Maria Luísa Serralheiro

Application of factorial design to the study of transesterification reactions using cutinase in AOT-reversed micelles

Abstract Fusarium solani pisi recombinant cutinase, solubilized in bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane-reversed micelles, was used to catalyze the transesterification reaction of butyl acetate with hexanol. Some relevant parameters for the cutinase activity such as the amount of water, temperature, buffer molarity, pH, surfactant, and hexanol and butyl acetate concentrations were studied in two factorial designs. The experimental planning included 2 (5−1) and 2 (3) factorial designs expanded further to a central composite design (CCD) according to Box 1 and Barker. 2 By knowing the system response to the experimental design, the effects of each factor were calculated and its interactions were determined. The response surface methodology was applied to the optimization of the transesterification in the system described. Cutinase displayed high activities for 490 mM hexanol and a temperature around 40–50°C. The molar ratio of water to surfactant, Wo , greatly influenced the activity and showed an optimum range between 5–8.

Dipeptide synthesis and separation in a reversed micellar membrane reactor

The synthesis of dipeptide AcPheLeuNH2 catalyzed by alpha-chymotrypsin encapsulated in TTAB/octanol/heptane reversed micelles was investigated in a tubular ceramic membrane reactor, operated in a batch mode. The reaction medium conditions (TTAB concentration, buffer molarity, and pH) were optimized using a factorial design in order to achieve maximum synthesis rates. Hydrated reversed micelles permeated through the membrane together with the substrate ester, dipeptide, and by-products. However, as a result of the low solubility of the peptide in the reaction medium, selective precipitation occurred, thus enabling the complete retention of the solid product by the ultrafiltration membrane and therefore an integration of a separation step in the biotransformation process. In spite of the continuous accumulation of solids inside the reactor, constant permeation flow rates could be maintained throughout the operation. The influence of alpha-chymotrypsin, TTAB, and water concentration on the kinetics and mass transfer of the system was also investigated. The behavior of the system during a continuous experiment was also evaluated.

Continuous production and simultaneous precipitation of a dipeptide in a reversed micellar membrane reactor

Abstract The dipeptide AcPheLeuNH 2 was continuously synthesized from acetyl phenylalanine ethyl ester (AcPheOEt) and leucinamide (LeuNH 2 ) by α-chymotrypsin encapsulated in TTAB/heptane/octanol reversed micelles in a ceramic membrane reactor. Simultaneous separation of the dipeptide was achieved by selective precipitation. Apart from the dipeptide, two by-products resulting from the hydrolysis and the transesterification of AcPheOEt with the cosolvent octanol were obtained. The dipeptide was recovered with high purity (higher than 92%) due to precipitation from the complex reaction medium. The influence of the concentration of the two substrates in performance parameters of the continuous reactor such as productivity, yields, conversion degree, selectivity, and operational stability was investigated. The integration of catalysis and separation in a ceramic membrane reactor led to a one-step process for the production of a dipeptide with a high purity.

Acetylcholinesterase inhibition, antioxidant activity and toxicity of Peumus boldus water extracts on HeLa and Caco-2 cell lines.

This work aimed to study the inhibition on acetylcholinesterase activity (AChE), the antioxidant activity and the toxicity towards Caco-2 and HeLa cells of aqueous extracts of Peumus Boldus. An IC(50) value of 0.93 mg/mL, for AChE inhibition, and EC(50) of 18.7 μg/mL, for the antioxidant activity, was determined. This activity can be attributed to glycosylated flavonoid derivatives detected, which were the main compounds, although boldine and other aporphine derivatives were also present. No changes in the chemical composition or the biochemical activities were found after gastrointestinal digestion. Toxicity of P. boldus decoction gave an IC(50) value 0.66 mg/mL for HeLa cells, which caused significant changes in the cell proteome profile.

Development of a new amperometric biosensor based on polyphenoloxidase and polyethersulphone membrane

An amperometric biosensor based on the enzyme polyphenoloxidase (PPO), which makes the bioelectrocatalysis of phenolic compounds, was developed and optimized using cathecol as substrate. Polyethersulphone membranes were used for enzyme immobilization. Polyphenoloxidase oxidizes monophenols (cresolase activity) and diphenols (catecholase activity) into the corresponding o-quinones; the o-quinones formed in the enzymatic catalysis are then reduced back to cathecol at 200 mV (vs. Ag, AgCl) at a platinum electrode. The polyphenoloxidase immobilized was from commercial origin or extracted from mushrooms. p-Cresol and phenol substrates were also tested. Reproducibility, response time, linearity, sensitivity, and stability of the biosensor were studied.

