Immobilized acetylcholinesterase in magnetic nanoparticles for in-line inhibition studies using a capillary electrophoresis system.

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.

Green synthesis of silica-coated magnetic nanocarriers for simultaneous purification-immobilization of ß-1,3-xylanase.

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.

Glucose-6-phosphate dehydrogenase immobilized onto magnetic beads (G6PDH-Mb) as a generator system for production of NADPH: Development and application in metabolism studies.

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.

A Comprehensive 2D-LC/MS Online Platform for Screening of Acetylcholinesterase Inhibitors.

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.

On-flow magnetic particle activity assay for the screening of human purine nucleoside phosphorylase inhibitors.

Human purine nucleoside phosphorylase (HsPNP) catalyzes reversible phosphorolysis of nucleosides and deoxynucleosides in the purine cascade. HsPNP has been a target on behalf of the development of new leads for the treatment of a variety of T-cell mediated disorders. Several studies on the HsPNP are focused on the identification of effective, safe, and selective inhibitors. Therefore, this study describes the development of direct, simple, reliable, and inexpensive enzymatic assays to screen HsPNP inhibitors. Initially, HsPNP was covalently immobilized on the surface of magnetic particles (MPs). Due to the versatility of the MPs as solid support for enzyme immobilization, two different methods to monitor the enzyme activity are presented. Firstly, the activity of HsPNP-MPs was assessed offline by HPLC-DAD quantifying the formed hypoxanthine. Then, HsPNP-MPs were trapped in a peek tube, furnishing a microreactor which was inserted on-flow in an HPLC-DAD system to monitor the enzyme activity by the hypoxanthine quantification. Kinetic assays provided KMapp values for the inosine substrate of 488.2 ± 49.1 and 1084 ± 111 µM for the offline and on-flow assays, respectively. For the first time, kinetic studies for Pi as substrate using the HsPNP-MPs exhibits a Michaelis-Menten kinetic, yielding KMapp values for offline and on-flow of 521.2 ± 62.9 µM and 601 ± 66.5 µM, respectively. Inhibition studies conducted with a fourth generation immucillin derivative (DI4G) were employed as proof of concept to validate the use of the HsPNP-MPs assays for screening purposes. Additionally, a small library containing 11 compounds was used to assess the selectivity of the developed assays. The results showed that both presented assays can be applied to selectively recognizing and characterizing HsPNP inhibitors. Particularly, the on-flow method exhibited a high throughput and performance because of its automation and represents an easy and practical approach to reuse the HsPNP-MPs. Besides, this novel enzyme activity assay model can be further applied to other biological targets.

Enzymatic production of lactulose by fed-batch and repeated fed-batch reactor.

The effects of the most significant operational variables on reactor performance of fed-batch and repeated fed-batch were evaluated in the lactulose production by enzymatic transgalactosylation. Feed flowrate in the fed stage (F) and fructose to lactose molar ratio (Fr/L) were the variables that mostly affected the values ​​of lactulose yield (YLu), lactulose productivity (πLu) and selectivity of transgalactosylation (SLu/TOS). Maximum YLu of 0.21 g lactulose per g lactose was obtained at 50% w/w inlet carbohydrates concentration (IC) of, 50 °C, Fr/L 8, F 1 mL⋅min-1, 200 IU∙gLactose-1 reactor enzyme load and pH 4.5. At these conditions the selectivity was 7.4, productivity was 0.71 gLu∙g-1∙h-1and lactose conversion was 0.66. The operation by repeated fed batch increases the efficiency of use of the biocatalysts (EB) and the accumulated productivity compared to batch and fed batch operation with the same biocatalyst. EB obtained was 4.13 gLu∙mgbiocatalyst protein-1, 10.6 times higher than in fed-batch.

LC-HRMS and acetylcholinesterase affinity assay as a workflow for profiling alkaloids in Annona salzmannii extract.

Structure-based molecular networking is useful as a dereplication strategy to identify known molecules, unknown close analogues, or compound families. On the other hand, the ligand fishing assay is a remarkable alternative to accelerate the screening process and to overcome the drawbacks of laborious experiments usually adopted in natural product research. The combination of these approaches contributes to high productivity in disclosing active metabolites and a decrease in lead time identification. To provide a valuable data base for the alkaloids of A. salzmannii bark herein we disclose thirty-one isoquinoline alkaloids including benzyltetrahydroisoquinolines, aporphines, proaporphines, and protoberberines. Among these, twenty-six have not been described for A. salzmannii including the unprecedented alkaloid N,O-dimethylcoclaurine N-oxide. In addition, norcoclaurine (1), norreticuline (13), N,O-dimethylcoclaurine N-oxide (15), and N-acetylasimilobine (24) are now reported for the first time as ligand for acetylcholinesterase.

