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The reaction mechanism of glycoside hydrolases belonging to family 1 (GH1) of carbohydrate-active enzymes classification, hydrolysing β-O-glycosidic bonds, is well characterised. This family includes several thousands of enzymes with more than 20 different EC numbers depending on the sugar glycone recognised as substrate. Most GH1 β-glycosidases bind their substrates with similar specificity through invariant amino acid residues. Despite extensive studies, the clear identification of the roles played by each of these residues in the recognition of different glycones is not always possible. We demonstrated here that a histidine residue, completely conserved in the active site of the enzymes of this family, interacts with the C2-OH of the substrate in addition to the C3-OH as previously shown by 3 D-structure determination.

The present study describes the first example of utilization of a natural clay mineral as catalyst in a process addressed to chemical valorization of poly-[bisphenol A carbonate] (PC; (1)) wastes. A natural clinochlore was investigated for the first time as the catalyst of the hydrolysis reaction of 1, a potential route to chemical recycling of wastes of this polymeric material. At 473 K, in tetrahydrofuran (THF) as the solvent, the mineral promoted effectively the depolymerization (up to 99%, after 6 h) of 1 by H2O and the selective (~99%) regeneration of the monomer bisphenol A (BPA, (2)). Temperature, catalyst loading, reaction time, H2O/PC weight ratio affected markedly the productivity of the process. The role of the catalyst was also focused: the experimental data showed that the exposed brucite-like sheets of clinochlore are involved in the hydrolysis reaction and take active part in promoting the depolymerization process.

In order to calibrate the model parameters, Sensitivity Analysis routines are mandatory to rank the parameters by their relevance and fix to nominal values the least influential factors. Despite the high number of works based on ADM1, very few are related to sensitivity analysis. In this study Global Sensitivity Analysis (GSA) and Uncertainty Quantification (UQ) for an ADM1-based Anaerobic Digestion Model have been performed. The modified version of ADM-based model selected in this study was presented by Esposito and co-authors in 2013. Unlike the first version of ADM1, focused on sewage sludge degradation, the model of Esposito is focused on organic fraction of municipal solid waste digestion. It his recalled that in many applications the hydrolysis is considered the bottleneck of the overall anaerobic digestion process when the input substrate is constituted of complex organic matter. In Esposito's model a surfaced based kinetic approach for the disintegration of complex organic matter is introduced. This approach allows to better model the disintegration step taking into account the effect of particle size distribution on the digestion process. This model needs thus GSA and UQ to pave the way for further improvements and reach a deep understanding of the main processes and leading input factors. Due to the large number of parameters to be analyzed a first preliminary screening analysis, with the Morris' Method, has been conducted. Since two quantities of interest (QoI) have been considered, the initial screening has been performed twice, obtaining two set of parameters containing the most influential factors in determining the value of each QoI. A surrogate of ADM1 model has been defined making use of the two defined quantities of interest. The output results from the surrogate model have been analyzed with Sobol' indices for the quantitative GSA. Finally, uncertainty quantification has been performed. By adopting kernel smoothing techniques, the Probability Density Functions of each quantity of interest have been defined.

In this work, natural phenolic compounds, such as Vanillic Acid (VA), Ferulic Acid (FA) and Thymol (Th), at very high concentrations, have been considered as pro-oxidant agents for Polylactic acid (PLA). Specifically, thin films of neat PLA and PLA-based systems containing 2 and 3 wt% of VA, FA and Th have been produced and subjected to accelerated degradation in different environmental conditions. Preliminary characterizations, through rheological, mechanical, optical and morphological analysis, of the formulated PLA-based systems show that the VA and FA, even less the Th, are able to exert a plasticizing action during the processing and subsequently, the PLA crystallinity and rigidity slightly decrease, while, the PLA ductility increase and its optical performance and morphology remain almost unchanged. To assess the pro-oxidant activity of the considered natural phenolic compounds, thin films of PLA and PLA-based systems containing VA, FA and Th have been subjected to accelerated degradation in different environmental conditions: (i) water-medium hydrolysis, (ii) photo-oxidation upon UVB exposure and (iii) thermo-oxidation upon thermal treatment in oven. All obtained results pointing out that the considered natural compounds, at these high concentrations, exert a clear pro-oxidant activity in PLA during the water-medium hydrolysis and the photo- and thermo-oxidative degradation. Moreover, the VA, FA and Th can be considered as suitable pro-degradant additives for PLA, also in order to control the bio-polyester degradation times.

