Repurposing designed mutants: a valuable strategy for computer-aided laccase engineering – the case of POXA1b

The broad specificity of laccases, a direct consequence of their shallow binding site, makes this class of enzymes a suitable template to build specificity toward putative substrates. In this work, a computational methodology that accumulates beneficial interactions between the enzyme and the substrate in productive conformations is applied to oxidize 2,4-diamino-benzenesulfonic acid with POXA1b laccase. Although the experimental validation of two designed variants yielded negative results, most likely due to the hard oxidizability of the target substrate, molecular simulations suggest that a novel polar binding scaffold was designed to anchor negatively charged groups. Consequently, the oxidation of three such molecules, selected as representative of different classes of substances with different industrial applications, significantly improved. According to molecular simulations, the reason behind such an improvement lies in the more productive enzyme–substrate binding achieved thanks to the designed polar scaffold. In the future, mutant repurposing toward other substrates could be first carried out computationally, as done here, testing molecules that share some similarity with the initial target. In this way, repurposing would not be a mere safety net (as it is in the laboratory and as it was here) but rather a powerful approach to transform laccases into more efficient multitasking enzymes.This work was funded by INDOX (KBBE-2013-7-613549) European project and CTQ2013-48287-R Spanish National Project. V. G. and E. M. acknowledge Università degli Studi di Napoli and Generalitat de Catalunya for their respective predoctoral fellowships.Peer ReviewedPostprint (author's final draft

Wild-Type and mutated laccases for a green industry

In the frame of white biotechnology, enzymes attract an enormous attention both for their potential and for the recent social and economical interest in green chemistry. Thanks to the improved knowledge of production biochemistry, fermentation processes, and recovery methods, an increasing number of enzymes can be affordably produced. The majority of currently used industrial enzymes are the hydrolytic ones, being used for the degradation of various natural substances. Also, various oxidative enzymes, primarily laccases are used in industries such as the starch, textile, detergent and baking industries, and they represent a second important group of enzymes. This project has been focused on the recombinant expression of the industrially attractive laccase POXA1b from Pleurotus ostreatus in Pichia pastoris and on exploiting its industrial application in order to substitute some chemical industrial processes with a better eco-friendly ones. An economical analysis of the recombinant POXA1b laccase production process, in terms of productivity and cost has been evaluated. Furthermore, computational analysis and site-specific mutagenesis of the POXA1b were performed to increase the enzymatic performances towards selected substrates. A POXA1b laccase immobilization process was optimized by statistic methods (Response Surface Methodology) in order to increment the operational stability of the enzyme in specific processes. Applications of the recombinant enzyme were then developed: (i) reduction of the phenol contents inside the fruit juices by immobilized laccase POXA1b; (ii) synthesis of a new eco-friendly dyes for the staining of different textile materials (nylon, cotton and wool) and (iii) synthesis of a new eco-friendly dye for proteins visualization on the polyacrilamide gels; (iv) cotton fiber functionalization with an anti-adhesive and anti-oxidant dye polymer

Optimizing enzymatic dyeing of wool and leather

This work reports on the environmental friendly enzymatic dyeing of wool and leather performed at low temperature and mild pH conditions without any dyeing auxiliaries. The substrates have been dyed with “in situ” generated pigment by means of laccase-catalyzed oxidative coupling of dye modifier 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and dye precursor 1,3-benzenediol in a batchwise process. The process reaction variables (laccase, precursor and modifier concentrations, temperature and dyeing time) were optimized by response surface methodology using an appropriate experimental design. The temperature, precursor concentration, interaction between precursor and modifier and time are the most important factors in the dyeing process. The best-optimized wool dyeing conditions (2 h reaction time, 50 μl laccase (0.1 U), 500 mM precursor, 10 mM modifier at 40 °C) were then successfully applied onto leather material. The enzymatic-dyeing optimized process can be successfully performed on wool and leather at low temperature and mild pH obtaining different hues and depths of shades by varying the modifier concentration and time. The colouring enzymatic system has a good reusability (which has a huge advantage in terms of cost reduction) and washing durability and is comparable in terms of fastness properties to the traditional dyeing process for both wool and leather.The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) for funding the project UID/CTM/00264/2019 and A. Zille contract IF/00071/2015

