Extending the computational and experimental analysis of lipase active site selectivity.

Molecular docking is an important computational analysis widely used to predict the interaction of enzymes with several starting materials for developing new valuable products from several starting materials, including oils and fats. In the present study, molecular docking was used as an efficient in silico screening tool to select biocatalysts with the highest catalytic performance in butyl esters production in a solvent-free system, an eco-friendly approach, via direct esterification of free fatty acids from Licuri oil with butanol. For such purpose, three commercial lipase preparations were used to perform molecular docking studies such as Burkholderia cepacia (BCL), Porcine pancreatic (PPL), and Candida rugosa (CRL). Concurrently, the results obtained in BCL and CRL are the most efficient in the esterification process due to their higher preference for catalyzing the esterification of lauric acid, the main fatty acid found in the licuri oil composition. Meanwhile, PPL was the least efficient because it preferentially interacts with minor fatty acids. Molecular docking with the experimental results indicated the better performance in the synthesis of esters was BCL. In conclusion, experimental results analysis shows higher enzymatic productivity in esterification reactions of 1294.83 µmol/h.mg, while the CRL and PPL demonstrated the lowest performance (189.87 µmol / h.mg and 23.96 µmol / h.mg, respectively). Thus, molecular docking and experimental results indicate that BCL is a more efficient lipase to produce fatty acids and esters from licuri oil with a high content of lauric acid. In addition, this study also demonstrates the application of molecular docking as an important tool for lipase screening to achieve more sustainable production of butyl esters with a view synthesis of biolubricants.

Improved L-Asparaginase Properties and Reusability by Immobilization onto Functionalized Carbon Xerogels.

Enzyme immobilization can offer a range of significant advantages, including reusability, and increased selectivity, stability, and activity. In this work, a central composite design (CCD) of experiments and response surface methodology (RSM) were used to study, for the first time, the L-asparaginase (ASNase) immobilization onto functionalized carbon xerogels (CXs). The best results were achieved using CXs obtained by hydrothermal oxidation with nitric acid and subsequent heat treatment in a nitrogen flow at 600 °C (CX-OX-600). Under the optimal conditions (81 min of contact time, pH 6.2 and 0.36 g/L of ASNase), an immobilization yield (IY) of 100 % and relative recovered activity (RRA) of 103 % were achieved. The kinetic parameters obtained also indicate a 1.25-fold increase in the affinity of ASNase towards the substrate after immobilization. Moreover, the immobilized enzyme retained 97 % of its initial activity after 6 consecutive reaction cycles. All these outcomes confirm the promising properties of functionalized CXs as support for ASNase, bringing new insights into the development of an efficient and stable immobilization platform for use in the pharmaceutical industry, food industry, and biosensors.

Experimental and Computational Analysis of Synthesis Conditions of Hybrid Nanoflowers for Lipase Immobilization.

This work presents a framework for evaluating hybrid nanoflowers using Burkholderia cepacia lipase. It was expanded on previous findings by testing lipase hybrid nanoflowers (hNF-lipase) formation over a wide range of pH values (5-9) and buffer concentrations (10-100 mM). The free enzyme activity was compared with that of hNF-lipase. The analysis, performed by molecular docking, described the effect of lipase interaction with copper ions. The morphological characterization of hNF-lipase was performed using scanning electron microscopy. Fourier Transform Infrared Spectroscopy performed the physical-chemical characterization. The results show that all hNF-lipase activity presented values higher than that of the free enzyme. Activity is higher at pH 7.4 and has the highest buffer concentration of 100 mM. Molecular docking analysis has been used to understand the effect of enzyme protonation on hNF-lipase formation and identify the main the main binding sites of the enzyme with copper ions. The hNF-lipase nanostructures show the shape of flowers in their micrographs from pH 6 to 8. The spectra of the nanoflowers present peaks typical of the amide regions I and II, current in lipase, and areas with P-O vibrations, confirming the presence of the phosphate group. Therefore, hNF-lipase is an efficient biocatalyst with increased catalytic activity, good nanostructure formation, and improved stability.