We report the first histidine-containing self-assembling tripeptide devoid of capping groups that forms a thermoreversible hydrogel under physiological conditions and catalyses hydrolysis of an ester, providing a minimalist building block for functional soft materials.

Urease uses a cluster of two NiII ions to activate a water molecule for urea hydrolysis. The key to this unsurpassed enzyme is a change in the conformation of a flexible structural motif, the mobile flap, which must be able to move from an open to a closed conformation to stabilize the chelating interaction of urea with the NiII cluster. This conformational change brings the imidazole side chain functionality of a critical histidine residue, αHis323, in close proximity to the site that holds the transition state structure of the reaction, facilitating its evolution to the products. Herein, we describe the influence of the solution pH in modulating the conformation of the mobile flap. High-resolution crystal structures of urease inhibited in the presence of N-(n-butyl)phosphoric triamide (NBPTO) at pH 6.5 and pH 7.5 are described and compared to the analogous structure obtained at pH 7.0. The kinetics of urease in the absence and presence of NBPTO are investigated by a calorimetric assay in the pH 6.0-8.0 range. The results indicate that pH modulates the protonation state of αHis323, which was revealed to have pKa =6.6, and consequently the conformation of the mobile flap. Two additional residues (αAsp224 and αArg339) are shown to be key factors for the conformational change. The role of pH in modulating the catalysis of urea hydrolysis is clarified through the molecular and structural details of the interplay between protein conformation and solution acidity in the paradigmatic case of a metalloenzyme.

Dicalcium phosphate dihydrate (DCPD) is one of the mineral phases indicated as possible precursors of biological apatites and it is widely employed in the preparation of calcium phosphate bone cements. Herein, we investigated the possibility to functionalize DCPD with aspartic acid (ASP) and poly-aspartic acid (PASP), as models of the acidic macromolecules of biomineralized tissues, and studied their influence on DCPD hydrolysis. To this aim, the synthesis of DCPD was performed in aqueous solution in the presence of increasing concentrations of PASP and ASP, whereas the hydrolysis reaction was carried out in physiological solution up to three days. The results indicate that it is possible to prepare DCPD functionalized with PASP up to a polyelectrolyte content of about 2.3 wt%. The increase of PASP content induces crystal aggregation, reduction of the yield of the reaction and of the thermal stability of the synthesized DCPD. Moreover, DCPD samples functionalized with PASP display a slower hydrolysis than pure DCPD. On the other hand, in the explored range of concentrations (up to 10 mM) ASP is not incorporated into DCPD and does not influence its crystallization nor its hydrolysis. At variance, when present in the hydrolysis solution, ASP, and even more PASP, delays the conversion into the more stable phases, octacalcium phosphate and/or hydroxyapatite. The greater influence of PASP on the synthesis and hydrolysis of DCPD can be ascribed to the cooperative action of the carboxylate groups and to its good fit with DCPD structure.

Bee pollen is an attractive resource in the field of alternative remedies and thanks to the content of carbohydrates, crude fibers, proteins and lipids must be considered as a supplementary food of high potential rate. In characterization of bee pollen with the aim to define its value in human nutrition, the amino acids profile is one of the most important attributes. In the present study, the determination of amino acids composition of different monofloral bee pollen samples was obtained by an approach combining microwave acidic hydrolysis (60 min at 150 °C instead of 22 h at 120 °C in conventional oven) followed by derivatization using 9-fluorenylmethylchloroformate (FMOC-Cl) and separation of amino acids derivatives using a Phenomenex Kinetex core-shell 5 μm C18 (150 x 4.6 mm i.d.) column under a ternary gradient elution. Separation of 19 amino acids was achieved in about 40 min and fluorimetric detection (λexc = 265 nm λem = 315 nm) allowed selective and sensitive quantitation with LOQ values ranging within 0.14–3.00 μg/mL. Interestingly, the present approach allowed determination of some amino acids e.g., tryptophan and trans-4-hydroxyproline that are often lost by other methods of analysis. Significant differences in the composition of the considered samples were found confirming the impact of botanical origin of the product on its nutritional value. Principal Component Analysis was applied to treat the obtained data, highlighting the importance for discrimination, of detecting low abundance amino acids. The proposed method can be used as an advantageous alternative to the existing ones for characterization of bee pollen as an important source of dietary proteins.

