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(1) Background: Producing active antimicrobial peptides with disulfide bonds in bacterial strains is challenging. The cytoplasm of Escherichia coli has a reducing environment, which is not favorable to the formation of disulfide bonds. Additionally, E. coli may express proteins as insoluble aggregates known as inclusion bodies and have proteolytic systems that can degrade recombinant peptides. Using E. coli strains like SHuffle and tagging the peptides with fusion proteins is a common strategy to overcome these difficulties. Still, the larger size of carrier proteins can affect the final yield of recombinant peptides. Therefore, a small fusion protein that can be purified using affinity chromatography may be an ideal strategy for producing antimicrobial peptides in E. coli. (2) Methods: In this study, we investigated the use of the small metal-binding protein SmbP as a fusion partner for expressing and purifying the antimicrobial peptide scygonadin in E. coli. Two constructs were designed: a monomer and a tandem repeat; both were tagged with SmbP at the N-terminus. The constructs were expressed in E. coli SHuffle T7 and purified using immobilized metal-affinity chromatography. Finally, their antimicrobial activity was determined against Staphylococcus aureus. (3) Results: SmbP is a remarkable fusion partner for purifying both scygonadin constructs, yielding around 20 mg for the monomer and 30 mg for the tandem repeat per 1 mL of IMAC column, reaching 95% purity. Both protein constructs demonstrated antimicrobial activity against S. aureus at MICs of 4 µM and 40 µM, respectively. (4) Conclusions: This study demonstrates the potential of SmbP for producing active peptides for therapeutic applications. The two scygonadin constructs in this work showed promising antimicrobial activity against S. aureus, suggesting they could be potential candidates for developing new antimicrobial drugs.

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This study aimed to develop and investigate the synthesis of 2-ethylhexyl oleate catalyzed by Candida antarctica lipase immobilized on magnetic poly(styrene-co-divinylbenzene) (STY-DVB-M) particles in a magnetically stabilized fluidized bed reactor (MSFBR) operated in continuous mode. The physical properties of the copolymer were characterized by Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The glass transition temperature was 85.68 °C, and the onset of thermal degradation occurred at 406.66 °C. Syntheses were performed at 50 °C using a space time of 12 h and a bed porosity of 0.892. Assays were conducted to assess the influence of magnetic field intensity (5 to 15 mT) on reaction yield, ester concentration, and productivity. The highest productivity was 0.850 ± 0.023 mmol g-1 h-1, obtained with a magnetic field intensity of 15 mT. An operational stability test was performed under these conditions, revealing a biocatalyst half-life of 2148 h (179 operation cycles) and a thermal deactivation constant of 3.23 × 10-4 h-1 (R2 = 0.9446). Computational simulations and mathematical modeling were performed using Scilab based on ping-pong bi-bi kinetics and molar balances of reaction species. The model provided consistent results of interstitial velocity and good prediction of reaction yields, with R2 = 0.926. These findings demonstrate that the studied technique can provide improvements in biocatalytic processes, representing a promising strategy for the enzymatic synthesis of 2-ethylhexyl oleate.

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This research evaluated H2TiO7 nanotubes (TiNTs) functionalized with 1 (1TiCN), 5 (2TiCN), and 10 (3TiCN) wt.% of chitosan for the removal of clonazepam by an adsorption/photocatalysis-conjugated method. The samples were immobilized on glass, and their mechanical stability was tested by washings. The functionalization of the samples was verified by the FTIR and DRS techniques. SEM images displayed nanotubes in the samples and thickness of 4.24 µm for the 2TiCN coating. The chemical composition of the 2TiCN coating was obtained by EDS. The XRD patterns evidenced chitosan and titanate phases in the functionalized samples. Furthermore, the 2TiCN coating was evaluated in the removal of clonazepam, reaching 80.79% (4.38 and 49.64% more than the TiNT and commercial TiO2 powders, respectively) after 240 min and being 6.88% more efficient after 4 reuses than the 2TiCN powders. OH- ions were the main oxidizing species found by scavenger tests. The surface area of 2TiCN (168.6 m2/g) was 2 times higher than that of TiNTs, and its bandgap (2.95 eV) was the lowest. Therefore, the 2TiCN coating is an excellent alternative to remove clonazepam.

