New liquid supports in the development of integrated platforms for the reuse of oxidative enzymes and polydopamine production

Polydopamine (PDA), a bioinspired polymer from mussel adhesive proteins, has attracted impressive attention as a novel coating for (nano) materials with an adequate conformal layer and adjustable thickness. Currently, PDA is obtained from dopamine chemical oxidation under alkaline conditions, limiting its use in materials sensible to alkaline environments. Envisaging a widespread use of PDA, the polymerization of dopamine by enzymatic catalysis allows the dopamine polymerization in a large range of pHs, overcoming thus the limitations of conventional chemical oxidation. Moreover, the conventional method of polymerization is a time-consuming process and produces PDA films with poor stability, which restricts its applications. On the other hand, the main bottleneck of enzyme-based biocatalytic processes is the high cost of the single use of the enzyme. In this work, laccase was used to catalyse dopamine polymerization. To improve its performance, a liquid support for integrating the laccase and its reuse together with the PDA production and recovery was developed using aqueous biphasic systems (ABS). Firstly, dopamine polymerization by laccase was optimized in terms of pH, temperature and initial dopamine concentration. It was demonstrated that the highest enzymatic polymerization of dopamine was achieved at pH 5.5, 30°C and 2 mg ml−1 of dopamine. Then, ABS composed of polymers, salts and ionic liquids were evaluated to optimize the laccase confinement in one phase while PDA is recovered in the opposite phase. The most promising ABS allowing the separation of laccase from the reaction product is composed of polypropylene glycol (400 g mol−1) and K2HPO4. The polymerization of dopamine in ABS leads to a remarkable improvement of polymerization of 3.9-fold in comparison to the conventional chemical PDA polymerization. The phase containing the confined laccase was reused for four consecutive reaction cycles, with a relative polymerization of 68.9% in the last cycle. The results of this work proved that ABS are a promising approach to create a liquid support for enzyme reuse allowing the process intensification efforts. The use of biocatalysts in ABS emerges as sustainable and alternative platforms from environmental and techno-economic points of view.publishe

Recovery of immunoglobulin G from rabbit serum using k-carrageenan-modified hybrid magnetic nanoparticles

Immunoglobulin G (IgG) has been used in the treatment of cancer, autoimmune diseases and neurological disorders, however, the current technologies to purify and recover IgG from biological media are of high-cost and time-consuming, resulting in high-cost products. In this sense, the search for cost-effective technologies to obtain highly pure and active IgG is highly required. The present work proposes a simple and efficient method for the purification and recovery of IgG from rabbit serum using magnetic iron oxide nanoparticles (magnetite, Fe3O4) coated with hybrid shells of a siliceous material modified with the anionic polysaccharide κ-carrageenan. Experimental parameters such as pH, contact time between the hybrid magnetic nanoparticles (HMNPs) and rabbit serum, and total protein concentration or dilution factor of serum were evaluated. The best results were achieved at pH 5.0, with a contact time of 60 min and using a rabbit serum with a total protein concentration of 4.8 mg·mL−1. Under these conditions, it was obtained an IgG purification factor and adsorption yield onto the HMNPs of 3.0 and 90%, respectively. The desorption of IgG from the HMNPs was evaluated using two strategies: a KCl aqueous solution and buffered aqueous solutions. Comparing to the initial rabbit serum, an IgG purification factor of 2.7 with a recovery yield of 74% were obtained using a buffered aqueous solution at pH 7.0. After desorption, the secondary structure of IgG and other proteins was evaluated by circular dichroism and no changes in the secondary structure were observed, meaning that the IgG integrity is kept after the adsorption and desorption steps. In summary, the application of HMNPs in the purification of IgG from serum samples has a high potential as a new downstream platform.in publicatio

Insights on the laccase extraction and activity in ionic-liquid-based aqueous biphasic systems

Due to their catalytic properties, selectivity, and efficiency, enzymes are excellent biocatalysts. In particular, laccases are versatile multi-copper oxidases with great interest for a wide plethora of biotechnological and environmental applications. Even though several laccase-catalysed processes have been reported at an industrial level, the high costs of their downstream processing required to provide biocatalysts with high purity levels, stability and activity remains one of the main drawbacks when economically evaluating the overall processes. Aqueous biphasic systems based on ionic liquids (ILs) can be foreseen as a promising alternative approach for the extraction and activity maintenance/improvement of enzymes, essentially due to the designer solvents ability of ionic liquids. However, to take advantage of this feature and to use the full potential of IL-based aqueous biphasic systems, it is necessary to understand the effect of ILs as phase-forming constituents and how they affect the enzymes extraction and activity. In order to overcome the lack of information on this topic in the literature, in this work, IL-based aqueous biphasic systems were investigated to extract and enhance the laccase activity, in order to gather evidences that could be used to improve the enzymes downstream processing. To this end, a wide screening of imidazolium-, pyridinium-, pyrrolidinium-, piperidinium-, tetraalkylphosphonium-, and tetraalkylammonium-based ILs as phase-forming components of ABS was carried out. Furthermore, these ILs were used to create ABS combined with salts, polymers and used as adjuvants in polymer-based ABS. Most ABS comprising ILs revealed to be highly efficient extraction platforms, allowing the complete extraction of laccase for all the conditions tested, and with an enzyme activity enhancement by more than 50%. Overall, the obtained results demonstrate that laccase preferentially partitions to the most hydrophilic phase in ABS comprising ILs, both used as adjuvants or as phase-forming components, corresponding to the phase in which the IL is enriched. Furthermore, the IL chemical structure of the IL plays a significant role in the enzyme activity, where ILs with a higher number of hydroxyl groups seem to be relevant to improve the laccase activity.publishe

