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Dr. Giuseppe Manco
Institute of Biochemistry and Cell Biology, National Research Council of Italy, Naples, Italy.

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0 Biosensors
0 Proteomics
0 Bio chemistry
0 Molecolar biology
0 Protein engineering (directed evolution)

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Review
Published: 18 February 2021 in International Journal of Molecular Sciences
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The DING proteins are ubiquitous in the three domains of life, from mesophiles to thermo- and hyperthermophiles. They belong to a family of more than sixty members and have a characteristic N-terminus, DINGGG, which is considered a “signature” of these proteins. Structurally, they share a highly conserved phosphate binding site, and a three dimensional organization resembling the “Venus Flytrap”, both reminding the ones of PstS proteins. They have unusually high sequence conservation, even between distantly related species. Nevertheless despite that the genomes of most of these species have been sequenced, the DING gene has not been reported for all the relative characterized DING proteins. Identity of known DING proteins has been confirmed immunologically and, in some cases, by N-terminal sequence analysis. Only a few of the DING proteins have been purified and biochemically characterized. DING proteins are heterogeneous for their wide range of biological activities and some show different activities not always correlated with each other. Most of them have been originally identified for different biological properties, or rather for binding to phosphate and also to other ligands. Their involvement in pathologies is described. This review is an update of the most recent findings on old and new DING proteins.

ACS Style

Elena Porzio; Maria Faraone Mennella; Giuseppe Manco. DING Proteins Extend to the Extremophilic World. International Journal of Molecular Sciences 2021, 22, 2035 .

AMA Style

Elena Porzio, Maria Faraone Mennella, Giuseppe Manco. DING Proteins Extend to the Extremophilic World. International Journal of Molecular Sciences. 2021; 22 (4):2035.

Chicago/Turabian Style

Elena Porzio; Maria Faraone Mennella; Giuseppe Manco. 2021. "DING Proteins Extend to the Extremophilic World." International Journal of Molecular Sciences 22, no. 4: 2035.

Review
Published: 07 February 2021 in Antioxidants
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PON1, PON2, and PON3 belong to a family of lactone hydrolyzing enzymes endowed with various substrate specificities. Among PONs, PON2 shows the highest hydrolytic activity toward many acyl-homoserine lactones (acyl-HL) involved in bacterial quorum-sensing signaling. Accordingly, defense against pathogens, such as Brevundimonas aeruginosa (B. aeruginosa), was postulated to be the principal function of PON2. However, recent findings have highlighted the importance of PON2 in oxidative stress control, inhibition of apoptosis, and the progression of various types of malignancies. This review focuses on all of these aspects of PON2.

ACS Style

Giuseppe Manco; Elena Porzio; Teresa Carusone. Human Paraoxonase-2 (PON2): Protein Functions and Modulation. Antioxidants 2021, 10, 256 .

AMA Style

Giuseppe Manco, Elena Porzio, Teresa Carusone. Human Paraoxonase-2 (PON2): Protein Functions and Modulation. Antioxidants. 2021; 10 (2):256.

Chicago/Turabian Style

Giuseppe Manco; Elena Porzio; Teresa Carusone. 2021. "Human Paraoxonase-2 (PON2): Protein Functions and Modulation." Antioxidants 10, no. 2: 256.

Journal article
Published: 31 July 2020 in Genes
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We identified two unstable variants in the third exon of α-globin genes: Hb Bernalda/Groene Hart (HBA1:c.358C>T), and Hb Caserta (HBA2:c.79G>A) in cis to Hb Sun Prairie (HBA2:c.391G>C), also named Hb Southern Italy. These mutations occurred in the H helix of the α-globin that is involved in heme contacting, specific recognition of α-hemoglobin-stabilizing protein (AHSP), and α1β1 interactions. The carriers showed α-thalassemia phenotype, but one also jaundice and cholelithiasis. Molecular identification of clusters of families in Southern Italy encouraged molecular characterization of mRNA, globin chain analyses, molecular modeling studies, and comparison with globin variants to understand the mechanisms causing the α-thalassemia phenotype. A normal amount of Hb Bernalda/Groene Hart mRNA were found, and molecular modeling highlighted additional H bonds with AHSP. For Hb Southern Italy, showing an unexpected α/β biosynthetic ratio typical of the β-thalassemia type, two different molecular mechanisms were shown: Reduction of the variant mRNA, likely due to the No-Go Decay for the presence of unused triplet ACG at cod 26, and protein instability due to the impairment of AHSP interaction. The UDP glucuronosyltransferase 1A (UGT1A1) genotyping was conclusive in the case of jaundice and cholelithiasis. Multiple approaches are needed to properly identify the mechanisms leading to unstable variants and the effect of a mutation.

ACS Style

Giovanna Cardiero; Gennaro Musollino; Maria Grazia Friscia; Rosario Testa; Lucrezia Virruso; Caterina Di Girgenti; Mercedes Caldora; Rosario Colella Bisogno; Carlo Gaudiano; Giuseppe Manco; Giuseppina Lacerra. Effect of Mutations on mRNA and Globin Stability: The Cases of Hb Bernalda/Groene Hart and Hb Southern Italy. Genes 2020, 11, 870 .

