ENZYME BIOPROSPECTING OF MICROBIAL GLYCOSIDASES AND DIOXYGENASES FOR BIOCATALYSIS.

Main aim of this PhD project is the bioprospecting of different microbial enzymatic activities, to evaluate their biotechnological potential in different bioconversion processes. The characterization of four extradiol ring cleavage dioxygenases (ERCDs) and of a α-L-rhamnosidase isolated from Novosphingobium sp. PP1Y (N. sp. PP1Y) is described. In the last part of the project, gut microorganisms have been used for the identification and characterization of novel glycosyl hydrolases able to degrade the arabinogalactan polysaccharide (AG) of M. tuberculosis cell wall. In this work, the marine microorganism N. sp. PP1Y and the gut microorganism Bacteroides finegoldii were used as source for oxygenases and glycosyl hydrolases. More in detail, the optimization of recombinant expression and purification of four novel ERCDs from strain PP1Y allowed to obtain in good yields the corresponding proteins and to carry out their characterization. The activity screening using different catecholic substrates confirmed that these enzymes are able to catalyze the ring cleavage of a variety of mono- and polycyclic aromatic hydrocarbons with different sizes and conformation. In addition, the bioconversion of catechol estrogens, which could be used as precursors in the production of different steroid families and hormones, was evaluated. The results described in this thesis confirmed that these enzymes can be foreseen as a valuable tool for the modification of complex hydroxylated heterocyclic aromatic compounds, which are a starting point in the production pipeline of many pharmacologically active molecules, such as steroid-like molecules. Noteworthy, site-specific cleavage and subsequent modification of aromatic substrates, obtained by enzymatic biocatalysts, is of great advantage for industrial applications when compared to the complex mixture of products that are released instead during chemical modification procedures. In the second part of the PhD project, the biochemical characterization of RHA-P, a bacterial α-L-rhamnosidase isolated from the microorganism Novosphingobium sp. PP1Y, was performed. The active site topology and substrate specificity of RHA-P were investigated by homology modeling. The enzyme, whose recombinant expression and purification was optimized, resulted to be appealing from a biotechnological point of view for the bioconversion and de-rhamnosylation of natural flavonoids. The biotechnological use of either wild-type or mutant rhamnosidases is currently a need for food and beverages industry to improve the organoleptic properties of processed vegetal products. Moreover, the possibility of using efficient whole cells biocatalysts for expressing RHA-P has been described. This is particularly interesting because whole cells biocatalysts have numerous advantages in industrial bioconversion processes, allowing costs and process steps reduction. Finally, the bioprospecting of novel GHs from microorganisms belonging to the human gut microbiome (HGM), able to degrade the AG of M. tuberculosis cell wall, have been carried out. The isolation and characterization of different galactofuranosidases is described, which are able to completely degrade the mycobacteria galactan moiety of AG. Moreover, evidences for the presence of arabinofuranosidases were found for another HGM bacterium, which may be used as biocatalysts for the complete degradation of mycobacteria AG polysaccharide

Flexible Packaging for High Pressure Treatments: Delamination Onset and Design Criteria of Multilayer Structures

Multi-layer flexible polymeric films employed for high pressure treatments of food packaging for pasteurization and sterilization frequently display delamination phenomena. This problem limits packaging reliability used for this treatment technology. This contribution is aimed at understanding the delamination phenomena of packaging structures under high pressures. Development of interlaminar stress fields, which promote localized delamination events, is here addressed by considering the case of mechanical failure of bi-layer structures. Analytical models and Finite Element based numerical simulations are exploited to this purpose. The theoretical and numerical results, that highlight the crucial role played by the mismatch of Young moduli and Poisson ratios of the laminated film sheets, are in full agreement with experimental findings on high pressure-treated food multilayer packages realized coupling different polymeric materials (i.e. polypropylene-polyethyleneterephthalate, polypropylene-cast polyamide and polypropylene-bioriented polyamide)

Sorption Thermodynamics of CO2, H2O, and CH3OH in a Glassy Polyetherimide: A Molecular Perspective

