Enzymatic Synthesis and Structural Modeling of Bio-Based Oligoesters as an Approach for the Fast Screening of Marine Biodegradation and Ecotoxicity.

Given the widespread use of esters and polyesters in products like cosmetics, fishing nets, lubricants and adhesives, whose specific application(s) may cause their dispersion in open environments, there is a critical need for stringent eco-design criteria based on biodegradability and ecotoxicity evidence. Our approach integrates experimental and computational methods based on short oligomers, offering a screening tool for the rapid identification of sustainable monomers and oligomers, with a special focus on bio-based alternates. We provide insights into the relationships between the chemical structure and properties of bio-based oligomers in terms of biodegradability in marine environments and toxicity in benchmark organisms. The experimental results reveal that the considered aromatic monomers (terephthalic acid and 2,5-furandicarboxylic acid) accumulate under the tested conditions (OECD 306), although some slight biodegradation is observable when the inoculum derives from sites affected by industrial and urban pollution, which suggests that ecosystems adapt to non-natural chemical pollutants. While clean seas are more susceptible to toxic chemical buildup, biotic catalytic activities offer promise for plastic pollution mitigation. Without prejudice to the fact that biodegradability inherently signifies a desirable trait in plastic products, nor that it automatically grants them a sustainable "license", this study is intended to facilitate the rational design of new polymers and materials on the basis of specific uses and applications.

Neuroprotective Properties of Cardoon Leaves Extracts against Neurodevelopmental Deficits in an In Vitro Model of Rett Syndrome Depend on the Extraction Method and Harvest Time.

This study investigates the bioactive properties of different extracts of cardoon leaves in rescuing neuronal development arrest in an in vitro model of Rett syndrome (RTT). Samples were obtained from plants harvested at different maturity stages and extracted with two different methodologies, namely Naviglio® and supercritical carbon dioxide (scCO2). While scCO2 extracts more hydrophobic fractions, the Naviglio® method extracts phenolic compounds and less hydrophobic components. Only the scCO2 cardoon leaves extract obtained from plants harvested in spring induced a significant rescue of neuronal atrophy in RTT neurons, while the scCO2 extract from the autumn harvest stimulated dendrite outgrowth in Wild-Type (WT) neurons. The scCO2 extracts were the richest in squalene, 3ß-taraxerol and lupeol, with concentrations in autumn harvest doubling those in spring harvest. The Naviglio® extract was rich in cynaropicrin and exerted a toxic effect at 20 µM on both WT and RTT neurons. When cynaropicrin, squalene, lupeol and 3ß-taraxerol were tested individually, no positive effect was observed, whereas a significant neurotoxicity of cynaropicrin and lupeol was evident. In conclusion, cardoon leaves extracts with high content of hydrophobic bioactive molecules and low cynaropicrin and lupeol concentrations have pharmacological potential to stimulate neuronal development in RTT and WT neurons in vitro.

Navigating within thiamine diphosphate-dependent decarboxylases: Sequences, structures, functional positions, and binding sites.

Thiamine diphosphate-dependent decarboxylases catalyze both cleavage and formation of CC bonds in various reactions, which have been assigned to different homologous sequence families. This work compares 53 ThDP-dependent decarboxylases with known crystal structures. Both sequence and structural information were analyzed synergistically and data were analyzed for global and local properties by means of statistical approaches (principle component analysis and principal coordinate analysis) enabling complexity reduction. The different results obtained both locally and globally, that is, individual positions compared with the overall protein sequence or structure, revealed challenges in the assignment of separated homologous families. The methods applied herein support the comparison of enzyme families and the identification of functionally relevant positions. The findings for the family of ThDP-dependent decarboxylases underline that global sequence identity alone is not sufficient to distinguish enzyme function. Instead, local sequence similarity, defined by comparisons of structurally equivalent positions, allows for a better navigation within several groups of homologous enzymes. The differentiation between homologous sequences is further enhanced by taking structural information into account, such as BioGPS analysis of the active site properties or pairwise structural superimpositions. The methods applied herein are expected to be transferrable to other enzyme families, to facilitate family assignments for homologous protein sequences.