A novel on-flow mass spectrometry-based dual enzyme assay.

This work describes a new simultaneous on-flow dual parallel enzyme assay based on immobilized enzyme reactors (ICERs) with mass spectrometry detection. The novelty of this work relies on the fact that two different enzymes can be screened at the same time with only one single sample injection and in less than 6 min. The system consisted of two immobilized capillary enzyme reactors (ICERs). More specifically, the ICERs comprised two different enzymes that were accommodated in parallel and were placed between a liquid chromatography (LC) system and a mass spectrometer (MS). The resulting system could be adapted to other types of enzyme reactors with different supports. All the elements in the system were interfaced by means of two 10-port/two-position switching valves. Different tubing dimensions allowed us to monitor the activity of each enzyme independently during the same analysis. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) bioreactors were chosen as proof of concept. Acetylcholine (ACh) was used as substrate; the area of its protonated enzymatic hydrolysis product ion, choline, [M+H]+m/z 104.0, was monitored in the presence and absence of the standard cholinesterase inhibitor galantamine. This method proved to be an interesting tool for fast, simultaneous, and independent label-free dual enzyme inhibitor assay.

Cathepsin D immobilized capillary reactors for on-flow screening assays.

The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (KM = 81.9 ±â€¯7.49 µmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts.

Enhanced long-chain fatty alcohol oxidation by immobilization of alcohol dehydrogenase from S. cerevisiae.

This work reports on the oxidation of long-chain aliphatic alcohols catalyzed by a stabilized alcohol dehydrogenase from S. cerevisiae (yeast alcohol dehydrogenase (YADH)). In particular, the oxidation of the fatty alcohol tetracosanol (C24H50O) to yield lignoceric acid (C23H47COOH) was studied. The immobilization of YADH onto glyoxyl agarose supports crosslinked with a polymer (polyethylenimine) produced a highly stable catalyst (60-fold higher than the soluble enzyme at 40 °C). Aliphatic alcohols with different chain lengths (ranging from 2 to 24 carbons) were studied as substrates for YADH. The activity of YADH with aliphatic alcohols with a chain length higher than five carbon atoms is reported for the first time. The activities obtained with the immobilized YADH were all similar in magnitude, even with long-chain fatty alcohols such as docosanol and tetracosanol. As far as the oxidation of tetracosanol is concerned, the best values of reaction rate and substrate conversion were obtained at pH = 8.2 and T = 58 °C. At these conditions, the soluble enzyme inactivated rapidly, precluding its use in batch reaction. However, using the immobilized YADH, up to three sequential reaction batches were performed by recovering the catalyst after each batch. Several applications in the green oleochemical industry, e.g., for making plasticizers, lubricants, detergents, and personal care products, may benefit from having novel and stable biocatalysts able to oxidize long-chain fatty alcohols.

Improved chemical synthesis of hydrophobic Aß peptides using addition of C-terminal lysines later removed by carboxypeptidase B.

Many amyloidogenic peptides are highly hydrophobic, introducing significant challenges to obtaining high quality peptides by chemical synthesis. For example, while good yield and purity can be obtained in the solid-phase synthesis of the Alzheimer's plaque peptide Aß40, addition of a C-terminal Ile-Ala sequence to generate the more toxic Aß42 molecule creates a much more difficult synthesis resulting in low yields and purities. We describe here a new method that significantly improves the Fmoc solid-phase synthesis of Aß peptides. In our method, Lys residues are linked to the desired peptide's C-terminus through standard peptide bonds during the synthesis. These Lys residues are then removed post-purification using immobilized carboxypeptidase B (CPB). With this method we obtained both Aß42 and Aß46 of superior quality that, for Aß42, rivals that obtained by recombinant expression. Intriguingly, the method appears to provide independent beneficial effects on both the total synthetic yield and on purification yield and final purity. Reversible Lys addition with CPB removal should be a generally useful method for making hydrophobic peptides that is applicable to any sequence not ending in Arg or Lys. As expected from the additional hydrophobicity of Aß46, which is extended from the sequence Aß42 by a C-terminal Thr-Val-Ile-Val sequence, this peptide makes typical amyloid at rates significantly faster than for Aß42 or Aß40. The enhanced amyloidogenicity of Aß46 suggests that, even though it is present in relatively low amounts in the human brain, it could play a significant role in helping to initiate Aß amyloid formation.

