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Supporting Information available at: https://chemistry-europe.onlinelibrary.wiley.com/action/downloadSupplement?doi=10.1002%2Fcssc.202202374&file=cssc202202374-sup-0001-misc_information.pdf, Aiming to reduce the toxicity and operational costs often associated to chemical processes, the enzymatic synthesis is applied herein as a sustainable route for producing polyesters. The use of NADES components as a source of monomers for the synthesis of polymers through lipase-catalysed esterification in an anhydrous medium is detailed for the first time. Three NADES composed by glycerol and an organic base, or acid, were used to produce polyesters, through polymerization reactions catalysed by Aspergillus oryzae lipase. High polyester conversion rates (above 70 \%), containing at least 20 monomeric units (glycerol: organic acid/base (1:1)), were observed by MALDI-TOF analysis. The NADES monomers' capacity for polymerization, along with their non-toxicity, cheap cost, and simplicity of production, sets up these solvents as a greener and cleaner approach for the synthesis of high value-added products., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit. David S. Freitas also thanks to FCT for funding (SFRH/BD/147190/2019)., info:eu-repo/semantics/publishedVersion

The world of cosmetics is an always-evolving field with constant updates on its formulation components. The current reality asks for an ever-increasing need for natural and sustainable replacements for synthetic compounds in all fields of modern consumer products. However, the research and development stages of finding these alternatives can be an expensive, time-consuming, and often wasteful process that turns this task into a laborious procedure. This study introduces the development of a computational methodology that will aid the research of silicone alternatives, disclosing their structural performance in a formulation. Additionally, an equilibration protocol was developed to measure the distribution and densities of these silicone alternatives to determine how they behave in relation to their counterparts, using molecular dynamics simulations. Two systems were tested, A and B, where the former is composed of one ester (Dipentaerythrityl Hexa C5 Acid Ester) and the latter by an ester combined with an alkane (Triheptanoin and C13-Isoalkane); all three molecules are commercially available and widely used. Both systems were subjected to a 3-step thermal regulation strategy. The systems went through an initial simulation at 25 °C and at 70 °C, then a temperature switch took place (25 °C « 70 °C), then a shock to 200 °C, and finally a Simulated Annealing protocol reaching 250 °C. In the end, all systems converged towards micelle-like structures. These results come to further ascertain the position of computational chemistry and Molecular Dynamics Simulations as an important part of R&D processes in modern sciences and investigation., This research was developed with the support of computing facilities pro vided by the project: “Search-ON2: Revitalization of HPC infrastructure of UMinho” (NORTE-07- 0162-FEDER-000086), co-funded by the North Portugal Regional Operational Programme (ON.2— O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF)., info:eu-repo/semantics/publishedVersion

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms232213999/s1., Onychomycosis is the most common nail fungal infection worldwide. There are several therapy options available for onychomycosis, such as oral antifungals, topicals, and physical treatments. Terbinafine is in the frontline for the treatment of onychomycosis; however, several adverse effects are associated to its oral administration. In this work, innovative keratin-based carriers encapsulating terbinafine were designed to overcome the drawbacks related to the use this drug. Therapeutic textiles functionalized with keratin-based particles (100% keratin; 80% keratin/20% keratin-PEG) encapsulating terbinafine were developed. The controlled release of terbinafine from the functionalized textiles was evaluated against different mimetic biologic solutions (PBS buffer—pH=7.4, micellar solution and acidic sweat solution—pH=4.3). The modification of keratin with polyethylene glycol (PEG) moieties favored the release of terbinafine at the end of 48 h for all the solution conditions. When the activity of functionalized textiles was tested against Trichophyton rubrum, a differentiated inhibition was observed. Textiles functionalized with 80% keratin/20% keratin-PEG encapsulating terbinafine showed a 2-fold inhibition halo compared with the textiles containing 100% keratin-encapsulating terbinafine. No activity was observed for the textiles functionalized with keratin-based particles without terbinafine. The systems herein developed revealed therapeutic potential towards nail fungal infections, taking advantage of keratin-based particles affinity to keratin structures and of the keratinase activity of T. rubrum., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020. AR thanks to Portuguese Foundation for Science and Technology (FCT) for is con tract under the CEEC-Individual—4th Edition with the reference 2021.02803.CEECIND., info:eu-repo/semantics/publishedVersion