Isolation and Characterization of Mercury-Resistant Bacteria From Sediments of Tagus Estuary (Portugal): Implications for Environmental and Human Health Risk Assessment

Mercury (Hg) contamination of aquatic systems has been recognized as a global and serious problem affecting both human and environmental health. In the aquatic ecosystems, mercurial compounds are microbiologically transformed with methylation responsible for generation of methylmercury (MeHg) and subsequent biomagnification in food chain, consequently increasing the risk of poisoning for humans and wildlife. High levels of Hg, especially MeHg, are known to exist in Tagus Estuary as a result of past industrial activities. The aim of this study was to isolate and characterize Hg-resistant bacteria from Tagus Estuary. Mercury-resistant (Hg-R) bacteria were isolated from sediments of two hotspots (Barreiro and North Channel) and one reserve area (Alcochete). Mercury contamination in these areas was examined and bacterial susceptibility to Hg compounds evaluated by determination of minimal inhibitory concentrations (MIC). The isolates characterization was based on morphological observation and biochemical testing. Bacteria characteristics, distribution, and Hg resistance levels were compared with metal levels. Barreiro and North Channel were highly contaminated with Hg, containing 126 and 18 μg/g total Hg, respectively, and in Alcochete, contamination was lower at 0.87 μg/g total Hg. Among the isolates there were aerobic and anaerobic bacteria, namely, sulfate-reducing bacteria, and Hg resistance levels ranged from 0.16 to 140 μg/ml for Hg2+ and from 0.02 to 50.1 μg/ml for MeHg. The distribution of these bacteria and the resistance levels were consistent with Hg contamination along the depth of the sediments. Overall, results show the importance of the characterization of Tagus Estuary bacteria for ecological and human health risk assessment.

Anaerobic reduction of a sulfonated azo dye, congo red, by sulfate-reducing bacteria

The capacity for anaerobic decolorization of a sulfonated azo dye, Congo Red, by a strain of a sulfate-reducing bacterium was evaluated. After optimizing the growth rate of the bacteria on a simple carbon source and terminal electron acceptor pair, lactate and sulfate, respectively, the effect of the dye concentration on their growth rate was analyzed. The decolorization rate was affected by the dye concentration in the growth medium. The azo-bond cleavage mechanism of reductive decolorization with the formation of benzidine was consistent with the results, as this metabolite was identified by high-performance liquid chromatography. Several fractions of the culture medium, including lysed cell extracts, were examined for the capacity to reduce the azo dye. This reduction capacity was found in the culture medium in which the cells had previously grown. The results showed that the mechanism of reductive decolorization of this sulfonated azo dye was extracellular and nonenzymatic, consistent with the production of sulfide anion by the microorganisms while growing on lactate and sulfate. The sulfide anions were the cause of the reduction leading to the disappearance of color in the medium. To increase the rate of decolorization, the presence of ferrous ion was also necessary together with the lactate and sulfate substrates.

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa.

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30–60 and 1,4-butanediol-diglycidyl ether: 16–36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a Mr of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and process-compatible alternative to other types of chromatography.

Irreversible thermoinactivation of α-chymotrypsin in buffer and water miscible organic solvent. Comparison with a reverse micellar system

Abstract The mechanism of irreversible thermoinactivation of α-chymotrypsin in buffer medium, dimethylformamide, dimethylsulfoxide/buffer and reverse micelles was studied. Experiments for the enzyme thermoinactivation in the different systems were accompanied by studies on protein structure alterations. The formation of free SH groups was followed during the stability tests. This formation is due to the cleavage of the enzyme primary structure which leads to small peptides. These were identified by SDS-PAGE, in the case of the buffer medium, and also by HPLC in the three other systems. An increase of SH groups with the residence time of the enzyme in the buffer and organic solvent/buffer system was observed. This was corroborated by the disappearance of the electrophoresis bands of the protein and also by HPLC analysis. In the latter technique, the peaks corresponding to α-chymotrypsin disappeared with the concomitant appearance of small peaks in the chromatogram. For the enzyme encapsulated in reverse micelles, the formation of free SH groups was not detected and the HPLC analysis revealed that the protein peak stayed intact during the residence time in this system. The thermoinactivation of chemically modified α-chymotrypsin by the introduction of dianhydride pyromellitic on the Lys residues of the protein was also studied in some of the systems. The results showed that the chemical modification stabilized the enzyme when the system used was buffer or organic solvent mixed with buffer being this stabilization partially due to a slower process of cysteine bond cleavage.

Characterization of monoclonal antibodies against altered (T103I) amidase from Pseudomonas aeruginosa

Monoclonal antibodies (MAbs) against mutant (T103I) amidase from Pseudomonas aeruginosa were raised by hybridoma technology. To select MAbs suitable for immunoaffinity chromatography, hybridoma clones secreting polyol-responsive MAbs (PR-MAbs) were screened that bind antigen tightly but release under mild and nondenaturing elution conditions. It was found that about 10% of enzyme-linked immunosorbent assay (ELISA)-positive hybridoma produce these MAbs as their ag-ab complex can be disrupted by propylene glycol in the presence of a suitable salt. Two of these hybridoma clones (F6G7 and E2A6) secreting PR-MAbs against mutant amidase were selected for optimization of experimental conditions for elution of amidase by using ELISA elution assay. These hybridoma cell lines secreted MAbs of IgM class that were purified in a single step by gel filtration chromatography, which revealed a single protein band on native polyacrylamide gel electrophoresis (PAGE). Specificity studies of this MAb revealed that it recognized specifically a common epitope on mutant and wild-type amidases as determined by direct ELISA. This MAb exhibited a higher affinity for denatured forms of wild-type and mutant amidases than for native forms as revealed by affinity constants (K), suggesting that it recognizes a cryptic epitope on an amidase molecule. Furthermore, MAb E2A6 inhibited about 60% of wild-type amidase activity, whereas it activated about 60% of mutant amidase (T103I) activity. The data presented in this work suggest that this MAb acts as a very useful probe to detect conformational changes in native and denatured amidases as well as to differentiate wild-type and mutant (T103I) amidases.