Activation of Leishmania (Leishmania) amazonensis arginase at low temperature by binuclear Mn(2+) center formation of the immobilized enzyme on a Ni(2+) resin

In Leishmania, arginase is responsible for the production of ornithine, a precursor of polyamines required for proliferation of the parasite. In this work, the activation kinetics of immobilized arginase enzyme from L. (L.) amazonensis were studied by varying the concentration of Mn(2+) applied to the nickel column at 23 degrees C. The intensity of the binding of the enzyme to the Ni(2+) resin was directly proportional to the concentration of Mn(2+). Conformational changes of the enzyme may occur when the enzyme interacts with immobilized Ni(2+), allowing the following to occur: (1) entrance of Mn(2+) and formation of the metal bridge; (2) stabilization and activation of the enzyme at 23 degrees C; and (3) an increase in the affinity of the enzyme to Ni(2+) after the Mn(2+) activation step. The conformational alterations can be summarized as follows: the interaction with the Ni(2+) simulates thermal heating in the artificial activation by opening a channel for Mn(2+) to enter. (C) 2010 Elsevier Inc. All rights reserved.

Immobilized Kidney 28-kDa Endostatin- Related (KES28kDa) Fragment Promotes Endothelial Cell Survival

Background/Objective: Renal ischemia-hypoxia is a leading cause of acute kidney injury (AKI). Ischemia causes extracellular matrix breakdown of the tubular basement membrane. Endostatin (ES) is the C-terminal fragment of collagen XVIII generated by proteolytic cleavage. Recent studies have demonstrated that ES expression is upregulated in ischemic kidneys. The present study aimed to characterize ES from ischemic kidneys. Methods: Ischemic renal failure was induced via 45 min of occlusion of the left renal artery and vein. After the ischemic period, blood was collected. Kidneys were harvested and used for immunohistochemical testing and protein extraction. Three-step purification was used. Soluble and immobilized purified ES were tested in cell viability and adhesion assays. Results: The soluble KES28kDa inhibited endothelial cell proliferation: 25 versus 12.5 mu g (p < 0.05); 12.5 versus 3.15 mu g (p < 0.05). Immobilization of KES28kDa supports endothelial cell survival over the control p = 0.021). Human umbilical vein endothelial cells plated on immobilized KES28kDa showed an increase in membrane ruffles and stress fibers. Conclusion: These data demonstrate the local synthesis of a 28-kDa ES-related fragment following AKI and suggest its role in endothelium survival. Copyright (C) 2010 S. Karger AG, Basel

Enzyme activity of horseradish peroxidase immobilized in chitosan matrices in alternated layers

The successful immobilization of enzymes such as horseradish peroxidase (HRP) in solid films is essential for applications in sensors and for fundamental studies aimed at identifying possible biotechnological devices. In this study we show that HRP can be immobilized in alternated layers with chitosan as the template material. The activity of HRP in HRP/chitosan films was preserved for several weeks, and could be detected optically upon monitoring the reaction with pyrogallol. The morphology of the film displayed stripes that disappeared after reaction with pyrogallol. Though the activity in the HRP/chitosan film was lower than in a homogeneous solution or in an LB film investigated earlier, the response was linear for a considerable period of time, which may be advantageous for sensing hydrogen peroxide. (C) 2009 Elsevier B.V. All rights reserved.

Controlled fabrication of gold nanoparticles biomediated by glucose oxidase immobilized on chitosan layer-by-layer films

The control of size and shape of metallic nanoparticles is a fundamental goal in nanochemistry, and crucial for applications exploiting nanoscale properties of materials. We present here an approach to the synthesis of gold nanoparticles mediated by glucose oxidase (GOD) immobilized on solid substrates using the Layer-by-Layer (LbL) technique. The LbL films contained four alternated layers of chitosan and poly(styrene sulfonate) (PSS), with GOD in the uppermost bilayer adsorbed on a fifth chitosan layer: (chitosan/PSS)(4)/(chitosan/GOD). The films were inserted into a solution containing gold salt and glucose, at various pHs. Optimum conditions were achieved at pH 9, producing gold nanoparticles of ca. 30 nm according to transmission electron microscopy. A comparative study with the enzyme in solution demonstrated that the synthesis of gold nanoparticles is more efficient using immobilized GOD. (C) 2009 Elsevier B.V. All rights reserved.

