Functionalization of Enzymatically Synthesized Rigid Poly(itaconate)s via Post-Polymerization aza-Michael Addition of Primary Amines

8The bulky 1,4-cyclohexanedimethanol was used as co-monomer for introducing rigidity in lipase synthetized poly(itaconates). Poly(1,4-cyclohexanedimethanol itaconate) was synthetized on a 14 g scale at 50°C, under solvent-free conditions and 70 mbar using only 135 Units of lipase B from Candida antarctica per gram of monomer. The mild conditions preserved the labile vinyl group of itaconic acid and avoided the decomposition of 1,4-cyclohexanedimethanol observed in chemical polycondensation. Experimental and computational data show that the enzymatic polycondensation proceeds despite the low reactivity of C1 of itaconic acid. The rigid poly(1,4-cyclohexanedimethanol itaconate) was investigated in the context of aza-Michael addition of hexamethylenediamine and 2-phenylethylamine to the vinyl moiety. The enzymatically synthesized linear poly(1,4-butylene itaconate) was studied as a comparison. The two oligoesters (Molecular Weights ranging from 720 to 2859 g mol-1) reacted on a gram scale, at 40-50°C, at atmospheric pressure and in solvent-free conditions. The addition of primary amines led to amine-functionalized oligoesters but also to chain degradation, and the reactivity of the poly(itaconate)s was influenced by the rigidity of the polymer chain. Upon the formation of the secondary amine adduct, the linear poly(1,4-butylene itaconate) undergoes fast intramolecular cyclization and subsequent degradation via pyrrolidone formation, especially in the presence of hexamethylenediamine. On the contrary, the bulky 1,4-cyclohexanedimethanol confers rigidity to poly(1,4-cyclohexanedimethanol itaconate), which hampers the intramolecular cyclization. Also the bulkiness of the amine and the use of solvent emerged as factors that affect the reactivity of poly(itaconate)s. Therefore, the possibility to insert discrete units of itaconic acid in oligoesters using biocatalysts under solvent-free mild conditions opens new routes for the generation of bio-based functional polymers or amine-triggered degradable materials, as a function of the rigidity of the polyester chain.partially_openopenAlice Guarneri, Viola Cutifani, Marco Cespugli, Alessandro Pellis, Roberta Vassallo, Fioretta Asaro, Cynthia Ebert, Lucia GardossiGuarneri, Alice; Viola, Cutifani; Cespugli, Marco; Alessandro, Pellis; Roberta, Vassallo; Asaro, Fioretta; Ebert, Cynthia; Gardossi, Luci

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 ,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

Establishment of Corbicula fluminea (O.F. M?ller, 1774) in Lake Maggiore: a spatial approach to trace the invasion dynamics

We analysed the dynamics of the invasive Asian basket clam Corbicula fluminea in Lake Maggiore (Italy), recorded for the first time in August 2010. In order to reveal the extent of its dispersal, we monitored 30 locations along the lake for presence/absence of clams. This assessment of population structure, density and biomass is based on quantitative samples collected along the southern shoreline at four sites with diverse types of habitats. In the present study, the on-going process of Corbicula invasion was analysed from a spatial and temporal perspective. We compared density and size structure of the population among the sites (spatial distribution). We attempted to trace the colonization dynamics of the clams, so the invasion dynamics were tentatively reconstructed from spatial distribution of size /age groups and the contribution of the last recruited cohort to total population density along the lake littoral zone. Results from our surveys conducted in 2010-2011 have demonstrated that the Asian clam was well-established in the lake, thus about one-third of the lake (i.e. the southern basin) was already colonized by C. fluminea in 2011. Size frequency distribution in autumn 2011 further illustrated reproduction events and new recruitments. Population densities in Lake Maggiore were among the highest ever recorded in an Italian lake. Both the rapid spread of Corbicula in the littoral area and the relatively high densities, even at the most recently invaded sites, infer the potential ecosystem impacts associated with a dominant invasive species. Data reported here are not intended to be exhaustive since they concern only two years of investigations, so more detailed studies on both the ecology and invasive habits of this new alien species in Lake Maggiore are planned. The spatial approach used in the present study may clarify the dynamics of this invasion. Future monitoring might help us to disentangle the effects of spatial variability versus temporal succession during the establishment of the invasive species

Effect of Binding Modules Fused to Cutinase on the Enzymatic Synthesis of Polyesters

open9In relation to the development of environmentally-friendly processing technologies for the continuously growing market of plastics, enzymes play an important role as green and sustainable biocatalysts. The present study reports the use of heterogeneous immobilized biocatalysts in solvent-free systems for the synthesis of aliphatic oligoesters with Mws and monomer conversions up to 1500 Da and 74%, respectively. To improve the accessibility of hydrophilic and hydrophobic substrates to the surface of the biocatalyst and improve the reaction kinetic and the chain elongation, two different binding modules were fused on the surface of cutinase 1 from Thermobifida cellulosilytica. The fusion enzymes were successfully immobilized (>99% of bound protein) via covalent bonding onto epoxy-activated beads. To the best of our knowledge, this is the first example where fused enzymes are used to catalyze transesterification reactions for polymer synthesis purposes.openFerrario, Valerio; Todea, Anamaria; Wolansky, Lisa; Piovesan, Nicola; Guarneri, Alice; Ribitsch, Doris; Guebitz, Georg M.; Gardossi, Lucia; Pellis, AlessandroFerrario, Valerio; Todea, Anamaria; Wolansky, Lisa; Piovesan, Nicola; Guarneri, Alice; Ribitsch, Doris; Guebitz, Georg M.; Gardossi, Lucia; Pellis, Alessandr

