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This study monitored the process and investigated the quality of compost obtained from different biomasses. Five blends of agri-food waste were composted by a laboratory pilot plant named COMPOSTER, that is designed to optimize biodegradation, and produce compost efficiently. The COMPOSTER consists of two 35-liter nearly adiabatic, aerated bioreactors that simulate an industrial process involving the typical sequence of mesophilic-thermophilic-mesophilic phases. It continuously monitors and records temperature, internal pressure, and biomass weight, while controlling and quantifying oxygen consumption and carbon dioxide emissions resulting from aerobic biodegradation. All composts were characterized for their main chemical, physical, and molecular features, as well as their suppressiveness against Fusarium oxysporum f.sp. lycopersici (FOL), tested on tomato seedlings. Optimized biodegradation yielded 50-60 % mature compost with a cumulative oxygen consumption ranging from 282 to 456 gO2 per kg of dry matter, with peaks of 2.55 gO2 per kg of volatile solids per hour, and carbon dioxide emissions of 22-36 % of the initial carbon content, with peaks of 5.89 g CO2 per kg of volatile solids per hour. Blends containing more ligno-cellulosic ingredients showed higher yields and lower CO2 emissions. Most of the nitrogen present initially was retained in the final compost; indeed, all mixtures exhibited an apparent nitrogen concentration increase due to carbon loss. Composting determined deep modifications in the molecular structure of the organic matter. 13C CPMAS-NMR and off-line thermochemolysis GC-MS analyses highlighted decomposition degree of polysaccharides and peptidic moieties, selective preservation of aliphatic and aromatic recalcitrant compounds, and optimal ongoing humification. All composts were non-phytotoxic, except for that including pepper crop residues, and all resulted rich in macro- and micro-elements for plant nutrition and proved to be active in controlling FOL disease. Compost comprising 81.2 % tomato crop waste exhibited the best growth performance and pathogen control on tomato. Mature, non-phytotoxic, nutrient-rich, and suppressive composts represent promising by-products that can be successfully recycled in agriculture, including high-value applications, leading to lower use of fertilizers and pesticides.
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The development of new natural antibiotics is considered as the heart of several investigations in the nutraceutical field. In this work, leaves of Quercus ilex L. treated by tropospheric ozone (O3) and nitrogen (N) deposition, exhibited a clear antimicrobial efficacy against five multi-drug resistant (MDR) bacterial strains (two gram-positive and three gram-negative). Under controlled conditions, it was studied how simulated N deposition influences the response to O3 and the antibacterial and antioxidant activity, and antioxidant performance. The extraction was performed by ultra-pure acetone using two different steps. A higher antioxidant activity was measured in the presence of interaction between O3 and N treatments on Quercus leaves. At the same time, all organic extracts tested have shown bacteriostatic activity against all the tested strains with a MIC comprised between 9 and 4 micrograms/mL, and a higher antioxidant efficacy shown by spectrophotometric assay. Stronger antimicrobial activity was found in the samples treated with O3, whereas N-treated plants exhibited an intermediate antibacterial performance. This performance is related to the stimulation of the non-enzymatic antioxidant system induced by the oxidative stress, which results in an increase in the production of antimicrobial bioactive compounds.
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A hydrotropic solution of maleic acid (MA) was exploited to fractionate giant reed (AD) and eucalypt (EUC). The pre-treatment was successful for AD, while it was unsatisfactory for EUC, likely due to unoptimized reaction conditions. Interestingly, lignin nanoparticles (LNP) were produced via spontaneous aggregation following spent liquor dilution. LNP were studied by a plethora of analytical techniques, such as thermogravimetry, electron microscopy, and Nuclear Magnetic Resonance spectroscopy (NMR). Notwithstanding LNP from both AD and EUC showed similar thermal behaviour and morphology, a greater content of aliphatic hydroxyl, carboxyl, guaiacyl and p-hydroxyphenyl moieties was reported for AD-LNP, whereas EUC-LNP had a larger amount of syringyl groups and a higher S/G ratio. Also, the 1H-DOSY NMR indicated the lower size of AD-LNP. Moreover, the LNP were found to negatively impact on the development of several human or plant pathogens, and their bioactivity was related to the occurrence of guaiacyl and p-hydroxyphenyl moieties and a lower the LNP size. We therefore found that MA delignification allows both to achieve high delignification efficiency and to obtain LNP with promising antibacterial effect. Such LNP may help counteracting the antibiotics resistance and sustain the quest for finding sustainable agrochemicals.