Investigating the Role of Conformational Effects on Laccase Stability and Hyperactivation under Stress Conditions.

Fungal laccase from Steccherinum ochraceum 1833 displays remarkable stability under different harsh conditions: organic/buffer mixtures, thermal treatment, and microwave radiation. The behavior is particularly significant in the light of the sharp inactivation observed for two different fungal laccases. Laccase from S. ochraceum 1833 also displays hyperactivation under mild thermal treatment (60 °C). Molecular dynamics simulations at 80 °C explained how this laccase retains the geometry of the electron transfer pathway, thereby assuring electron transfer through the copper ions and thus maintaining its catalytic activity at high temperature. Spectroscopic studies revealed that the thermal activation corresponds to specific conformational changes in the protein. The results indicate that this laccase is potentially applicable under denaturing conditions that might be beneficial for the biotransformation of recalcitrant substrates.

Efficient immobilisation of industrial biocatalysts: criteria and constraints for the selection of organic polymeric carriers and immobilisation methods.

Efficient immobilisation protocols are the result of perfect matching of factors depending on the enzyme, the process and the support for immobilisation. Physical-chemical phenomena, such as partition, solvation and diffusion, strongly affect the efficiency of the biocatalyst in each specific reaction system. Therefore, tailored solutions must be developed for each specific process of interest. Indeed, direct investigation of what occurs at the molecular level in a reaction catalysed by an immobilised enzyme is a quite formidable task and observed differences in the performance of immobilised biocatalysts must be interpreted very carefully. In any study dealing with enzyme immobilisation the prerequisite is the rigorous planning and reporting of experiments, being aware of the complexity of these multi-phase systems.

Understanding Marine Biodegradation of Bio-Based Oligoesters and Plasticizers.

The study reports the enzymatic synthesis of bio-based oligoesters and chemo-enzymatic processes for obtaining epoxidized bioplasticizers and biolubricants starting from cardoon seed oil. All of the molecules had MW below 1000 g mol-1 and were analyzed in terms of marine biodegradation. The data shed light on the effects of the chemical structure, chemical bond lability, thermal behavior, and water solubility on biodegradation. Moreover, the analysis of the biodegradation of the building blocks that constituted the different bio-based products allowed us to distinguish between different chemical and physicochemical factors. These hints are of major importance for the rational eco-design of new benign bio-based products. Overall, the high lability of ester bonds was confirmed, along with the negligible effect of the presence of epoxy rings on triglyceride structures. The biodegradation data clearly indicated that the monomers/building blocks undergo a much slower process of abiotic or biotic transformations, potentially leading to accumulation. Therefore, the simple analysis of the erosion, hydrolysis, or visual/chemical disappearance of the chemical products or plastic is not sufficient, but ecotoxicity studies on the effects of such small molecules are of major importance. The use of natural feedstocks, such as vegetable seed oils and their derivatives, allows the minimization of these risks, because microorganisms have evolved enzymes and metabolic pathways for processing such natural molecules.

Rational Guidelines for the Two-Step Scalability of Enzymatic Polycondensation: Experimental and Computational Optimization of the Enzymatic Synthesis of Poly(glycerolazelate).