Fully renewable polyesters via polycondensation catalyzed by Thermobifida cellulosilytica cutinase 1: an integrated approach

The present study addresses comprehensively the problem of producing polyesters through sustainable processes while using fully renewable raw materials and biocatalysts. Polycondensation of bio-based dimethyl adipate with different diols was catalyzed by cutinase 1 from Thermobifida cellulosilytica (Thc_cut1) under solvent free and thin-film conditions. The biocatalyst was immobilized efficiently on a fully renewable cheap carrier based on milled rice husk. A multivariate factorial design demonstrated that Thc_cut1 is less sensitive to the presence of water in the system and it works efficiently under milder conditions (50 \ub0C; 535 mbar) when compared to lipase B from Candida antarctica (CaLB), thus enabling energy savings. Experimental and computational investigations of cutinase 1 from Thermobifida cellulosilytica (Thc_cut1) disclosed structural and functional features that make this serine-hydrolase efficient in polycondensation reactions. Bioinformatic analysis performed with the BioGPS tool pointed out functional similarities with CaLB and provided guidelines for future engineering studies aiming, for instance, at introducing different promiscuous activities in the Thc_cut1 scaffold. The results set robust premises for a full exploitation of enzymes in environmentally and economically sustainable enzymatic polycondensation reactions

Detection of circulating immunosuppressive cytokines in malignant pleural mesothelioma patients for prognostic stratification.

Abstract Background No data on circulating biomarkers for the prognostic stratification of Malignant Pleural Mesothelioma (MPM) patients are available. We prospectively explored the prognostic role of circulating monocyte and cytokine levels and their dynamic change during chemotherapy. Patients and Methods MPM patients receiving a first line treatment based on a platinum compound plus pemetrexed were eligible. Blood samples were collected at the baseline and at the end of induction chemotherapy. CCL-2, IL-10 and TGF-β levels in plasma were quantified by Enzyme-Linked Immunosorbent Assay (ELISA); white blood cells, monocytes and platelets were evaluated by blood count test. Results Thirty-one patients were included in the study. Median overall survival (OS) was 12.13 months versus 9.6 months in patients with lower and higher monocytes count, respectively (p value = 0.02). We further stratified patients according to a combined score based on the association of IL-10, TGF-β levels and monocytes count. High combined score was associated with shorter OS and PFS in univariate and multivariate analysis. Chemotherapy induced an increase in monocytes, IL-10, but not TGF-β levels. Conclusion The prognostic value of circulating levels of multiple immunosuppressive cytokines and inflammatory cells should be confirmed in a wider validation set of MPM patients

Use of scalp cooling device to prevent alopecia for early breast cancer patients receiving chemotherapy: A prospective study

Chemotherapy-induced alopecia (CIA) affects the majority of patients receiving chemotherapy (CT) for early breast cancer. It is a highly distressing side effect of CT, with psychological and social impact. Primary aim of the present analysis was to assess the efficacy of scalp cooling with DigniCap® in preventing CIA. Success rate was defined as patients' self-reported hair loss <50% according to Dean scale. In this analysis, we reported success rate at 3 weeks after the first CT course and at 3 weeks after the last CT course. Secondary endpoints included self-reported tolerability and patients' judgment on scalp cooling performance. Consecutive early breast cancer patients admitted to Istituto Oncologico Veneto who were recommended to receive neoadjuvant or adjuvant CT, were eligible to undergo scalp cooling during the CT administration within this study. 135 patients were included: 74% received adjuvant CT and 26% neoadjuvant CT (P < .001). The type of CT was: docetaxel-cyclophosphamide (26%), paclitaxel (23%), epirubicin-cyclophosphamide followed by paclitaxel (32%), and paclitaxel followed by epirubicincyclophosphamide (19%). The rate of success in preventing alopecia was 77% (104/135) at 3 weeks from the start of CT and 60% (81/135) at 3 weeks from the end of treatment. Higher success rates were reported in non-anthracycline (71%) compared to anthracycline-containing CT regimens (54%; P < 0.001). Premature discontinuation of scalp cooling was reported in 29/135 patients (21.5%), including withdrawal for alopecia (16/29), for low scalp cooling tolerability (8/29) or both (5/29). Scalp cooling was generally well tolerated. These results overall suggest that the use of scalp cooling is effective in preventing alopecia in the majority of early breast cancer patients receiving neoadjuvant or adjuvant CT, especially for patients undergoing a taxane-based non-anthracycline regimen