Enzyme Activity of Catalase Immobilized in Langmuir-Blodgett Films of Phospholipids

A major challenge for producing low cost biosensors based on nanostructured films with control of molecular architectures is to preserve the catalytic activity of the immobilized biomolecules. In this study, we show that catalase (HRP) keeps its activity if immobilized in Langmuir-Blodgett (LB) films of dipalmitoyl phosphatidylglycerol (DPPG). The incorporation of catalase into a DPPG monolayer at the at interface was demonstrated with surface pressure and surface potential isotherms, in addition to polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). According to the PM-IRRAS data. catalase was not denatured upon adsorption on a preformed DPPG monolayer and could be transferred onto a solid substrate. The catalytic activity of catalase in a mixed LB film with DPPG was ca. 13% higher than in solution. The control of molecular architecture and choice of a suitable phospholipid matrix allows catalase-containing LB films to be used in sensing hydrogen peroxide.

Kinetic resolution of a drug precursor by Burkholderia cepacia lipase immobilized by different methodologies on superparamagnetic nanoparticles

Burkholderia cepacia lipase was immobilized on superparamagnetic nanoparticles using three different methodologies (adsorption, chemisorption with carboxibenzaldehyde and chemisorption with glutaraldehyde) and employed in the kinetic resolution of a chiral drug precursor, (RS)-2-bromo-1-(phenyl)ethanol, via enantioselective acetylation reaction. An excellent improvement of lipase catalytical performance was observed. Free B. cepacia lipase gave the ester (S)-2 with poor E-value <30, and after its immobilization to magnetic nanoparticles the E-value was up to >200. The effect of several reaction parameters in the kinetic resolution was studied. The best results for kinetic resolution were obtained using vinyl acetate as acetyl donor and toluene as solvent, typically yielding the ester in high enantiomeric excess (>99%) and E-value (E > 200). Of the three tested immobilization methods, chemisorption with glutaraldehyde was the best one in terms of temperature stability and yield product. (C) 2010 Elsevier B.V. All rights reserved.

Degradation and adsorption of AZO RR239 dye in aqueous solution by nanoscale zerovalent iron particles immobilized on sawdust

Carra sawdust pretrated with formaldehyde was used to adsorb RR239 (reactive azo dye) at varying pH and zerovalent iron (ZVI) dosage. Modeling of kinetic results shows that sorption process is best described by the pseudo-second-order model. Batch experiments suggest that the decolorization efficiency was strongly enhanced with the presence of ZVI and low solution pH. The kinetics of dye sorption by mixed sorbent (5 g of sawdust and 180 mg of ZVI) at pH 2.0 was rapid, reaching more than 90% of the total discoloration in three minutes.

Amperometric detection of benzoyl peroxide in pharmaceutical preparations using carbon paste electrodes with peroxidases naturally immobilized on coconut fibers

This paper describes the applications of anew carbon paste electrode containing fibers of coconut (Cocus nucifera L) fruit, which are very rich in peroxidase enzymes naturally immobilized on its structure. The new sensor was applied for the amperometric quantification of benzoyl peroxide in facial creams and dermatological shampoos. The amperometric measurements were performed in 0.1 mol L(-1) phosphate buffer (pH 5.2), at 0.0 V (versus Ag/AgCl). On these conditions, benzoyl peroxide was rapidly determined in the 5.0-55 mu mol L(-1), with a detection limit of 2.5 mu mol L(-1) (s/n = 3), response time of 4.1 s (90% of the steady state) and sensitivity limit of 0.33 A mol L(-1) cm(-2). The amperometric results are in good agreement with those obtained by spectrophotometric technique, used as a standard method. (C) 2009 Elsevier B.V. All rights reserved.