The lipase-catalyzed polycondensation of azelaic acid and glycerol is investigated according to a Design-of-Experiment approach that helps to elucidate the effect of experimental variables on monomer conversion, Mn and regioselectivity of acylation of glycerol. Chemometric analysis shows that after 24 h the reaction proceeds regardless of the presence of the enzyme. Accordingly, the biocatalyst was removed after a first step of synthesis and the chain elongation continued at 80 °C. That allowed the removal of the biocatalyst and the preservation of its activity: pre-requites for efficient applicability at industrial scale. The experimental study, combined with docking-based computational analysis, provides rational guidelines for the optimization of the regioselective acylation of glycerol. The process is scaled up to 73.5 g of monomer. The novelty of the present study is the rigorous control of the reaction conditions and of the integrity of the immobilized biocatalyst, which serve to avoiding any interference of free enzyme or fines released in the reaction mixture. The quantitative analysis of the effect of experimental conditions and the overcoming of some major technical bottlenecks for the scalability of enzymatic polycondensation opens new scenarios for industrial exploitation.

2,5-Furandicarboxaldehyde as a bio-based crosslinking agent replacing glutaraldehyde for covalent enzyme immobilization.

In the quest for a bio-based and safer substitute for glutaraldehyde, we have investigated 2,5 diformylfuran (DFF) as bifunctional crosslinking agent for the covalent immobilization of glucoamylase on amino-functionalized methacrylic resins. Immobilization experiments and systematic comparison with glutaraldehyde at four different concentrations for the activation step showed that DFF leads to comparable enzymatic activities at all tested concentrations. Continuous flow experiment confirms a similar long term stability of the immobilized formulations obtained with the two crosslinkers. The NMR study of DFF in aqueous solution evidenced a much simpler behaviour as compared to glutaraldehyde, since no enolic forms can form and only a mono-hydrated form was observed. Unlike in the case of glutaraldehyde, DFF reacts covalently with the primary amino groups via imine bond formation only. Nevertheless, the stability of the covalent immobilization was confirmed also at acidic pH (4.5), most probably because of the higher stability of the imine bonds formed with the aromatic aldehydes. In terms of toxicity DFF has the advantage of being poorly soluble in water and, more importantly, poorly volatile as compared to glutaraldehyde, which displays severe respiratory toxicity. We have performed preliminary ecotoxicity assays using Aliivibrio fischeri, a marine bacterium, evidencing comparable behaviour (below the toxicity threshold) for both dialdehydes at the tested concentrations.

Renewable polymers and plastics: Performance beyond the green.

Renewable bio-based polymers are one of the effective answers that the bioeconomy offers to solve the environmental emergency connected to plastics and more specifically fossil-based plastics. Previous studies have shown that more than 70 % of the natural capital cost associated with plastic derives from the extraction and processing of fossil raw materials and that the price of fossil plastic would be on average 44 % higher if such impact was fully paid by businesses. The disclosure of the hidden costs of plastics will contribute to dispelling the myth of the expensiveness of renewable polymers. Nevertheless, the adoption of bio-based plastics in the market must be motivated by their functional properties and not merely by their green credentials. This article highlights some successful examples of synergies between chemistry and biotechnology in achieving a new generation of bio-based monomers and polymers. Their success is justified by the combination of scientific advances with positive environmental and social fallouts.

Azelaic Acid: A Bio-Based Building Block for Biodegradable Polymers.

Azelaic acid is a dicarboxylic acid containing nine C atoms, industrially obtained from oleic acid. Besides its important properties and pharmacological applications, as an individual compound, azelaic acid has proved to be a valuable bio-based monomer for the synthesis of biodegradable and sustainable polymers, plasticizers and lubricants. This review discusses the studies and the state of the art in the field of the production of azelaic acid from oleic acid, the chemical and enzymatic synthesis of bio-based oligo and polyester and their properties, including biodegradability and biocompostability.

The bioeconomy in Italy and the new national strategy for a more competitive and sustainable country.