In this paper, the sorption thermodynamics of low-molecular-weight penetrants in a glassy polyetherimide, endowed with specific interactions, is addressed by combining an experimental approach based on vibrational spectroscopy with thermodynamics modeling. This modeling approach is based on the extension of equilibrium theories to the out-of-equilibrium glassy state. Specific interactions are accounted for in the framework of a compressible lattice fluid theory. In particular, the sorption of carbon dioxide, water, and methanol is illustrated, exploiting the wealth of information gathered at a molecular level from Fourier-transform infrared (FTIR) spectroscopy to tailor thermodynamics modeling. The investigated penetrants display a different interacting characteristic with respect to the polymer substrate, which reflects itself in the sorption thermodynamics. For the specific case of water, the outcomes from molecular dynamics simulations are compared with the results of the present analysis

Sorption of CO2, CH4 and Their Mixtures in Amorphous Poly(2,6-dimethyl-1,4-phenylene)oxide (PPO)

Sorption of pure CO2 and CH4 and CO2/CH4 binary gas mixtures in amorphous glassy Poly(2,6-dimethyl-1,4-phenylene) oxide (PPO) at 35 °C up to 1000 Torr was investigated. Sorption experiments were carried out using an approach that combines barometry with FTIR spectroscopy in the transmission mode to quantify the sorption of pure and mixed gases in polymers. The pressure range was chosen to prevent any variation of the glassy polymer density. The solubility within the polymer of the CO2 present in the gaseous binary mixtures was practically coincident with the solubility of pure gaseous CO2, up to a total pressure of the gaseous mixtures equal to 1000 Torr and for CO2 mole fractions of ~0.5 mol mol-1 and ~0.3 mol mol-1. The Non-Equilibrium Thermodynamics for Glassy Polymers (NET-GP) modelling approach has been applied to the Non-Random Hydrogen Bonding (NRHB) lattice fluid model to fit the solubility data of pure gases. We have assumed here that no specific interactions were occurring between the matrix and the absorbed gas. The same thermodynamic approach has been then used to predict the solubility of CO2/CH4 mixed gases in PPO, resulting in a deviation lower than 9.5% from the experimental results for CO2 solubility

Gas Sorption and Diffusion in Amorphous and Semicrystalline Nanoporous Poly(2,6-dimethyl-1,4-phenylene)oxide

In this contribution is presented an analysis of mass transport properties of low molecular weight compounds in amorphous PPO and in two semicrystalline PPOs obtained by treating with benzene and carbon tetrachloride the amorphous sample. It is found that semicrystalline samples are endowed with larger gas sorption capacity and diffusivity as compared to the amorphous ones: this behavior has been attributed prevalently to the nanoporous nature of the crystalline phases induced by treatment with solvents. In particular, sorption experiments, carried out at 30 °C with methane, carbon dioxide, propane and propylene, have shown that both semicrystalline PPOs display rather interesting features which make them suitable for use as membrane materials in gas separation processes, in view of the relatively high values of solubility and diffusivity. Moreover, these peculiar sorption and mass transport properties have been found to be virtually unaffected by thermal aging: in fact, sorption experiments conducted on amorphous and semicrystalline PPO after treatment at 65 °C for three months showed that sorption and transport properties of aged samples are the same as for the untreated ones. This is an important feature to ensure the stability of performances in membrane applications

High-yield production in Escherichia coli and convenient purification of a candidate vaccine against SARS-CoV-2

Objectives: The aim of the present work was to identify a time-saving, effective, and low-cost strategy to produce in Escherichia coli a protein chimera representing a fusion anti-SARS-CoV-2 candidate vaccine, consisting of immunogenic and antigenic moieties. Results: We overexpressed in E. coli BL21(DE3) a synthetic gene coding for CRM197-RBD, and the target protein was detected in inclusion bodies. CRM197-RBD was solubilized with 1 % (w/v) of the anionic detergent N-lauroylsarcosine (sarkosyl), the removal of which from the protein solution was conveniently accomplished with Amberlite XAD-4. The detergent-free CRM197-RBD was then separated from contaminating DNA using polyethylenimine (PEI), and finally purified from PEI by salting out with ammonium sulfate. Structural (CD spectrum) and functional (DNase activity) assays revealed that the CRM197-RBD chimera featured a native and active conformation. Remarkably, we determined a yield of purified CRM197-RBD equal to 23 mg per litre of culture. Conclusions: To produce CRM197-RBD, we devised the use of sarkosyl as an alternative to urea to solubilize the target protein from E. coli inclusion bodies, and the easy removal of sarkosyl by means of Amberlite XAD-4