Antifungal potential of the new copper(II)-theophylline/1,10-phenanthroline complex against drug-resistant Candida species.

Candida spp. are the commonest fungal pathogens worldwide. Antifungal resistance is a problem that has prompted the discovery of novel anti-Candida drugs. Herein, 25 compounds, some of them containing copper(II), cobalt(II) and manganese(II) ions, were initially evaluated for inhibiting the growth of reference strains of Candida albicans and Candida tropicalis. Eight (32%) of the compounds inhibited the proliferation of these yeasts, displaying minimum inhibitory concentrations (MICs) ranging from 31.25 to 250 µg/mL and minimum fungicidal concentration (MFCs) from 62.5 to 250 µg/mL. Drug-likeness/pharmacokinetic calculated by SwissADME indicated that the 8 selected compounds were suitable for use as topical drugs. The complex CTP, Cu(theo)2phen(H2O).5H2O (theo = theophylline; phen = 1,10-phenanthroline), was chosen for further testing against 10 medically relevant Candida species that were resistant to fluconazole/amphotericin B. CTP demonstrated a broad spectrum of action, inhibiting the growth of all 20 clinical fungal isolates, with MICs from 7.81 to 62.5 µg/mL and MFCs from 15.62 to 62.5 µg/mL. Conversely, CTP did not cause lysis in erythrocytes. The toxicity of CTP was evaluated in vivo using Galleria mellonella and Tenebrio molitor. CTP had no or low levels of toxicity at doses ranging from 31.25 to 250 µg/mL for 5 days. After 24 h of treatment, G. mellonella larvae exhibited high survival rates even when exposed to high doses of CTP (600 µg/mL), with the 50% cytotoxic concentration calculated as 776.2 µg/mL, generating selectivity indexes varying from 12.4 to 99.4 depending on each Candida species. These findings suggest that CTP could serve as a potential drug to treat infections caused by Candida species resistant to clinically available antifungals.

Active Cu(II), Mn(II) and Ag(I) 1,10-phenanthroline/1,10-phenanthroline-5,6-dione/dicarboxylate chelates: effects on Scedosporium.

Background: Scedosporium/Lomentospora species are human pathogens that are resistant to almost all antifungals currently available in clinical practice. Methods: The effects of 16 1,10-phenanthroline (phen)/1,10-phenanthroline-5,6-dione/dicarboxylate chelates containing Cu(II), Mn(II) and Ag(I) against Scedosporium apiospermum, Scedosporium minutisporum, Scedosporium aurantiacum and Lomentospora prolificans were evaluated. Results: To different degrees, all of the test chelates inhibited the viability of planktonic conidial cells, displaying MICs ranging from 0.029 to 72.08 µM. Generally, Mn(II)-containing chelates were the least toxic to lung epithelial cells, particularly [Mn2(oda)(phen)4(H2O)2][Mn2(oda)(phen)4(oda)2].4H2O (MICs: 1.62-3.25 µM: selectivity indexes >64). Moreover, this manganese-based chelate reduced the biofilm biomass formation and diminished the mature biofilm viability. Conclusion: [Mn2(oda)(phen)4(H2O)2][Mn2(oda)(phen)4(oda)2].4H2O opens a new chemotherapeutic avenue for the deactivation of these emergent, multidrug-resistant filamentous fungi.

Metals have been used to treat microbial infections for centuries. In this context, the effects of 16 metal-based compounds against the human pathogens Scedosporium apiospermum, Scedosporium minutisporum, Scedosporium aurantiacum and Lomentospora prolificans were tested. All the 16 metal-based compounds were able to interfere with the viability of these fungal pathogens to different degrees. Among the 16 compounds, a manganese-containing compound presented the best activity against the fungal species and it presented the least toxicity to a human lung cell line. In addition, this manganese-containing compound reduced the ability of fungal cells to come together and form a type of community called biofilm. In conclusion, the manganese-containing compound presents a promising option against the multidrug-resistant filamentous fungi species belonging to the Scedosporium/Lomentospora genera.