Enantioselective transesterification catalysis by Candida antarctica lipase immobilized on superparamagnetic nanoparticles

Lipase B from Candida antarctica can be directly immobilized onto functionalized superparamagnetic nanoparticles, preserving its enzymatic activity in the enantioselective transesterification of secondary alcohols, with excellent results in terms of enantiomeric discrimination. The immobilized enzyme can be easily recovered with a magnet, allowing its reuse with negligible loss of activity. (C) 2009 Elsevier Ltd. All rights reserved

Hydrolytic Activity of Free and Immobilized Cellulase

Cellulase is an enzymatic complex which synergically promotes the degradation of cellulose to glucose. The adsorption behavior of cellulase from Trichoderma reesei onto Si wafers or amino-terminated surfaces was investigated by means of ellipsometry and atomic force microscopy (AFM) as a function of temperature. Upon increasing temperature from (24 +/- 1) to (60 +/- 1) degrees C, adsorption of cellulase became faster and more pronounced and the mean roughness of cellulase adsorbed layers increased. In the case of cellulase adsorbed onto Si wafers, Arrhenius`s plot allowed us to estimate the adsorption energy as 24.2 kJ mol(-1). The hydrolytic activity of free cellulase and cellulase immobilized onto Si wafers was tested using cellulose dispersions as substrates. The incubation temperature ranged from (37 +/- 1) to (60 +/- 1) degrees C. The highest efficiency was observed at (60 +/- 1) degrees C. The amount of glucose produced by free cellulase was similar to 20% higher than that obtained from immobilized cellulase. However, immobilizing cellulase onto Si wafers proved to be advantageous because they could be reused six times while retaining their original activity level. Such an effect was attributed to surface hydration, which prevents enzyme denaturation. The hydrolytic activity of cellulase immobilized onto amino-terminated surfaces was slightly lower than that observed for cellulase adsorbed onto Si wafers, and reuse was not possible.

Catalytic properties of thioredoxin immobilized on superparamagnetic nanoparticles

Thioredoxin (Trx1), a very important protein for regulating intracellular redox reactions, was immobilized on iron oxide superparamagnetic nanoparticles previously coated with 3-aminopropyltriethoxysilane (APTS) via covalent coupling using the EDC (1-ethyl-3-{3-dimethylaminopropyl}carbodiimide) method. The system was extensively characterized by atomic force microscopy, vibrational and magnetic techniques. In addition, gold nanoparticles were also employed to probe the exposed groups in the immobilized enzyme based on the SERS (surface enhanced Raman scattering) effect, confirming the accessibility of the cysteines residues at the catalytic site. For the single coated superparamagnetic nanoparticle, by monitoring the enzyme activity with the Ellman reagent, DTNB=5,5`-dithio-bis(2-15 nitrobenzoic acid), an inhibitory effect was observed after the first catalytic cycle. The inhibiting effect disappeared after the application of an additional silicate coating before the AFTS treatment, reflecting a possible influence of unprotected iron-oxide sites in the redox kinetics. In contrast, the doubly coated system exhibited a normal in-vitro kinetic activity, allowing a good enzyme recovery and recyclability. (C) 2011 Elsevier Inc. All rights reserved.

Catalytic ethanolysis of soybean oil with immobilized lipase from Candida antarctica and (1)H NMR and GC quantification of the ethyl esters (biodiesel) produced

