Effect of Microwave Radiation on Enzymatic and Chemical Peptide Bond Synthesis on Solid Phase

Peptide bond synthesis was performed on PEGA beads under microwave radiations. Classical chemical coupling as well as thermolysin catalyzed synthesis was studied, and the effect of microwave radiations on reaction kinetics, beads' integrity, and enzyme activity was assessed. Results demonstrate that microwave radiations can be profitably exploited to improve reaction kinetics in solid phase peptide synthesis when both chemical and biocatalytic strategies are used

Criteria for Engineering Cutinases: Bioinformatics Analysis of Catalophores

Cutinases are bacterial and fungal enzymes that catalyze the hydrolysis of natural cutin, a three-dimensional inter-esterified polyester with epoxy-hydroxy fatty acids with chain lengths between 16 and 18 carbon atoms. Due to their ability to accept long chain substrates, cutinases are also effective in catalyzing in vitro both the degradation and synthesis of several synthetic polyesters and polyamides. Here, we present a bioinformatics study that intends to correlate the structural features of cutinases with their catalytic properties to provide rational basis for their effective exploitation, particularly in polymer synthesis and biodegradation. The bioinformatics study used the BioGPS method (Global Positioning System in Biological Space) that computed molecular descriptors based on Molecular Interaction Fields (MIFs) described in the GRID force field. The information was used to generate catalophores, spatial representations of the ability of each enzymatic active site to establish hydrophobic and electrostatic interactions. These tools were exploited for comparing cutinases to other serine-hydrolases enzymes, namely lipases, esterases, amidases and proteases, and for highlighting differences and similarities that might guide rational engineering strategies. Structural features of cutinases with their catalytic properties were correlated. The \u201ccatalophore\u201d of cutinases indicate shared features with lipases and esterases

Understanding potentials and restrictions of solvent-free enzymatic polycondensation of itaconic acid: an experimental and computational analysis

6siItaconic acid is a chemically versatile unsaturated diacid that can be produced by fermentation and potentially it can replace petrol based monomers such as maleic and fumaric acids in the production of curable polyesters or new biocompatible functionalized materials. Unfortunately, due to the presence of the unsaturated C=C bond, polycondensation of itaconic acid is hampered by cross reactivity and isomerization. Therefore, enzymatic polycondensations would respond to the need of mild and selective synthetic routes for the production of novel bio-based polymers. The present work analyses the feasibility of enzymatic polycondensation of diethyl itaconate and, for the first time, provides comprehensive solutions embracing both the formulation of the biocatalyst, the reaction conditions and the choice of the co-monomers. Computational docking was used to disclose the structural factors responsible for the low reactivity of dimethyl itaconate and to identify possible solutions. Surprisingly, experimental and computational analysis revealed that 1,4-butanediol is an unsuitable co-monomer for the polycondensation of dimethyl itaconate whereas the cyclic and rigid 1,4-cyclohexanedimethanol promotes the elongation of the oligomers.partially_openembargoed_20160430Corici, Livia; Pellis, Alessandro; Ferrario, Valerio; Ebert, Cynthia; Cantone, Sara; Gardossi, LuciaCorici, Livia; Pellis, Alessandro; Ferrario, Valerio; Ebert, Cynthia; Cantone, Sara; Gardossi, Luci

Nature Inspired Solutions for Polymers: Will Cutinase Enzymes Make Polyesters and Polyamides Greener?

5siThe polymer and plastic sectors are under the urge of mitigating their environmental impact. The need for novel and more benign catalysts for polyester synthesis or targeted functionalization led, in recent years, to an increasing interest towards cutinases due to their natural ability to hydrolyze ester bonds in cutin, a natural polymer. In this review, the most recent advances in the synthesis and hydrolysis of various classes of polyesters and polyamides are discussed with a critical focus on the actual perspectives of applying enzymatic technologies for practical industrial purposes. More specifically, cutinase enzymes are compared to lipases and, in particular, to lipase B from Candida antarctica, the biocatalyst most widely employed in polymer chemistry so far. Computational and bioinformatics studies suggest that the natural role of cutinases in attacking natural polymers confer some essential features for processing also synthetic polyesters and polyamides.openopenFerrario, Valerio; Pellis, Alessandro; Cespugli, Marco; Guebitz, Georg; Gardossi, LuciaFerrario, Valerio; Pellis, Alessandro; Cespugli, Marco; Guebitz, Georg; Gardossi, Luci

Interim Evaluation of the Bio-based Industries Joint Undertaking (2014-2016) operating under Horizon 2020

The Bio-Based Industries Joint Undertaking (BBI JU) is a public-private partnership (PPP)1 between the European Commission and the Bio-based Industries Consortium (BIC). The Council Regulation (EU) No 560/2014 sets the basis for the establishment of the Bio-based Industries Joint Undertaking (BBI JU).2 BIC developed the Strategic Innovation and Research Agenda (SIRA) based on extensive consultation with public and private stakeholders. As per all the seven JUs, BBI JU awards Horizon 2020 funding for projects based on competitive calls. Five JUs were already set up in 2007-2008 under the Seventh Framework Programme (FP7), whereas Bio-based Industries (BBI) is one of the newly established JUs under Horizon 2020 with the specific aim of developing sustainable and competitive bio-based industries in Europe. BBI JU set out financial commitments from both the EU and from the industry members in order to provide funding for large-scale, longer-term and high risk/reward research. The objectives of BBI JU may best be achieved by the Partnerships and, most importantly, by bringing together of companies, universities, research centres, innovative SMEs and other groups and organisations around the topic of the bio-based economy, which is of great industrial and social relevance. BBI JU is expected to be a concrete example of the European Union's efforts towards strengthening its competitiveness through scientific excellence, industry led research, openness and innovation. According to Article 32(3) of the Horizon 2020 Regulation, the Commission must provide an in-depth assessment of all JUs. Article 11(1) of BBI JU regulation3 provides the main legal basis for this interim evaluation, which was carried out by a group of five independent experts who analysed the activities of BBI JU in the period 2014-2016. The evaluation takes place at an early stage, less than three years after the adoption of Regulation (EU) No. 560/2014, which established the BBI JU. It covers five main evaluation criteria: relevance, efficiency, effectiveness, coherence and EU added value.4,5 Although at the time point of this interim evaluation none of the research projects funded by BBI JU had been completed, qualitative input in combination with quantitative information, as was available, were used to assess the effectiveness of implementation and the main achievements so far

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 ,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

Towards feasible and scalable solvent-free enzymatic polycondensations: integrating robust biocatalysts with thin film reactions

There is an enormous potential for synthesizing novel bio-based functionalized polyesters under environmentally benign conditions by exploiting the catalytic efficiency and selectivity of enzymes. Despite the wide number of studies addressing in vitro enzymatic polycondensation, insufficient progress has been documented in the last two decades towards the preparative and industrial application of this methodology. The present study analyses bottlenecks hampering the practical applicability of enzymatic polycondensation that have been most often neglected in the past, with a specific focus on solvent-free processes. Data here presented elucidate how classical approaches for enzyme immobilization combined with batch reactor configuration translate into insufficient mass transfer as well as limited recyclability of the biocatalyst. In order to overcome such bottlenecks, the present study proposes thin-film processes employing robust covalently immobilized lipases. The strategy was validated experimentally by carrying out the solvent-free polycondensation of esters of adipic and itaconic acids. The results open new perspectives for enlarging the applicability of biocatalysts in other viscous and solvent-free syntheses

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