Designing protein nano-construct in ionic liquid: a boost in efficacy of cytochrome C under stresses.

Herein, we present a simple approach to fabricate protein nanoconstructs by complexing cytochrome C (Cyt C) with silk nanofibrils (SNF) and choline dihydrogen phosphate ionic liquid (IL). The peroxidase activity of the IL modified Cyt C nanoconstruct (Cyt C + SNF + IL) increased significantly (2.5 to 10-fold) over unmodified Cyt C and showed enhanced catalytic activity and stability under harsh conditions, proving its potential as a suitable protein packaging strategy.

Integrated Approach to Extract and Purify Proteins from Honey by Ionic Liquid-Based Three-Phase Partitioning.

The purification of value-added compounds by three-phase partitioning (TPP) is a promising alternative to conventional processes since the target compound can be easily recovered from the liquid-liquid interphase. Although this technique has been successfully applied to the recovery of proteins, the minimization of the use of salts and solvents must be pursued to improve the overall process sustainability. Accordingly, we have here investigated the use of biobased glycine-betaine ionic liquids (IL) directly with honey, a carbohydrate-rich matrix, as phase-forming components of TPP systems. These ILTPP systems were applied in the purification of major royal jelly proteins (MRJPs) from honey. The results obtained show that MRJPs mostly precipitate in the ILTPP interphase, with a recovery yield ranging between 82.8% and 97.3%. In particular, MRJP1 can be obtained with a purity level up to 90.1%. Furthermore, these systems allow the simultaneous separation of antioxidants and carbohydrates to different liquid phases. The proposed approach allows the separation of proteins, antioxidants, and carbohydrates from honey in a single step, while using only ILs and a real carbohydrate-rich matrix, thus being sustainable TPP processes.

Presenting B-DNA as macromolecular crowding agent to improve efficacy of cytochrome c under various stresses.

Existence of numerous biomolecules results in biological fluids to be extremely crowded. Thus, Macromolecular crowding is an essential phenomenon to sustain active conformation of proteins in biological systems. Herein, double helical deoxyribonucleic acid (B-DNA) is presented for the first time as a biomacromolecular crowding system for sustainable packaging of cytochrome c (Cyt C). The peroxidase activity of Cyt C was investigated in the presence of various concentrations of B-DNA (from salmon milt). At an optimized concentration of 0.125 mg/mL B-DNA, an 11-fold higher catalytic activity was found than in native Cyt C with improved stability. Molecular docking and spectroscopic analyses revealed that electrostatic and H-bonding are the main interactions between DNA and Cyt C that affect the structural stability and activity of the protein. Moreover, the catalytic activity and stability of the protein were further investigated in the presence of severe process conditions by UV-visible, circular dichroism, and Fourier-transform infrared spectroscopies. Molecularly crowded Cyt C showed significantly higher activity and stability under severe environments such as high temperature (110 °C), oxidative stress, high pH (pH 10) and biological (trypsin) and chemical denaturants (urea) compared to bare Cyt C. The observed results support the suitability of DNA-based macromolecular crowding media as a viable and effective stabilizer of proteins against multiple stresses.

Evaluation of lipase access tunnels and analysis of substance transport in comparison with experimental data.