AMA Style

Giovanna Cardiero, Gennaro Musollino, Maria Grazia Friscia, Rosario Testa, Lucrezia Virruso, Caterina Di Girgenti, Mercedes Caldora, Rosario Colella Bisogno, Carlo Gaudiano, Giuseppe Manco, Giuseppina Lacerra. Effect of Mutations on mRNA and Globin Stability: The Cases of Hb Bernalda/Groene Hart and Hb Southern Italy. Genes. 2020; 11 (8):870.

Chicago/Turabian Style

Giovanna Cardiero; Gennaro Musollino; Maria Grazia Friscia; Rosario Testa; Lucrezia Virruso; Caterina Di Girgenti; Mercedes Caldora; Rosario Colella Bisogno; Carlo Gaudiano; Giuseppe Manco; Giuseppina Lacerra. 2020. "Effect of Mutations on mRNA and Globin Stability: The Cases of Hb Bernalda/Groene Hart and Hb Southern Italy." Genes 11, no. 8: 870.

Journal article
Published: 07 May 2020 in Cell Death & Disease
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The activity of human paraoxonase 2 (PON2) is rapidly reduced in cells incubated with the bacterial quorormone 3-Oxo-dodecanoyl Homoserine Lactone (3OC12HSL), an observation that led to hypothesize a fast PON2 post-translational modification (PTM). Recently, we detected a 3OC12HSL-induced PTM in a cell-free system in which a crude extract from 3OC12HSL-treated HeLa cells was able to inactivate and ubiquitinate at position 144 a recombinant PON2. Here we show the occurrence of this and new PTMs on PON2 in HeLa cells. PTMs were found to gather nearby the two SNPs, A148G, and S311C, that are related to type-2 diabetes and its complications. Furthermore, we detected a PTM nearby a 12 amino acids region that is deleted in PON2 Isoform 2. An in vitro mutation analysis showed that the SNPs and the deletion are involved in PON2 activity and suggested a role of PTMs on its modulation, while a SAXS analysis pointed to Isoform 2 as being largely unstructured, compared to the wild type. Besides, we discovered a control of PON2 expression via a putative mRNA operon involving the Wilms tumor 1 associated protein (WTAP) and the E3 ubiquitin ligase (E3UbL) baculoviral IAP repeat-containing 3 (BIRC3).

ACS Style

Teresa Maria Carusone; Giovanna Cardiero; Mariangela Cerreta; Luigi Mandrich; Oscar Moran; Elena Porzio; Giuliana Catara; Giuseppina Lacerra; Giuseppe Manco. WTAP and BIRC3 are involved in the posttranscriptional mechanisms that impact on the expression and activity of the human lactonase PON2. Cell Death & Disease 2020, 11, 324 -17.

AMA Style

Teresa Maria Carusone, Giovanna Cardiero, Mariangela Cerreta, Luigi Mandrich, Oscar Moran, Elena Porzio, Giuliana Catara, Giuseppina Lacerra, Giuseppe Manco. WTAP and BIRC3 are involved in the posttranscriptional mechanisms that impact on the expression and activity of the human lactonase PON2. Cell Death & Disease. 2020; 11 (5):324-17.

Chicago/Turabian Style

Teresa Maria Carusone; Giovanna Cardiero; Mariangela Cerreta; Luigi Mandrich; Oscar Moran; Elena Porzio; Giuliana Catara; Giuseppina Lacerra; Giuseppe Manco. 2020. "WTAP and BIRC3 are involved in the posttranscriptional mechanisms that impact on the expression and activity of the human lactonase PON2." Cell Death & Disease 11, no. 5: 324-17.

Journal article
Published: 02 March 2020 in Sensors
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Pesticides represent some of the most common man-made chemicals in the world. Despite their unquestionable utility in the agricultural field and in the prevention of pest infestation in public areas of cities, pesticides and their biotransformation products are toxic to the environment and hazardous to human health. Esterase-based biosensors represent a viable alternative to the expensive and time-consuming systems currently used for their detection. In this work, we used the esterase-2 from Alicyclobacillus acidocaldarius as bioreceptor for a biosensing device based on an automated robotic approach. Coupling the robotic system with a fluorescence inhibition assay, in only 30 s of enzymatic assay, we accomplished the detection limit of 10 pmol for 11 chemically oxidized thio-organophosphates in solution. In addition, we observed differences in the shape of the inhibition curves determined measuring the decrease of esterase-2 residual activity over time. These differences could be used for the characterization and identification of thio-organophosphate pesticides, leading to a pseudo fingerprinting for each of these compounds. This research represents a starting point to develop technologies for automated screening of toxic compounds in samples from industrial sectors, such as the food industry, and for environmental monitoring.

ACS Style

Giovanni Paolo Cetrangolo; Janis Rusko; Carla Gori; Paola Carullo; Giuseppe Manco; Marco Chino; Ferdinando Febbraio. Highly Sensitive Detection of Chemically Modified Thio-Organophosphates by an Enzymatic Biosensing Device: An Automated Robotic Approach. Sensors 2020, 20, 1365 .

AMA Style

Giovanni Paolo Cetrangolo, Janis Rusko, Carla Gori, Paola Carullo, Giuseppe Manco, Marco Chino, Ferdinando Febbraio. Highly Sensitive Detection of Chemically Modified Thio-Organophosphates by an Enzymatic Biosensing Device: An Automated Robotic Approach. Sensors. 2020; 20 (5):1365.

