Enzymes as catalysts in polymer chemistry

2009/2010The use of enzymes in synthetic chemistry is attracting the interest of many researchers thanks to their extraordinary efficiency under mild conditions, high stereo- regio- and chemoselectivity and low environmental impact. Their application in the field of polymer chemistry has provided new synthetic strategies for useful polymers. The advantages coming from the use of enzymes are mainly: i) the possibility to synthesize polymers with novel properties and difficult to produce by conventional chemical catalysts; ii) the improvement of the synthetic processes in terms of sustainability, for instance by avoiding toxic catalysts. In particular hydrolases have demonstrated to be efficient catalysts in the development of highly structured polyesters with low polydispersity characterized by the presence of different functionalisations. Although generally the enzymatic processes lead to the formation of oligomers of limited Mn, these can be successively chemically polymerized to synthesize new innovative products with high Mn. Hydrolases enzymes offer also a great opportunity for using non-petrochemical renewable resources as starting substrates thus contributing to global sustainability of polymerization processes. Also oxidases have proven to be efficient catalysts in polymer chemistry thanks to their capability to catalyze oxidation of phenolic groups. This radicalic reaction can be exploited either for: i) degrading lignin; ii) functionalizing lignin phenolic moieties thus improving their chemical and physical properties; iii) polymerize phenol derivatives or phenol based oligomers. Unfortunately, the use of enzymes in polymer chemistry on industrial scale is still hindered by many factors among which the most significant are: i) the instability of many commercial immobilized preparations under process conditions, ii) the high costs of both the biocatalyst and the monomers from renewable resources and iii) the limited availability of information concerning the main factors which affect the scalability and the industrialization of this kind of enzymatic processes. At this regard, the work described in Chapter 2 of the thesis exploits the catalytic potential of lipase B from Candida antarctica in polymerization processes. In collaboration with the group of Professor Karl Hult (KTH “Royal Institute of Technology”, Stockholm) a new synthetic route for the enzymatic synthesis of new innovative highly structured allyl functionalized polyester of industrial interest has been developed. Finally, products obtained have been efficiently employed in thiol-ene chemistry for films production. Chapter 3 deals with the synthesis of functionalized polyesters starting from renewable feedstock which can be employed as new starting materials for the production of surfactants for cosmetic and pharma industry. Continuing on the study of lipase catalyzed polyester synthesis, in Chapter 4 particular attention has been given to the feasibility of the biocatalysed technology at industrial level, namely addressing the problem of biocatalyst’s stability and formulation. In particular a highly stable covalently immobilized preparation of CALB, developed in collaboration with SPRIN Technologies, has been exploited for the synthesis of polyesters demonstrating the advantages coming from covalent immobilization over the other commercially available CALB adsorbed preparations, namely higher recyclability and absence of enzyme leaching. On this respect, one-step and multistep processes for the synthesis of polyesters employing adipic acid and 1,4-butanediol have been the subject of a deep investigation. A comparative analysis describes the effect of enzyme leaching from adsorbed preparations during polycondensation reactions evidencing meanwhile the stability of the covalent enzymatic preparation under harsh conditions. Within the context of this study characterization of the synthesized polymers has been feasible thanks to the collaboration with the group of Dr. Mario Malinconico at the “Istituto di Chimica e Tecnologia dei Polimeri” ICTP-CNR, Pozzuoli (NA). Moreover, a new methodology for polymer characterization which integrates DOSY NMR and GROMACS simulation techniques has been developed in collaboration with CBM (“Cluster in Biomedicine”, Area Science Park, Trieste). The obtained results open new perspectives for the study of polymer behavior in specific media. The final part of the work (Chapter 5) focuses on the potential applicability of laccases under non-conventional conditions, thus exploring new routes for more efficient lignin degradation. The most important obstacle to technological and commercial application of laccases is the limited number of enzymes readily available for industrial applications. At