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Anaerobic bioreactors are an efficient technology for the biodegradation of emerging contaminants in environmental matrices. In this work, a horizontal-flow anaerobic immobilized biomass (HAIB) bioreactor was used to remove caffeine (CAF), which is frequently found in various aqueous matrices. The acrylic bench top bioreactor, with dimensions of 100 × 5.00 cm, was operated with a hydraulic retention time (HRT) of 12 h, during 45 weeks, under mesophilic conditions. The operation was performed in 4 phases: without CAF addition (phase I); CAF spiked at 300 µg L-1 (phase II); CAF at 600 µg L-1 (phase III); and CAF at 900 µg L-1 (phase IV). Samples of bioreactor influent and effluent were analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). The bioreactor removed organic matter (OM) and CAF with efficiencies of 88 and 93%, respectively. The first-order apparent removal constant (Kapp) values for OM and CAF were 0.419 and 0.304 h-1, respectively. Five transformation products (TPs) were identified, with m/z 243, 227, 211, and 181 (two products). The HAIB bioreactor is a suitable system for the removal of CAF present in wastewater, even at a concentration level of µg L-1.

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Acylases catalyze the hydrolysis of amide bonds. Penicillin G acylase (PGA) is used for the semi-synthesis of penicillins and cephalosporins. Although protein immobilization increases enzyme stability, the design of immobilized systems is difficult and usually it is empirically performed. We describe a novel application of our strategy for the Rational Design of Immobilized Derivatives (RDID) to produce optimized acylase-based immobilized biocatalysts for enzymatic bioconversion. We studied the covalent immobilization of the porcine kidney aminoacylase-1 onto aldehyde-based supports. Predictions of the RDID1.0 software and the experimental results led to the selection of glyoxyl-Sepharose CL 4B support and pH 10.0. One of the predicted clusters of reactive amino groups generates an enzyme-support configuration with highly accessible active sites, contributing with 82% of the biocatalyst's total activity. For Escherichia coli PGA, the predictions and experimental results show similar maximal amounts of immobilized protein and activity at pH 8.0 and 10.0 on glyoxyl-Sepharose CL 10B. However, thermal stability of the immobilized derivative is higher at pH 10.0 due to an elevated probability of multipoint covalent attachment. In this case, two clusters of amino groups are predicted to be relevant for PGA immobilization in catalytically competent configurations at pH 10.0, showing accessible active sites and contributing with 36% and 44% of the total activity, respectively. Our results support the usefulness of the RDID strategy to model different protein engineering approaches (site-directed mutagenesis or obtainment of fusion proteins) and select the most promising ones, saving time and laboratory work, since the in silico-designed modified proteins could have higher probabilities of success on bioconversion processes.

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Nucleoside Hydrolases (NH) are considered a target for the development of new antiprotozoal agents. The development of new and automated screening assays for the identification of NH inhibitors can accelerate the first stages of the drug discovery process. In this work, NH from Leishmania donovani (LdNH) was covalently immobilized onto magnetic particles (LdNH-MPs) and trapped by magnets into a TFE tube to yield an immobilized enzyme reactor (IMER). For an automated assay, the LdNH-MP-IMER was connected in-line to an analytical column in an HPLC-DAD system to monitor the enzyme activity through quantification of the product hypoxanthine. Kinetic studies provided a KM value of 2079 ± 87 µmol.L-1 for the inosine substrate. Validation of the LdNH-MP-IMER for onflow screening purposes was performed with a library containing 12 quinolone ribonucleosides. Among them, three were identified as new competitive LdNH inhibitors, with Ki values between 83.5 and 169.4 µmol.L-1. This novel in-line screening assay has proven to be reliable, fast, low cost, and applicable to large libraries of compounds.