Monte Carlo Techniques for Drug Design: The Success Case of PELE

This chapter summarizes the most representative software packages that readily allow running Monte Carlo (MC) simulations in relevant scenarios for drug design. It explores in detail the Protein Energy Landscape Exploration (PELE) program, providing first the main characteristics of the technique, followed by an analysis of the different application studies in mapping protein‐ligand interactions. The ligand, formed by a rigid core and a set of rotatable side chains, is perturbed by translating and rotating it. PELE creates a list of perturbation poses, and then chooses the one with the lowest system energy. PELE was originally designed to map ligand migration pathways: its first applications aimed at finding exit pathways starting from ligand‐bound crystallographic structures. Additional applied studies have centered on modeling enzymatic mechanisms and engineering; the same techniques applied in mapping protein‐drug interactions can be used in the study of substrate recognition by enzymes.Along the development of PELE in the last years, we gratefully acknowledge financial support from the European Union (in particular from the ERC program) and from the Catalan and Spanish Governments. In addition we want to thank all present and past members from the EAPM lab. at BSC for their dedication and work.Peer ReviewedPostprint (author's final draft

Vulnerabilidade das microrregiões do Nordeste brasileiro à pandemia do novo coronavírus (SARS-CoV-2)

Report from “Observatório COVID19 - Grupo: Redes de Contágio – Laboratório de Estudos de Defesa” for the Northeast Region of Brazil. We combined data on confirmed cases of the new coronavirus (SARS-CoV-2) as reported by public authorities by 02/04 and structural analyses of road transport networks within and between the nine States of Northeast Brazil to predict the potential influence of the 187 micro-regions on the geographic propagation of the pandemic.  Este é o primeiro relatório do Observatório COVID1920 - Grupo: Redes de Contágio – Laboratório de Estudos de Defesa para a região Nordeste do Brasil. Combinamos dados de casos confirmados do novo coronavírus (SARS-CoV-2) para o Nordeste, conforme disponível até o dia 02/04, com análises estruturais da rede de rotas rodoviárias intra e interestaduais para estimarmos a vulnerabilidade e potencial influência das microrregiões nordestinas na propagação da doença

Potenzialità e limiti per la valorizzazione delle carni di suini di razza autoctona Nero Pugliese

L’obbiettivo della tesi ha riguardato l’analisi delle potenzialità e i limiti dell’allevamento e dei prodotti ottenuti dalla razza suina autoctona Nero Pugliese, discendente dalla grande famiglia “Apulo-Calabrese”. Nella prima parte sono elencate le principali razze del panorama suinicolo italiano utilizzate per la produzione di suino pesante, ovvero Large White, Landrace e Duroc. Sono successivamente prese in esame le sei razza autoctone italiane (Casertana, Cinta Senese, Mora Romagnola, Apulo-Calabrese, Nero Siciliano e Sarda) attualmente riconosciute e registrate a livello nazionale. Ciascuna razza è stata descritta nello specifico con particolare riferimento alle origini e alle loro caratteristiche morfologiche. La parte principale della tesi riguarda le caratteristiche e l’area nella quale sono attualmente allevati i suini di razza Nero Pugliese. In questo contesto, è stata considerata un’azienda locale (Salumi Martina Franca s.r.l.) produttrice di salumi provenienti da questa razza, e gli studi presenti in letteratura sulla qualità del suino Apulo-calabrese. Sono stati considerati gli esiti del progetto Suis, che ha previsto una serie di azioni innovative per rendere più sostenibile la biodiversità genetica e gastronomica delle razze suine italiane autoctone. La parte finale ha riguardato gli esiti di un questionario, utile a comprendere le aspettative del consumatore nei confronti dei salumi prodotti con carni ottenute da suini di razza Nero Pugliese in termini di proprietà organolettiche, genuinità, principi nutrivi, biodiversità, benessere dell'animale e valorizzazione del territorio. Sulla base dei risultati ottenuti, è stata realizzata un’analisi swot per identificare le principali limitazioni ed i punti di forza come strumento per l’adozione di strategie per valorizzare il suino Nero Pugliese nel contesto locale e nazionale

How does coevolution in mutualistic networks shape the fitness and persistence of species?