Tyrosinase immobilized enzyme reactor: development and evaluation.

Immobilized enzyme reactors of tyrosinase (tyr-IMERs) for use on-line in HPLC system were prepared by different procedures and then compared. The enzyme, obtained from Agaricus bisporus, was immobilized on epoxy-silica which was prepared using different conditions. Enzyme immobilization was conducted by both in situ and in batch techniques. The different procedures were compared in terms of protein and activity retention, IMERs activity, kinetics and stability. The influence of immobilization procedure on enzyme activity and the behavior of the IMERs against a standard inhibitor were also investigated. In situ immobilization on epoxy-silica, synthesized using microwave assistance, provided the best conditions to prepare tyrosinase IMERs. The tyr-IMERs were successfully tested with known and potential inhibitors of tyrosinase, and the results showed that they can be used for the screening of inhibitors of that enzyme.

Diffusional restrictions in glyoxyl-agarose immobilized penicillin G acylase of different particle size and protein loading / Restricciones para la difusión de penicilina G acilasa inmovilizada en glicosil agarosa

Particle size and enzyme protein loading are design parameters of enzyme immobilization affecting biocatalyst performance that can be varied within broad margins. Their effect on mass transfer limitations at different bulk penicillin G concentrations has been studied with glyoxyl agarose immobilized penicillin G acylase biocatalysts of average particle size of 5·10-5m and 10·10-4m at protein loadings from 15 to 130 mg/g gel. Internal diffusional restrictions were evaluated for such biocatalysts: Thiele modulus varied from 1.17 for the small particles at the lower protein load to 5.84 for the large particles at the higher protein load. Effectiveness factors at different bulk substrate concentrations were determined for all biocatalysts, values ranging from 0.78 for small particle size at 25 mM penicillin G to 0.15 for large particle size at 2 mM penicillin G. Enzyme protein loading had a strong impact on the effectiveness factors of immobilized penicillin G acylase, being it more pronounced in the case of large particle size biocatalysts. At conditions in which 6-aminopenicillanic acid is industrially produced, all biocatalysts tested were mass-transfer limited, being this information valuable for reactor design and performance evaluation.

Bioconversão de sacarose em ácido glicônico e frutose usando reator com membrana / Sucrose bioconversion into gluconic acid and fructose using a membrane reactor

A conversão enzimática da sacarose pela ação sucessiva da invertase e da glicose oxidase (GOD), permite obter produtos de maior valor agregado, a saber, frutose e o ácido glicônico, dois produtos de amplo uso na indústria farmacêutica, alimentícia e química. Foi estudada a aplicação da invertase imobilizada em resinas aniônicas do tipo Dowex® (um copolímero de poliestireno-divinilbenzeno) sobre a hidrólise da sacarose bem como a oxidação da glicose pela glicose oxidase solúvel ou imobilizada no mesmo suporte em separado (sistema bifásico), utilizando-se um reator de membrana acoplado à membrana de ultrafiltração (100kDa) ou de microfitração (5µm). Posteriormente, avaliou-se o desempenho de ambas as formas de enzimas, solúveis ou imobilizadas num sistema monofásico empregando o mesmo reator...

The enzymatic conversion of sucrose through a successive action of invertase and glucose oxidase (GOO) allows the obtainment of products with higher commercial value, fructose and gluconic acid, which are widely used in pharmaceutical, food and chemical industries. Invertase and GOO immobilized on Dowex® anionic resin (a polystyrene divinylbenzene copolymer) as well as soluble GOD were used in a membrane bioreactor (MS) for sucrose hydrolysis and glucose oxidation. The MB was coupled with a UF-membrane (100kDa) or a MF-membrane (5µm). The bioconversion was conducted in two steps (biphasic system) as well as in one step (monophasic system). The bioconversion operated in a biphasic system permitted obtaining a fructose syrup with a concentration of about 70% through a separation of glucose and fructose using a cationic resin, 50W:8-100. As for the monophasic system, the yield of 96.6% and 67.4% for soluble and immobilized forms were attained respectively. No leakage of the enzymes from the support allowed the use of a microfiltration membrane, adding advantages to the membrane bioreactor operation.