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Lignina , Nanopartículas , Humanos , Lignina/química , Espectroscopía de Resonancia MagnéticaRESUMEN
Sustainable active food packaging is essential to reduce the use of plastics, preserve food quality and minimize the environmental impact. Humic substances (HS) are rich in redox-active compounds, such as quinones, phenols, carboxyl, and hydroxyl moieties, making them functional additives for biopolymeric matrices, such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Herein, composites made by incorporating different amounts of HS into PHBV were developed using the electrospinning technology and converted into homogeneous and continuous films by a thermal post-treatment to obtain a bioactive and biodegradable layer which could be part of a multilayer food packaging solution. The morphology, thermal, optical, mechanical, antioxidant and barrier properties of the resulting PHBV-based films have been evaluated, as well as the antifungal activity against Aspergillus flavus and Candida albicans and the antimicrobial properties against both Gram (+) and Gram (-) bacterial strains. HS show great potential as natural additives for biopolymer matrices, since they confer antioxidant, antimicrobial, and antifungal properties to the resulting materials. In addition, barrier, optical and mechanical properties highlighted that the obtained films are suitable for sustainable active packaging. Therefore, the electrospinning methodology is a promising and sustainable approach to give biowaste a new life through the development of multifunctional materials suitable in the active bio-packaging.
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Embalaje de Alimentos , Sustancias Húmicas , Ácidos Pentanoicos , Antifúngicos/farmacología , Antioxidantes/farmacología , PoliésteresRESUMEN
As a result of the accumulation of plastic in the environment, microplastics have become part of the food chain, boosting the resistance of fungi and bacteria which can frequently encounter human beings. Employing photocatalytic degradation is a possible route towards the removal of chemical and biological pollutants, such as plastics and microplastic wastes as well as microorganisms. Using biowaste materials to design hybrid nanoparticles with enhanced photocatalytic and antimicrobial features would uphold the principles of the circular bioeconomy. Here, two unexpensive semiconductors-namely titanium dioxide (TiO2) and zinc oxide (ZnO) - were synthetized through solvothermal synthesis and combined with humic substances deriving from agrifood biomass. The preparation led to hybrid nanoparticles exhibiting enhanced ROS-generating properties for simultaneous applications as antimicrobial agents against different bacterial and fungal strains and as photoactive catalysts to degrade polylactic acid (PLA) microplastics under UVA and solar irradiation. In comparison to bare nanoparticles, hybrid nanoparticles demonstrated higher antibacterial and antimycotic capabilities toward various pathogenic microorganisms as well as advanced photocatalytic activity in the degradation of PLA with a carbonyl index reduction in the range of 15-23%, thus confirming a noteworthy ability in microplastics photodegradation under UVA and solar irradiation.
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Antibacterianos , Nanopartículas Multifuncionales , Humanos , Antibacterianos/farmacología , Microplásticos , Plásticos , Sustancias Húmicas , Óxidos , Poliésteres/farmacología , Titanio/farmacología , Titanio/químicaRESUMEN
Natural bioactive compounds represent a new frontier of antimicrobial molecules, and the marine ecosystem represents a new challenge in this regard. In the present work, we evaluated the possibility of changes in the antibacterial activity of protamine-like (PL) proteins, the major nuclear basic protein components of Mytilus galloprovincialis sperm chromatin, after the exposure of mussels to subtoxic doses of chromium (VI) (1, 10, and 100 nM) and mercury (1, 10, and 100 pM) HgCl2, since these metals affect some properties of PL. After exposure, we analyzed the electrophoretic pattern of PLs by both acetic acid-urea polyacrylamide gel electrophoresis (AU-PAGE) and SDS-PAGE and determined the MIC and MBC of these proteins on different gram+ and gram- bacteria. PLs, particularly after mussels were exposed to the highest doses of chromium and mercury, showed significantly reduced antibacterial activity. Just at the highest doses of exposure to the two metals, changes were found in the electrophoretic pattern of PLs, suggesting that there were conformational changes in these proteins, which were confirmed by the fluorescence measurements of PLs. These results provide the first evidence of a reduction in the antibacterial activity of these proteins following the exposure of mussels to these metals. Based on the results, hypothetical molecular mechanisms that could explain the decrease in the antibacterial activity of PLs are discussed.