Italy has the third largest bioeconomy in Europe (€330 billion annual turnover, 2 million employees), making it a core pillar of the national economy. Its sectors of excellence are food and biobased products, and it is a consistent presence in research and innovation projects funded by the EU Horizon 2020 programme (Societal Challenges 2) and the European Public Private Partnership "Biobased industry" (BBI-JU). The bioeconomy reduces dependence on fossil fuels and finite materials, loss of biodiversity and changing land use. It contributes to environmental regeneration, spurs economic growth and supports jobs in rural, coastal and abandoned industrial areas, leveraging local contexts and traditions. In 2017 the Italian government promoted the development of a national Bioeconomy Strategy (BIT), recently updated (BIT II) to interconnect more efficiently the pillars of the national bioeconomy: production of renewable biological resources, their conversion into valuable food/feed, biobased products and bio-energy, and transformation and valorization of bio-waste streams. BIT II aims to improve coordination between Ministries and Italian regions in alignment of policies, regulations, R&I funding programmes and infrastructures investment. The goal is a 15 % increase in turnover and employment in the Italian bioeconomy by 2030. Based on Italy's strategic geopolitical position in the Mediterranean basin, BIT II also includes actions to improve sustainable productivity, social cohesion and political stability through the implementation of bioeconomy strategies in this area. This paper provides an insight into these strategies and discusses the strengths and weaknesses of the sectors involved and the measures, regulatory initiatives and monitoring actions undertaken.

Fructose production by inulinase covalently immobilized on sepabeads in batch and fluidized bed bioreactor.

The present work is an experimental study of the performance of a recently designed immobilized enzyme: inulinase from Aspergillus sp. covalently immobilized on Sepabeads. The aim of the work is to test the new biocatalyst in conditions of industrial interest and to assess the feasibility of the process in a fluidized bed bioreactor (FBBR). The catalyst was first tested in a batch reactor at standard conditions and in various sets of conditions of interest for the process. Once the response of the catalyst to different operating conditions was tested and the operational stability assessed, one of the sets of conditions tested in batch was chosen for tests in FBBR. Prior to reaction tests, preliminary fluidization tests were realized in order to define an operating range of admissible flow rates. As a result, the FBR was run at different feed flow rates in a closed cycle configuration and its performance was compared to that of the batch system. The FBBR proved to be performing and suitable for scale up to large fructose production.

Thermal Upgrade of Enzymatically Synthesized Aliphatic and Aromatic Oligoesters.

The enzymatic synthesis of polyesters in solventless systems is an environmentally friendly and sustainable method for synthetizing bio-derived materials. Despite the greenness of the technique, in most cases only short oligoesters are obtained, with limited practical applications or requiring further chemical processing for their elongation. In this work, we present a catalyst-free thermal upgrade of enzymatically synthesized oligoesters. Different aliphatic and aromatic oligoesters were synthesized using immobilized Candida antarctica lipase B (iCaLB) as the catalyst (70 °C, 24 h) yielding poly(1,4-butylene adipate) (PBA, Mw = 2200), poly(1,4-butylene isophthalate) (PBI, Mw = 1000), poly(1,4-butylene 2,5-furandicarboxylate) (PBF, Mw = 600), and poly(1,4-butylene 2,4-pyridinedicarboxylate) (PBP, Mw = 1000). These polyesters were successfully thermally treated to obtain an increase in Mw of 8.5, 2.6, 3.3, and 2.7 folds, respectively. This investigation focused on the most successful upgrade, poly(1,4-butylene adipate), then discussed the possible effect of di-ester monomers as compared to di-acids in the thermally driven polycondensation. The herein-described two-step synthesis method represents a practical and cost-effective way to synthesize higher-molecular-weight polymers without the use of toxic metal catalysts such as titanium(IV) tert-butoxide, tin(II) 2-ethylhexanoate, and in particular, antimony(IV) oxide. At the same time, the method allows for the extension of the number of reuses of the biocatalyst by preventing its exposure to extreme denaturating conditions.

Synbeads porous-rigid methacrylic support: application to solid phase peptide synthesis and characterization of the polymeric matrix by FTIR microspectroscopy and high resolution magic angle spinning NMR.