The catalytic ethanolysis of soybean oil with commercial immobilized lipase type B from Candida antarctica to yield ethyl esters (biodiesel) has been investigated. Transesterification was monitored with respect to the following parameters: quantity of biocatalyst, reaction time, amount of water added and turnover of lipase. The highest yields of biodiesel (87% by (1)H NMR; 82.9% by GC) were obtained after a reaction time of 24 h at 32 degrees C in the presence of lipase equivalent to 5.0% (w/w) of the amount of soybean oil present. The production of ethyl esters by enzymatic ethanolysis was not influenced by the addition of water up to 4.0% (v/v) of the alcohol indicating that it is possible to use hydrated ethanol in the production of biodiesel catalyzed by lipase. The immobilized enzyme showed high stability under moderate reaction conditions and retained its activity after five production cycles. The (1)H NMR methodology elaborated for the quantification of biodiesel in unpurified reaction mixtures showed good correlations between the signal areas of peaks associated with the alpha-methylene groups of the ethyl esters and those of the triacyl-glycerides in residual soybean oil. Monoacylglycerides, diacylglycerides and triglycerides could also be detected and quantified in the crude biodiesel using (1)H NMR spectroscopic and GC-FID chromatographic methods. The biodiesel production by enzymatic catalysis was promising. In this case, was produced a low concentration of glycerol (0.74%) and easily removed by water extraction. (C) 2010 Elsevier B.V. All rights reserved.

Electroanalytical Method for the Analysis of Methyldopa In Pharmaceutical Tablets Using a Crude Extract of Laccase from Pycnoporus sanguineus as Oxidizing Agent

Several colorimetric and chromatographic methods have been used for the identification and quantification of methyldopa (MA) in pharmaceutical formulations and clinical samples. However, these methods are time- and reagent-consuming, which stimulated our efforts to develop a simple, fast, and low-cost alternative method. We carried out an electroanalytical method for the determination of MA in pharmaceutical formulations using the crude enzymatic extract of laccase from Pycnoporus sanguineus as oxidizing agent. This method is based on the biochemical oxidation of MA by laccase (LAC), both in solution, followed by electrochemical reduction on glassy carbon electrode surface. This method was employed for the determination of MA in pure and pharmaceutical formulations and compared with the results obtained using the official method. A wide linear curve from 23 x 10(-5) to 1 x 10(-4) mol L(-1) was found with a detection limit calculated from 43 x 10(-6) mol L(-1).

Immobilized analogues of sunflower trypsin inhibitor-1 constitute a versatile group of affinity sorbents for selective isolation of serine proteases

Sunflower trypsin inhibitor-1 (SFI-1), a natural 14-residue cyclic peptide, and some of its synthetic acyclic variants are potent protease inhibitors displaying peculiar inhibitory profiles. Here we describe the synthesis and use of affinity sorbents prepared by coupling SFTI-1 analogues to agarose resin. Chymotrypsinand trypsin-like proteases could then be selectively isolated from pancreatin; similarly, other proteases were obtained from distinct biological sources. The binding capacity of [Lys5]-SFTI-1-agarose for trypsin was estimated at over 10 mg/mL of packed gel. SFTI-1-based resins could find application either to improve the performance of current purification protocols or as novel protease-discovery tools in different areas of biological investigation. (C) 2009 Elsevier B.V. All rights reserved.

Effect of agitation on the performance of an anaerobic sequencing batch biofilm reactor in the treatment of dairy effluents

Agitation rate is an important parameter in the operation of Anaerobic Sequencing Biofilm Batch Reactors (ASBBRs), and a proper agitation rate guarantees good mixing, improves mass transfer, and enhances the solubility of the particulate organic matter. Dairy effluents have a high amount of particulate organic matter, and their anaerobic digestion presents inhibitory intermediates (e. g., long-chain fatty acids). The importance of studying agitation in such batch systems is clear. The present study aimed to evaluate how agitation frequency influences the anaerobic treatment of dairy effluents. The ASBBR was fed with wastewater from milk pasteurisation process and cheese manufacture with no whey segregation. The organic matter concentration, measured as chemical oxygen demand (COD), was maintained at approximately 8,000 mg/L. The reactor was operated with four agitation frequencies: 500 rpm, 350 rpm, 200 rpm, and no agitation. In terms of COD removal efficiency, similar results were observed for 500 rpm and 350 rpm (around 90%) and for 200 rpm and no agitation (around 80%). Increasing the system`s agitation thus not only improved the global efficiency of organic matter removal but also influenced volatile acid production and consumption and clearly modified this balance in each experimental condition.