An ongoing challenge in olive oil analytics is the development of a reliable procedure that can draw the consensus of all interested parties regarding the quantification of concentrations above the required minimum value of 5 mg of bioactive “olive oil polyphenols” per 20 g of the oil, to fulfill the health claim introduced by the European Commission (EC) Regulation 432/2012. An in-house validated ultra-high performance liquid chromatography (UHPLC) protocol fit for this purpose is proposed. It relies on quantification of the total hydroxytyrsol (Htyr) and tyrosol (Tyr) content in the virgin olive oil (VOO) polar fraction (PF) before and after acidic hydrolysis of their bound forms. PF extraction and hydrolysis conditions were as previously reported. The chromatographic run lasts ~1/3 of the time needed under high performance liquid chromatography (HPLC) conditions, this was also examined. Eluent consumption for the same piece of information was 6-fold less. Apart from being cost effective, a larger number of samples can be analyzed daily with less environmental impact. Two external curves, detection at 280 nm and correction factors for molecular weight difference are proposed. The method, which is fit for purpose, is selective, robust with satisfactory precision (percentage relative standard deviation (%RSD) values < 11%) and recoveries higher than 87.6% for the target analytes (Htyr, Tyr). Standard operational procedures are easy to apply in the olive oil sector., This work was developed in the context of the project OLEUM ‘Advanced solutions for assuring authenticity and quality of olive oil at global scale’ funded by the European Commission within the Horizon 2020 Programme (2014–2020, grant agreement [no. 635690]). The information and views set out in this article are those of the author(s) and do not necessarily reflect the official opinion of the European Union. Neither the European Union institutions and bodies nor any person acting on their behalf may be held responsible for the use which may be made of the information contained therein., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Food and feed safety are of paramount relevance in everyday life. The awareness that different chemicals, e.g., those largely used in agriculture, could present both environmental problems and health hazards, has led to a large limitation of their use. Chemicals were also the main tool in a control of fungal pathogens and their secondary metabolites, mycotoxins. There is a drive to develop more environmentally friendly, “green”, approaches to control mycotoxin contamination of foodstuffs. Different mushroom metabolites showed the potential to act as control agents against mycotoxin production. The use of a polysaccharide, Tramesan, extracted from the basidiomycete Trametes versicolor, for controlling biosynthesis of aflatoxin B1 and ochratoxin A, has been previously discussed. In this study, oligosaccharides obtained from Tramesan were evaluated. The purified exopolysaccharide of T. versicolor was partially hydrolyzed and separated by chromatography into fractions from disaccharides to heptasaccharides. Each fraction was individually tested for mycotoxin inhibition in A. flavus and A. carbonarius. Fragments smaller than seven units showed no significant effect on mycotoxin inhibition, heptasaccharides showed inhibitory activity of up to 90% in both fungi. These results indicated that these oligosaccharides could be used as natural alternatives to crop protection chemicals for controlling these two mycotoxins.

Protein hydrolysates are largely used as plant biostimulants for boosting crop growth, and improving crop tolerance to abiotic stresses and fruit quality. Protein hydrolysate-based biostimulants are mostly produced by chemical hydrolysis starting from animal wastes. However, an innovative process of enzymatic hydrolysis of legume-derived proteins has been recently introduced by few companies. The objective of this study was to evaluate the energy use and environmental impact of the production processes of enzymatically-produced protein hydrolysate starting from lupine seeds and protein hydrolysate obtained from chemical hydrolysis of leather wastes through the application of life cycle assessment (LCA). The LCA method was applied through the software GEMIS “Global Emission Model for Integrated Systems”, elaborated at L’Oko-Institute in Germany, and the parameters taken into account were: CO2 emissions in g per kg of protein hydrolysate; the consumption of fossil energy expressed in MJ per kg of protein hydrolysate; and water consumption reported in kg per kg of protein hydrolysate. In the case of legume-derived protein hydrolysate, the evaluation of the energy use and the environmental impact started from field production of lupine grains and ended with the industrial production of protein hydrolysate. In the case of animal-derived protein hydrolysate, the LCA method was applied only in the industrial production process, because the collagen is considered a waste product of the leather industry. The type of hydrolysis is the step that most affects the energy use and environmental impact on the entire industrial production process. The results obtained in terms of CO2 emissions, fossil energy consumption and water use through the application of LCA showed that the production process of the animal-derived protein hydrolysate was characterized by a higher energy use (+26%) and environmental impact (+57% of CO2 emissions) in comparison with the enzymatic production process of lupine-derived protein hydrolysate. In conclusion, the production of legume-derived protein hydrolysate by enzymatic hydrolysis is more environmentally friendly than the production of animal-derived protein hydrolysate through chemical hydrolysis.

