No abstract

Grounding of thousands of newly fledged petrels and shearwaters (family Procellariidae) in built‐up areas due to artificial light is a global problem. Due to their anatomy these grounded birds find it difficult to take off from built‐up areas and many fall victim to predation, cars, dehydration or starvation. This research investigated a combination of several factors that may influence the number of Manx Shearwater Puffinus puffinus groundings in a coastal village of Scotland located close to a nesting site for this species. A model was developed that used meteorological variables and moon cycle to predict the daily quantity of birds that were recovered on the ground. The model, explaining 46.32% of the variance of the data, revealed how new moon and strong onshore winds influence grounding. To a lesser extent, visibility conditions can also have an effect on grounding probabilities. The analysis presented in this study can improve rescue campaigns of not only Manx Shearwaters but also other species attracted to the light pollution by predicting conditions leading to an increase in the number of groundings. It could also inform local authorities when artificial light intensity needs to be reduced.

Cell-penetrating peptides (CPPs) have been applied as novel transport systems with the ability to facilitate the delivery of peptides, proteins, and oligonucleotides into cells. Herein, we designed different fusion proteins composed by pig odorant binding protein (OBP-I) and three CPPs, namely Tat, pVEC and Pep-1. A new methodology using liposomes as reservoirs and OBP:CPPs as carriers was developed as an advanced system to capture odorant molecules. 1-aminoanthracene (1-AMA) was used as a model molecule to evaluate the transduction ability of OBP:CPPs into the reservoirs. The transduction efficiency was dependent on the initial capacity of OBP:CPPs to bind 1-AMA and on the penetration of liposomes promoted by the CPPs. An encapsulation efficiency of 42% was obtained with OBP:Tat fusion protein. The presence of Tat peptide increased the 1-AMA transduction of 1.3 and 2.5 fold compared with Pep-1 and pVEC, respectively. This work expands the application of OBPs and CPPs on the design of promising capture and delivery systems for textile and cosmetic applications.

In this work we explored PEGylation as an efficient strategy to improve esterase's catalytic performance. For this, we PEGylated three esterases, namely lipase from Candida antarctica B (CALB), lipase from Thermomyces lanuginosus (TL) and cutinase from Fusarium solani pisi (CUT) and evaluated their catalytic performance by using the biosynthesis of poly(ethylene glutarate) as model reaction. After PEGylation with a 5 kDa aldehyde‐PEG, CALB and cutinase revealed an increase of activity against p‐nitrophenyl butyrate hydrolysis (2‐fold of increase for CALB and 4‐fold of increase for cutinase). Unmodified and PEGylated lipase TL displayed however similar activity results. The polymerase activity of native and PEGylated esterases was also assessed. The data revealed a higher polymerase activity for the lipase TL and cutinase PEGylated forms (88 % conversion for PEG‐lipase TL and 34 % for PEG‐cutinase). Molecular dynamics were used to evaluate the effect of PEG on the geometry of the active site of enzymes with lid domain (TL and CALB). These studies corroborate the experimental data revealing a more open active site cavity for the PEGylated catalysts facilitating the catalysis.

In this work, the anchorage of pig odorant binding protein (OBP-I) into liposomal membrane was promoted by the fusion of OBP-I with the anchor SP-DS3 peptide and with the (GQ) spacer. The presence of the (GQ) spacer in the construct confers flexibility to the protein and increases the distance between the OBP binding site and the liposomal surface. The engineered proteins, OBP::SP-DS3 and OBP::(GQ)::SP-DS3, were produced in Escherichia coli BL21(DE3) and characterized by circular dichroism spectroscopy and MALDI-TOF. The functionalization of liposomes with the OBP proteins was performed through ethanol injection, and similar liposomal anchorage (∼92-97%) was found for both OBP constructs. The effect of OBPs' proximity to the liposomes membrane on 1-aminoanthracene (1-AMA, model ligand) transduction was evaluated by measuring the amount of 1-AMA transduced into liposomes by fluorescence spectroscopy. While protein flexibility, given by the presence of the (GQ) spacer, seems to influence the binding efficiency, ∼45% for OBP::(GQ)::SP-DS3 and ∼29% for OBP::SP-DS3, the distance between the proteins' binding site and the liposomal membrane determines their ability to transduce the 1-AMA into the liposomes (∼23% for OBP::SP-DS3 and ∼19% for OBP::(GQ)::SP-DS3). The anchorage capacity and proximity effect were confirmed by an experimental control where the wild-type (wt) OBP was added to the liposomes, resulting in low 1-AMA transduction (∼3.5%) and low binding to OBPwt (∼9%). These findings evidence the effect of anchorage, carrier protein's flexibility, and proximity as key features for the entrapment of molecules into the liposomal membrane. The developed OBP-based devices are thus promising anchorage systems for the capture and storage of odors with potential applications in textile and cosmetic industries.