Tailoring the partitioning of proteins using ionic liquids as adjuvants in polymer-polymer aqueous biphasic systems

Aqueous biphasic systems (ABS) are promising for proteins purification; however, when dealing with samples comprising several proteins, the selectivity towards a target protein is difficult to achieve. In this work, the addition of ionic liquids (ILs) as adjuvants (5 and 10 wt%) in ABS composed of polyethyleneglycol (PEG) and Dextran was investigated to tailor proteins’ partitioning between the coexisting phases. The liquid-liquid phase diagrams were determined at 298 K, followed by partition studies of three proteins (bovine serum albumin (BSA), immunoglobulin G (IgG), and cytochrome C (Cyt C)). Partition coefficients of IgG and BSA indicate the preference of both proteins to the dextran-rich phase, whereas Cyt C has no preferential partitioning between the phases. The addition of chloride-based ILs as adjuvants allows to increase or decrease the partition coefficients, thus tailoring the proteins partitioning between the phases. BSA partitioning essentially depends on the IL content in each phase, whereas Cyt C and IgG partitioning is ruled by the ILs chemical structure and established interactions. Molecular docking was carried out to address the ILs effect on the proteins partitioning, supporting experimental observations, while identifying the specific interactions occurring. The partition of each protein in polymer-salt ABS with ILs as adjuvants was determined, demonstrating the higher tailoring ability of polymer-polymer ABS when adding ILs. Finally, the partition of each protein in presence of the remaining ones was determined, as well as the selectivity of the studied systems to separate each pair of proteins, paving the way for their use in liquid-liquid chromatography.publishe

A globally applicable PCR-based detection and discrimination of BK and JC polyomaviruses

BKV and JCV belong to the Polyomaviridae family and are opportunistic agents associated with complications in immunocompromised individuals. Although a single screening assay for both viruses would be convenient, the diversity of BKV and JCV serotypes and genotypes is a methodological challenge. In this paper, we developed a PCR method able to detect and segregate BKV and JCV, despite these genetic discrepancies. A duplex semi-nested PCR (duplex snPCR) was designed to target a conserved region (639nt-1516nt) within the VP2 gene. In the first PCR, a primer set common to all BKV and JCV serotypes/ genotypes was used, followed by a semi-nested PCR with internal primers for BKV and JCV segregation. The limit of detection of the duplex snPCR was as low as 10 copies of BKV or JCV plasmids/µL. Specific products were observed when JCV and BKV plasmids were mixed in the same reaction. In field sample testing, the duplex snPCR detected and distinguished both viruses in different biological samples. Results were confirmed by Sanger’s sequencing. The geographical complexity of BKV and JCV serotypes and genotypes imposes limits to a simple and universal method that could detect each virus. However, we describe here a sensitive and reliable PCR technique for BKV and JCV diagnosis that overcomes these limitations and could be universally applied

Rarity of monodominance in hyperdiverse Amazonian forests.

Tropical forests are known for their high diversity. Yet, forest patches do occur in the tropics where a single tree species is dominant. Such "monodominant" forests are known from all of the main tropical regions. For Amazonia, we sampled the occurrence of monodominance in a massive, basin-wide database of forest-inventory plots from the Amazon Tree Diversity Network (ATDN). Utilizing a simple defining metric of at least half of the trees ≥ 10 cm diameter belonging to one species, we found only a few occurrences of monodominance in Amazonia, and the phenomenon was not significantly linked to previously hypothesized life history traits such wood density, seed mass, ectomycorrhizal associations, or Rhizobium nodulation. In our analysis, coppicing (the formation of sprouts at the base of the tree or on roots) was the only trait significantly linked to monodominance. While at specific locales coppicing or ectomycorrhizal associations may confer a considerable advantage to a tree species and lead to its monodominance, very few species have these traits. Mining of the ATDN dataset suggests that monodominance is quite rare in Amazonia, and may be linked primarily to edaphic factors

Geographic patterns of tree dispersal modes in Amazonia and their ecological correlates