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssuschemeng.2c01422., Quercus suber cork generates bioactive components, such as phenolic acids, terpenoids, and tannins, with excellent biological properties, including antioxidant, anti-inflammatory, and antiaging activities. Aiming to design environmentally benign processes to eliminate, or reduce, the use of toxic chemicals, we propose the green extraction of bioactives from cork using natural deep eutectic solvents (NADES). Several deep eutectic mixtures were developed, through the mixture of natural compounds, namely, lactic acid, glycerol, ethylene glycol, sodium citrate, and sodium lactate, chosen according to their origin, toxicity, biocompatibility, polarity, and pH. The results revealed higher extraction yields when using NADES instead of harsh solvents like dioxane, with the extraction process governed by several physical--chemical parameters, including pH, polarity, viscosity, and density, and also by the method of extraction. Acidic NADES composed of lactic acid and glycerol, or sodium citrate, extracted a greater amount of aromatic compounds, terpenoids, and fatty acids and their derivatives. More basic eutectic mixtures, composed of sodium lactate and a polyol (ethylene glycol or glycerol), extracted predominantly low molecular weight polar compounds. The extracts range encompassed by the developed NADES, together with the associated nontoxicity, low price, and ease of preparation, establish these solvents as a green approach to extract high added-value compounds from cork., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit. David S. Freitas also thanks FCT for funding (SFRH/BD/147190/2019)., info:eu-repo/semantics/publishedVersion

The present disclosure relates to the development of Natural Deep Eutectic Solvents (NADES) using natural products, like sugars, organic bases and organic acids, as starting compounds. These solvents can be used for the extraction of bioactive compounds from natural sources, such as cork; agricultural wastes, including grape seed and peels; tomato; olive oil; and plants (teas, eucalyptus, lavender, or others), and from fish skin and bones. The extractives could then be further formulated with active topical cosmetic components to prepare cosmetic compositions., info:eu-repo/semantics/publishedVersion

The demand for adequate and ecologically acceptable procedures to produce the most differentiated products has been growing in recent decades, with enzymes being excellent examples of the advances achieved so far. Lipases are astonishing catalysts with a vast range of applications including the synthesis of esters, flavours, biodiesel, and polymers. The broad specificity of the substrates, as well as the regio-, stereo-, and enantioselectivity, are the differentiating factors of these enzymes. Structural modification is a current approach to enhance the activity of lipases. Chemical modification of lipases to improve catalytic performance is of great interest considering the increasingly broad fields of application. Together with the physical immobilization onto solid supports, different strategies have been developed to produce catalysts with higher activity and stability. In this review, practical insights into the different strategies developed in recent years regarding the modification of lipases are described. For the first time, the impact of the modifications on the activity and stability of lipases, as well as on the biotechnological applications, is fully compiled., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit. Jennifer Noro also thanks FCT for funding (SFRH/BD/121673/2016)., info:eu-repo/semantics/publishedVersion

The laccase-assisted grafting of poly(tyrosine) is performed aiming to impart shrink-resistant properties to the wool materials. Laccase is able to catalyze the oxidation of tyrosine into dityrosine efficiently. When the reaction is performed using poly(ethylene glycol) (PEG) as the template, the tyrosine oxidation gives rise to differentiated oxidation products, including di-, tri-, and tetra-tyrosine, promoting a higher grafting onto the wool fibers. The characterization of the grafted wool fibers and fabrics reveals some loss of crystallinity (about 4% of crystallization index) and thermal resistance (decreasing from 232°C to 224°C) resulting from the grafting process. However, the new bonds established between the new oxidation products and the amino groups of the wool promote an improvement, not only of the shrinkage properties (1.87% of area shrinkage) but also of their mechanical behavior (increasing by 45.28%). The strategy herein presented demonstrates the potentiality to be applied as a green process for wool anti-shrinkage without compromising the fibers performance., This study was supported by National Natural Science Foundation of China (Grant No. 52003108) and the Fundamental Research Funds for the Central Universities (JUSRP121027). This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit., info:eu-repo/semantics/publishedVersion