Ruta chalepensis L. (Rutaceae) leaf extract was investigated for its chemical profile and antioxidant and hypoglycaemic properties. The antioxidant effects were investigated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), Chi-carotene bleaching, and metal chelating activity assays. The carbohydrate-hydrolysing enzymes inhibition assay was used to test the hypoglycaemic potential. R. chalepensis showed a high content of hesperidin and rutin with values of 591.9 and 266.7 mg/g dry extract, respectively. The extract exhibited a promising protection of lipid peroxidation (IC50 value of 16.9 mu g/mL) and inhibited both alpha-amylase and alpha-glucosidase enzymes in a concentration-dependent manner. The highest activity was found against alpha-amylase (IC50 value of 69.0 mu g/mL). The obtained results support the use of R. chalepensis leaves as healthy food ingredients.

Waste lettuce leaves - from the "fresh cut vegetable" industry - were pretreated with the deep eutectic solvent (DES) made of choline chloride - glycerol. Reaction time (3-16 h) and the operation temperature (80-150 °C) were investigated. Enzymatic glucose and xylose yields of 94.9% and 75.0%, respectively were obtained when the biomass was pretreated at 150 °C for 16 h. Sugars contained in the biomass hydrolysate were fermented in batch cultures of Clostridium acetobutylicum DSMZ 792. The energy consumption and the energy efficiency related to the DES pretreatment were calculated and compared to the most common lignocellulosic pretreatment processes reported in the literature. The DES pretreatment process was characterized by lower energy required (about 28% decrease and 72% decrease) than the NAOH pretreatment and steam explosion process respectively. The Net Energy Ratio (NER) value related to butanol production via DES biomass pretreatment was assessed.

Catalytic conversion of actual biomass to valuable chemicals is a crucial issue in green chemistry. This review discusses on the recent approach in the levulinic acid (LA) formation from three prominent generations of biomasses. Our paper highlights the impact of the nature of different types of biomass and their complex structure and impurities, different groups of catalyst, solvents, and reaction system, and condition and all related pros and cons for this process.

Although the term nanozymes was coined by us in 2004 to highlight the enzyme-like properties of gold nanoparticles passivated with a monolayer of Zn(II)-complexes in the cleavage of phosphate diesters, systems resembling those metallo-nanoparticles, like micelles and vesicles, have been the subject of investigation since the mid-eighties of the last century. This paper reviews what has been done in the field and compares the different nanosystems highlighting the source of catalysis and frequent misconceptions found in the literature.

A chemical investigation of the interaction mechanisms of nicotine with the metal(II) ions Pb $$ ^{2+} $$ , Fe $$ ^{2+} $$ , and Cu $$ ^{2+} $$ is reported. The complex formation between nicotine and metal(II) has been investigated at 25.00 $$ \pm $$ 0.02 $$^{\circ }$$ C, in constant ionic medium (as sodium perchlorate, or sodium chloride), by UV/Vis spectrophotometric and potentiometric methods. The experimental method consists of coulometric or volumetric titrations. The protolysis constants of nicotine have been determined under the same experimental conditions. The pH investigated spans between 3 and 10. By using the specific ion interaction theory the conditional constants of nicotine have been extrapolated at zero ionic strength. The results of the graphical and numerical methods adopted indicate, for all the systems investigated, the formation of a predominating Me(II)/nicotine mononuclear complexes.

Halomonas pantelleriensis DSM9661Τ is a Gram-negative haloalkaliphilic bacterium isolated from the sand of the volcanic Venus mirror lake, closed to seashore in the Pantelleria island in the south of Italy. It is able to optimally grow in media containing 3-15% (w/v) total salt and at pH between 9-10. To survive in these harsh conditions the bacterium has developed several strategies that probably concern the bacteria outer membrane, a barrier regulating the exchange with the environment. In such a context the lipopolysaccharides (LPSs), which are among the major constituent of the Gram-negative outer membrane, are thought to contribute to the restrictive membrane permeability properties. The structure of the lipid A family derived from the lipopolysaccharide of Halomonas pantelleriensis DSM 9661T is reported herein. The lipid A was obtained from the purified LPS by mild acid hydrolysis. The lipid A, which contains different numbers of fatty acids residues, and its partially deacylated derivatives were completely characterized by means of ESI FT-ICR mass spectrometry and chemical analysis. Preliminary immunological assays were performed, and a comparison with the lipid A structure of the phylogenetic proximal Halomonas magadiensis is also reported.