubMed ID: 29772758	2018
Galactomannan degradation by thermophilic enzymes: a hot topic for biotechnological applications. World journal of microbiology & biotechnology\| PubMed ID: 30701316	2019
A physicochemical investigation on the metal binding properties of TtSmtB, a thermophilic member of the ArsR/SmtB transcription factor family. International journal of biological macromolecules\| PubMed ID: 31356933	2019
A peroxiredoxin of Thermus thermophilus HB27: Biochemical characterization of a new player in the antioxidant defence. International journal of biological macromolecules\| PubMed ID: 32165200	2020
Prebiotic properties of Bacillus coagulans MA-13: production of galactoside hydrolyzing enzymes and characterization of the transglycosylation properties of a GH42 β-galactosidase. Microbial cell factories\| PubMed ID: 33736637	2021

Danila Limauro

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