Porous and rigid methacrylic Synbeads were optimized and applied efficiently to the solid phase peptide synthesis with the objective of improving significantly volumetric yields (0.33 mol/L calculated on the basis of maximum chemical accessibility, i.e. the maximum number of functional groups that can be acylated by FmocCl) as compared to swelling commercial polymers (from 0.06 to 0.12 mol/L). The effects of the density of functional groups and spacer length were investigated obtaining a chemical accessibility of the functional groups up to 1 mmol/g(dry). High resolution magic angle spinning (HR-MAS) was exploited to evidence the presence of "solution-like" flexible linkers anchored on the rigid methacrylic backbone of Synbeads and to study the degree of functionalization by the Wang linker. To demonstrate the efficiency of the optimized Synbeads, the peptides Somatostatin and Terlipressin were synthesized. In the case of Somatostatin, final synthetic yields of 45 and 60% were achieved by following the HCTU/DIPEA and DIC/HOBt routes respectively, with the HPLC purity always higher than 83%. In the case of Terlipressin, the synthesis was carried out in parallel on Synbeads and also on TentaGel, ChemMatrix, and PS-DVB for comparison (DIC/HOBt route). The profiles describing the synthetic efficiency demonstrated that Synbeads leads to synthetic efficiency (86%) comparable to PS-DVB (96%) or ChemMatrix (84%). In order to gain a more precise picture of chemical and morphological features of Synbeads, their matrix was also characterized by exploiting innovative approaches based on FTIR microspectroscopy with a conventional source and with synchrotron radiation. A uniform distribution of the functional groups was evidenced through a detailed chemical mapping.

Integrating computational and experimental methods for efficient biocatalytic synthesis of polyesters.

The research on biocatalyzed polycondensation has delivered an array of polyesters having molecular weights below 20,000gmol-1 but characterized by controlled structures and desired functionalities. Their unique catalytic efficiency under mild conditions enables enzymes to catalyze the polycondensation of monomers bearing labile lateral moieties that can be easily accessed via post-polymerization modifications. Despite this great potential, nowadays biocatalysts are not employed for polycondensation on industrial scale due to some bottlenecks related to the formulation of biocatalysts and the process configuration, which make the enzymatic technology non-economic. Recycling the enzymatic catalysts is not only a matter of producing an active and robust formulation, but it also requires the optimal integration of such biocatalyst within a specific reactor and process configuration that must enable efficient mass-transfer while preserving the integrity of the enzymatic preparation. In this chapter, we describe examples of integrated experimental-computational approaches for the rational planning and implementation of enzymatic polycondensation using lipase B from Candida antarctica and cutinase 1 from Thermobifida cellulosilytica. They rely on molecular visualization, molecular modeling and chemometrics, which are methods requiring very modest computational power and approachable by operators who do not have specific computational background. The examples also address the sustainability issue, by describing solvent-free processes involving bio-based monomers and biocatalysts immobilized on renewable carriers.

Evolving biocatalysis to meet bioeconomy challenges and opportunities.

The unique selectivity of enzymes, along with their remarkable catalytic activity, constitute powerful tools for transforming renewable feedstock and also for adding value to an array of building blocks and monomers produced by the emerging bio-based chemistry sector. Although some relevant biotransformations run at the ton scale demonstrate the success of biocatalysis in industry, there is still a huge untapped potential of catalytic activities available for targeted valorization of new raw materials, such as waste streams and CO2. For decades, the needs of the pharmaceutical and fine chemistry sectors have driven scientific research in the field of biocatalysis. Nowadays, such consolidated advances have the potential to translate into effective innovation for the benefit of bio-based chemistry. However, the new scenario of bioeconomy requires a stringent integration between scientific advances and economics, and environmental as well as technological constraints. Computational methods and tools for effective big-data analysis are expected to boost the use of enzymes for the transformation of a new array of renewable feedstock and, ultimately, to enlarge the scope of biocatalysis.

