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- A Flow Injection Methodology for Acetamide Determination Using a Tubular Bioreactor and na Ammonium Sensor †Publication . Veríssimo, Marta I. S.; Silva, Nelson A. F.; Karmali, Amin; Gomes, Maria Teresa S. R.Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative bacterium quite versatile that grows in the soil, in coastal marine habitats, as well as in the tissues of plants and animals. P. aeruginosa is the source of amidase (acylamide amidohydrolase E.C. 3.5.1.4) which catalyzes the hydrolysis of a small range of short aliphatic amides into the corresponding carboxylic acids and ammonia. A low cost piezoelectric quartz crystal coated with a selective membrane for ammonium was used to detect the reaction product. Conversion of amide into the correspondent amine was achieved both with cell-free extract of P. Aeruginosa or the whole cells. This conversion was first performed in batch and later on injected into the sensor system where a buffer carrier was flowing over the coated crystal. Another approach consisted in incorporating a conversion reactor with the immobilized cell-free extract of P. Aeruginosa in the FIA system. Amide solutions were injected and carried by the buffer stream through the reactor and then directed to the sensor. Different supports were used for immobilization, such as calcium alginate beads, glass beads and the inside walls of a hollow glass column. The best arrangement allowed acetamide determination without sensitivity lost for 1-month period.
- A sensitive microplate assay for lipase activity measurement using olive oil emulsion substrate: modification of the copper soap colorimetric methodPublication . Mustafa, Ahmad; Karmali, Amin; Abdelmoez, WaelThe present work involves a sensitive high-throughput microtiter plate based colorimetric assay for estimating lipase activity using cupric acetate pyridine reagent (CAPR). In the first approach, three factors two levels factorial design methodology was used to evaluate the interactive effect of different parameters on the sensitivity of the assay method. The optimization study revealed that the optimum CAPR concentration was 7.5% w/v, the optimum solvent was heptane and the optimum CAPR pH was 6. In the second approach, the optimized colorimetric microplate assay was used to measure lipase activity based on enzymatic hydrolysis of olive oil emulsion substrate at 37°C and 150 rpm. The emulsion substrates were formulated by using olive oil, triton X-100 (10% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 1:1:1 in the case of Candida sp. lipase. While in the case of immobilized lipozyme RMIM, The emulsion substrates were formulated by using olive oil, triton X-100 (1% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 2:1:1. Absorbance was measured at 655 nm. The stability of this assay (in terms of colored heptane phase absorbance readings) retained more than 92.5% after 24 h at 4°C compared to the absorbance readings measured at zero time. In comparison with other lipase assay methods, beside the developed sensitivity, the reproducibility and the lower limit of detection (LOD) of the proposed method, it permits analyzing of 96 samples at one time in a 96-well microplate. Furthermore, it consumes small quantities of chemicals and unit operations.
- Investigation of structural effects and behaviour of pseudomonas aeruginosa amidase encapsulated in reserved micellesPublication . Fragosa, Ana; Pacheco, Rita; Karmali, AminThe acetohydroxamic acid synthesis reaction was studied using whole cells, cell-free extract and purified amidase from the strains of Pseudomonas aeruginosa L10 and A13 entrapped in a reverse micelles system composed of cationic surfactant tetradecyltrimethyl ammonium bromide. The specific activity of amidase, yield of synthesis and storage stability were determined for the reversed micellar system as well as for free amidase in conventional buffer medium. The results have revealed that amidase solutions in the reverse micelles system exhibited a substantial increase in specific activity, yield of synthesis and storage stability. In fact, whole cells from P. aeruginosa L10 and AI3 in reverse micellar medium revealed an increase in specific activity of 9.3- and 13.9-fold, respectively, relatively to the buffer medium. Yields of approximately 92% and 66% of acetohydroxamic acid synthesis were obtained for encapsulated cell free extract from P. aeruginosa L10 and A13, respectively. On the other hand, the half-life values obtained for the amidase solutions encapsulated in reverse micelles were overall higher than that obtained for the free amidase solution in buffer medium. Half-life values obtained for encapsulated purified amidase from P. aeruginosa strain L10 and encapsulated cell-free extract from P. aeruginosa strain AI3 were of 17.0 and 26.0 days, respectively. As far as the different sources biocatalyst are concerned, the data presented in this work has revealed that the best results, in both storage stability and biocatalytic efficiency, were obtained when encapsulated cell-free extract from P. aeruginosa strain AI3 at 14/0 of 10 were used. Conformational changes occurring upon encapsulation of both strains enzymes in reverse micelles of TAB in heptane/octanol were additionally identified by FTIR spectroscopy which clarified the biocatalysts performances.
