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.

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.

Gene delivery using dendrimer/pDNA complexes immobilized in electrospun fibers using the layer-by-layer technique

Tissue engineering is an important branch of regenerative medicine that uses cells, materials (scaffolds), and suitable biochemical and physicochemical factors to improve or replace specific biological functions. In particular, the control of cell behavior (namely, of cell adhesion, proliferation and differentiation) is a key aspect for the design of successful therapeutical approaches. In this study, poly(lactic-co-glycolic acid) (PLGA) fiber mats were prepared using the electrospinning technology (the fiber diameters were in the micrometer range). Furthermore, the electrospun fiber mats thus formed were functionalized using the layer-by- layer (LbL) technique with chitosan and alginate (natural and biodegradable polyelectrolytes having opposite charges) as a mean for the immobilization of pDNA/dendrimer complexes. The polyelectrolyte multilayer deposition was confirmed by fluorescence spectroscopy using fluorescent-labeled polyelectrolytes. The electrospun fiber mats coated with chitosan and alginate were successfully loaded with complexes of pDNA and poly(amidoamine) (PAMAM) dendrimers (generation 5) and were able of releasing them in a controlled manner along time. In addition, these mats supported the adhesion and proliferation of NIH 3T3 cells and of human mesenchymal stem cells (hMSCs) in their surface. Transfection experiments using a pDNA encoding for luciferase showed the ability of the electrospun fiber mats to efficiently serve as gene delivery systems. When a pDNA encoding for bone morphogenetic protein-2 (BMP-2) was used, the osteoblastic differentiation of hMSCs cultured on the surface of the mats was promoted. Taken together, the results revealed that merging the electrospinning technique with the LbL technique, can be a suitable methodology for the creation of biological active matrices for bone tissue engineering.

Isomaltulose production from Sucrose by Protaminobacter rubrum immobilized in calcium alginate

Different culture conditions for Protaminobacter rubrum and enzymatic reaction parameters were evaluated with the goal of improving isomaltulose production. P. rubrum was grown in a medium with 1% (w/v) cane molasses and 0.5% yeast extract and achieved a maximum cell yield Y(x/s) of 0.295 g of cells/g sucrose and a specific growth rate (mu) of 0.192 h(-1). The immobilization of P. rubrum cells was carried out with calcium alginate, glutaraldehyde and polyethyleneimine. Stabile immobilized cell pellets were obtained and used 24 times in batch processes. Enzymatic conversion was carried out at different sucrose concentrations and in pH 6 medium with 70% (w/v) sucrose at 30 degrees C an isomaltulose yield of 89-94% (w/v) was obtained. The specific activity of the P. rubrum immobilized pellets in calcium alginate at 30 degrees C ranged from 1.6 to 4.0 g isomaltulose g(-1) pellet h(-1), respectively with 70% and 65% sucrose solution, while in lower sucrose concentration had higher specific activities presumably due to substrate inhibition of the isomaltulose synthase in higher sucrose concentrations. (C) 2009 Elsevier Ltd. All rights reserved.

Protein Hydrolysis by Immobilized and Stabilized Trypsin

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Sucrose hydrolysis by gelatin-immobilized inulinase from Kluyveromyces marxianus var. bulgaricus

The crude cell-free medium from a culture of Kluyveromyces marxianus var. bulgaricus was immobilized in a gelatin-water support, with an immobilization yield of 82.60% for inulinase activity. The optimum pH for both free and immobilized inulinase was the same (3.5) and the optimum temperatures were 55 degrees C for the free and 60 degrees C for the immobilized enzyme. The Arrhenius plots were linear and activation energies were 56.20 (free enzyme) and 20.27 kj/mol K (immobilized enzyme). The kinetic parameters were calculated by Lineweaver-Burk plots and the V-max and K-m were 37.60 IU/mg protein and 61.83 mM for the free inulinase and 31.45 IU/mg protein and 149.28 mM for the immobilized enzyme, respectively. The operational stability of the immobilized inulinase was studied in a continuous fixed-bed column reactor for 33 days, at the end of which the sucrose conversion was 58.12%. (c) 2008 Elsevier Ltd. All rights reserved.

Synergistic effect in the catalytic activity of lipase Rhizomucor miehei immobilized on zeolites for the production of biodiesel

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hydrolysis of whey lactose by immobilized β-Galactosidase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Spectroscopic characterization of schiff base-copper complexes immobilized in smectite clays

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Production of low methoxyl pectin using immobilized pectin methylesterase silica from acerola (Malpighia glabra L.)

The aim of this work was to develop an efficient reactor for the production of low methoxyl pectin, using pectinmethylesterase (PME, EC 3.1.1.11) from acerola immobilized on silica. The immobilized enzyme was used in up to 50 successive bioconversion runs at 50 degrees C with an efficiency loss of less than 20%. The fixed-bed reactor (6.0 x 1.5 cm) was prepared using PME immobilized in glutaraldehyde-activated silica operated at 50 degrees C with an optimum flow rate of 10 mL h(-1). The bioconversion yield was shown to strongly depend on the nature of the enzymatic preparation. An efficiency of 44% was achieved when concentrated PME was used, compared with only 30% with purified PME, both after an 8-h run. The process described could provide the basis for the development of a commercial-scale process. (c) 2006 Society of Chemical Industry.

Production of fructooligosaccharides by the mycelia of Aspergillus japonicus immobilized in calcium alginate

This work describes fructose oligosaccharide (FOS) production by the immobilized mycelia (IM) of a strain of Aspergillus japonicus, isolated from soil. The microorganism was inoculated into 50 mi of medium composed of sugar cane molasses (5.0% of total sugars); yeast powder; 2.0%; K2HPO4, 0.5%; NaNO3, 0.2%; MgSO4. 7H(2)O, 0.05%; KCl, 0.05%, final pH 5.0, and the flasks were agitated in an orbital shaker at 200 rpm for 60 h, at 30 degrees C. The beta-fructofuranosidase activity (Uf), transfructosylating activity (Ut), hydrolyzing activity (Uh), and FOS production were analyzed by high performance liquid chromatography. FOS production was performed in a batch process in a 2-l jar fermenter by IM in calcium alginate beads. The optimum pH and temperature were 5.0-5.6 and 55 degrees C, respectively No loss of activity was observed when the mycelium was maintaned at 60 degrees C for 60 min. Maximum production was obtained using 5.75% (cellular weight/volume) of mycelia (122.4 Ut g(-1)) and 65% sucrose solution (w:v) for 4 h of reaction when the final product reached 61.28% of fetal FOS containing GF(2) (30.56%), GF(3) (26.45%), GF(4) (4.27%), sucrose (9.6%) and glucose (29.10%). In the assay conditions, 23 batches were performed without loss of activity of the IM, showing that the microorganism and the process utilized have potential for industrial applications. (C) 1998 Elsevier B.V. Ltd. All rights reserved.