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In the present research work, the photocatalytic evaluation of NiTiO3 nanoparticles immobilized on glass plates by the spin-coating procedure was carried out in the degradation of the recalcitrant herbicide 2,6-dichlorobenzamide (BAM). The concentrations of Ni employed to synthesize NiTiO3 nanoparticles were 1 wt% (1TESNi) and 2 wt% (2TESNi). The stability of coatings was evaluated by several washings and thermal treatments, which were verified by UV-vis analyses. The morphology of the coatings was studied by scanning electron microscopy (SEM-EDS). The coatings displayed thickness values of 1.35 and 2.56 µm for TiO2 and 1TESNi, respectively. The crystalline phases of the coatings were analyzed by X-ray diffraction (XRD), confirming the presence of NiTiO3 and other phases related to TiO2. The bandgap of 1TESNi, compared with the bare TiO2, was reduced from 2.96 to 2.40 eV as a consequence of Ni addition. The TiO2, 1TESNi and 2TESNi coatings were evaluated in the photodegradation of BAM using visible-light for 240 min. The highest effectiveness was displayed by the 1TESNi coating, obtaining degradation of 92.56% after 240 min. Also, the photocatalytic efficiency of the 1TESNi coating was only reduced 1.99% after 3 reuse cycles in the BAM degradation. The scavenger tests revealed that the main oxidizing species involved in the reaction were the •OH- and •O2- radicals. The 1TESNi coating showed the highest photocatalytic efficiency because of its absorption in the visible-light region, valuable surface area and electronic charge separation. Thus, these advantageous features guarantee that NiTiO3 coatings are an efficient method for degrading recalcitrant herbicides from drinking water using a practical way to recover and reuse photocatalysts.

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Enzyme assays can be performed with the capillary electrophoresis technique (CE) in many approaches, such as the immobilized enzyme micro-reactor. Acetylcholinesterase is a promising enzyme to be used when pursuing such a method, as it has already been explored in the proposal of similar methods of miniaturizing enzyme assays. The present work proposes a novel enzyme micro-reactor, based on the anchorage of the enzyme on magnetic nanoparticles of MnFe2O4, with chitosan and glutaraldehyde as the cross-linker in the capillary by means of an arrange of neodymium magnets. The calculated Km of the enzyme evaluated by this method was 1.12 mmol L-1, comparable to other studies in the literature that utilizes immobilized enzymes. Also, IC50 for neostigmine was assessed in 3 different micro-reactors, with an average of 29.42 ± 3.88 µmol L-1. In terms of the micro-reactor stability, it was possible to perform at least 25 experiments with assembled micro-reactor. The method was applied to hydroalcoholic extracts of 7 plant species. Plinia cauliflora had the best result, with 42.31 ± 6.81% of enzyme inhibition in a concentration of 100 mg L-1.

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The increasing worries by the inadequate use of energy and the preservation of nature are promoting an increasing interest in the production of biolubricants. After discussing the necessity of producing biolubricants, this review focuses on the production of these interesting molecules through the use of lipases, discussing the different possibilities (esterification of free fatty acids, hydroesterification or transesterification of oils and fats, transesterification of biodiesel with more adequate alcohols, estolides production, modification of fatty acids). The utilization of discarded substrates has special interest due to the double positive ecological impact (e.g., oil distillated, overused oils). Pros and cons of all these possibilities, together with general considerations to optimize the different processes will be outlined. Some possibilities to overcome some of the problems detected in the production of these interesting compounds will be also discussed.

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The ε4 allele of the apolipoprotein E gene (APOE4) constitutes the main genetic risk factor for late-onset Alzheimer disease (AD). High amounts of pure apolipoprotein E4 (ApoE4), in a rapid and reproducible fashion, could be of value for studying its pathophysiological roles in AD. The aim of the present work was to optimize a preparative method to obtain highly purified recombinant ApoE4 (rApoE4) with full biological activity. rApoE4 was expressed in the E. Coli BL21(D3) strain and a soluble form of the protein was purified by a combination of affinity and size-exclusion chromatography that precluded a denaturation step. The structural integrity and the biochemical activity of the purified rApoE4 were confirmed by circular dichroism and a lipid-binding assay. Several biological parameters affected by rApoE4, such as mitochondrial morphology, mitochondrial membrane potential and reactive oxygen species production were studied in CNh cells, a neuronal cell line, and neurodifferentiation and dendritogenesis were analyzed in the SH-SY5Y neuroblastoma cell line. The improved rApoE4 purification technique reported here enables the production of highly purified protein that retain the structural properties and functional activity of the native protein, as confirmed by tests in two different neuronal cell lines in culture.