Interações ecológicas positivas mutualismos - são uma das principais forças que sustentam os ecossistemas mais ricos da Terra. No entanto, entender como mutualismos sustentam a biodiversidade da Terra é desafiador por três motivos principais. Primeiro, na natureza, dezenas a milhares de espécies interagem formando redes mutualísticas. Em segundo, essas interações mutualísticas podem dar origem a pressões seletivas recíprocas entre as espécies envolvidas, resultando em mudanças evolutivas recíprocas i.e. coevolução. A coevolução é um processo chave que molda os atributos de espécies, os quais, por sua vez, mediam como mutualismos influenciam a principal medida biológica que determina a persistência das espécies: a aptidão média de seus indivíduos, i.e, a capacidade de sobrevivência e se reprodução dos indivíduos de uma espécie. Terceiro, os efeitos da coevolução na aptidão média e persistência das espécies podem se manifestar em diferentes escalas espaciais. Nesta tese, abordamos esses desafios para entendermos como a coevolução em redes mutualísticas molda a aptidão média e a persistência das espécies em diferentes escalas espaciais. Primeiro, mostramos que, em redes mutualísticas locais, os efeitos evolutivos diretos entre as espécies podem se propagar e influenciar indiretamente outras espécies na rede. Esses efeitos evolutivos indiretos dificultam a capacidade das espécies de se adaptar tanto aos seus parceiros mutualísticos quanto a outras fontes de pressões seletivas no ambiente, moldando a aptidão média das espécies. Em seguida, integramos a coevolução mutualística em escala local com a dinâmica de colonização e extinção de metacomunidades em uma escala regional. Nossos resultados mostram que a coevolução mutualística local pode homogeneizar os atributos das espécies através da paisagem, facilitando colonizações, expandindo a distribuição, e aumentando a persistência mesmo quando as espécies estão mal adaptadas ao ambiente abiótico local. Juntos, nossos resultados mostram que a coevolução em redes mutualísticas é uma grande força que molda o a aptidão média e a persistência das espécies através de diferentes escalas espaciais.Mutualistic interactions - ecological interactions with a net positive effect to both interacting species - are one of the major forces that sustains many of Earth\'s richest ecosystems. Yet, understanding how does mutualistic interactions shape Earth\'s biodiversity is challenging for three main reasons. First, in mutualistic communities from dozen to thousands of species interact forming mutualistic networks. Second, these mutualistic interactions can give rise to reciprocal selective pressures between interacting species, resulting in reciprocal evolutionary changes - coevolution. Coevolution is a key process that drive species traits that, ultimately, mediate the net effect of mutualistic interactions on the main biological currency that determines the persistence of species: the fitness of its individuals, i.e. the ability of the individuals of a given species to survive and reproduce. Third, the effects of coevolution on the average fitness and persistence of species can manifest across different ecological scales, for instance, across different spatial scales. Here, we tackle these challenges to understand how coevolution in mutualistic networks shape the average fitness and persistence of species across different spatial scales. We first show that in local mutualistic networks the direct evolutionary effects between species can cascade and indirectly affect other species in the network. These indirect effects hinder the ability of species to adapt to both mutualistic partners and other sources of selective pressures in the environment, shaping species average fitness. Then, we proceeded to integrate mutualistic coevolution at a local scale, with the colonization and extinction dynamics of metacommunities at a regional scale. Our results show that local mutualistic coevolution can homogenize species traits across landscapes, facilitating colonization, range expansions and persistence even when species are maldapted to the local abiotic environment. Together, our results show that coevolution in mutualistic networks can be a major force that shape the average fitness and persistence of species across different spatial scales