Nicotinamide coenzyme biomimetics for biocatalysis

In the last century, the use of enzymes for manufacturing of bulk and fine chemicals has spiked. Enabling redox reactions, oxidoreductases are mostly employed for the generation of chiral centres due to their exquisite enantioselectivity. These biocatalysts demand the presence of coenzymes to mediate the electron transfer, with nicotinamide adenine dinucleotide being the most common redox coenzyme. Both its unphosphorylated (NAD) and phosphorylated (NADP) forms are expensive and unstable, particularly in their reduced state. Born as NAD(P) models to be used for mechanistic studies, nicotinamide coenzyme biomimetics (NCBs) retain the dihydropyridine core of the natural coenzymes but lack of the dinucleotide moiety. While being a simpler version of NAD(P), NCBs maintain the redox capacity of the natural pyridine coenzyme and, in the last decade, gained popularity as alternative and cheaper coenzymes for oxidoreductases. In this thesis we further explore the use of NCBs with multiple oxidoreductase classes and debate their future in the applied biocatalysis sector.Chapter 1 provides the background for this Ph.D. thesis. Redox biocatalysts, their chemistry and their coenzyme requirements are here described. A more in depth description of nicotinamide adenine dinucleotide coenzymes (NAD(P)) and the development of their synthetic biomimetics (NCBs) is included. Moreover, the relevance of this project is outlined.Chapter 2 revolves around coenzyme alternatives to the natural nicotinamide, flavin and S-adenosyl-L-methionine coenzymes. An overview of the application of the artificial coenzymes with different types of redox enzymes is given by reviewing recent literature. As described, artificial coenzymes can be cost effective and can be employed to tune reaction rates or the type of reaction. Special attention is given to NCBs, as these biomimetics have been proven to be particularly effective with flavin-dependent oxidoreductases.Chapter 3 offers a detailed picture of the diverse catalytic mechanisms and chemistries performed by flavoprotein monooxygenases (FPMOs) and halogenases. It is also explained which FPMOs can be coupled to NCBs for the regeneration of their flavin coenzyme.In Chapter 4 we show the coupling of four group A FPMOs with a panel of NCBs for the regioselective hydroxylation of phenolic compounds. Because of product inhibition, one of the FPMOs showed meagre conversions with NCBs, whereas the other three enzymes accepted NCBs as electron donors with 100% product yield. Intriguingly, each enzyme preferred a specific NCB as hydride donor and formed only minor amounts of the by-product hydrogen peroxide. Steady state kinetics revealed that the biomimetics do have weak binding and relatively low catalytic efficiency compared to the preferred natural coenzymes. Based on this behaviour we propose a “kiss and ride” mechanism for these FAD-dependent aromatic hydroxylases.Oxidoreductases that contain a Rossmann-like domain for NAD(P) binding poorly accept NCBs as coenzymes. This is particularly true for solely NAD(P)-dependent oxidoreductases, of which only a few were reported to accept the artificial coenzymes, although with low activity. To solve this scarcity, Chapter 5 and Chapter 6 focus on the discovery of flavin-independent redox biocatalysts that can recognize NCBs as their coenzymes.Chapter 5 deals with flavin- and zinc-independent double bond reductases (DBRs). DBRs are a promising class of biocatalysts for the asymmetric hydrogenation of activated alkenes, and their catalytic mechanism as well as their substrate scope and applications are reviewed in this Chapter. Starting from a previously reported activity of a DBR with several NCBs, sequence alignment was employed to identify two putative DBRs, which are presumably also able to catalyze their reaction in presence of the biomimetics. Together with the previously reported DBR, we screened these enzymes for the reduction of phenylpropenal substrates with three NCBs to find that moderate conversions were achieved in presence of NCBs (≤ 10%), with an underlying preference for N1-benzyl substituted biomimetics. Intriguingly, the activity of these DBRs with NCBs depended also on the substrate used in the reaction.Chapter 6 follows a different computational approach, based on catalophores, to find putative NCB-dependent oxidoreductases. By mapping the active sites of biocatalysts reported to use NCBs as their hydride donors, and subsequently mining the Protein Data Bank for enzymes showing similar active sites in terms of physical-chemical properties, we were able to identify four NADP-dependent enzymes and test them for activity with NCBs. The best result was obtained with an aldehyde dehydrogenase, which yielded 6% conversion over 24 h. Although product formation was confirmed, a ketoreductase gave very meagre activity in presence of NCBs. Interestingly, a possible new reactivity was found for a double bond reductase.Although the achieved conversions were not optimal, Chapter 5 and Chapter 6 serve as a starting point for the future optimization of the interactions between the newly identified NCB-accepting oxidoreductases and the synthetic coenzymes, as well as for the future discovery of other NCB-dependent enzymes.Finally, by merging the results described in the previous Chapters, this thesis is concluded by a general discussion (Chapter 7) that places our findings in a broader context and includes unpublished data. At first, an analysis regarding the multiple uses of NCBs with redox enzymes is given. Further accent is put on the crucial challenges that NCBs have to face in order to be widely applied for biocatalysis such as their stability and their in vitro recycling. Lastly, the use of NAD(P)H and NCBs for photobiocatalysis is introduced and preliminary data are shown together with future suggestions for this exciting new branch of biocatalysis

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 fossilbased 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 biobased 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 fallout