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This work provides the basis for implementing a continuous treatment system using a bacterial consortium for wastewater containing a pesticide mixture of iprodione (IPR) and chlorpyrifos (CHL). Two bacterial strains (Achromobacter spanius C1 and Pseudomonas rhodesiae C4) isolated from the biomixture of a biopurification system were able to efficiently remove pesticides IPR and CHL at different concentrations (10 to 100 mg L-1) from the liquid medium as individual strains and free consortium. The half-life time (T1/2) for IPR and CHL was determined for individual strains and a free bacterial consortium. However, when the free bacterial consortium was used, a lower T1/2 was obtained, especially for CHL. Based on these results, an immobilized bacterial consortium was formulated with each bacterial strain encapsulated individually in alginate beads. Then, different inoculum concentrations (5, 10, and 15% w/v) of the immobilized consortium were evaluated in batch experiments for IPR and CHL removal. The inoculum concentration of 15% w/v demonstrated the highest pesticide removal. Using this inoculum concentration, the packed-bed bioreactor with an immobilized bacterial consortium was operated in continuous mode at different flow rates (30, 60, and 90 mL h-1) at a pesticide concentration of 50 mg L-1 each. The performance in the bioreactor demonstrated that it is possible to efficiently remove a pesticide mixture of IPR and CHL in a continuous system. The metabolites 3,5-dichloroaniline (3,5-DCA) and 3,5,6-trichloro-2-pyridinol (TCP) were produced, and a slight accumulation of TCP was observed. The bioreactor was influenced by TCP accumulation but was able to recover performance quickly. Finally, after 60 days of operation, the removal efficiency was 96% for IPR and 82% for CHL. The findings of this study demonstrate that it is possible to remove IPR and CHL from pesticide-containing wastewater in a continuous system.

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Tailoring magnetic nanocarriers with tunable properties is of great significance for the development of multifunctional candidate materials in numerous fields. Herein, we report a one-pot biomimetic silicification-based method for the synthesis of silica-coated magnetic nanoparticles. The synthesis process was mild, low cost, and highly efficient, which took only about 21 min compared with 4.5-120 h in other literature. Then, the carriers had been characterized by VSM, SEM, TEM, XRD, FT-IR, and EDS to confirm their function. To evaluate the usefulness of the carriers, they were adopted to couple the purification and immobilization of ß-1,3-xylanase from the cell lysate in a single step with high immobilization yield (92.8 %) and high activity recovery (82.4 %). The immobilized enzyme also retained 58.4 % of the initial activity after 10 cycles and displayed good storage properties, and improved thermal stability, which would be promising in algae biomass bioconversion as well as other diverse applications.

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U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.

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The purpose of this study was the production of maltobionic acid, in the form of sodium maltobionate, by Z. mobilis cells immobilized in polyurethane. The in situ immobilized system (0.125-0.35 mm) was composed of 7 g polyol, 3.5 g isocyanate, 0.02 g silicone, and 7 g Z. mobilis cell, at the concentration of 210 g/L. The bioconversion of maltose to sodium maltobionate was performed with different cell concentrations (7.0-9.0 gimobilized/Lreaction_medium), temperature (30.54-47.46 °C), pH (5.55-7.25), and substrate concentration (0.7-1.3 mol/L). The stability of the immobilized system was evaluated for 24 h bioconversion cycles and storage of 6 months. The maximum concentration of sodium maltobionate was 648.61 mmol/L in 34.34 h process (8.5 gdry_cell/Lreaction_medium) at 39 °C and pH 6.30. The immobilized system showed stability for 19 successive operational cycles of 24 h bioconversion and 6 months of storage, at 4 °C or 22 °C.