Perspiration as a response to daily activity and physical exercise results in unpleasant odors that cause social unrest and embarrassment. To tackle it, functional textiles incorporating fragrances could be an effective clothing deodorizing product. This work presents two strategies for the release of β-citronellol from functionalized cotton with carbohydrate-binding module (CBM)-based complexes (OBP::GQ 2 0 ::CBM/β-citronellol−approach 1 and CBM::GQ 20 ::SP-DS3-liposome/β-citronellol−approach 2). CBM from Cellulomonas f imi was fused with the odorantbinding protein (OBP::GQ 20 ::CBM) and with an anchor peptide with affinity to the liposome membrane (CBM::GQ 20 ::SP-DS3). In approach 1, OBP fusion protein served as a fragrance container, whereas in approach 2, the fragrance was loaded into liposomes with a higher cargo capacity. The two strategies showed a differentiated β-citronellol release profile triggered by an acidic sweat solution. OBP::GQ 20 ::CBM complex revealed a fast release (31.9% and 25.8% of the initial amount, after 1.5 and 24 h of exposure with acidic sweat solution, respectively), while the CBM::GQ 20 ::SP-DS3-liposome complex demonstrated a slower and controlled release (5.9% and 10.5% of the initial amount, after 1.5 and 24 h of exposure with acidic sweat solution, respectively). Both strategies revealed high potential for textile functionalization aimed at controlled release of fragrances. The OBP::GQ 20 ::CBM/β-citronellol complex is ideal for applications requiring fast release of a high amount of fragrance, whereas the CBM::GQ 20 ::SP-DS3-liposome/βcitronellol complex is more suitable for prolonged and controlled release of a lower amount of β-citronellol.

In this work, zein, a by-product of the agricultural industry of corn, was used to impart hydrophobicity and antimicrobial activity to textiles. The cotton textiles were functionalized with zein in two different forms, free or as particles. The optimized coating conditions which rendered the highest hydrophobic character to the textiles were found to be 50 g/L zein in the free form prepared with 70% ethanol. To enhance the antimicrobial activity of the coated textiles, ellagic acid was encapsulated into the zein particles. These presented stable diameters, between 300 and 450 nm, and positive surface charge revealing high encapsulation efficiency. After textiles coating, the controlled release of ellagic acid was tested against different mimetic biologic solutions. The release profile was dependent on the ethanol concentration, on the sweat solution pH and on the external media used during dialysis. The functionalized textiles revealed differentiated antimicrobial activity against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). For E. coli the activity was mainly related with the action of zein while for S. aureus a combined effect between the protein and the ellagic acid was observed. The results herein presented pave the way for the development of new zein-based products for a wide range of applications.

Succinic acid is a platform chemical that plays an important role as precursor for the synthesis of many valuable bio-based chemicals. Its microbial production from renewable resources has seen great developments, specially exploring the use of yeasts to overcome the limitations of using bacteria. The objective of the present work was to screen for succinate-producing isolates, using a yeast collection with different origins and characteristics. Four strains were chosen, two as promising succinic acid producers, in comparison with two low producers. Genome of these isolates was analysed, and differences were found mainly in genes SDH1, SDH3, MDH1 and the transcription factor HAP4, regarding the number of single nucleotide polymorphisms and the gene copy-number profile. Real-time PCR was used to study gene expression of 10 selected genes involved in the metabolic pathway of succinic acid production. Results show that for the non-producing strain, higher expression of genes SDH1, SDH2, ADH1, ADH3, IDH1 and HAP4 was detected, together with lower expression of ADR1 transcription factor, in comparison with the best producer strain. This is the first study showing the capacity of natural yeast isolates to produce high amounts of succinic acid, together with the understanding of the key factors associated, giving clues for strain improvement.