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A series of novel 4-acetyl-1,3,4-oxadiazole derivatives was designed and synthesized for their biological evaluation in vitro against Trypanosoma cruzi and Leishmania mexicana. Additionally, compounds were evaluated by molecular docking on the cruzain of T. cruzi (TcCz) and the cysteine protease B (CPB) of L. mexicana (LmCPB) to know their potential mechanism of binding. Compound OX-12 had better trypanocidal activity against NINOA (IC50= 10.5 µM) and A1 (IC50= 21.7 µM) T. cruzi strains that reference drug benznidazole (IC50= 30.3 µM and 39.8 µM, respectively). Compound OX-2 had the best biological activity against L. mexicana in M379 (IC50= 11.9 µM) and FCQEPS (IC50= 34.0 µM) strains that the reference drug glucantime (IC50 ˃120 µM). All the compounds showed important interactions with residues on the active site of TcCz (Gly66, Trp26, Leu67, and Ala138) and LmCPB (Gly67, Asn62, Leu68, and Ala140). Finally, the molecular dynamics simulations of the compound OX-12 shown moderate stability from 40 to 115 ns with an RMSD value of 6.5 Å. Meanwhile, compound OX-2 showed a minor stability in complex with CPB from 25 to 200 ns of simulation (RMSD <9 Å). These results encourage to develop more potent and efficient trypanocidal and leishmanicidal agents using the 1,3,4-oxadiazole scaffold.

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Dendritic cells (DC) along with macrophages are the main host cells of the intracellular parasite Leishmania. DC traverse a process of maturation, passing through an immature state with phagocytic ability to a mature one where they can modulate the immune response through the secretion of cytokines. Several studies have demonstrated that Leishmania inhibits DC maturation. Nevertheless, when cells are subjected to a second stimulus such as LPS/IFN-γ, they manage to mature. In the maturation process of DC, several signaling pathways have been implicated, importantly MAPK. On the other hand, Akt is a signaling pathway deeply involved in cell survival. Some Leishmania species have shown to activate MAPK and Akt in different cells. The aim of this work was to investigate the role of ERK and Akt in the maturation of monocyte-derived DC (moDC) infected with L. mexicana. moDC were infected with L. mexicana metacyclic promastigotes, and the phosphorylation of ERK and Akt, the expression of MHCII and CD86 and IL-12 transcript, and secretion were determined in the presence or absence of an Akt inhibitor. We showed that L. mexicana induces a sustained Akt and ERK phosphorylation, while the Akt inhibitor inhibits it. Moreover, the infection of moDC downregulates CD86 expression but not MHCII, and the Akt inhibitor reestablishes CD86 expression and 12p40 production. Thus, L. mexicana can modulate DC maturation though Akt signaling.

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Feruloyl esterases (FAEs) are versatile enzymes able to release hydroxycinnamic acids or synthesize their ester derivatives, both molecules with interesting biological activities such as: antioxidants, antifungals, antivirals, antifibrotic, anti-inflammatory, among others. The importance of these molecules in medicine, food or cosmetic industries provides FAEs with several biotechnological applications as key industrial biocatalysts. However, FAEs have some operational limitations that must be overcome, which can be addressed through different protein engineering approaches to enhance their thermal stability, catalytic efficiencies, and selectivity. This review aims to present a brief historical tour through the mutagenesis strategies employed to improve enzymes performance and analyze the current protein engineering strategies applied to FAEs as interesting biocatalysts. Finally, an outlook of the future of FAEs protein engineering approaches to achieve successful industrial biocatalysts is given.

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Halophilic microorganisms are potentially capable as platforms to produce low-cost biosurfactants. However, the robustness of bioprocesses is still a challenge and, therefore, it is essential to understand the effects of microbiological culture conditions through bioreactor engineering. Based on a design of experiments (DOE) and a response surface methodology (RSM) tailored and taken from the literature, the present work focuses on the evaluation of a composite central design (CCD) under batch cultures in stirred-tank bioreactors with the halophilic bacteria Salibacterium sp. 4CTb in order to determine the operative conditions that favor mass transfer and optimize the production of a lipopeptide. The results obtained showed profiles highlighting the most favorable culture conditions, which lead to an emulsification index (E24%) higher than 70%. Moreover, through the behavior of dissolved oxygen (DO), it was possible to experimentally evaluate the higher volumetric coefficient of mass transfer in the presence of lipopeptide (kLa = 31 1/h) as a key criterion for the synthesis of the biosurfactant on further cell expansion.