Tese de Doutoramento em Engenharia Química e Biológica, A necessidade de implementação de processos mais verdes e amigos do ambiente, continua a ser uma temática de grande importância para a comunidade científica. Das diferentes estratégias disponíveis para modificar e estabilizar compostos hidrofóbicos, o uso de enzimas para sua síntese e de nanoemulsões para o seu encapsulamento, são as abordagens mais comuns. Nesta tese, foram desenvolvidas nanoemulsões compostas por oligossacarídeos lipofílicos para a encapsulação de um composto hidrofóbico, o metotrexato (MTX). Enzimas, nomeadamente lipases ou proteases, foram aplicadas como catalisadores para a biossíntese de compostos hidrofóbicos com o objetivo de desenvolver alternativas ecológicas para futuras aplicações industriais. Primeiramente, a síntese de ciclo-oligossacarídeos lipofílicos (β-ciclodextrina, γ-ciclodextrina e ciclosoforaose) foi realizada através da ligação de cadeias de palmitoílo aos grupos hidroxilo. As estruturas hidrofóbicas desenvolvidas revelaram propriedades emulsificantes quando submetidas a ultrassons. A sua estabilidade, baixo tamanho e baixa polidispersividade demonstraram o alto potencial destas emulsões como novos dispositivos para o encapsulamento e libertação do MTX. Posteriormente, foram usadas duas abordagens enzimáticas para produzir pró-drogas de MTX. Inicialmente, lipases da Thermomyces lanuginosus (TL) e da Cândida antarctica B (CALB) foram utilizadas como catalisadores para a reação do MTX com triacilgliceróis e ciclodextrinas. Um meio aquoso, sob a ação de ultrassons, revelou ser uma estratégia eficaz para a síntese de pró-drogas de MTX, com o acoplamento de compostos não tóxicos. Na segunda abordagem, foi utilizada a α-quimiotripsina do pâncreas de bovino para a auto-polimerização do MTX. A protease revelou capacidade de produzir oligómeros de MTX até um máximo de 6 unidades. Foi assim estabelecida uma nova via para a produção de uma pró-droga de MTX polimérico, salientando o potencial desta protease para a catálise de substratos não naturais. Ao longo desta tese, foi também avaliado o efeito da modificação química de enzimas nas suas propriedades catalíticas. Numa primeira abordagem, as modificações foram realizadas através da PEGilação das lipases TL, CALB e de uma cutinase da fusarium solani pisi. Verificou-se que enquanto a PEGilação melhorou a atividade hidrolítica da CALB e da cutinase, a atividade da lipase TL permaneceu inalterada. O efeito dessa modificação foi também avaliado na atividade de polimerase das enzimas, na síntese de um poliéster, o poli(etileno glutarato). Das três enzimas estudadas, a lipase TL PEGuilada foi responsável pela maior produção de polímero, juntamente com um maior grau de polimerização (DP), em comparação com a forma nativa da enzima. Ambas as formas da CALB exibiram atividade de polimerase semelhante. O efeito da PEGuilação na cutinase não foi significativo, sendo detetado um pequeno aumento na conversão do polímero, em comparação com a enzima nativa, porém com menor DP. A segunda abordagem, implementada para a melhoria das propriedades catalíticas das lipases, baseou-se na ligação de pequenos ligandos hidrofóbicos, aldeídos e isotiocianatos, à lipase TL. O efeito dos ligandos na atividade hidrolítica e na estabilidade da enzima foi extensivamente estudado, tendo os dados revelado maior estabilidade a diferentes temperaturas e pHs, após a sua modificação. Além disso, a lipase modificada com 4 cadeias de dodecil (a partir do dodecil aldeído) apresentou excelente atividade catalítica, avaliada por reação com substratos de diferentes tamanhos de cadeia alifática (p-nitrofenil acetato a p-nitrofenil palmitato). Posteriormente, foi avaliada a atividade das enzimas na transesterificação do p-nitrofenil palmitato e na esterificação do ácido oleico, usando como substratos álcoois com diferentes tamanhos de cadeia (de metanol a eicosanol). A lipase modificada com 4 cadeias de dodecil, apresentou uma atividade superior, para ambas as reações, em comparação com a lipase nativa. Este incremento foi diretamente proporcional ao tamanho do álcool utilizado como