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Reduced nicotinamide adenine dinucleotide phosphate (NADPH) participates in several anabolic and catabolic pathways, being essential in numerous biochemical reactions involving energy release. Most of these reactions require a high amount of NADPH, which can be expensive from an industry point of view. Thus, biotechnology industries developed a great interest in NADPH production. Currently, there are different ways to obtain NADPH in situ, however, the most common is by enzymatic reactions, known as generator systems. Although this approach can be beneficial in terms of cost, the major drawback is the impossibility of reusing the enzyme. To overcome this, enzyme immobilization is a proven alternative. Herein, we report the use of glucose-6-phosphate dehydrogenase immobilized onto magnetic beads (G6PDH-Mb) through glutaraldehyde coupling to produce high amounts of NADPH. The G6PDH-Mbs were kinetically characterized showing a sigmoidal curve. Besides, the stability was evaluated, and their reuse was demonstrated for a period superior to 40 days. The G6PDH-Mb was used to in situ production of the NADPH metabolism experiments, using human liver microsome solutions and either albendazole or fiscalin B as model targets. The production of in vitro metabolites from albendazole and fiscalin B was evaluated by comparing the use of NADPH generated in situ with those obtained by commercial NADPH. Moreover, the activity of the G6PDH-Mb was monitored after using it for five consecutive albendazole metabolism reactions, with only a minor decrease in the enzyme activity (3.58 ± 1.67%) after the fifth time of use. The higher concentration obtained when using the designed G6PDH-Mb generator system demonstrated proof of the concept and its applicability.

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Genome editing using the CRISPR/Cas9 system is one of the trendiest methodologies in the scientific community. Many genome editing approaches require recombinant Streptococcus pyogenes Cas9 (SpCas9) at some point during their application, for instance, for in vitro validation of single guide RNAs (SgRNAs) or for the DNA-free editing of genes of interest. Hereby, we provide a simple and detailed expression and purification protocol for SpCas9 as a protein fused to GFP and MBP. This protocol improves protein yield and simplifies the purification process by overcoming the frequently occurring obstacles such as plasmid loss, inconsistent protein expression levels, or inadequate protein binding to affinity resins. On average, this protocol yields 10 to 30 mg of purified, active, His6−MBP−SpCas9 NLS−GFP protein. The purity addressed through SDS-PAGE is > 80%.

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Vinasse is a high pollutant liquid residue from bioethanol production. Due to its toxicity, most vinasse is used not disposed of in water bodies but employed for the fertigation of sugarcane crops, potentially leading to soil salinization or heavy metal deposition. The anaerobic digestion of vinasse for energy production is the main alternative to fertigation, but the process cannot eliminate colored compounds such as melanoidins, caramels, or phenolic compounds. The treatment of raw vinasse with white-rot fungi could remove colored and persistent toxic compounds, but is generally considered cost-ineffective. We report the treatment of vinasse by an autochthonous Trametes sp. strain immobilized in polyurethane foam and the concomitant production of high titers of laccase, a high value-added product that could improve the viability of the process. The reuse of the immobilized biomass and the discoloration of raw vinasse, the concentration of phenolic compounds, BOD and COD, and the phytotoxicity of the treated vinasse were measured to assess the viability of the process and the potential use of treated vinasse in fertigation or as a complementary treatment to anaerobic digestion. Under optimal conditions (vinasse 0.25X, 30 °C, 21 days incubation, 2% glucose added in the implantation stage), immobilized Trametes sp. causes a decrease of 75% in vinasse color and total phenolic compounds, reaching 1082 U L-1 of laccase. The fungi could be used to treat 0.50X vinasse (BOD 44,400 mg O2 L-1), causing a 26% decolorization and a 30% removal of phenolic compounds after 21 days of treatment with maximum laccase titers of 112 U L-1, while reducing COD and BOD from 103,290 to 42,500 mg O2 L-1 (59%) and from 44,440 to 21,230 mg O2 L-1 (52%), respectively. The re-utilization of immobilized biomass to treat 0.50X vinasse proved to be successful, leading to the production of 361 U L-1 of laccase with 77% decolorization, 61% degradation of phenolic compounds, and the reduction of COD and BOD by 75% and 80%, respectively. Trametes sp. also reduced vinasse phytotoxicity to Lactuca sativa seedlings. The obtained results show that the aerobic treatment of vinasse by immobilized Trametes sp. is an interesting technology that could be employed as a sole treatment for the bioremediation of vinasse, with the concomitant the production of laccase. Alternatively, the methodology could be used in combination with anaerobic digestion to achieve greater decolorization and reduction of phenolic compounds, melanoidins, and organic load.