Lipases (E.C. 3.1.1.3) have buried active sites and used access tunnels in the transport of substrates and products for biotransformation processes. Computational methods are used to predict the trajectory and energy profile of ligands through these tunnels, and they complement the experimental methodologies because they filter data, optimizing laboratory time and experimental costs. Access tunnels of Burkholderia cepacia lipase (BCL), Candida rugosa lipase (CRL), and porcine pancreas lipase (PPL) and the transport of fatty acids, alcohols and esters through the tunnels were evaluated using the online server CaverWeb V1.0, and server calculation results were compared with experimental data (productivity). BCL showed higher productivity with palmitic acid-C16:0 (4029.95 µmol/h mg); CRL obtained productivity for oleic acid-C18:1 (380.80 µmol/h mg), and PPL achieved productivity for lauric acid-C12:0 (71.27 µmol/h mg). The highest probability of transport for BCL is through the tunnels 1 and 2, for CRL through the tunnel 1, and for PPL through the tunnels 1, 2, 3 and 4. Thus, the best in silico result was the transport of the substrates palmitic acid and ethanol and product ethyl palmitate in tunnel 1 of BCL. This result corroborates with the best result for the productivity data (higher productivity for BCL with palmitic acid-4029.95 µmol/h mg). The combination of in silico evaluation and experimental data gave similar results, demonstrating that in silico approaches are a promising alternative for reducing screening tests and minimizing laboratory time in the bio-catalysis area by identifying the lipases with the greatest reaction potential, as in the case of this proposal.

Enhanced Enzyme Reuse through the Bioconjugation of L-Asparaginase and Silica-Based Supported Ionic Liquid-like Phase Materials.

L-asparaginase (ASNase) is an amidohydrolase that can be used as a biopharmaceutical, as an agent for acrylamide reduction, and as an active molecule for L-asparagine detection. However, its free form displays some limitations, such as the enzyme's single use and low stability. Hence, immobilization is one of the most effective tools for enzyme recovery and reuse. Silica is a promising material due to its low-cost, biological compatibility, and tunable physicochemical characteristics if properly functionalized. Ionic liquids (ILs) are designer compounds that allow the tailoring of their physicochemical properties for a given task. If properly designed, bioconjugates combine the features of the selected ILs with those of the support used, enabling the simple recovery and reuse of the enzyme. In this work, silica-based supported ionic liquid-like phase (SSILLP) materials with quaternary ammoniums and chloride as the counterion were studied as novel supports for ASNase immobilization since it has been reported that ammonium ILs have beneficial effects on enzyme stability. SSILLP materials were characterized by elemental analysis and zeta potential. The immobilization process was studied and the pH effect, enzyme/support ratio, and contact time were optimized regarding the ASNase enzymatic activity. ASNase-SSILLP bioconjugates were characterized by ATR-FTIR. The bioconjugates displayed promising potential since [Si][N3444]Cl, [Si][N3666]Cl, and [Si][N3888]Cl recovered more than 92% of the initial ASNase activity under the optimized immobilization conditions (pH 8, 6 × 10-3 mg of ASNase per mg of SSILLP material, and 60 min). The ASNase-SSILLP bioconjugates showed more enhanced enzyme reuse than reported for other materials and immobilization methods, allowing five cycles of reaction while keeping more than 75% of the initial immobilized ASNase activity. According to molecular docking studies, the main interactions established between ASNase and SSILLP materials correspond to hydrophobic interactions. Overall, it is here demonstrated that SSILLP materials are efficient supports for ASNase, paving the way for their use in the pharmaceutical and food industries.

Rutin-Functionalized Multi-Walled Carbon Nanotubes: Molecular Docking, Physicochemistry and Cytotoxicity in Fibroblasts.

Multi-Walled Carbon Nanotubes (MWCNT) have been functionalized with rutin through three steps (i. reaction step; ii. purification step; iii. drying step) and their physicochemical properties investigated with respect to morphological structure, thermal analysis, Fourier Transform Infrared Spectroscopy (FTIR), and cytotoxicity. The molecular docking suggested the rutin-functionalized MWCNT occurred by hydrogen bonds, which was confirmed by FTIR assays, corroborating the results obtained by thermal analyses. A tubular shape, arranged in a three-dimensional structure, could be observed. Mild cytotoxicity observed in 3T3 fibroblasts suggested a dose-effect relationship after exposure. These findings suggest the formation of aggregates of filamentous structures on the cells favoring the cell penetration.

Designing biopolymer-based artificial peroxidase for oxidative removal of dibenzothiophene from a model diesel fuel.