substrato. Estas estratégias demonstraram a grande potencialidade das enzimas modificadas na biossíntese de produtos industriais de alto valor acrescentado., The pursuit for green and environmentally friendly processes is still a great challenge among the scientific community. From the different strategies available to modify and stabilize hydrophobic compounds, the use of enzymes for their synthesis and of nanoemulsions for their encapsulation, are the most common approaches. In this thesis, nanoemulsions composed by lipophilic oligosaccharides were developed for the encapsulation of an hydrophobic compound, methotrexate (MTX). Enzymes, namely lipases or proteases, were applied as catalysts for the biosynthesis of hydrophobic compounds aiming to develop new eco-friendly routes for future industrial applications. Firstly, the synthesis of lipophilic cyclo-oligosaccharides (β-cyclodextrin, γ-cyclodextrin and cyclosophoraose) was carried out through the chemical coupling of palmitoyl chains to the hydroxylic groups. The hydrophobic structures showed emulsifying properties under ultrasonic energy. Their stability, narrow size, and low polydispersity demonstrated the high potential as new nanodevices for encapsulation and delivery of MTX. Afterward, two different enzymatic approaches were used to produce MTX-prodrugs. In a first approach, lipases from Thermomyces lanuginosus (TL) and from Candida antarctica B (CALB) were applied as catalysts for the reaction of MTX with triacylglycerols and cyclodextrins. Aqueous medium, together with ultrasounds showed to be a successful route for the synthesis of MTX ester prodrugs, with the coupling of non-toxic compounds. The second approach included the use α-chymotrypsin from bovine pancreas for the self-polymerization of MTX. The protease revealed the ability to produce oligomers up to 6 MTX units. A novel pathway to produce a polymeric MTX prodrug was established, unravelling the potential of this protease for the catalysis of non-natural substrates. Along this thesis, the effect of the chemical modification of enzymes on their catalytic properties was also assessed. In a first approach, the chemical modifications were performed throughout PEGylation of lipase TL, CALB and cutinase from fusarium solani pisi. The data revealed that whereas PEGylation improved the hydrolytic activity of CALB and cutinase, it did not alter the activity of lipase TL. The effect of this modification was also evaluated throughout their polymerase activity in the synthesis of a polyester, poly(ethylene glutarate). From the three enzymes, PEGylated lipase TL was responsible for the highest polymer production with the highest degree of polymerization (DP), in comparison to the native form. Both CALB forms displayed similar polymerase activity. The effect of PEGylation on cutinase was not significant being detected a small increase of polymer conversion, however with lower DP, comparing with the native enzyme. The second approach for the improvement of lipases’ catalytic properties covered the grafting of small hydrophobic aldehydes and isothiocyanates to the lipase TL. The effect of the linkers was extensively studied in terms of hydrolytic activity and stability, and the data obtained revealed higher temperature and pH stability after modification. Moreover, the lipase grafted with 4 dodecyl chains (from dodecyl aldehyde) showed outstanding catalytic activity, evaluated against a panoply of substrates differing in the aliphatic chain size (p-nitrophenyl acetate to p-nitrophenyl palmitate). Afterward, the enzymes’ activity in the transesterification of p-nitrophenyl palmitate, and in the esterification of oleic acid was evaluated. Chain size differentiated alcohols (methanol to eicosanol) were tested as substrates of the reactions. The modified lipase (with 4 dodecyl chains) showed superior activity for both reactions comparing to the native lipase. This increment was more directly proportional to the size of the alcohol used for the reaction. These strategies revealed high potentiality for the use of modified enzymes on the biosynthesis of industrial added-value products.