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Mercurio , Mytilus , Contaminantes Químicos del Agua , Animales , Masculino , Protaminas/farmacología , Protaminas/metabolismo , Mercurio/toxicidad , Cromo/toxicidad , Cromo/metabolismo , Ecosistema , Semen/metabolismo , Proteínas Nucleares/metabolismo , Metales/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Contaminantes Químicos del Agua/metabolismoRESUMEN
Exploring opportunities for biowaste valorization, herein, humic substances (HS) were combined with gelatin, a hydrophilic biocompatible and bioavailable polymer, to obtain 3D hydrogels. Hybrid gels (Gel HS) were prepared at different HS contents, exploiting physical or chemical cross-linking, through 1-ethyl-(3-3-dimethylaminopropyl)carbodiimide (EDC) chemistry, between HS and gelatin. Physicochemical features were assessed through rheological measurements, X-ray diffraction, attenuated total reflectance (ATR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and scanning electron microscopy (SEM). ATR and NMR spectroscopies suggested the formation of an amide bond between HS and Gel via EDC chemistry. In addition, antioxidant and antimicrobial features toward both Gram(-) and Gram(+) strains were evaluated. HS confers great antioxidant and widespread antibiotic performance to the whole gel. Furthermore, the chemical cross-linking affects the viscoelastic behavior, crystalline structures, water uptake, and functional performance and produces a marked improvement of biocide action.
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Gelatina , Hidrogeles , Gelatina/química , Hidrogeles/farmacología , Hidrogeles/química , Sustancias Húmicas , Antioxidantes/farmacología , Reactivos de Enlaces Cruzados/química , Antibacterianos/farmacologíaRESUMEN
Helicobacter pylori (H. pylori) is a common human pathogen causing inflammation. Recent studies have suggested a sophisticated interplay between mitochondria, innate immunity and inflammatory response, thus proposing mitochondrial disfunction as the hallmark of severe inflammatory disorders. In this study, humic substances isolated from composted fennel residues (HS-FEN) were tested as potential therapeutical strategy to restore the mitochondrial physiology and control the inflammation associated with H. pylori infection. The molecular features of HS-FEN were characterized by infrared spectrometry, thermochemolysis-GC/MS, NMR spectroscopy, and high-performance size-exclusion chromatography (HPSEC), which revealed the presence of aromatic polyphenolic components arranged in a rather stable conformation. In vitro results showed antioxidant and anti-inflammatory properties of HS-FEN, that was found to increase the expression level of OPA-1 and SOD-2 genes and in AGS cells stimulated with H. pylori culture filtrate (Hpcf) and concomitantly decrease the expression level of Drp-1 gene and IL-12, IL-17 and G-CSF proteins. The hydrophobic features of HS, their conformational arrangement and large content of bioactive molecules may explain the beneficial effects of HS-FEN, that may potentially become an interesting source of anti-inflammatory agents capable to counteract or prevent the H. pylori-related inflammatory disorders.
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Foeniculum , Infecciones por Helicobacter , Helicobacter pylori , Humanos , Sustancias Húmicas , Infecciones por Helicobacter/complicaciones , Inflamación/metabolismo , Antiinflamatorios/farmacología , Células Epiteliales/metabolismo , Mucosa Gástrica/metabolismoRESUMEN
Photoaging is the premature aging of the skin caused by repeated exposure to ultraviolet (UV) rays. The harmful effects of UV rays-from the sun or from artificial sources-alter normal skin structures and cause visible damage, especially in the most exposed areas. Fighting premature aging is one of the most important challenges of the medical landscape. Additionally, consumers are looking for care products that offer multiple benefits with reduced environmental and economic impact. The growing requests for bioactive compounds from aromatic plants for pharmaceutical and cosmetic applications have to find new sustainable methods to increase the effectiveness of new active formulations derived from eco-compatible technologies. The principle of sustainable practices and the circular economy favor the use of bioactive components derived from recycled biomass. The guidelines of the European Commission support the reuse of various types of organic biomass and organic waste, thus transforming waste management problems into economic opportunities. This review aims to elucidate the main mechanisms of photoaging and how these can be managed using natural renewable sources and specific bioactive derivatives, such as humic extracts from recycled organic biomass, as potential new actors in modern medicine.