Naturally occurring peroxidases are important for living organisms and have manifold utility in industries. However, lack of stability in harsh reaction conditions hinders wide applicability of such enzymes. Thus, suitable alternative is vital which can endure severe reaction conditions. As a substitute of natural peroxidase, herein, biopolymer-based polyelectrolyte complexes (PECs) coordinated with Fen+ is proposed as macromolecular peroxidase mimicking systems. Three PECs were engineered via complexation of protonated chitosan and alginate with Fe2+ (Fe2+-PEC), Fe3+ (Fe3+-PEC), and Fe3O4 (Fe3O4-PEC), respectively. Computational study showed the Fe3+-PEC was highly stable with abundant electrostatic and intramolecular hydrogen bonding interactions. The versatility of the Fe-PECs as artificial peroxidase biocatalysts was probed by two types of peroxidase assays - ABTS oxidation in buffer systems (pH 4.0 and 7.0) and pyrogallol oxidation in organic solvents (acetonitrile, ethyl acetate and toluene). Overall, Fe3+-PEC showed remarkably high peroxidase activity both in aqueous buffers and in organic solvents, whereas, Fe3O4-PEC showed least catalytic activity. Finally, as a proof of concept, the ability of the biocatalyst to carry out deep oxidative desulphurization was demonstrated envisaging removal of dibenzothiophene from model fossil fuel in a sustainable way.

Computational and experimental analysis on the preferential selectivity of lipases for triglycerides in Licuri oil.

In the present study, we demonstrated the use of molecular docking as an efficient in silico screening tool for lipase-triglyceride interactions. Computational simulations using the crystal structures from Burkholderia cepacia lipase (BCL), Thermomyces lanuginosus lipase (TLL), and pancreatic porcine lipase (PPL) were performed to elucidate the catalytic behavior with the majority triglycerides present in Licuri oil, as follows: caprilyl-dilauryl-glycerol (CyLaLa), capryl-dilauryl-glycerol (CaLaLa), capryl-lauryl-myristoyl-glycerol (CaLaM), and dilauryl-myristoyl-glycerol (LaLaM). The computational simulation results showed that BCL has the potential to preferentially catalyze the major triglycerides present in Licuri oil, demonstrating that CyLaLa, (≈25.75% oil composition) interacts directly with two of the three amino acid residues in its catalytic triad (Ser87 and His286) with the lowest energy (-5.9 kcal/mol), while other triglycerides (CaLaLa, CaLaM, and LaLaM) interact with only one amino acid (His286). In one hard, TLL showed a preference for catalyzing the triglyceride CaLaLa also interacting with His286 residue, but, achieving higher binding energies (-5.3 kcal/mol) than found in BCL (-5.7 kcal/mol). On the other hand, PPL prefers to catalyze only with LaLaM triglyceride by His264 residue interaction. When comparing the computational simulations with the experimental results, it was possible to understand how BCL and TLL display more stable binding with the majority triglycerides present in the Licuri oil, achieving conversions of 50.86 and 49.01%, respectively. These results indicate the production of fatty acid concentrates from Licuri oil with high lauric acid content. Meanwhile, this study also demonstrates the application of molecular docking as an important tool for lipase screening to reach a more sustainable production of fatty acid concentrates from vegetable oils.

Anti-Tumor Efficiency of Perillylalcohol/ß-Cyclodextrin Inclusion Complexes in a Sarcoma S180-Induced Mice Model.

The low solubility and high volatility of perillyl alcohol (POH) compromise its bioavailability and potential use as chemotherapeutic drug. In this work, we have evaluated the anticancer activity of POH complexed with ß-cyclodextrin (ß-CD) using three complexation approaches. Molecular docking suggests the hydrogen-bond between POH and ß-cyclodextrin in molar proportion was 1:1. Thermal analysis and Fourier-transform infrared spectroscopy (FTIR) confirmed that the POH was enclosed in the ß-CD cavity. Also, there was a significant reduction of particle size thereof, indicating a modification of the ß-cyclodextrin crystals. The complexes were tested against human L929 fibroblasts after 24 h of incubation showing no signs of cytotoxicity. Concerning the histopathological results, the treatment with POH/ß-CD at a dose of 50 mg/kg promoted approximately 60% inhibition of tumor growth in a sarcoma S180-induced mice model and the reduction of nuclear immunoexpression of the Ki67 antigen compared to the control group. Obtained data suggest a significant reduction of cycling cells and tumor proliferation. Our results confirm that complexation of POH/ß-CD not only solves the problem related to the volatility of the monoterpene but also increases its efficiency as an antitumor agent.