Chicago/Turabian Style

Giovanni Paolo Cetrangolo; Janis Rusko; Carla Gori; Paola Carullo; Giuseppe Manco; Marco Chino; Ferdinando Febbraio. 2020. "Highly Sensitive Detection of Chemically Modified Thio-Organophosphates by an Enzymatic Biosensing Device: An Automated Robotic Approach." Sensors 20, no. 5: 1365.

Journal article
Published: 01 March 2020 in International Journal of Molecular Sciences
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Increasing attention is more and more directed toward the thermostable Phosphotriesterase-Like-Lactonase (PLL) family of enzymes, for the efficient and reliable decontamination of toxic nerve agents. In the present study, the DNA Staggered Extension Process (StEP) technique was utilized to obtain new variants of PLL enzymes. Divergent homologous genes encoding PLL enzymes were utilized as templates for gene recombination and yielded a new variant of SsoPox from Saccharolobus solfataricus. The new mutant, V82L/C258L/I261F/W263A (4Mut) exhibited catalytic efficiency of 1.6 × 105 M−1 s−1 against paraoxon hydrolysis at 70°C, which is more than 3.5-fold and 42-fold improved in comparison with C258L/I261F/W263A (3Mut) and wild type SsoPox, respectively. 4Mut was also tested with chemical warfare nerve agents including tabun, sarin, soman, cyclosarin and VX. In particular, 4Mut showed about 10-fold enhancement in the hydrolysis of tabun and soman with respect to 3Mut. The crystal structure of 4Mut has been solved at the resolution of 2.8 Å. We propose that, reorganization of dimer conformation that led to increased central groove volume and dimer flexibility could be the major determinant for the improvement in hydrolytic activity in the 4Mut.

ACS Style

Yoko Suzumoto; Orly Dym; Giovanni N. Roviello; Franz Worek; Joel L. Sussman; Giuseppe Manco. Structural and Functional Characterization of New SsoPox Variant Points to the Dimer Interface as a Driver for the Increase in Promiscuous Paraoxonase Activity. International Journal of Molecular Sciences 2020, 21, 1683 .

AMA Style

Yoko Suzumoto, Orly Dym, Giovanni N. Roviello, Franz Worek, Joel L. Sussman, Giuseppe Manco. Structural and Functional Characterization of New SsoPox Variant Points to the Dimer Interface as a Driver for the Increase in Promiscuous Paraoxonase Activity. International Journal of Molecular Sciences. 2020; 21 (5):1683.

Chicago/Turabian Style

Yoko Suzumoto; Orly Dym; Giovanni N. Roviello; Franz Worek; Joel L. Sussman; Giuseppe Manco. 2020. "Structural and Functional Characterization of New SsoPox Variant Points to the Dimer Interface as a Driver for the Increase in Promiscuous Paraoxonase Activity." International Journal of Molecular Sciences 21, no. 5: 1683.

Journal article
Published: 07 November 2019 in Sensors
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Organophosphate (OP) pesticides are widely used in the agricultural field and in the prevention of pest infestation in private and public areas of cities. Despite their unquestionable utility, several of these compounds demonstrate toxic effects to the environment and human health. In particular, the occurrence of some organophosphate pesticides is correlated to the incidence of nervous system disorders, especially in children. The detection of pesticide residues in the human body represents an important task to preserve human health. In our work we propose the use of esterase-based biosensors as a viable alternative to the expensive and time-consuming systems currently used for their detection in human fluids. Using the esterase-2 activity, coupled with a fluorescence inhibition assay, we are able to detect very low concentration levels of diethyl (4-nitrophenyl) phosphate (paraoxon) in the range of the femtomole (fmol). Method robustness tests indicate the stability of esterase-2 in a diluted solution of 4% human urine, and we are able to accurately determine concentration levels of paraoxon in the range from 0.1 to 2 picomoles (pmol). The system sensitivity for OP detection is calculated at 524 ± 14.15 fmol of paraoxon recognized at 10% of inhibition, with an estimated limit of quantification of 262 ± 8.12 pmol mL−1. These values are comparable with the most recent analysis methods based on mass spectrometry carried out on human samples for pesticide detection. This research represents a starting point to develop cheap and fast testing methods for a rapid screening of toxic substances in human samples.

ACS Style

Giovanni Paolo Cetrangolo; Carla Gori; Janis Rusko; Sara Terreri; Giuseppe Manco; Amelia Cimmino; Ferdinando Febbraio. Determination of Picomolar Concentrations of Paraoxon in Human Urine by Fluorescence-Based Enzymatic Assay. Sensors 2019, 19, 4852 .

AMA Style

Giovanni Paolo Cetrangolo, Carla Gori, Janis Rusko, Sara Terreri, Giuseppe Manco, Amelia Cimmino, Ferdinando Febbraio. Determination of Picomolar Concentrations of Paraoxon in Human Urine by Fluorescence-Based Enzymatic Assay. Sensors. 2019; 19 (22):4852.

Chicago/Turabian Style

Giovanni Paolo Cetrangolo; Carla Gori; Janis Rusko; Sara Terreri; Giuseppe Manco; Amelia Cimmino; Ferdinando Febbraio. 2019. "Determination of Picomolar Concentrations of Paraoxon in Human Urine by Fluorescence-Based Enzymatic Assay." Sensors 19, no. 22: 4852.