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BACKGROUND: An important goal of modern medicine is the development of products deriving from natural sources to improve environmental sustainability. In this study, humic substances (HS) and compost teas (CTs) extracted from artichoke (ART) and coffee grounds (COF) as recycled biomasses were employed on Ocimum basilicum plants to optimize the yield of specific metabolites with nutraceutical and antibacterial features by applying sustainable strategies. METHODS: The molecular characteristics of compost derivates were elucidated by Nuclear Magnetic Resonance spectroscopy to investigate the structure-activity relationship between organic extracts and their bioactive potential. Additionally, combined untargeted and targeted metabolomics workflows were applied to plants treated with different concentrations of compost extracts. RESULTS: The substances HS-ART and CT-COF improved both antioxidant activity (TEAC values between 39 and 55 µmol g-1) and the antimicrobial efficacy (MIC value between 3.7 and 1.3 µg mL-1) of basil metabolites. The metabolomic approach identified about 149 metabolites related to the applied treatments. Targeted metabolite quantification further highlighted the eliciting effect of HS-ART and CT-COF on the synthesis of aromatic amino acids and phenolic compounds for nutraceutical application. CONCLUSIONS: The combination of molecular characterization, biological assays, and an advanced metabolomic approach, provided innovative insight into the valorization of recycled biomass to increase the availability of natural compounds employed in the medical field.
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Long-term exposure to air pollution has been associated with the development of some inflammatory processes related to skin. The goal of modern medicine is the development of new products with antiflammatory action deriving from natural sources to improve environmental and economic sustainability. In this study, two different humic acids (HA) were isolated from from lignite (HA-LIG) and composted artichoke wastes (HA-CYN) and characterized by infrared spectrometry, NMR spectroscopy, thermochemolysis-GC/MS, and high-performance size-exclusion chromatography (HPSEC), while their antiflammatory activity was evaluated on HaCaT cells. Spectroscopic results showing the predominance of apolar aliphatic and aromatic components in HA-LIG, whereas HA-CYN revealed a presence of polysaccharides and polyphenolic lignin residues. The HA application on human keratinocyte pre-treated with Urban Dust revealed a general increase of viability suggesting a protective effect of humic matter due to the content of aromatic, phenolic and lignin components. Conversely, the gene expression of IL-6 and IL-1ß cytokines indicated a significant decrease after application of HA-LIG, thus exhibiting a greater antiflammatory power than HA-CYN. The specific combination of HA protective hydrophobic components, viable conformational arrangements, and content of bioactive molecules, suggests an innovative applicability of humic matter in dermatology as skin protectors from environmental irritants and as antiflammatory agents.
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Antiinflamatorios no Esteroideos/aislamiento & purificación , Antiinflamatorios no Esteroideos/farmacología , Carbón Mineral , Compostaje , Sustancias Húmicas , Antiinflamatorios no Esteroideos/química , Supervivencia Celular , Cromatografía en Gel , Carbón Mineral/análisis , Cromatografía de Gases y Espectrometría de Masas , Células HaCaT , Humanos , Sustancias Húmicas/análisis , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Queratinocitos/citología , Espectroscopía de Resonancia MagnéticaRESUMEN
BACKGROUND: Developing eco-friendly antioxidant and antimicrobial substances originating from biomass residues has recently attracted considerable interest. In this study, two lignosulfonates and various oxidized water-soluble lignins were investigated for their antioxidant properties, as assessed by ABTS, DPPH and Folin-Ciocalteu methods, and their antimicrobial activity against some bacterial strains responsible for human pathologies. RESULTS: The lignosulfonates showed the largest antiradical/antimicrobial capacity, whereas the other substrates were less effective. The observed antioxidant/antibacterial properties were positively correlated with lignin aromatic/phenolic content. The positive correlation between antiradical and antimicrobial activities suggests that lignin scavenging capacity was also involved in its antibacterial activity. A greater antimicrobial performance was generally observed against Gram-positive bacterial strains, and it was attributed to the intrinsic larger susceptibility of Gram-positive bacteria to lignin phenols. A significant though lesser inhibitory activity was also found against Escherichia coli. CONCLUSION: Our results confirmed the dependence of lignin antioxidant/antibacterial power on its extraction method and chemical structure, as well as on the type of bacterial strains. Identifying the relationship between lignin molecular composition and its antioxidant/antibacterial features represents an advance on the potential future use of renewable and eco-compatible lignin materials in nutraceutical, pharmaceutical and cosmetic sectors. © 2021 Society of Chemical Industry.