- Pseudomonas aeruginosa amidase: Aggregation in recombinant Escherichia coliPublication . Borges, Patrícia; Pacheco, Rita; Karmali, AminThe effect of cultivation parameters such as temperature incubation, IPTG induction and ethanol shock on the production of Pseudomonasaeruginosa amidase (E.C.3.5.1.4) in a recombinant Escherichia coli strain in LB ampicillin culture medium was investigated. The highest yield of solubleamidase, relatively to other proteins, was obtained in the condition at 37 degrees C using 0.40 mM IPTG to induce growth, with ethanol. Our results demonstrate the formation of insoluble aggregates containing amidase, which was biologically active, in all tested growth conditions. Addition of ethanol at 25 degrees C in the culture medium improved amidase yield, which quantitatively aggregated in a biologically active form and exhibited in all conditions an increased specific activity relatively to the soluble form of the enzyme. Non-denaturing solubilization of the aggregated amidase was successfully achieved using L-arginine. The aggregates obtained from conditions at 37 degrees C by Furier transform infrared spectroscopy (FTIR) analysis demonstrated a lower content of intermolecular interactions, which facilitated the solubilization step applying non-denaturing conditions. The higher interactions exhibited in aggregates obtained at suboptimal conditions compromised the solubilization yield. This work provides an approach for the characterization and solubilization of novel reported biologically active aggregates of this amidase.
- A novel colorimetric assay of beta-D glucans in basidiomycete strains by alcian blue dye in a 96-well microtiter platePublication . Semedo, Madga Sofia Cardoso Nobre; Karmali, Amin; Fonseca, LuísBasidiomycete strains synthesize several types of beta-D-glucans, which play a major role in the medicinal properties of mushrooms. Therefore, the specific quantification of these beta-D-glucans in mushroom strains is of great biochemical importance. Because published assay methods for these beta-D-glucans present some disadvantages, a novel colorimetric assay method for beta-D-glucan with alcian blue dye was developed. The complex formation was detected by following the decrease in absorbance in the range of 620 nm and by hypsochromic shift from 620 to 606 nm (similar to 14 nm) in UV-Vis spectrophotometer. Analysis of variance was used for optimization of the slope of the calibration curve by using the assay mixture containing 0.017% (w/v) alcian blue in 2% (v/v) acetic acid at pH 3.0. The high-throughput colorimetric assay method on microtiter plates was used for quantification of beta-D-glucans in the range of 0-0.8 mu g, with a slope of 44.15 x 10(-2) and a limit of detection of 0.017 mu g/well. Recovery experiments were carried out by using a sample of Hericium erinaceus, which exhibited a recovery of 95.8% for beta-1,3-D-glucan. The present assay method exhibited a 10-fold higher sensitivity and a 59-fold lower limit of detection compared with the published method with congo red beta-D-glucans of several mushrooms strains were isolated from fruiting bodies and mycelia, and they were quantified by this assay method. This assay method is fast, specific, simple, and it can be used to quantify beta-D-glucans from other biological sources. (C) 2015 American Institute of Chemical Engineers
- Bioconversion of D-glucose into D-glucosone by Glucose 2-oxidase from Coriolus versicolor at Moderate PressuresPublication . Karmali, Amin; Coelho, JoseGlucose 2-oxidase (pyranose oxidase, pyranose: oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor catalyses the oxidation of D-glucose at carbon 2 in the presence of molecular O(2) producing D-glucosone (2-keto-glucose and D-arabino-2-hexosulose) and H(2)O(2). It was used to convert D-glucose into D-glucosone at moderate pressures (i.e. up to 150 bar) with compressed air in a modified commercial batch reactor. Several parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, [enzyme], [glucose], pH, temperature, nature of fluid and the presence of catalase. Glucose 2-oxidase was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. The rate of bioconversion of D-glucose increased with the pressure since an increase in the pressure with compressed air resulted in higher rates of conversion. On the other hand, the presence of catalase increased the rate of reaction which strongly suggests that H(2)O(2) acted as inhibitor for this reaction. The rate of bioconversion of D-glucose by glucose 2-oxidase in the presence of either nitrogen or supercritical CO(2) at 110 bar was very low compared with the use of compressed air at the same pressure. The optimum temperature (55 degrees C) and pH (5.0) of D-glucose bioconversion as well as kinetic parameters for this enzyme were determined under moderate pressure. The activation energy (E(a)) was 32.08 kJmol(-1) and kinetic parameters (V(max), K(m), K(cat) and K(cat)/K(m)) for this bioconversion were 8.8 Umg(-1) protein, 2.95 mM, 30.81 s(-1) and 10,444.06 s(-1)M(-1), respectively. The biomass of C. versicolor as well as the cell-free extract containing glucose 2-oxidase activity were also useful for bioconversion of D-glucose at moderate pressures. The enzyme was apparently stable at moderate pressures since such pressures did not affect significantly the enzyme activity.
