Integration of bioinformatics analysis and experimental biocatalysis for a comprehensive approach to the synthesis of renewable polyesters

La crescente domanda di poliesteri funzionalizzabili ha accresciuto l\u2019interesse nello sviluppo di nuove strade per la sintesi biocatalizzata di polimeri, dove gli enzimi sono in grado di rispondere alla sfida di combinare condizioni di reazione sostenibili dal punto di vista ambientale con l\u2019alta selettivit\ue0 ed efficienza della catalisi. Gli enzimi sono un\u2019attrattiva sostenibile ai catalizzatori tossici usati nelle policondensazione, come quelli metallici, stagno in particolare. L\u2019obiettivo di questa tesi \ue8 quello di integrare approcci sperimentali e bioinformatici per lo studio di nuovi biocatalizzatori per le policondensazioni. Una valida alternativa \ue8 infatti rappresentata dagli enzimi, i quali consentono riciclabilit\ue0, assenza di contaminazione del prodotto grazie all\u2019immobilizzazione della proteina. Attualmente, i supporti per l\u2019immobilizzazione sono di natura non rinnovabile (metacrilici e stirenici). In questa tesi (Capitolo 3) si \ue8 esplorata la possibilit\ue0 di utilizzare la lolla di riso \u2013 un economico materiale di scarto lignocellulosico - a questo proposito. Verr\ue0 proposto un confronto tra metodi chemoenzimatici per la funzionalizzazione della lolla, con l\u2019obiettivo di ottenere un supporto di immobilizzazione rinnovabile capace di rispondere alle sfide della green chemistry. Il metodo enzimatico utilizza un sistema laccasi-mediatore con l\u2019inserimento di un linker diamminico. Questo approccio consente di evitare l\u2019utilizzo del periodato di sodio, che \ue8 responsabile di importanti alterazioni nella struttura morfologica della lolla, come dimostrato da microscopia SEM. Candida antarctica Lipasi B e due asparaginasi sono state immobilizzate e testate. La lipasi immobilizzata \ue8 stata utilizzata per sintetizzare un poliestere con l\u2019acido itaconico. Mentre le lipasi sono la pi\uf9 comune scelta per le reazioni di policondensazione, il nostro gruppo si \ue8 concentrato sullo studio di nuove serin idrolasi da utilizzare in questo campo, nello specifico, le cutinasi. Questa classe di enzimi \ue8 gi\ue0 stata utilizzata per catalizzare la sintesi efficiente di poliesteri con monomeri biobased, lavorando in condizioni sostenibili dal punto di vista ambientale. Uno studio bioinformatico approfondito delle cutinasi verr\ue0 proposto nel Capitolo 4 utilizzando i descrittori GRID-based di BioGPS. Il software ha consentito di proiettare una selezione di cutinasi su un modello UPCA (Unsupervised Pattern Cognition Analysis) precedentemente studiato da questo gruppo di ricerca, confermando che l\u2019ambiente chimico-fisico pre-organizzato di Cutinasi 1 da Thermobifida cellulosilytica \ue8 molto simile a quello di Candida antarctica Lipasi B ed \ue8 in grado di offrire ulteriori vantaggi in termini di tipologie di substrato accettati grazie al suo sito attivo molto superficiale. BioGPS \ue8 stato usato anche per generare il \u201ccataloforo\u201d di differenti sottoclassi di serin idrolasi, permettendo di estrarre le caratteristiche minime proprie di ciascuna di esse. Utilizzando il \u201ccataloforo\u201d e studi di dinamica molecolare \ue8 stato possibile chiarire le ragioni alla base delle caratteristiche vantaggiose delle cutinasi nella sintesi di poliesteri.The rising demand for advanced polyesters, displaying new functional properties, has boosted the development of new biocatalysed routes for polymer synthesis, where enzymes concretely respond to the challenge of combining benign conditions with high selectivity and