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Antioxidantes , Lignina , Antibacterianos/química , Antibacterianos/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Bacterias , Bacterias Grampositivas , Humanos , Lignina/farmacología , Fenoles/química , Extractos Vegetales/química , Extractos Vegetales/farmacologíaRESUMEN
Humic acids (HA) are considered a promising and inexpensive source for novel multifunctional materials for a huge range of applications. However, aggregation and degradation phenomena in aqueous environment prevent from their full exploitation. A valid strategy to address these issues relies on combining HA moieties at the molecular scale with an inorganic nanostructured component, leading to more stable hybrid nanomaterials with tunable functionalities. Indeed, chemical composition of HA can determine their interactions with the inorganic constituent in the hybrid nanoparticles and consequently affect their overall physico-chemical properties, including their stability and functional properties in aqueous environment. As a fundamental contribution to HA materials-based technology, this study aims at unveiling this aspect. To this purpose, SiO2 nanoparticles have been chosen as a model platform and three different HAs extracted from composted biomasses, manure (HA_Man), artichoke residues (HA_Art) and coffee grounds (HA_Cof), were employed to synthetize hybrid HA-SiO2 nanoparticles through in-situ sol-gel synthesis. Prepared samples were submitted to aging in water to assess their stability. Furthermore, antioxidant properties and physico-chemical properties of both as prepared and aged samples in aqueous environment were assessed through Scanning Electron Microscopy (SEM), N2 physisorption, Simultaneous Thermogravimetric (TGA) and Differential Scanning Calorimetric (DSC) Analysis, Fourier Transform Infrared (FT-IR), Nuclear Magnetic Resonance (NMR), Electron Paramagnetic Resonance (EPR) spectroscopies. The experimental results highlighted that hybrid HA-SiO2 nanostructures acted as dynamic systems which exhibit structural supramolecular reorganization during aging in aqueous environment with marked effects on physico-chemical and functional properties, including improved antioxidant activity. Obtained results enlighten a unique aspect of interactions between HA and inorganic nanoparticles that could be useful to predict their behavior in aqueous environment. Furthermore, the proposed approach traces a technological route for the exploitation of organic biowaste in the design of hybrid nanomaterials, providing a significant contribution to the development of waste to wealth strategies based on humic substances.
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Sustancias Húmicas , Nanoestructuras , Anciano , Humanos , Sustancias Húmicas/análisis , Masculino , Dióxido de Silicio , Espectroscopía Infrarroja por Transformada de Fourier , AguaRESUMEN
The acknowledgement of bioactive functions of compost teas promotes the research on characteristics and potential application of these heterogeneous water-soluble extracts from recycled biomasses. In this work, compost teas were isolated from on-farm composts made with agro-industrial residues of artichoke, pepper and coffee husks with the aim to evaluate the structural-activity relationship of dissolved bioactive molecules. The molecular features of compost teas were determined by 13C-CPMAS NMR spectroscopy, Infrared spectroscopy, and off-line pyrolysis-Gas Chromatography/Mass Spectrometry. Bioactivity of different compost teas was tested on Basil seeds germination, while the antioxidant capacity was measured by ABTS and DDPH spectrophotometric assays. The antimicrobial activity was measured against some pathogenic human bacterial strains. The seed germination experiment showed no phytotoxic effects and a significant increase of both root and epicotyls upon application of coffee husks and pepper CT samples. The same compost teas revealed the largest antioxidant activity and a clear antimicrobial effect determined by MIC (Minimal Inhibitory Concentration) against some gram-negative bacterial strains such as Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae. The bioactivity of CT-samples was related to their general hydrophobic features and to specific molecular composition. In particular, 13C-CPMAS NMR spectra and off-line thermochemolysis GC-MS highlighted a close correlation between radical scavenger activity and antibacterial bioactive functions with bio-available soluble aromatic compounds, such as lignin and phenols derivatives. The antioxidant and, antibacterial properties of compost teas from green composts encourages an innovative potential application of these eco-friendly products not only in agricultural applications but also in nutraceutical and pharmaceutical fields.