- Adsorption of myoglobin on calixarenes and biocatalysis in organic mediaPublication . Semedo, Magda C.; Karmali, Amin; Barata, Patrícia; Prata, José V.Derivatives of p-tert-butylcalix[4,6,8]arene carboxylic acids were used for selective adsorption of myoglobin.Amixtureofmyoglobin,laccaseandperoxidase wasusedforextractionwithcalixarenesandonlymyoglobin was selectively extracted to organic media. Myoglobin and Mb c–calixarene exhibited pseudoactivity of peroxidase in aqueous and organic media. This protein-calixarene complex exhibited the highest specific activity of 1.37 × 10−1 U.mg protein−1 at initial pH 6.5 of myoglobin aqueous solution. Apparent kinetic parameters (V max, K m, k cat and k cat/K m) for the pseudoperoxidase activity were determined in organic media for different initial pH values of myoglobin aqueous solution by Michaelis-Menten plot. The stability of this complex was studied for different initial pH values and t1/2 values were obtained in the range of 3.5–5.2 days. The extracted Mb c in organic media was recovered into fresh aqueous solutions at alkaline pH with a recovery of pseudoperoxidase activity of over 100%.
- Development of a biosensor for urea assay based on amidase inhibition, using an ion-selective electrodePublication . Barbosa, Ana Rita Dantas Balsemão; Karmali, AminA biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; anion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensorresponse and showed that 30 mu L of cell-free extract containing 7.47 mg protein mL(-1), 2 mu L of glutaraldehyde (5%, v/v) and 10 mu L of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation ofmembranes in urea. The biosensor exhibited a linear response in the range of 4.0-10.0 mu M urea, a detection limit of 2.0 mu M for urea, a response timeof 20 s, a sensitivity of 58.245 % per mu M urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.
- Optimized Production of Hydroxamic Acid Derivatives with Antioxidant and Anticholinergic Potential by Immobilized Pseudomonas aeruginosa CellsPublication . Bernardo, Marisa; Reis, Telma; Minhalma, Miguel; Karmali, Amin; Serralheiro, Maria Luísa; Pacheco, RitaIn this study were investigated, the synthesis, acetylcholinesterase inhibition and antioxidant activity of a series of hydroxamic acid derivatives (HAD), with different chemical group characteristics, such as aliphatic (acetohydroxamic acid and butyryl hydroxamic acid), aromatic (benzohydroxamic acid and phenylalanine hydroxamic acid) and amino acid (glycine hydroxamic acid and alanine hydroxamic acid). It was observed that these HAD compounds present very promising activity as acetylcholinesterase (AChE) inhibitors and as antioxidants. The aliphatic HAD demonstrated to have a higher inhibitory activity of AChE than amino acid or aromatic HAD. As for the antioxidant activity, a high antioxidant potential was found for all the compounds with EC50 values ranging from 0.19 µM to 1.65 µM. Aiming these applications, a biocatalysis approach was used to obtain these HADs with optimal reactional conditions. In this study, reverse micelles with immobilized Pseudomonas aeruginosa intact cells containing amidase were used as a biocatalyst to catalyze the acyltransferase reaction of the corresponding substrate amide and hydroxylamine to obtain various HAD and this was achieved for the first time with yields of approximately 100 %.
- Optimisation and economic assessment of lipase-catalysed production of monoesters using Rhizomucor miehei lipase in a solvent-free systemPublication . Ahmad, Mustafa; Karmali, Amin; Abdelmoez, WaelEnzymatic production of glycerin monolaurate (GML) was carried out by lipase catalysed esterification of lauric acid and glycerin in a solvent-free media. Response surface methodology (RSM), based on 5-level and 3-variable composite design, was employed to study the interactive effects of reaction temperature (48-60 degrees C), enzyme load (1-4% w/w), and glycerin to lauric acid molar ratio (1:1-4:1) on glycerin laurate yield. The optimum conditions obtained were a temperature of 60 degrees C, an enzyme load of 4%, and a glycerin to lauric acid molar ratio of 4:1. The maximum predicted and experimental conversion values were 92.26% and 93.23%, respectively. Utilisation of Lipozyme RM IM (Rhizomucor miehei lipase) allowed the formation of a mixture consisting of 50% monoglyceride, 34.6% diglyceride and 8A% triglyceride, which fulfills the requirements established by the World Health Organization (WHO) for use as a food emulsifier. In addition, the Lipozyme RM IM maintained more than 90% of its original activity after being used for six cycles. Finally, an economic study was performed to interrogate the feasibility of the proposed enzymatic manufacturing process. The obtained results were Compared with the traditional catalysed chemical process. The economic feasibility study revealed that the needed amount of Lipozyme RM IM was 2.5 kg/t of feedstock. The high conversion achieved in a short reaction time (1 h), as well as the proven operational stability of Lipozyme RM IM, revealed a promising potential for this green and sustainable route in practical applications.