efficient catalysis. Enzymes are attractive sustainable alternatives to toxic catalysts used in polycondensation, such as metal catalysts and tin in particular. Moreover, they enable the synthesis of functional polyesters that are otherwise not easily accessible by using traditional chemical routes. The aim of the present thesis is to integrate experimental and bioinformatics approaches in order to study new biocatalysts to be used in polycondensations. A valid alternative to metal catalysts is represented by enzymes. Biocatalyst recyclability and avoidance of product contamination are usually obtained via enzyme immobilization on solid carriers. Nowadays, non-renewable petrochemical-based supports are used for this purpose, namely methacrylic and styrenic resins. In this thesis (Chapter 3), rice husk - a waste product of rice milling available worldwide at a negligible price - has been explored as an innovative and fully renewable lignocellulosic carrier endowed with morphological complexity and chemical versatility that makes it prone to multiple and benign chemo-enzymatic modifications. A comparison of chemical and enzymatic methods for the functionalization of rice husk has been carried out, enabling the development of a renewable immobilization carrier suitable for responding to the looming challenge of green chemistry. The enzymatic method relies on laccase oxidation using laccase from Trametes spec. and TEMPO-radical mediator, followed by the insertion of a diamine spacer. As compared to the classical cellulose oxidation performed via sodium periodate, the enzymatic method offers the advantage of preserving the morphology of rice husk, as demonstrated by SEM microscopy. Laccase oxidation also assures benign operative conditions. Candida antarctica Lipase B, and two commercially available formulations of asparaginase, were immobilized and tested. In the first case, the lipase was successfully applied in the polycondensation of the biobased monomer dimethyl itaconate whereas the immobilized asparaginases were applied in the hydrolysis of asparagine, a precursor of the toxic acrylamide in food. In addition, lignin removal via alkaline hydrogen peroxide bleaching has been tested as a method for increasing the specific activity of the immobilized formulation. While lipases being the most common alternative for polycondensation reactions, our research group focused on the study of a novel class of serine hydrolases to be used in these kind of reactions, namely the cutinases. The cutinase class proved to catalyse the efficient polycondensation of biobased monomers working in mild conditions in terms of pressure and temperature. A thorough bioinformatics study was carried out based on GRID-based BioGPS descriptors (Chapter 4). BioGPS allowed to project a selection of cutinases on a Unsupervised Pattern Cognition Analysis (UPCA) model previously published by this research group, confirming that the pre-organized physicochemical environment in the active site of Cutinase 1 from Thermobifida cellulosilytica is very similar to the one of Candida antarctica Lipase B, while offering increased capabilities in terms of the size of the substrate accepted, thanks to a superficial and wide active site. The said software was used also to generate the \u201ccatalophor\u201d of different serine hydrolase subfamilies, enabling to extract the structural features that distinguish the various sub-families of serine hydrolases. Exploiting the \u201ccatalophor\u201d tool and molecular dynamics studies it was possible to shed light on the particular behaviour that makes cutinases an advantageous biocatalyst to be used in polycondensation reactions