Computational methods to rationalize experimental strategies in biocatalysis.

Computational methods are more and more widely applied in biocatalysis to gain rational guidelines, to orient experimental planning and, ultimately, to avoid expensive and time-consuming experiments. In this respect, molecular modelling, multivariate statistical analysis and chemometrics in general are useful computational tools, although they follow completely different investigative approaches.

In silico prediction of medium effects on esterification equilibrium using the COSMO-RS method.

This paper presents a new approach for predicting solvent effects on esterification reactions of industrial importance in the field of biocatalysis. The COSMO-RS method has been used to calculate the activity coefficients of the chemical species involved in various reactions, carried out in different solvents. For comparison we also used the traditional UNIFAC method. Three lipase-catalyzed esterifications were considered: (1) 1-dodecanoic acid with menthol in n-hexane, n-heptane, cyclohexane, 2,2,4-trimethylpentane, toluene, acetonitrile, and 2-methyl-2-butanol; (2) 1-dodecanoic acid and 1-dodecanol in n-hexane, n-heptane, cyclohexane, 2,2,4-trimethylpentane, and toluene; and (3) glycerol and n-octanoic acid in acetonitrile, benzene, and toluene and in the neat reaction mixture (without any solvent). Predicted activities were used to calculate the thermodynamic equilibrium ratio. This should be independent of medium, and the variation in COSMO-RS values is at most 9-fold (corresponding to a DeltaG degrees of about 5.5 kJ/mol, which would still be a very useful prediction) and often only 2-fold (corresponding to less than 2 kJ/mol or 0.5 kcal/mol, therefore comparable with experimental error). UNIFAC is weaker, especially when important roles are played by conformational freedom, intramolecular interactions, strong polar effects, and charge distribution of molecules in the mixture. The relative percent deviations from the mean of equilibrium constants in different solvents range between 17 and 49 for COSMO-RS versus 32 to 65 for UNIFAC. The COSMO-RS method opens up new perspectives for the development of theoretical models for solvent selection with general applicability.

The Closure of the Cycle: Enzymatic Synthesis and Functionalization of Bio-Based Polyesters.

The polymer industry is under pressure to mitigate the environmental cost of petrol-based plastics. Biotechnologies contribute to the gradual replacement of petrol-based chemistry and the development of new renewable products, leading to the closure of carbon circle. An array of bio-based building blocks is already available on an industrial scale and is boosting the development of new generations of sustainable and functionally competitive polymers, such as polylactic acid (PLA). Biocatalysts add higher value to bio-based polymers by catalyzing not only their selective modification, but also their synthesis under mild and controlled conditions. The ultimate aim is the introduction of chemical functionalities on the surface of the polymer while retaining its bulk properties, thus enlarging the spectrum of advanced applications.

Exploring mild enzymatic sustainable routes for the synthesis of bio-degradable aromatic-aliphatic oligoesters.

The application of Candida antarctica lipase B in enzyme-catalyzed synthesis of aromatic-aliphatic oligoesters is here reported. The aim of the present study is to systematically investigate the most favorable conditions for the enzyme catalyzed synthesis of aromatic-aliphatic oligomers using commercially available monomers. Reaction conditions and enzyme selectivity for polymerization of various commercially available monomers were considered using different inactivated/activated aromatic monomers combined with linear polyols ranging from C2 to C12 . The effect of various reaction solvents in enzymatic polymerization was assessed and toluene allowed to achieve the highest conversions for the reaction of dimethyl isophthalate with 1,4-butanediol and with 1,10-decanediol (88 and 87% monomer conversion respectively). Mw as high as 1512 Da was obtained from the reaction of dimethyl isophthalate with 1,10-decanediol. The obtained oligomers have potential applications as raw materials in personal and home care formulations, for the production of aliphatic-aromatic block co-polymers or can be further functionalized with various moieties for a subsequent photo- or radical polymerization.