Aim: To investigate the geographic patterns and ecological correlates in the geographic distribution of the most common tree dispersal modes in Amazonia (endozoochory, synzoochory, anemochory and hydrochory). We examined if the proportional abundance of these dispersal modes could be explained by the availability of dispersal agents (disperser-availability hypothesis) and/or the availability of resources for constructing zoochorous fruits (resource-availability hypothesis). Time period: Tree-inventory plots established between 1934 and 2019. Major taxa studied: Trees with a diameter at breast height (DBH) ≥ 9.55 cm. Location: Amazonia, here defined as the lowland rain forests of the Amazon River basin and the Guiana Shield. Methods: We assigned dispersal modes to a total of 5433 species and morphospecies within 1877 tree-inventory plots across terra-firme, seasonally flooded, and permanently flooded forests. We investigated geographic patterns in the proportional abundance of dispersal modes. We performed an abundance-weighted mean pairwise distance (MPD) test and fit generalized linear models (GLMs) to explain the geographic distribution of dispersal modes. Results: Anemochory was significantly, positively associated with mean annual wind speed, and hydrochory was significantly higher in flooded forests. Dispersal modes did not consistently show significant associations with the availability of resources for constructing zoochorous fruits. A lower dissimilarity in dispersal modes, resulting from a higher dominance of endozoochory, occurred in terra-firme forests (excluding podzols) compared to flooded forests. Main conclusions: The disperser-availability hypothesis was well supported for abiotic dispersal modes (anemochory and hydrochory). The availability of resources for constructing zoochorous fruits seems an unlikely explanation for the distribution of dispersal modes in Amazonia. The association between frugivores and the proportional abundance of zoochory requires further research, as tree recruitment not only depends on dispersal vectors but also on conditions that favour or limit seedling recruitment across forest types

Geography and ecology shape the phylogenetic composition of Amazonian tree communities

AimAmazonia hosts more tree species from numerous evolutionary lineages, both young and ancient, than any other biogeographic region. Previous studies have shown that tree lineages colonized multiple edaphic environments and dispersed widely across Amazonia, leading to a hypothesis, which we test, that lineages should not be strongly associated with either geographic regions or edaphic forest types.LocationAmazonia.TaxonAngiosperms (Magnoliids; Monocots; Eudicots).MethodsData for the abundance of 5082 tree species in 1989 plots were combined with a mega-phylogeny. We applied evolutionary ordination to assess how phylogenetic composition varies across Amazonia. We used variation partitioning and Moran's eigenvector maps (MEM) to test and quantify the separate and joint contributions of spatial and environmental variables to explain the phylogenetic composition of plots. We tested the indicator value of lineages for geographic regions and edaphic forest types and mapped associations onto the phylogeny.ResultsIn the terra firme and várzea forest types, the phylogenetic composition varies by geographic region, but the igapó and white-sand forest types retain a unique evolutionary signature regardless of region. Overall, we find that soil chemistry, climate and topography explain 24% of the variation in phylogenetic composition, with 79% of that variation being spatially structured (R2 = 19% overall for combined spatial/environmental effects). The phylogenetic composition also shows substantial spatial patterns not related to the environmental variables we quantified (R2 = 28%). A greater number of lineages were significant indicators of geographic regions than forest types.Main ConclusionNumerous tree lineages, including some ancient ones (>66 Ma), show strong associations with geographic regions and edaphic forest types of Amazonia. This shows that specialization in specific edaphic environments has played a long-standing role in the evolutionary assembly of Amazonian forests. Furthermore, many lineages, even those that have dispersed across Amazonia, dominate within a specific region, likely because of phylogenetically conserved niches for environmental conditions that are prevalent within regions

Geography and ecology shape the phylogenetic composition of Amazonian tree communities

Aim: Amazonia hosts more tree species from numerous evolutionary lineages, both young and ancient, than any other biogeographic region. Previous studies have shown that tree lineages colonized multiple edaphic environments and dispersed widely across Amazonia, leading to a hypothesis, which we test, that lineages should not be strongly associated with either geographic regions or edaphic forest types. Location: Amazonia. Taxon: Angiosperms (Magnoliids; Monocots; Eudicots). Methods: Data for the abundance of 5082 tree species in 1989 plots were combined with a mega-phylogeny. We applied evolutionary ordination to assess how phylogenetic composition varies across Amazonia. We used variation partitioning and Moran\u27s eigenvector maps (MEM) to test and quantify the separate and joint contributions of spatial and environmental variables to explain the phylogenetic composition of plots. We tested the indicator value of lineages for geographic regions and edaphic forest types and mapped associations onto the phylogeny. Results: In the terra firme and várzea forest types, the phylogenetic composition varies by geographic region, but the igapó and white-sand forest types retain a unique evolutionary signature regardless of region. Overall, we find that soil chemistry, climate and topography explain 24% of the variation in phylogenetic composition, with 79% of that variation being spatially structured (R$^{2}$ = 19% overall for combined spatial/environmental effects). The phylogenetic composition also shows substantial spatial patterns not related to the environmental variables we quantified (R$^{2}$ = 28%). A greater number of lineages were significant indicators of geographic regions than forest types. Main Conclusion: Numerous tree lineages, including some ancient ones (>66 Ma), show strong associations with geographic regions and edaphic forest types of Amazonia. This shows that specialization in specific edaphic environments has played a long-standing role in the evolutionary assembly of Amazonian forests. Furthermore, many lineages, even those that have dispersed across Amazonia, dominate within a specific region, likely because of phylogenetically conserved niches for environmental conditions that are prevalent within regions