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

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

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

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

A multi-pronged approach targeting SARS-CoV-2 proteins using ultra-large virtual screening.

The unparalleled global effort to combat the continuing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic over the last year has resulted in promising prophylactic measures. However, a need still exists for cheap, effective therapeutics, and targeting multiple points in the viral life cycle could help tackle the current, as well as future, coronaviruses. Here, we leverage our recently developed, ultra-large-scale in silico screening platform, VirtualFlow, to search for inhibitors that target SARS-CoV-2. In this unprecedented structure-based virtual campaign, we screened roughly 1 billion molecules against each of 40 different target sites on 17 different potential viral and host targets. In addition to targeting the active sites of viral enzymes, we also targeted critical auxiliary sites such as functionally important protein-protein interactions

A community effort in SARS-CoV-2 drug discovery.

peer reviewedThe COVID-19 pandemic continues to pose a substantial threat to human lives and is likely to do so for years to come. Despite the availability of vaccines, searching for efficient small-molecule drugs that are widely available, including in low- and middle-income countries, is an ongoing challenge. In this work, we report the results of an open science community effort, the "Billion molecules against Covid-19 challenge", to identify small-molecule inhibitors against SARS-CoV-2 or relevant human receptors. Participating teams used a wide variety of computational methods to screen a minimum of 1 billion virtual molecules against 6 protein targets. Overall, 31 teams participated, and they suggested a total of 639,024 molecules, which were subsequently ranked to find 'consensus compounds'. The organizing team coordinated with various contract research organizations (CROs) and collaborating institutions to synthesize and test 878 compounds for biological activity against proteases (Nsp5, Nsp3, TMPRSS2), nucleocapsid N, RdRP (only the Nsp12 domain), and (alpha) spike protein S. Overall, 27 compounds with weak inhibition/binding were experimentally identified by binding-, cleavage-, and/or viral suppression assays and are presented here. Open science approaches such as the one presented here contribute to the knowledge base of future drug discovery efforts in finding better SARS-CoV-2 treatments.R-AGR-3826 - COVID19-14715687-CovScreen (01/06/2020 - 31/01/2021) - GLAAB Enric

Rice Husk as an Inexpensive Renewable Immobilization Carrier for Biocatalysts Employed in the Food, Cosmetic and Polymer Sectors

The high cost and environmental impact of fossil-based organic carriers represent a critical bottleneck to their use in large-scale industrial processes. The present study demonstrates the applicability of rice husk as inexpensive renewable carrier for the immobilization of enzymes applicable sectors where the covalent anchorage of the protein is a pre-requisite for preventing protein contamination while assuring the recyclability. Rice husk was oxidized and then functionalized with a di-amino spacer. The morphological characterization shed light on the properties that affect the functionalization processes. Lipase B from Candida antarctica (CaLB) and two commercial asparaginases were immobilized covalently achieving higher immobilization yield than previously reported. All enzymes were immobilized also on commercial epoxy methacrylic resins and the CaLB immobilized on rice husk demonstrated a higher efficiency in the solvent-free polycondensation of dimethylitaconate. CaLB on rice husk appears particularly suitable for applications in highly viscous processes because of the unusual combination of its low density and remarkable mechanical robustness. In the case of the two asparaginases, the biocatalyst immobilized on rice husk performed in aqueous solution at least as efficiently as the enzyme immobilized on methacrylic resins, although the rice husk loaded a lower amount of protein

Nature inspired solutions for polymers: will cutinase enzymes make polyesters and polyamides greener?

The 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

Rational guidelines for the two-step scalability of enzymatic polycondensation: experimental and computational optimization of the enzymatic synthesis of poly(glycerolazelate)

8si: The lipase catalyzed polycondensation of azelaic acid and glycerol was investigated according to a Design of Experiment approach that allowed to understand the effect of the experimental variables on monomer conversion, M n and regioselectivity of acylation of glycerol. The chemometric analysis showed that after 24h 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, provided rational guidelines for the optimization of the regioselective acylation of glycerol. Overall, the process was scaled up to 73.5 g of monomer. The novelty of the present study stays in the rigorous control of the reaction conditions and of the integrity of the immobilized biocatalyst, thus avoiding any interference of free enzyme or fines released in the reaction mixture.  The quantitative analysis of the effect of the experimental conditions and the overcoming of the major technical bottlenecks for the scalability of enzymatic polycondensation opens new scenarios for its industrial exploitation.noneopenTodea, Anamaria; Fortuna, Sara; Ebert, Cynthia; Asaro, Fioretta; Tomada, Stefano; Cespugli, Marco; Hollan, Fabio; Gardossi, LuciaTodea, Anamaria; Fortuna, Sara; Ebert, Cynthia; Asaro, Fioretta; Tomada, Stefano; Cespugli, Marco; Hollan, Fabio; Gardossi, Luci