Journal article
Published: 17 November 2018 in Journal of Hazardous Materials
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Organophosphates (OPs) are highly toxic compounds used as pesticides and nerve agents. The devastating effects, reported in different studies, on the environment and human health indicate a serious scenario for both instantaneous and long terms effects. Bio-based strategies for OPs degradation seem the most promising solutions, particularly when extremophiles enzymes are used. These systems permit OPs degradation with high efficiency and specificity under mild conditions. However, as frequently observed, enzymes can easily lose activity in batch systems, so that a strategy to improve biocatalyst stability is highly needed, in order to develop continuous systems. In this work, for the first time, a continuous biocatalytic system for organophosphates (OPs) detoxification has been proposed by using a triple mutant of the thermostable phosphotriesterase (named SsoPox) isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. The enzyme was covalently immobilized on polymeric membranes to develop a biocatalytic membrane reactor (BMR) able to hydrolyse a pesticide (paraoxon) contained in water. High paraoxon degradation (about 90%) and long term stability (1 year) were obtained when the enzyme was covalently immobilized on hydrophilic membranes. On the contrary, the enzyme in batch system completely loses its activity within few months after its solubilisation in buffer.

ACS Style

G. Vitola; R. Mazzei; T. Poerio; Elena Porzio; Giuseppe Manco; Ida Perrotta; F. Militano; L. Giorno. Biocatalytic membrane reactor development for organophosphates degradation. Journal of Hazardous Materials 2018, 365, 789 -795.

AMA Style

G. Vitola, R. Mazzei, T. Poerio, Elena Porzio, Giuseppe Manco, Ida Perrotta, F. Militano, L. Giorno. Biocatalytic membrane reactor development for organophosphates degradation. Journal of Hazardous Materials. 2018; 365 ():789-795.

Chicago/Turabian Style

G. Vitola; R. Mazzei; T. Poerio; Elena Porzio; Giuseppe Manco; Ida Perrotta; F. Militano; L. Giorno. 2018. "Biocatalytic membrane reactor development for organophosphates degradation." Journal of Hazardous Materials 365, no. : 789-795.

Journal article
Published: 13 September 2018 in Scientific Reports
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Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development of an electrochemical biosensor and a decontamination formulation, respectively. The features of the developed tools have been tested in an ad-hoc fabricated chamber, to mimic an alarming situation of exposure to a nerve agent. Choosing ethyl-paraoxon as nerve agent simulant, a limit of detection (LOD) of 0.4 nM, after 5 s of exposure time was obtained. Furthermore, an optimized enzymatic formulation was used for a fast and efficient environmental detoxification (>99%) of the nebulized nerve agent simulants in the air and on surfaces. Crucial, large-scale experiments have been possible thanks to production of grams amounts of pure (>90%) enzymes.

ACS Style

Elena Porzio; Francesca Bettazzi; Luigi Mandrich; Immacolata Del Giudice; Odile F. Restaino; Serena Laschi; Ferdinando Febbraio; Valentina De Luca; Maria G. Borzacchiello; Teresa M. Carusone; Franz Worek; Antonio Pisanti; Piero Porcaro; Chiara Schiraldi; Mario De Rosa; Ilaria Palchetti; Giuseppe Manco. Innovative Biocatalysts as Tools to Detect and Inactivate Nerve Agents. Scientific Reports 2018, 8, 13773 .

AMA Style

Elena Porzio, Francesca Bettazzi, Luigi Mandrich, Immacolata Del Giudice, Odile F. Restaino, Serena Laschi, Ferdinando Febbraio, Valentina De Luca, Maria G. Borzacchiello, Teresa M. Carusone, Franz Worek, Antonio Pisanti, Piero Porcaro, Chiara Schiraldi, Mario De Rosa, Ilaria Palchetti, Giuseppe Manco. Innovative Biocatalysts as Tools to Detect and Inactivate Nerve Agents. Scientific Reports. 2018; 8 (1):13773.

Chicago/Turabian Style

Elena Porzio; Francesca Bettazzi; Luigi Mandrich; Immacolata Del Giudice; Odile F. Restaino; Serena Laschi; Ferdinando Febbraio; Valentina De Luca; Maria G. Borzacchiello; Teresa M. Carusone; Franz Worek; Antonio Pisanti; Piero Porcaro; Chiara Schiraldi; Mario De Rosa; Ilaria Palchetti; Giuseppe Manco. 2018. "Innovative Biocatalysts as Tools to Detect and Inactivate Nerve Agents." Scientific Reports 8, no. 1: 13773.

Journal article
Published: 01 August 2018 in Journal of Hepatology
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Acute liver failure is a rapidly progressive and life-threatening deterioration of liver function resulting in high mortality and morbidity. We found in experimental mouse models of acute liver failure that two metabolic enzymes, namely pyruvate dehydrogenase complex and lactic dehydrogenase, translocate to the nucleus resulting in detrimental gene expression. Treatment with an inhibitor of these two enzymes was found to reduce liver damage and to improve survival.

ACS Style

Rosa Ferriero; Edoardo Nusco; Rossella De Cegli; Annamaria Carissimo; Giuseppe Manco; Nicola Brunetti-Pierri. Pyruvate dehydrogenase complex and lactate dehydrogenase are targets for therapy of acute liver failure. Journal of Hepatology 2018, 69, 325 -335.