this regard, thanks to the collaboration with the group of Prof. Ludmila Golovleva (Institute of Biochemistry and Physiology of Microorganisms, “Russian Academy of Science”, Puschino, Russia), we have studied the stability and activity of three different fungal laccases in the conditions of interest for lignin processing, namely in organic/buffer mixtures and under microwaves radiation. The proof of concept for the application of the considered laccases in lignin valorization has been demonstrated assessing lignin over laccases mediated oxidation in organic/buffer mixtures media. Moreover, a laccase endowed with remarkable higher stability as compared to the other tested has been identified. The enzyme is currently under study for understanding the structural basis of its stability. In conclusion, the work of this thesis demonstrates that the lipase catalyzed synthesis of polyesters is a mature technology ready to be employed at industrial scale for those specific applications where the chemical properties of products or the sustainability of the process represent crucial issues in the classical chemical processes. Moreover, the new properties of the immobilized lipase employed in the present study induce to believe that the problem of biocatalyst stability and recyclability has been finally overcome. Concerning the application of laccases, the valorization of lignin in terms of production of bio-based chemicals is still a very new field of research, although it is expected to become one of the major topics for the future development of the “Sustainable Chemistry”.1 The present study intends to be a contribution for the identification of suitable biocatalysts and more efficient conditions for the degradation and modification of one of the most abundant biopolymers in Nature. The high stability of the laccase here reported induces to pursue in their characterization under non-conventional reaction conditions on different types of lignin. 1 J. E. Holladay, J. J. Bozell, J. F. White, D. Johnson, “Top Value-Added Chemicals from Biomass”, 2007, U.S. Department of Energy.(http://www1.eere.energy.gov/biomass/pdfs/pnnl-16983.pdf).L'impiego di enzimi nella chimica di sintesi sta attirando l'interesse di molti ricercatori, ciò è dovuto in particolare alla loro straordinaria efficienza in condizioni di reazione blande, alla loro elevata stereo-, regio- e chemoselettività e al loro basso impatto ambientale. L’ applicazione di biocatalizzatori nel campo della chimica dei polimeri ha fornito nuove strategie sintetiche per lo sviluppo di materiali innovativi. I principali vantaggi derivanti dall'impiego di enzimi sono: i) la possibilità di sintetizzare polimeri con nuove proprietà e difficili da ottenere mediante processi chimici convenzionali ed ii) il miglioramento dei processi di sintesi in termini di sostenibilità ambientale evitando l’impiego di catalizzatori tossici. Le idrolasi in particolare hanno dimostrato di essere catalizzatori efficienti per lo sviluppo di poliesteri altamente strutturati e a bassa polidispersità, caratterizzati inoltre dalla presenza di varie funzionalizzazioni chimiche terminali. Anche se generalmente i processi enzimatici portano alla formazione di oligomeri a basso peso molecolare, questi possono essere successivamente polimerizzati chimicamente per formare nuovi prodotti innovativi ad alto Mn. Le idrolasi offrono inoltre l’opportunità di impiegare monomeri provenienti da risorse rinnovabili contribuendo così alla sostenibilità ambientale dei processi di polimerizzazione. Anche le ossidasi hanno dimostrato di essere catalizzatori efficienti e facilmente impiegabili nella chimica dei polimeri grazie alla loro capacità di catalizzare l'ossidazione radicalica di gruppi fenolici. Queste reazioni radicaliche possono trovare diverse applicazioni come ad esempio: i) nella degradazione della lignina; ii) nella funzionalizzazione dei gruppi fenolici della lignina che consentano di migliorarne le proprietà chimiche e fisiche; iii) nella polimerizzazione di derivati fenolici o di oligomeri a base di fenolo. Sfortunatamente, l'impiego di enzimi su scala industriale è ancora ostacolato da una serie di fattori tra i quali i più significativi sono: i) l'instabilità delle preparazioni enzimatiche commercialmente disponibili nelle condizioni di processo, ii) gli alti costi del biocatalizzatore e dei monomeri provenienti da risorse rinnovabili e iii) la limitata disponibilità di informazioni riguardanti i principali fattori che influenzano la scalabilità e l'industrializzazione di questo tipo di processi enzimatici. A questo proposito, il lavoro descritto nel capitolo 2 di questa tesi sfrutta le potenzialità catalitiche della lipasi B da Candida antarctica nei processi di polimerizzazione. In particolare è stata sviluppata, in collaborazione