Catalytic Combustion of Waste Streams Coming from the Solvent Recovery Stage of a Packaging Industry

Esters and alcohols are widely used as solvents in the packaging industry. They contribute to the increase of volatile organic compound (VOC) emissions and, as such, represent a serious air pollution problem that must be faced. To this end, two main strategies can be pursued: solvent recovery and/or incineration, the former being of course preferred. Solvent recovery consists of an activated carbon plant, which adsorbs the post-printing exhaust solvents, and a distillation system, which separates the recovered solvent mixture. The waste streams coming from the solvent recovery stage need to be disposed, implying additional costs as well as safety issues. In this work, a low-cost perovskite-based catalyst, doped with a low amount of noble metal (Pt) and supported over a monolithic reactor, was used to run lab-scale combustion tests for waste streams coming from the solvent recovery stage of the packaging industry Icimendue (www.icimen.com). Results of experimental tests support the validity of catalytic combustion of the waste streams as an alternative approach to their disposal

A Hyphenated Approach Combining Pressure-Decay and in Situ FT-NIR Spectroscopy to Monitor Penetrant Sorption and Concurrent Swelling in Polymers

A new hyphenated technique based on simultaneous in situ FT-NIR spectroscopy and pressure-decay measurements has been implemented to study sorption of low-molecular-weight compounds in polymeric membranes and the induced swelling of the matrix. The FT-NIR measurements are performed in the transmission mode and, besides sorption equilibrium and kinetics, allow also the straightforward measurement of polymer swelling. The pressure decay method is used to provide quantitative information on the concentration of penetrant sorbed in the polymer. This measurement, once combined with the photometric data, allows an accurate estimation of the molar absorptivity of the analytical peaks as well. To validate the new experimental approach, sorption of CO2 in polydimethylsiloxane at 35 °C and at pressures up to 9 bar has been investigated and the results are compared with available literature data

Modelling sorption thermodynamics and mass transport of n-hexane in a propylene-ethylene elastomer

Optimization of post polymerization processes of polyolefin elastomers (POE) involving solvents is of considerable industrial interest. To this aim, experimental determination and theoretical interpretation of the thermodynamics and mass transport properties of POE-solvent mixtures is relevant. Sorption behavior of n-hexane vapor in a commercial propylene-ethylene elastomer (V8880 Vistamaxx™ from ExxonMobil, Machelen, Belgium) is addressed here, determining experimentally the sorption isotherms at temperatures ranging from 115 to 140 °C and pressure values of n-hexane vapor up to 1 atm. Sorption isotherms have been interpreted using a Non Random Lattice Fluid (NRLF) Equation of State model retrieving, from data fitting, the value of the binary interaction parameter for the n-hexane/V8880 system. Both the cases of temperature-independent and of temperature-dependent binary interaction parameter have been considered. Sorption kinetics was also investigated at different pressures and has been interpreted using a Fick’s model determining values of the mutual diffusivity as a function of temperature and of n-hexane/V8880 mixture composition. From these values, n-hexane intra-diffusion coefficient has been calculated interpreting its dependence on mixture concentration and temperature by a semi-empiric model based on free volume arguments

Survey on Adsorption of Low Molecular Weight Compounds in Cu-BTC Metal–Organic Framework: Experimental Results and Thermodynamic Modeling

This contribution aims at providing a critical overview of experimental results for the sorption of low molecular weight compounds in the Cu-BTC Metal–Organic Framework (MOF) and of their interpretation using available and new, specifically developed, theoretical approaches. First, a literature review of experimental results for the sorption of gases and vapors is presented, with particular focus on the results obtained from vibrational spectroscopy techniques. Then, an overview of theoretical models available in the literature is presented starting from semiempirical theoretical approaches suitable to interpret the adsorption thermodynamics of gases and vapors in Cu-BTC. A more detailed description is provided of a recently proposed Lattice Fluid approach, the Rigid Adsorbent Lattice Fluid (RALF) model. In addition, to deal with the cases where specific self- and cross-interactions (e.g., H-bonding, Lewis acid/Lewis base interactions) play a role, a modification of the RALF model, i.e., the RALFHB model, is introduced here for the first time. An extension of both RALF and RALFHB is also presented to cope with the cases in which the heterogeneity of the rigid adsorbent displaying a different kind of adsorbent cages is of relevance, as it occurs for the adsorption of some low molecular weight substances in Cu-BTC MOF