AMA Style

Rosa Ferriero, Edoardo Nusco, Rossella De Cegli, Annamaria Carissimo, Giuseppe Manco, Nicola Brunetti-Pierri. Pyruvate dehydrogenase complex and lactate dehydrogenase are targets for therapy of acute liver failure. Journal of Hepatology. 2018; 69 (2):325-335.

Chicago/Turabian Style

Rosa Ferriero; Edoardo Nusco; Rossella De Cegli; Annamaria Carissimo; Giuseppe Manco; Nicola Brunetti-Pierri. 2018. "Pyruvate dehydrogenase complex and lactate dehydrogenase are targets for therapy of acute liver failure." Journal of Hepatology 69, no. 2: 325-335.

Article
Published: 06 April 2018 in Journal of Chemical Technology & Biotechnology
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Organophosphorus (OP) compounds are highly toxic molecules mainly used as pesticides. OP compounds also include nerve gasses used in the past as chemical warfare agents and collectively we refer to OP pesticides and nerve gasses as nerve agents (NA). An intensive, widespread use of pesticides since the 20th century has resulted in the emergence of an urgent global issue concerning both environment and human health. In addition, past terroristic acts and the recent dramatic events in Syria highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and detoxification of these compounds in stored bulks, on critical surfaces and media (food, water and air) and for in vivo prophylaxes and therapies. OP compounds, act as covalent inhibitors of acetylcholinesterase (AChE) in nerve system of vertebrates, thus posing a substantial threat to the ecosystem. In order to address a strong demand for the establishment of environmental monitoring system and remediation process for NA , an increasing number of studies have been focused on the enzymatic degradation in vitro . Use of enzymes for detoxification and decontamination of toxic NA could provide a long-term benefit as it is environmentally friendly compared to conventional methods such as chemical treatments and incineration. This review presents an overview of current state of enzymatic detoxification research against NA. This includes the detailed characterization and protein engineering for the improvement in NA-degrading activities of such enzymes. Research on biosensors for NA detection and identification yet important in the field has not be treated in this review. Instead a special attention has been paid to the Phosphotriesterase-Like-Lactonase (PLL) enzyme family. Several PLL enzymes have been isolated from hyperthermophilic Archaea or thermophilic/extremophilic Bacteria, and exhibit exceptional thermal stability. Extremophilic PLLs therefore hold promise for the potential industrial application towards NA detoxification.

ACS Style

Giuseppe Manco; Elena Porzio; Yoko Suzumoto. Enzymatic detoxification: a sustainable means of degrading toxic organophosphate pesticides and chemical warfare nerve agents. Journal of Chemical Technology & Biotechnology 2018, 93, 2064 -2082.

AMA Style

Giuseppe Manco, Elena Porzio, Yoko Suzumoto. Enzymatic detoxification: a sustainable means of degrading toxic organophosphate pesticides and chemical warfare nerve agents. Journal of Chemical Technology & Biotechnology. 2018; 93 (8):2064-2082.

Chicago/Turabian Style

Giuseppe Manco; Elena Porzio; Yoko Suzumoto. 2018. "Enzymatic detoxification: a sustainable means of degrading toxic organophosphate pesticides and chemical warfare nerve agents." Journal of Chemical Technology & Biotechnology 93, no. 8: 2064-2082.

Journal article
Published: 20 March 2018 in BMC Biotechnology
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Thermostable phosphotriesterase-like lactonases (PLLs) are able to degrade organophosphates and could be potentially employed as bioremediation tools and bioscavengers. But nowadays their manufacturing in high yields is still an issue that limits their industrial applications. In this work we aimed to set up a high yield production and purification biotechnological process of two recombinant PLLs expressed in E. coli, the wild type SacPox from Sulfolobus acidocaldarius and a triple mutated SsoPox C258L/I261F/W263A, originally from Sulfolobus solfataricus. To follow this aim new induction approaches were investigated to boost the enzyme production, high cell density fermentation strategies were set-up to reach higher and higher enzyme yields up to 22-L scale, a downstream train was studied to meet the requirements of an efficient industrial purification process. Physiological studies in shake flasks demonstrated that the use of galactose as inducer increased the enzyme concentrations up to 4.5 folds, compared to the production obtained by induction with IPTG. Optimising high cell density fed-batch strategies the production and the productivity of both enzymes were further enhanced of 26 folds, up to 2300 U·L− 1 and 47.1 U·L− 1·h− 1 for SacPox and to 8700 U·L− 1 and 180.6 U·L− 1·h− 1 for SsoPox C258L/I261F/W263A, and the fermentation processes resulted scalable from 2.5 to 22.0 L. After being produced and extracted from the cells, the enzymes were first purified by a thermo-precipitation step, whose conditions were optimised by response surface methodology. A following ultra-filtration process on 100 and 5 KDa cut-off membranes drove to a final pureness and a total recovery of both enzymes of 70.0 ± 2.0%, suitable for industrial applications. In this paper, for the first time, a high yield biotechnological manufacturing process of the recombinant enzymes SacPox and SsoPox C258L/I261F/W263A was set-up. The enzyme production was boosted by combining a new galactose induction approach with high cell density fed-batch fermentation strategies. An efficient enzyme purification protocol was designed coupling a thermo-precipitation step with a following membrane-based ultra-filtration process.