con il gruppo del prof. Karl Hult (KTH “Royal Institute of Technology”, Stoccolma), una nuova via sintetica biocatalizzata per la produzione di poliesteri innovativi altamente strutturati recanti funzionalizzazione allilica, che trovano ampia applicabilità industriale. I prodotti così ottenuti sono stati successivamente impiegati con successo nella chimica dei tioleni per la produzione di films. Il capitolo 3 è invece dedicato alla sintesi di poliesteri funzionalizzati a partire da materie prime rinnovabili; i prodotti così sviluppati trovano potenziale applicazione come tensioattivi per l'industria cosmetica e farmaceutica. Proseguendo lo studio della sintesi biocatalizzata di poliesteri, nel capitolo 4 particolare attenzione è stata data alla fattibilità dei processi biocatalizzati a livello industriale affrontando il problema della stabilità del biocatalizzatore. In particolare, una preparazione di CALB immobilizzata covalentemente su supporti polimerici, sviluppata in collaborazione con SPRIN Technologies, è stata impiegata nella sintesi di poliesteri dimostrando i vantaggi dovuti all’ immobilizzazione covalente rispetto alle altre preparazioni adsorbite commercialmente disponibili, ovvero l’elevata riciclabilità del biocatalizzatore e la mancanza di rilascio di enzima nel prodotto finale. A tal proposito, la sintesi one-step e multistep di poliesteri a partire da acido adipico e 1,4-butandiolo è stata oggetto di una profonda indagine. Il lavoro riporta un' analisi comparativa che descrive l'effetto del rilascio dell’ enzima durante le reazioni di policondensazione evidenziando nel contempo la straordinaria stabilità della preparazione enzimatica covalente. La caratterizzazione dei polimeri così ottenuti è stata possibile grazie alla preziosa collaborazione con il gruppo del Dott. Mario Malinconico (Istituto di Chimica e Tecnologia dei Polimeri "ICTP-CNR, Pozzuoli (NA)). Al termine del capitolo 4 è stata sviluppata, in collaborazione con CBM ("Cluster in Biomedicine", Area Science Park, Trieste), una nuova metodologia per la caratterizzazione dei polimeri che integra DOSY NMR e tecniche di simulazione GROMACS. I risultati ottenuti da questo lavoro aprono nuove prospettive per lo studio del comportamento dei polimeri in solventi organici. La parte finale del lavoro di tesi (capitolo 5) si concentra sull’ applicabilità di laccasi in condizioni non convenzionali, esplorando nuove vie per i processi di degradazione della lignina. Il principale ostacolo nell’impiego tecnologico delle laccasi su scala industriale è il limitato numero di enzimi commercialmente disponibili per tali applicazioni. A questo proposito, grazie alla collaborazione con il gruppo della Prof.ssa Ludmila Golovleva (Istituto di Biochimica e Fisiologia dei Microorganismi, "Russian Academy of Science", Puschino, Russia), è stato possibile studiare la stabilità e l'attività di tre diverse laccasi fungine nelle condizioni di interesse per la trasformazione della lignina, vale a dire in miscele di solvente organico e sotto irradiazione di microonde. L’applicabilità delle laccasi considerate nella valorizzazione della lignina è stata dimostrata valutando l’ ossidazione della lignina nelle condizioni di interesse. E’stata quindi identificata una laccasi caratterizzata da una stabilità particolarmente elevata, l' enzima è attualmente in studio per la comprensione delle basi strutturali che gli conferiscono tali caratteristiche. In conclusione, questo lavoro di tesi dimostra che la sintesi di poliesteri CALB catalizzata è una tecnologia matura, pronta per essere impiegata su scala industriale laddove le proprietà chimiche dei prodotti o la sostenibilità ambientale del processo rappresentano ostacoli non sormontabili impiegando le calssiche metodologie sintetiche. Inoltre l’elevata stabilità della lipasi immobilizzata covalentemenete induce a credere che il problema della stabilità del biocatalizzatore e di conseguenza del suo riciclaggio è stato definitivamente superato. L'applicazione delle laccasi nella valorizzazione della lignina per lo sviluppo di prodotti chimici biobased è un campo di ricerca relativamente nuovo ma destinato a diventare uno dei settori più importanti per lo sviluppo della "chimica sostenibile".1 Il presente studio intende essere un contributo per l'identificazione del biocatalizzatore e delle condizioni di reazione più adatte per la degradazione e la trasformazione della lignina, uno dei biopolimeri più abbondanti presenti in natura. L’elevata stabilità della laccasi identificata in questo lavoro induce inoltre a proseguire con gli studi di applicabilità di questo enzima in mezzi non convenzionali. 1 J. E. Holladay, J. J. Bozell, J. F. White, D. Johnson, “Top Value-Added Chemicals from Biomass”, 2007, U.S. Department of Energy.)XXIII Ciclo198

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

Production of hybrid granitic magma at the advancing front of basaltic underplating: Inferences from the Sesia Magmatic System (south-western Alps, Italy)

The Permian Sesia Magmatic System of the southwestern Alps displays the plumbing system beneath a Permian caldera, including a deep crustal gabbroic complex, upper crustal granite plutons and a bimodal volcanic field dominated by rhyolitic tuff filling the caldera. Isotopic compositions of the deep crustal gabbro overlap those of coeval andesitic basalts, whereas granites define a distinct, more radiogenic cluster (Sri 480.708 and 0.710, respectively). AFC computations starting from the best mafic candidate for a starting melt show that Nd and Sr isotopic compositions and trace elements of andesitic basalts may be modeled by reactive bulk assimilation of 4830% of partially depleted crust and 4815%\u201330% gabbro fractionation. Trace elements of the deep crustal gabbro cumulates require a further 4860% fractionation of the andesitic basalt and loss of 4840% of silica-rich residual melt. The composition of the granite plutons is consistent with a mixture of relatively constant proportions of residual melt delivered from the gabbro and anatectic melt. Chemical and field evidence leads to a conceptual modelwhich links the production of the two granitic components to the evolution of theMafic Complex. During the growth of the Mafic Complex, progressive incorporation of packages of crustal rocks resulted in a roughly steady state rate of assimilation. Anatectic granite originates in the hot zone of melting crust located above the advancing mafic intrusion. Upward segregation of anatectic melts facilitates the assimilation of the partially depleted restite by stoping. At each cycle of mafic intrusion and incorporation, residual and anatectic melts are produced in roughly constant proportions, because the amount of anatectic melt produced at the roof is a function of volume and latent heat of crystallization of the underplated mafic melt which in turn produces proportional amounts of hybrid gabbro cumulates and residualmelt. Such a process can explain the restricted range in isotopic compositions of most rhyolitic and granitic rocks of the Permo-Carboniferous province of Europe and elsewhere

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

U-Pb zircon SHRIMP data from the Cana Brava layered complex: new constraints for the mafic-ultramafic intrusions of Northern Goiás, Brazil

The Cana Brava Complex is the northernmost and less-known layered intrusion of a discontinuous belt of mafic-ultramafic massifs within the Brasilia Belt, which also comprises the Niquelândia and Barro Alto complexes. Available geochronological determination by means of different systematics (K/Ar, Ar/Ar, Rb/Sr, Sm/Nd and U/Pb) provide a range of possible ages (time span from 3.9 Ga to 450 Ma), hence a precise and statistically reliable age for the Cana Brava Complex is still lacking. Also, preliminary isotopic and geochemical data of the Cana Brava Complex suggest a significant crustal contamination, which could have affected bulk-rock Sr and Nd systematics resulting in meaningless age determinations. In this paper, we present new U-Pb SHRIMP zircon analyses from four samples of different units of the Cana Brava Complex which suggest that the intrusion occurred during the Neoproterozoic, between 800 and 780 Ma, i.e. at the same age of Niquelândia. Discordant older 206Pb/238U ages are provided by inherited zircons, and match the age of the metamorphism of the encasing Palmeirópolis Sequence