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The intracellular parasite Leishmania mexicana inhibits camptothecin (CPT)-induced apoptosis of monocyte-derived dendritic cells (moDC) through the down-regulation of p38 and JNK phosphorylation, while the kinase Akt is maintained active for 24 h. In addition, the infection of moDC with L. mexicana promastigotes increases the protein presence of the antiapoptotic protein Bcl-xL. In the present work, we aimed to investigate the role of Akt in the inhibition of apoptosis of moDC by L. mexicana and in the modulation of the expression of the antiapoptotic proteins Bcl-2, Mcl-1 and Bcl-xL. moDC were infected with L. mexicana metacyclic promastigotes and treated with CPT, an Akt inhibitor, or both and the mitochondrial outer membrane permeabilization (MOMP) and protein presence of active caspase 3, Bcl-2, Mcl-1 and Bcl-xL were evaluated. Our results show that the specific inhibition of Akt reverts the apoptosis protective effect exerted by L. mexicana on moDC reflected by a reduction in MOMP, caspase 3 activation, and upregulation of Bcl-xL. Interestingly, we also found that the infection of moDC with L. mexicana promastigotes induces a decrease in Bcl-2 along with an isoform change of Mcl-1, this independently to Akt activity. We demonstrated that Akt is deeply involved in the inhibition of apoptosis of moDC by L. mexicana.

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Amycolatopsis sp. ATCC 39116 catabolizes ferulic acid by the non-oxidative deacetylation and ß-oxidation pathways to produce vanillin and vanillic acid, respectively. In submerged culture, vanillin productivity decreased more than 8-fold, when ferulic, p-coumaric, and caffeic acids were employed in pre-cultures of the microorganism in order to activate the ferulic acid catabolic pathways, resulting in a carbon redistribution since vanillic acid and guaiacol productivities increased more than 5-fold compared with control. In contrast, in surface culture, the effects of ferulic and sinapic acids in pre-cultures were totally opposite to those of the submerged culture, directing the carbon distribution into vanillin formation. In surface culture, more than 30% of ferulic acid can be used as carbon source for other metabolic processes, such as ATP regeneration. In this way, the intracellular ATP concentration remained constant during the biotransformation process by surface culture (100 µg ATP/mg protein), demonstrating a high energetic state, which can maintain active the non-oxidative deacetylation pathway. In contrast, in submerged culture, it decreased 3.15-fold at the end of the biotransformation compared with the initial content, showing a low energetic state, while the NAD+/NADH ratio (23.15) increased 1.81-fold. It seems that in submerged culture, low energetic and high oxidative states are the physiological conditions that can redirect the ferulic catabolism into ß-oxidative pathway and/or vanillin oxidation to produce vanillic acid.

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Feruloyl esterases synthesize butyl hydroxycinnamates, molecules possessing interesting biological properties, nonetheless, they exhibit a low stability under synthesis conditions in organic solvents, restricting its use. To enhance its operational stability in synthesis, we immobilized type A feruloyl esterase from Aspergillus niger (AnFAEA) using several carrier-bound and carrier-free strategies. The most active biocatalysts were: 1) AnFAEA immobilized on epoxy-activated carriers (protein load of 0.6 mgenzyme x mg-1carrier) that recovered 91 % of the initial hydrolytic activity, and 2) AnFAEA aggregated and cross-linked in the presence of 5 mg of BSA and 15 mM of glutaraldehyde (AnFAEA-amino-CLEAs), which exhibited 385 % of its initial hydrolytic activity; both using 4-nitrophenyl butyrate as substrate. The AnFAEA-amino-CLEAs were 12.7 times more thermostable at 60 °C than the AnFAEA immobilized on epoxy-activated carrier, thus AnFAEA-amino-CLEAs were selected for further characterization. Interestingly, during methyl sinapate hydrolysis (pH 7.2 and 30 °C), AnFAEA-amino-CLEAs KM was 15 % higher, while during butyl sinapate synthesis the KM was reduced in 63 %, both compared with the soluble enzyme. The direct esterification of butyl sinapate at solvent free conditions using sinapic acid 50 mM, reached 95 % conversion after 24 h employing AnFAEA-amino-CLEAs, which could be used for 10 cycles without significant activity losses, demonstrating their outstanding operational stability.