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Compostaje , Agricultura , Antibacterianos/toxicidad , Humanos , Lignina , SueloRESUMEN
Humic acids (HAs) provide an important bio-source for redox-active materials. Their functional chemical groups are responsible for several properties, such as metal ion chelating activity, adsorption ability towards small molecules and antibacterial activity, through reactive oxygen species (ROS) generation. However, the poor selectivity and instability of HAs in solution hinder their application. A promising strategy for overcoming these disadvantages is conjugation with an inorganic phase, which leads to more stable hybrid nanomaterials with tuneable functionalities. In this study, we demonstrate that hybrid humic acid/titanium dioxide nanostructured materials that are prepared via a versatile in situ hydrothermal strategy display promising antibacterial activity against various pathogens and behave as selective sequestering agents of amoxicillin and tetracycline antibiotics from wastewater. A physicochemical investigation in which a combination of techniques were utilized, which included TEM, BET, 13C-CPMAS-NMR, EPR, DLS and SANS, shed light on the structure-property-function relationships of the nanohybrids. The proposed approach traces a technological path for the exploitation of organic biowaste in the design at the molecular scale of multifunctional nanomaterials, which is useful for addressing environmental and health problems that are related to water contamination by antibiotics and pathogens.
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Sustancias Húmicas , Nanoestructuras , Antibacterianos/farmacología , Sustancias Húmicas/análisis , Titanio , Aguas ResidualesRESUMEN
Protamine-like proteins (PL-II, PL-III and PL-IV) represent the major basic nuclear component of Mytilus galloprovincialis L sperm chromatin. The present study investigates the effects induced on the properties of PL-II protein after exposure of Mytilus galloprovincialis L for 24â¯h to 1.5 and 5⯵M CdCl2. We found cadmium accumulation in protamine-like proteins with a linear grow up with the exposition dose. In particular, after 5⯵M CdCl2 mussels exposure, the mobility of PL-II band changed in SDS-PAGE, suggesting structural rearrangement in presence of cadmium. Structural analysis using fluorescent probes, indicated that at 5⯵M CdCl2 the complete conformational change of PL-II protein was reached. In the same condition of mussels exposure of 5⯵M CdCl2, PL-II protein changed its DNA binding mode, which determined a closer DNA binding, because higher amount of NaCl were required for PL-II protein release by sperm nuclei. These results supported the hypothesis that mussel exposure to this CdCl2 dose, although lower to toxic ones, affects the properties of this protein and as a consequence chromatin organization of spermatozoa that is essential for the success of fertilization.
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Cadmio/toxicidad , Mytilus/efectos de los fármacos , Proteínas Nucleares/metabolismo , Espermatozoides/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Proteínas de Unión al ADN , Electroforesis en Gel de Poliacrilamida , Masculino , Proteínas Nucleares/química , Conformación Proteica , Espermatozoides/metabolismoRESUMEN
Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics, and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoBL, an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C)p53pAnt, an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as a function of pH, temperature, and incubation time. In spite of the differences in the amino acid sequence (GTGDCGKY, GTGDCHVA, GSGTDCGSR, SQGSDCGSR) we obtained for all the proteins a cleavage efficiency of about 70-80% after 24 h incubation at 60 °C and pH 2. All the peptides were produced with very good yield (5-16 mg/L of LB cultures), high purity (>96%), and the expected content of free thiol groups (1 mol per mole of peptide). Furthermore, (C)GKY20 was modified with PyMPO-maleimide, a commercially available fluorophore bearing a thiol reactive group, and with 6-hydroxy-2-cyanobenzothiazole, a reagent specific for N-terminal cysteines, with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue.