SMARTBOX is a project funded by the Bio-Based Industries Joint Undertaking (BBI JU), a public-private partnership between the European Union (H2020 program) and the Bio-based Industries Consortium (BIC). The aim of the project is the development of new economic and bio-based processes for high-value compounds production starting from lignocellulose. The production processes, that SMARTBOX wants to realize, are built on the exploitation of the synergy between two highly selective technologies: Reductive Catalytic Fractionation and oxidative biocatalysis The work done in the thesis internship consisted in providing the crystallographic structure of the enzymes within an engineering program supported by a computational platform to predict the beneficial mutations for the stability, activity and selectivity of the biocatalyst. Two enzymes that are at different development levels have been treated during the internship. The first is HMFO, an enzyme that catalyzes all the reaction steps for HMF conversion into FDCA, an important monomer for bioplastics. This enzyme has already been engineered before, but in order to continue with further optimization, the structure of the HMFO8xb mutant, the most performance variant to date, was requested. The second enzyme is to be applied in the conversion of isoeugenol to vanillin. In this case it was required to identify a variant of IEMO that had all the characteristics to represent the starting enzyme to be evolved. The choice fell on the homologue NOV1 because, albeit less active on isougenol compared to the IEMO variants, it has no stability problems and its structure was already determined.
SMARTBOX è un progetto finanziato dalla Bio-Based Industries Joint Undertaking (BBI JU), un partenariato pubblico-privato tra l'Unione europea (programma H2020) e il Bio-based Industries Consortium (BIC). L’obiettivo del progetto è quello di sviluppare nuovi processi biobased ed economici per la produzione di molecole di interesse industriale partendo dalla lignocellulosa. I processi produttivi che SMARTBOX vuole realizzare sono progettati per sfruttare la sinergia tra due tecnologie altamente selettive: frazionamento catalitico riduttivo e biocatalisi ossidativa. Il lavoro svolto nell’internato di tesi consisteva nel fornire la struttura cristallografica degli enzimi all’interno di un programma di ingegnerizzazione supportata da una piattaforma computazionale per la predizione di mutazioni potenzialmente benefiche per la stabilità, l’attività e la selettività del biocatalizzatore. Sono stati trattati, in particolare, due enzimi che si trovano a livelli di sviluppo differenti. Il primo è HMFO, enzima che catalizza tutti gli step di reazione per la conversione HMF in FDCA, un importante monomero per bioplastiche. Questo enzima è già stato ingegnerizzato precedentemente ma per poter proseguire con una ulteriore ottimizzazione è stata richiesta la struttura del mutante HMFO8xb, la variante più prestazionale ad oggi ottenuta. Il secondo enzima è da applicare nella conversione di isoeugenolo in vanillina. In questo caso era richiesto di individuare una variante di IEMO che presentasse tutte le caratteristiche per rappresentare l’enzima di partenza da evolvere. La scelta è ricaduta sulla proteina omologa NOV1 in quanto, sebbene meno attiva sull’isoeugenolo rispetto alle varianti IEMO, non possiede problemi di stabilità e la sua struttura era già stata determinata.
Ingegnerizzazione guidata computazionalmente di biocatalizzatori ossidativi applicabili in processi di valorizzazione della biomassa
CAROLI, JONATAN
2019/2020
Abstract
SMARTBOX is a project funded by the Bio-Based Industries Joint Undertaking (BBI JU), a public-private partnership between the European Union (H2020 program) and the Bio-based Industries Consortium (BIC). The aim of the project is the development of new economic and bio-based processes for high-value compounds production starting from lignocellulose. The production processes, that SMARTBOX wants to realize, are built on the exploitation of the synergy between two highly selective technologies: Reductive Catalytic Fractionation and oxidative biocatalysis The work done in the thesis internship consisted in providing the crystallographic structure of the enzymes within an engineering program supported by a computational platform to predict the beneficial mutations for the stability, activity and selectivity of the biocatalyst. Two enzymes that are at different development levels have been treated during the internship. The first is HMFO, an enzyme that catalyzes all the reaction steps for HMF conversion into FDCA, an important monomer for bioplastics. This enzyme has already been engineered before, but in order to continue with further optimization, the structure of the HMFO8xb mutant, the most performance variant to date, was requested. The second enzyme is to be applied in the conversion of isoeugenol to vanillin. In this case it was required to identify a variant of IEMO that had all the characteristics to represent the starting enzyme to be evolved. The choice fell on the homologue NOV1 because, albeit less active on isougenol compared to the IEMO variants, it has no stability problems and its structure was already determined.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/11718