ACS Style

Odile Francesca Restaino; Maria Giovanna Borzacchiello; Ilaria Scognamiglio; Luigi Fedele; Alberto Alfano; Elena Porzio; Giuseppe Manco; Mario De Rosa; Chiara Schiraldi. High yield production and purification of two recombinant thermostable phosphotriesterase-like lactonases from Sulfolobus acidocaldarius and Sulfolobus solfataricus useful as bioremediation tools and bioscavengers. BMC Biotechnology 2018, 18, 18 .

AMA Style

Odile Francesca Restaino, Maria Giovanna Borzacchiello, Ilaria Scognamiglio, Luigi Fedele, Alberto Alfano, Elena Porzio, Giuseppe Manco, Mario De Rosa, Chiara Schiraldi. High yield production and purification of two recombinant thermostable phosphotriesterase-like lactonases from Sulfolobus acidocaldarius and Sulfolobus solfataricus useful as bioremediation tools and bioscavengers. BMC Biotechnology. 2018; 18 (1):18.

Chicago/Turabian Style

Odile Francesca Restaino; Maria Giovanna Borzacchiello; Ilaria Scognamiglio; Luigi Fedele; Alberto Alfano; Elena Porzio; Giuseppe Manco; Mario De Rosa; Chiara Schiraldi. 2018. "High yield production and purification of two recombinant thermostable phosphotriesterase-like lactonases from Sulfolobus acidocaldarius and Sulfolobus solfataricus useful as bioremediation tools and bioscavengers." BMC Biotechnology 18, no. 1: 18.

Original paper
Published: 11 January 2018 in Extremophiles
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DING proteins represent a new group of 40 kDa-related members, ubiquitous in living organisms. The family also include the DING protein from Sulfolobus solfataricus, functionally related to poly(ADP-ribose) polymerases. Here, the archaeal protein has been compared with the human Phosphate-Binding Protein and the Pseudomonas fluorescence DING enzyme, by enzyme assays and immune cross-reactivity. Surprisingly, as the Sulfolobus enzyme, the Human and Pseudomonas proteins display poly(ADP-ribose) polymerase activity, whereas a phosphatase activity was only present in Sulfolobus and human protein, despite the conserved phosphate-binding site residues in Pseudomonas DING. All proteins were positive to anti-DING antibodies and gave a comparable pattern of anti-poly(ADP-ribose) polymerase immunoreactivity with two bands, at around 40 kDa and roughly at the double of this molecular mass. The latter signal was present in all Sulfolobus enzyme preparations and proved not due to either a contaminant or a precursor protein, but likely being a dimeric form of the 40 kDa polypeptide. The common immunological and partly enzymatic behavior linking human, Pseudomonas and Sulfolobus DING proteins, makes the archaeal protein an important model system to investigate DING protein function and evolution within the cell.

ACS Style

Elena Porzio; Anna De Maio; Teresa Ricciardi; Carmela Mistretta; Giuseppe Manco; Maria Rosaria Faraone-Mennella. Comparison of the DING protein from the archaeon Sulfolobus solfataricus with human phosphate-binding protein and Pseudomonas fluorescence DING counterparts. Extremophiles 2018, 22, 177 -188.

AMA Style

Elena Porzio, Anna De Maio, Teresa Ricciardi, Carmela Mistretta, Giuseppe Manco, Maria Rosaria Faraone-Mennella. Comparison of the DING protein from the archaeon Sulfolobus solfataricus with human phosphate-binding protein and Pseudomonas fluorescence DING counterparts. Extremophiles. 2018; 22 (2):177-188.

Chicago/Turabian Style

Elena Porzio; Anna De Maio; Teresa Ricciardi; Carmela Mistretta; Giuseppe Manco; Maria Rosaria Faraone-Mennella. 2018. "Comparison of the DING protein from the archaeon Sulfolobus solfataricus with human phosphate-binding protein and Pseudomonas fluorescence DING counterparts." Extremophiles 22, no. 2: 177-188.

Journal article
Published: 10 October 2017 in Biomedical Journal of Scientific & Technical Research
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ACS Style

Giuseppe Manco. New Insight in Human Lactonase PON2. Biomedical Journal of Scientific & Technical Research 2017, 1, 1 .

AMA Style

Giuseppe Manco. New Insight in Human Lactonase PON2. Biomedical Journal of Scientific & Technical Research. 2017; 1 (5):1.

Chicago/Turabian Style

Giuseppe Manco. 2017. "New Insight in Human Lactonase PON2." Biomedical Journal of Scientific & Technical Research 1, no. 5: 1.

Journal article
Published: 01 March 2017 in Journal of Industrial Microbiology and Biotechnology
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Thermostable phosphotriesterase-like lactonases (PLLs) from extremophile archaea, like SsoPox from Sulfolobus solfataricus, are attractive biotechnological tools with industrial applications as organophosphate decontaminants, but their manufacturing still remains an unresolved issue because of the high costs and the low production yields. In this paper, for the first time, an efficient biotechnological process for the production and purification of a recombinant, engineered PLL, SsoPox W263F, expressed in E. coli, has been set up by studying new induction strategies, by designing high cell density cultivations and a new membrane-based downstream process. In fed batches, the enzyme production was boosted of 69-fold up to 4660.0 U L−1 using galactose as inducer in the replacement of IPTG; the process was scalable from 2.5 up to 150 L. By coupling a single thermo-precipitation step and an ultrafiltration process, a total enzyme recovery of 77% with a purity grade of almost 80% was reached.