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Background: Aspergillus ochraceus was isolated from coffee pulp and selected as an interesting hydroxycinnamoyl esterase strain producer, using an activity microplate high-throughput screening method. In this work, we purified and characterized a new type C A. ochraceus feruloyl esterase (AocFaeC), which synthesized specifically butyl hydroxycinnamates in a ternary solvent system. Results: AocFaeC was produced by solid state fermentation, reaching its maximal activity (1.1 U/g) after 48 h of culture. After purification, the monomeric protein (34 kDa) showed a specific activity of 57.9 U/mg towards methyl ferulate. AocFaeC biochemical characterization confirmed its identity as a type C feruloyl esterase and suggested the presence of a catalytic serine in the active site. Its maximum hydrolytic activity was achieved at 40°C and pH 6.5 and increased by 109 and 77% with Ca2+ and Mg2+, but decreased by 90 and 45% with Hg2+ and Cu2+, respectively. The initial butyl ferulate synthesis rate increased from 0.8 to 23.7 nmol/min after transesterification condition improvement, using an isooctane:butanol:water ternary solvent system, surprisingly the synthesis activity using other alcohols was negligible. At these conditions, the synthesis specific activities for butyl p-coumarate, sinapinate, ferulate, and caffeate were 87.3, 97.6, 168.2, and 234 U/µmol, respectively. Remarkably, AocFaeC showed 5 folds higher butyl caffeate synthesis rate compared to type B Aspergillus niger feruloyl esterase, a well-known enzyme for its elevated activity towards caffeic acid esters. Conclusions: Type C feruloyl esterase from A. ochraceus is a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

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Solid-state fermentation (SSF) has been largely employed during the last three decades to produce different biomolecules of industrial interest, particularly enzymes. Through the use of agroindustrial wastes as SSF substrates, an economic process of lipases production can be achieved. In this chapter we describe a comprehensive SSF method for producing an economical preparation of Rhizomucor miehei lipase, employing sugarcane bagasse and used vegetal oil as substrates. To demonstrate the usefulness of the lipase produced by this method, we utilized directly the dried fermented solid, as a heterogeneous biocatalyst for the ethanolysis of different fats and oils. Final ethyl ester conversions (>90%, 24 h) were similar with those obtained using a commercial immobilized Rhizomucor miehei lipase at our best conditions. In this work we demonstrated that SSF is an easy and economical method for the production of lipases that can be used directly as heterogeneous biocatalysts for biodiesel production, employing low-cost feedstocks.

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Dendritic cells (DCs) are one of the principal host cells of the obligate intracellular parasite Leishmania that can survive and reproduce within cells due to the ability to regulate different cellular events, including apoptosis. Inhibition of host cell apoptosis is a strategy employed by multiple pathogens to ensure their survival in the infected cell. We have previously reported that Leishmania mexicana promastigotes and amastigotes inhibit camptothecin-induced apoptosis of monocyte-derived dendritic cells (moDCs) through the downregulation of p38 and JNK phosphorylation. The upregulation of glutathione (GSH), the most important regulator of reactive oxygen species (ROS) concentration, has proven to protect cells from apoptosis through the inhibition of JNK1. Another mechanism employed by cells for the protection of apoptosis is the expression of anti-apoptotic proteins of the Bcl-2 family. The aim of this study was to determine if GSH, ROS, and Bcl-xL participate in the inhibition of camptothecin-induced apoptosis of moDC by L. mexicana promastigotes. GSH quantification assays showed that camptothecin and BSO (an inhibitor of glutathione synthesis) strongly decreased intracellular GSH concentration in moDC, while infection with L. mexicana promastigotes had no effect in the level of GSH. On the other hand, infection with L. mexicana promastigotes of BSO- and camptothecin-treated moDC diminished the concentration of ROS and induced the expression of the anti-apoptotic protein Bcl-xL. Our findings suggest that inhibition of camptothecin-induced apoptosis of moDC by L. mexicana promastigotes is preferentially regulated by the expression of anti-apoptotic proteins of the Bcl-2 family rather than by the redox status of the cell.

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