Enzymatic transesterification of coconut oil by using immobilized lipase on biochar: An experimental and molecular docking study.

Guava seed biochar appears as a new alternative of the effective support to the immobilization of Burkholderia cepacia lipase (BCL) by physical adsorption. The objective of this work was to evaluate the potential of this immobilized biocatalyst in the transesterification reaction of crude coconut oil and ethanol and to understand the mechanism of the reaction through the study of molecular docking. The best loading of BCL was determined to be 0.15 genzyme /gsupport having a hydrolytic activity of 260 U/g and 54% immobilization yield. The products of transesterification reaction produced a maximum yield at 40 °C under different reaction conditions. The monoacylglycerols (MAGs) conversion of 59% was using substrate molar ratio oil:ethanol of 1:7 with the reaction time of 24 H. In addition, the highest ethyl esters yield (48%) had the molar ratio of 1:7 with the reaction time of 96 H and maximum conversion of diacylglycerols (DAGs) was 30% with the molar ratio of 1:6 with the reaction time of 24 H. Molecular Docking was applied to clarify the mechanisms of transesterification reaction at the molecular level. MAGs and DAGs are compounds with excellent emulsifying properties used in industrial production of several bioproducts such as cosmetic, pharmaceuticals, foods, and lubricants.

Lipase activation by molecular bioimprinting: The role of interactions between fatty acids and enzyme active site.

Bioimprinting is an easy, sustainable and low-cost technique that promotes a printing of potential substrates on enzyme structure, inducing a more selective and stable conformation. Bioimprinting promotes conformational changes in enzymes, resulting in better catalytic performance. In this work, the effect of bioimprinting of Burkholderia cepacia lipase (BCL) and porcine pancreatic extracts (PPE) with four different fatty acids (lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), and stearic acid (C18:0)) was investigated. The results demonstrated that the better bioimprinting effect was in BCL with lauric acid in esterification reaction, promoting BCL activation in which relative enzyme activity was 70 times greater than nonimprinted BCL. Bioimprinting results were influenced by the carbon chain length of fatty acids imprinted in the BCL, in which the effects were weaker with the chain increase. Molecular docking was performed to better understand the bioimprinting method. The results of these simulations showed that indeed all fatty acids were imprinted in the active site of BCL. However, lauric acid presented the highest imprinting preference in the active site of BCL, resulting in the highest relative activity. Furthermore, Fourier transform infrared (FTIR) analysis confirmed important variations in secondary structure of bioimprinting BCL with lauric acid, in which there was a reduction in the α-helix content and an increase in the ß-sheet content that facilitated substrate access to the active site of BCL and led higher rigidity, resulting in high activity. Bioimprinted BCL with lauric acid showed excellent operational stability in esterification reaction, maintaining its original relative activity after five successive cycles. Thus, the results show that bioimprinting of BCL with lauric acid is a successful strategy due to its high catalytic activity and reusability.

Design for preparation of more active cross-linked enzyme aggregates of Burkholderia cepacia lipase using palm fiber residue.

A new design of cross-linked enzyme aggregates (CLEAs) of Burkholderia cepacia lipase (BCL) based mainly on the use of lignocellulosic residue of palm fiber as an additive was proposed. Different parameters for the preparation of active CLEAs in the hydrolysis of olive oil, such as precipitation agents, crosslinking agent concentration, additives, and coating agents were investigated. The highest activity yield (121.1 ± 0.1%) and volumetric activity (1578.1 ± 2.5 U/mL) were achieved for CLEAs prepared using the combination of a coating step with Triton® X-100 and polyethyleneimine plus the use of palm fiber as an additive. The variations of the secondary structures of BCL-CLEAs were analyzed by second-derivative infrared spectra, mainly indicating a reduction of the α-helix structure, which was responsible for the lipase activation in the supramolecular structure of the CLEAs. Thus, these results provided evidence of an innovative design of BCL-CLEAs as a sustainable and biocompatible opportunity for biotechnology applications.