ACS Style

Odile Francesca Restaino; Maria Giovanna Borzacchiello; Ilaria Scognamiglio; Elena Porzio; Giuseppe Manco; Luigi Fedele; Cinzia Donatiello; Mario De Rosa; Chiara Schiraldi. Boosted large-scale production and purification of a thermostable archaeal phosphotriesterase-like lactonase for organophosphate decontamination. Journal of Industrial Microbiology and Biotechnology 2017, 44, 363 -375.

AMA Style

Odile Francesca Restaino, Maria Giovanna Borzacchiello, Ilaria Scognamiglio, Elena Porzio, Giuseppe Manco, Luigi Fedele, Cinzia Donatiello, Mario De Rosa, Chiara Schiraldi. Boosted large-scale production and purification of a thermostable archaeal phosphotriesterase-like lactonase for organophosphate decontamination. Journal of Industrial Microbiology and Biotechnology. 2017; 44 (3):363-375.

Chicago/Turabian Style

Odile Francesca Restaino; Maria Giovanna Borzacchiello; Ilaria Scognamiglio; Elena Porzio; Giuseppe Manco; Luigi Fedele; Cinzia Donatiello; Mario De Rosa; Chiara Schiraldi. 2017. "Boosted large-scale production and purification of a thermostable archaeal phosphotriesterase-like lactonase for organophosphate decontamination." Journal of Industrial Microbiology and Biotechnology 44, no. 3: 363-375.

Book chapter
Published: 28 April 2016 in Biotechnology of Extremophiles:
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Organophosphate compounds, as most pesticides and chemical warfare agents, first appeared in the US after 1930s and became widespread after World War II. At present, enzymatic detoxification of organophosphate compounds represents an important issue worldwide, due to their permanent and excessive use that has led in many places to the contamination of soil and water. In the last years our research group focused the attention on the enzymes belonging to amidohydrolase superfamily. In particular, a new family of lactonases with promiscuous phosphotriesterase activity, dubbed PTE-like Lactonases (PLLs), has been discovered. We report here an overview of the actual use of organophosphate compounds and the hydrolytic enzymes able to degrade them. In the PLL family there are enzymes that hydrolyze pesticides, show high thermal resistance and, therefore, are very attractive from a biotechnology point of view. The combination of different in vitro evolution methods represents a successful approach to increase their promiscuous phosphotriesterase activity in order to obtain efficient detoxification enzymatic tools.

ACS Style

Elena Porzio; Immacolata Del Giudice; Giuseppe Manco. Engineering of Extremophilic Phosphotriesterase-Like Lactonases for Biotechnological Applications. Biotechnology of Extremophiles: 2016, 471 -503.

AMA Style

Elena Porzio, Immacolata Del Giudice, Giuseppe Manco. Engineering of Extremophilic Phosphotriesterase-Like Lactonases for Biotechnological Applications. Biotechnology of Extremophiles:. 2016; ():471-503.

Chicago/Turabian Style

Elena Porzio; Immacolata Del Giudice; Giuseppe Manco. 2016. "Engineering of Extremophilic Phosphotriesterase-Like Lactonases for Biotechnological Applications." Biotechnology of Extremophiles: , no. : 471-503.

Journal article
Published: 20 October 2015 in Biotechnology and Bioengineering
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In vitro evolution of enzymes represents a powerful device to evolve new or to improve weak enzymatic functions. In the present work a semi-rational engineering approach has been used to design an efficient and thermostable organophosphate hydrolase, starting from a lactonase scaffold (SsoPox from Sulfolobus solfataricus). In particular, by in vitro evolution of the SsoPox ancillary promiscuous activity, the triple mutant C258L/I261F/W263A has been obtained which, retaining its inherent stability, showed an enhancement of its hydrolytic activity on paraoxon up to 300-fold, achieving absolute values of catalytic efficiency up to 105 M−1s−1. The kinetics and structural determinants of this enhanced activity were thoroughly investigated and, in order to evaluate its potential biotechnological applications, the mutant was tested in formulations of different solvents (methanol or ethanol) or detergents (SDS or a commercial soap) for the cleaning of pesticide-contaminated surfaces. Biotechnol. Bioeng. 2015;9999: 1–11.

ACS Style

Immacolata Del Giudice; Rossella Coppolecchia; Luigia Merone; Elena Porzio; Teresa Maria Carusone; Luigi Mandrich; Franz Worek; Giuseppe Manco. An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination. Biotechnology and Bioengineering 2015, 113, 724 -734.

AMA Style

Immacolata Del Giudice, Rossella Coppolecchia, Luigia Merone, Elena Porzio, Teresa Maria Carusone, Luigi Mandrich, Franz Worek, Giuseppe Manco. An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination. Biotechnology and Bioengineering. 2015; 113 (4):724-734.

Chicago/Turabian Style

Immacolata Del Giudice; Rossella Coppolecchia; Luigia Merone; Elena Porzio; Teresa Maria Carusone; Luigi Mandrich; Franz Worek; Giuseppe Manco. 2015. "An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination." Biotechnology and Bioengineering 113, no. 4: 724-734.