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Aptamers are affinity-based oligonucleotide ligands raised against a target molecule, which might be of proteic or other nature. Aptamers are developed by using a reiterative in vitro selection procedure, named SELEX, in which the target is exposed to a combinatorial oligonucleotide combinatorial library. Target bound oligonucleotides are eluted, and PCR amplified followed by the next SELEX round. The process is repeated until no further increase in target binding affinity and specificity is achieved. Selected aptamers are identified and immobilized for protein purification. In view of their stability against denaturation and capability of renaturation, low costs of production, easiness of modification and stabilization, oligonucleotide aptamers are excellent tools as high-affinity ligands for applications of protein purification.

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The continuous interest in discovering new bioactive molecules derived from natural products (NP) has stimulated the development of improved screening assays to help overcome challenges in NP-based drug discovery. Here, we describe a unique platform for the online screening of acetylcholinesterase inhibitors without the need for pre-treating the sample. In the current study, we have demonstrated the ability to combine reversed-phase separation with a capillary immobilized enzyme reactor (cIMER) in two-dimensional liquid chromatography system coupled with mass spectrometry detection. We systematically investigated the effects of method parameters that are of practical significance and are known to affect the enzyme assay and interfere in the analysis such as: bioreactor dimensions, loop sizes, amount of immobilized enzyme, second dimension flow rates, reaction time, substrate concentration, presence of organic modifier, limit of detection and signal suppression. The performance of this new platform was evaluated using a mixture containing three known AChE inhibitors (tacrine, galanthamine and donepezil) and an ethanolic extract obtained from the dry bulbs of Hippeastrum calyptratum (Amaryllidaceae) was investigated to provide a proof of concept of the applicability of the platform for the analysis of complex mixtures such as those derived from NPs.

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This study aimed the enzymatic decyl esters production by hydroesterification, a two-step process consisting of hydrolysis of refined soybean (RSBO) or used soybean cooking (USCO) oils to produce free fatty acids (FFA) and further esterification of purified FFA. Using free lipase from Candida rugosa (CRL), about 98% hydrolyses for both oils have been observed after 180 min of reaction using a CRL loading of 50 U g-1 of reaction mixture, 40 °C, and a mechanical stirring of 1500 rpm. FFA esterification with decanol in solvent-free systems was performed using lipase from Thermomyces lanuginosus (TLL) immobilized by physical adsorption on silica particles extracted from rice husk, an agricultural waste. For such purpose, non-functionalized (SiO2) or functionalized rice husk silica bearing octyl (Octyl-SiO2) or phenyl (Phe-SiO2) groups have been used as immobilization supports. Protein amounts between 22 and 28 mg g-1 of support were observed. When used in the esterification, they enabled a FFA conversion of 81.3-87.6% after 90-300 min of reaction. Lipozyme TL IM, a commercial immobilized TLL, exhibited similar performance compared to TLL-Octyl-SiO2 (FFA conversion ≈90% after 90-120 min of reaction). However, high operational stability after fifteen successive esterification batches was observed only for TLL immobilized on Octyl-SiO2 (activity retention of ≈90% using both FFA sources). The produced decyl esters presented good characteristics as potential biolubricants according to standard methods (ASTM) and thermal analysis.

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