Comparing the Cana Brava and Niquelândia complexes: large mafic-ultramafic intrusions in the lower crust and contamination processes

Mafic-ultramafic complexes offer a unique opportunity to study how intrusions of mantlederived melts growth into the deep crust and interact with the country rocks. The Cana Brava and Niquelândia complexes are two mafic-ultramafic bodies which outcrop within the Brasilia Belt (Goias, central Brazil) and that intruded the metavolcanicmetasedimentary sequences of Palmeiropolis and Indaianopolis during a Neoproterozoic continental rifting. The two complexes are parts, together with the Barro Alto complex, of a ~350 km NNE-trend belt of layered bodies which were exhumed during the Gondwana formation. New field, geochemical and isotopic data give new constraints on the model of growth of these complexes and the interactions between parent melts and the lower crust. Field evidences suggest that the complexes grow via multiple-melt intrusions under hyper- to subsolidus shear conditions. During the complex growth, the upper metavolcanic-metasedimentary sequence was delaminated and xenoliths were incorporated and deformed within the crystal mush. The increase of the 87Sr/86Sr(790) along the complex stratigraphy, coupled with a decrease of the εNd(790), provides evidences of strong crustal contamination by the embedded xenoliths. The enrichment in most incompatible elements (e.g. K, Ba and LREE) and hydrous phases (biotite and amphibole) in rocks containing more xenoliths supports also the crustal contamination. The almost linear trend of isotopic contamination suggests that this process involved all the magma colum, similarly to AFC. However, the increase abundance of incompatible elements and H2O contents toward xenoliths-rich bands provide for a local effect of contamination

The growth of large mafic intrusions: Comparing Niquelandia and Ivrea igneous complexes

The Niquelandia Complex, Brazil, is one of the world's largest mafic-ultramafic plutonic complexes. Like the Mafic Complex of the Ivrea-Verbano Zone, it is affected by a pervasive high-T foliation and shows hypersolidus deformation structures, contains significant inclusions of country-rock paragneiss, and is subdivided into a Lower and an Upper Complex. In this paper, we present new SHRIMP U-Pb zircon ages that provide compelling evidence that the Upper and the Lower Niquelandia Complexes formed during the same igneous event at ca. 790 Ma. Coexistence of syn-magmatic and high-T subsolidus deformation structures indicates that both complexes grew incrementally as large crystal mush bodies which were continuously stretched while fed by pulses of fresh magma. Syn-magmatic recrystallization during this deformation resulted in textures and structures which, although appearing metamorphic, are not ascribable to post-magmatic metamorphic event(s), but are instead characteristic of the growth process in huge and deep mafic intrusions such as both the Niquelandia and Ivrea Complexes. Melting of incorporated country-rock paragneiss continued producing hybrid rocks during the last, vanishing stages of magmatic crystallization. This resulted in the formation of minor, late-stage hybrid rocks, whose presence obscures the record of the main processes of interaction between mantle magmas and crustal components, which may be active at the peak of the igneous events and lead to the generation of eruptible hybrid magmas. (C) 2012 Elsevier B.V. All rights reserved.Research Support Foundation of the State of Sao Paulo (FAPESP)Brazilian National Research Council (CNPq

Zircon ages in granulite facies rocks: decoupling from geochemistry above 850 °C?