Journal article
Published: 28 July 2015 in Extremophiles
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The enzymatic regioselective hydrolysis of (a) acetylated mono- to tetrasaccharides of different nature, (b) of acetylated aryl glycosides and (c) of different acetylated nucleosides was studied enlarging the portfolio of substrates that can be employed by the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius. The reactions were optimised to the extent that the amount of enzyme needed was lowered of two orders of magnitude with respect to the previously reported reactions, namely from 4000 to 40 U of enzyme per reaction. New additional solvents were screened and dramatic changes in regioselectivity were observed depending on the amount and type of solvent used. For example, in the presence of 10 % DMF, only two α-d-glucose products 6-OH and 4,6-OH (in a 76:24 ratio) were detected, whereas with 25 % DMF, at least four products of similar amount were observed. This versatility adds specific value to the biocatalyst making possible the design of biocatalytic reactions with different hydrophobic ester substrates. As an additional remarkable example, EST2 catalysed with a good yield and high regioselectivity the hydrolysis of p-nitrophenyl β-d-xylopyranoside triacetate producing only the monoacetylated derivative with acetyl group in 3-O-position, in 2 min. The results with nucleosides as substrates are particularly interesting. The peracetates of 3′,5′-di-O-acetylthymidine are converted almost quantitatively (95 %) to the monoacetylated derivative possessing free secondary OH; this regioselectivity is complementary to hydrolysis/alcoholysis reactions catalysed by CAL-B lipase or to other microbial hydrolytic biocatalysts, generally giving products with free primary OH groups. A docking analysis was undertaken with all analysed substrates suggesting a structural interpretation of the results. In most of cases, the best pose of the selected substrate was in line with the observed regioselectivity.

ACS Style

Angela Pennacchio; Luigi Mandrich; Giuseppe Manco; Antonio Trincone. Enlarging the substrate portfolio of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius. Extremophiles 2015, 19, 1001 -1011.

AMA Style

Angela Pennacchio, Luigi Mandrich, Giuseppe Manco, Antonio Trincone. Enlarging the substrate portfolio of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius. Extremophiles. 2015; 19 (5):1001-1011.

Chicago/Turabian Style

Angela Pennacchio; Luigi Mandrich; Giuseppe Manco; Antonio Trincone. 2015. "Enlarging the substrate portfolio of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius." Extremophiles 19, no. 5: 1001-1011.

Research article
Published: 23 February 2015 in PLOS ONE
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Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

ACS Style

Yu Zhang; Jiao An; Guang-Yu Yang; Aixi Bai; Baisong Zheng; Zhiyong Lou; Geng Wu; Wei Ye; Hai-Feng Chen; Yan Feng; Giuseppe Manco. Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426. PLOS ONE 2015, 10, e0115130 .

AMA Style

Yu Zhang, Jiao An, Guang-Yu Yang, Aixi Bai, Baisong Zheng, Zhiyong Lou, Geng Wu, Wei Ye, Hai-Feng Chen, Yan Feng, Giuseppe Manco. Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426. PLOS ONE. 2015; 10 (2):e0115130.

Chicago/Turabian Style

Yu Zhang; Jiao An; Guang-Yu Yang; Aixi Bai; Baisong Zheng; Zhiyong Lou; Geng Wu; Wei Ye; Hai-Feng Chen; Yan Feng; Giuseppe Manco. 2015. "Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426." PLOS ONE 10, no. 2: e0115130.

Journal article
Published: 09 February 2015 in Sensors
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Organophosphates are organic substances that contain a phosphoryl or a thiophosphoryl bond. They are mainly used around the world as pesticides, but can also be used as chemical warfare agents. Their detection is normally entrusted to techniques like GC- and LC-MS that, although sensitive, do not allow their identification on site and in real time. We have approached their identification by exploiting the high-affinity binding of these compounds with the esterase 2 from Alicyclobacillus acidocaldarius. Using an in silico analysis to evaluate the binding affinities of the enzyme with organophosphate inhibitors, like paraoxon, and other organophosphate compounds, like parathion, chlorpyriphos, and other organophosphate thio-derivatives, we have designed fluorescence spectroscopy experiments to study the quenching of the tryptophan residues after esterase 2 binding with the organophosphate pesticides. The changes in the fluorescence signals permitted an immediate and quantitative identification of these compounds from nano- to picomolar concentrations. A fluorescence based polarity-sensitive probe (ANS) was also employed as a means to understand the extent of the interactions involved, as well as to explore other ways to detect organophosphate pesticides. Finally, we designed a framework for the development of a biosensor that exploits fluorescence technology in combination with a sensitive and very stable bio-receptor.

ACS Style

Paola Carullo; Giovanni Paolo Cetrangolo; Luigi Mandrich; Giuseppe Manco; Ferdinando Febbraio. Fluorescence Spectroscopy Approaches for the Development of a Real-Time Organophosphate Detection System Using an Enzymatic Sensor. Sensors 2015, 15, 3932 -3951.

AMA Style

Paola Carullo, Giovanni Paolo Cetrangolo, Luigi Mandrich, Giuseppe Manco, Ferdinando Febbraio. Fluorescence Spectroscopy Approaches for the Development of a Real-Time Organophosphate Detection System Using an Enzymatic Sensor. Sensors. 2015; 15 (2):3932-3951.

Chicago/Turabian Style

Paola Carullo; Giovanni Paolo Cetrangolo; Luigi Mandrich; Giuseppe Manco; Ferdinando Febbraio. 2015. "Fluorescence Spectroscopy Approaches for the Development of a Real-Time Organophosphate Detection System Using an Enzymatic Sensor." Sensors 15, no. 2: 3932-3951.