Towards the differential diagnosis of prostate cancer by the pre-treatment of human urine using ionic liquids.

Prostate specific antigen (PSA) is the most widely used clinical biomarker for the diagnosis and monitoring of prostate cancer. Most available techniques for PSA quantification in human fluids require extensive sample processing and expensive immunoassays that are often unavailable in developing countries. The quantification of PSA in serum is the most common practice; however, PSA is also present in human urine, although less used in diagnosis. Herein we demonstrate the use of ionic-liquid-based aqueous biphasic systems (IL-based ABS) as effective pre-treatment strategies of human urine, allowing the PSA detection and quantification by more expedite equipment in a non-invasive matrix. If properly designed, IL-based ABS afford the simultaneous extraction and concentration of PSA (at least up to 250-fold) in the IL-rich phase. The best ABS not only allow to concentrate PSA but also other forms of PSA, which can be additionally quantified, paving the way to their use in differential prostate cancer diagnosis.

ß-Cyclodextrin/Isopentyl Caffeate Inclusion Complex: Synthesis, Characterization and Antileishmanial Activity.

Isopentyl caffeate (ICaf) is a bioactive ester widely distributed in nature. Our patented work has shown promising results of this molecule against Leishmania. However, ICaf shows poor solubility, which limits its usage in clinical settings. In this work, we have proposed the development of an inclusion complex of ICaf in ß-cyclodextrin (ß-CD), with the aim to improve the drug solubility, and thus, its bioavailability. The inclusion complex (ICaf:ß-CD) was developed applying three distinct methods, i.e., physical mixture (PM), kneading (KN) or co-evaporation (CO) in different molar proportions (0.25:1, 1:1 and 2:1). Characterization of the complexes was carried out by thermal analysis, Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and molecular docking. The ICaf:ß-CD complex in a molar ratio of 1:1 obtained by CO showed the best complexation and, therefore, was selected for further analysis. Solubility assay showed a marked improvement in the ICaf:ß-CD (CO, 1:1) solubility profile when compared to the pure ICaf compound. Cell proliferation assay using ICaf:ß-CD complex showed an IC50 of 3.8 and 2.7 µg/mL against L. amazonesis and L. chagasi promastigotes, respectively. These results demonstrate the great potential of the inclusion complex to improve the treatment options for visceral and cutaneous leishmaniases.

Continuous flow reactor based with an immobilized biocatalyst for the continuous enzymatic transesterification of crude coconut oil.

Here, we have assessed the use of one packed bed or two packed bed reactors in series in which Burkholderia cepacia lipase (BCL) was immobilized on protic ionic liquid (PIL)-modified silica and used as a biocatalyst for the transesterification of crude coconut oil. Reaction parameters including volumetric flow, temperature, and molar ratio were evaluated. The conversion of transesterification reaction products (ethyl esters) was determined using gas chromatography and the quantities of intermediate products (diglyceride and monoglyceride [MG]) were assessed using high-performance liquid chromatography. Packed bed reactors in series produced ethyl esters with the greatest efficiency, achieving 65.27% conversion after 96 H at a volumetric flow rate of 0.50 mL Min-1 at 40 °C and a 1:9 molar ratio of oil to ethanol. Further, within the first 24 H of the reaction, increased MG (54.5%) production was observed. Molecular docking analyses were performed to evaluate the catalytic step of coconut oil transesterification in the presence of BCL. Molecular docking analysis showed that triglycerides have a higher affinity energy (-5.7 kcal mol-1 ) than the smallest MG (-6.0 kcal mol-1 ), therefore, BCL catalyzes the conversion of triglycerides rather than MG, which is consistent with experimental results.