Granulite facies rocks frequently show a large spread in their zircon ages, the interpretation of which raises questions: Has the isotopic system been disturbed? By what process(es) and conditions did the alteration occur? Can the dates be regarded as real ages, reflecting several growth episodes? Furthermore, under some circumstances of (ultra-)high-temperature metamorphism, decoupling of zircon U–Pb dates from their trace element geochemistry has been reported. Understanding these processes is crucial to help interpret such dates in the context of the P–T history. Our study presents evidence for decoupling in zircon from the highest grade metapelites (> 850 °C) taken along a continuous high-temperature metamorphic field gradient in the Ivrea Zone (NW Italy). These rocks represent a well-characterised segment of Permian lower continental crust with a protracted high-temperature history. Cathodoluminescence images reveal that zircons in the mid-amphibolite facies preserve mainly detrital cores with narrow overgrowths. In the upper amphibolite and granulite facies, preserved detrital cores decrease and metamorphic zircon increases in quantity. Across all samples we document a sequence of four rim generations based on textures. U–Pb dates, Th/U ratios and Ti-in-zircon concentrations show an essentially continuous evolution with increasing metamorphic grade, except in the samples from the granulite facies, which display significant scatter in age and chemistry. We associate the observed decoupling of zircon systematics in high-grade non-metamict zircon with disturbance processes related to differences in behaviour of non-formula elements (i.e. Pb, Th, U, Ti) at high-temperature conditions, notably differences in compatibility within the crystal structure

PRELIMINARY U-Pb LA-ICPMS ZIRCON ANALYSES FROM THE GOIAS COMPLEXES: SHRIMP COMPARISON AND INTRUSION AGE

Cana Brava, Niquelàndia and Barro Alto are three mafic-ultramafic layered intrusions (from N to S) which form a - 350 km, NNE-trend belt within the Brasilia Belt (Goiés state, Brazil). Presently, their intrusion ages and geologica! evolution are stili debated. The Niquelàndia and Barro Alto complexes are forrned by two main sequences: the upper sequence and the lower one. Some authors suggest that the two sequences represent two separate intrusions: the upper sequence would be a Mesoproterozoic intrusion at - 1.3 Ga, whereas the lower sequence a Neoproterozic one at - 790 Ma. According to this interpretation, the two sequences were re-crystallized by Neoproterozoic metamorphism and exhumed and juxtaposed during the Brazilian event of formation of the Gondwana continent. Another model suggests that the two sequences are part of the same intrusion, which occurred during the Neoproterozoic and was exhumed during the Brazilian event. New U-Pb SHRIMP-II zircon analyses were perforrned at the Universidade de Sào Paulo from samples from Cana Brava and Barro Alto, the two least-known complexes in order to clarify the sequence of events that led to their forrnation. Analyses were then replicated at the CIGS of the Università di Modena e Reggio Emilia using a X Series" quadrupole ICP-MS coupled with a New Wave UP-213 Nd:YAG laser ablation system. Zircons were sampled through a 40 micron spot (static mode), using a He flux of 0.6 l/min, with an energy density of - 6 J/cm2. Daily instrument calibration was perforrned with the NIST 610 standard, monitoring also the oxide production rate {232Th160/232Th << 0.01 %). Laser-induced elemental fractionation was corrected by repeated analyses of the standard zircon TEMORA2 (Black et al., 2004). A secondary reference materia! (zircon CZ3) was used to check the precision and accuracy of the corrections. Our LA-ICP-MS data are preliminary, but very promising being the accuracy of the measured ratio within the SHRIMP variability. We are currently working to improve the precision of our methodology, which however is now comparable with literature LA-ICP-MS data (propagated 2SE - 2-6%; Horstwood et al., 2008). Overall, the isotopic data of Cana Brava and Barro Alto complexes previde for a coeval Neoproterozoic intrusion age at - 790 Ma. These ages are consistent with those reported in literature for Niquelàndia. Mesoproterozoic ages, consistent with the forrnation age of the metavolcanic-metasedimentary sequence in magmatic contaci with the complexes, were found in inherited zircon cores. Our data clearly show that the Goiàs complexes are formed by single bodies intruded during the Neoproterozoic at -790 Ma and that the hypothesis of two separate intrusions juxtaposed by tectonic must be discarded