Development of computational methods for the simulation of enzymes under operational conditions

2008/2009The thesis work is about the development of new techniques in order to simulate enzymes in real operational conditions. The work is focused on the most wiedely used enzyme class: lipases. The developed techniques were also applied on a completly different enzyme in order to confirm the validity of the methodologies.XXII Ciclo197

New technology based on clamping for high gradient radio frequency photogun

High gradient rf photoguns have been a key development to enable several applications of high quality electron beams. They allow the generation of beams with very high peak current and low transverse emittance, satisfying the tight demands for free-electron lasers, energy recovery linacs, Compton/Thomson sources and high-energy linear colliders. In the present paper we present the design of a new rf photogun recently developed in the framework of the SPARC_LAB photoinjector activities at the laboratories of the National Institute of Nuclear Physics in Frascati (LNF-INFN, Italy). This design implements several new features from the electromagnetic point of view and, more important, a novel technology for its realization that does not involve any brazing process. From the electromagnetic point of view the gun presents high mode separation, low peak surface electric field at the iris and minimized pulsed heating on the coupler. For the realization, we have implemented a novel fabrication design that, avoiding brazing, strongly reduces the cost, the realization time and the risk of failure. Details on the electromagnetic design, low power rf measurements and high power radiofrequency and beam tests performed at the University of California in Los Angeles (UCLA) are discussed in the paper

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

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

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

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

Enlarging the tools for efficient enzymatic polycondensation: structural and catalytic features of cutinase 1 from Thermobifida cellulosilytica

9siCutinase 1 from Thermobifida cellulosilytica is reported for the first time as an efficient biocatalyst in polycondensation reactions. Under thin film conditions the covalently immobilized enzyme catalyzes the synthesis of oligoesters of dimetil adipate with different polyols leading to higher Mw (~1900) and Mn (~1000) if compared to lipase B from Candida antarctica or cutinase from Humicola insolens. Computational analysis discloses the structural features that make this enzyme readily accessible to substrates and optimally suited for covalent immobilization. As lipases and other cutinase enzymes, it presents hydrophobic superficial regions around the active site. However, molecular dynamics simulations indicate the absence of interfacial activation, similarly to what already documented for lipase B from Candida antarctica. Notably, cutinase from Humicola insolens displays a “breathing like” conformational movement, which modifies the accessibility of the active site. These observations stimulate wider experimental and bioinformatics studies aiming at a systematic comparison of functional differences between cutinases and lipases.partially_openembargoed_20161210Pellis, Alessandro; Ferrario, Valerio; Zartl, Barbara; Brandauer, Martin; Gamerith, Caroline; Herrero-Acero, Enrique; Ebert, Cynthia; Gardossi, Lucia; Guebitz, GeorgPellis, Alessandro; Ferrario, Valerio; Zartl, Barbara; Brandauer, Martin; Gamerith, Caroline; Herrero Acero, Enrique; Ebert, Cynthia; Gardossi, Lucia; Guebitz, Geor

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

RF Design of the X-band Linac for the EuPRAXIA@SPARC_LAB Project

We illustrate the RF design of the X-band linac for the upgrade of the SPARC_LAB facility at INFN-LNF (EuPRAXIA@SPARC_LAB). The structures are travelling wave (TW) cavities, working on the 2π/3 mode, fed by klystrons with pulse compressor systems. The tapering of the cells along the structure and the cell profiles have been optimized to maximize the effective shunt impedance keeping under control the maximum value of the modified Poynting vector, while the couplers have been designed to have a symmetric feeding and a reduced pulsed heating. In the paper we also present the RF power distribution layout of the accelerating module and a preliminary mechanical design

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