Multipoint covalent immobilization of lipase on chitosan hybrid hydrogels: influence of the polyelectrolyte complex type and chemical modification on the catalytic properties of the biocatalysts

This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TLL) by multipoint covalent immobilization of the enzyme on chitosan-based matrices. The resulting biocatalysts were tested for synthesis of biodiesel by ethanolysis of palm oil. Different hydrogels were prepared: chitosan alone and in polyelectrolyte complexes (PEC) with kappa-carrageenan, gelatin, alginate, and polyvinyl alcohol (PVA). The obtained supports were chemically modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to increase support hydrophobicity, followed by activation with different agents such as glycidol (GLY), epichlorohydrin (EPI), and glutaraldehyde (GLU). The chitosan-alginate hydrogel, chemically modified with TNBS, provided derivatives with higher apparent hydrolytic activity (HA(app)) and thermal stability, being up to 45-fold more stable than soluble lipase. The maximum load of immobilized enzyme was 17.5 mg g(-1) of gel for GLU, 7.76 mg g(-1) of gel for GLY, and 7.65 mg g(-1) of gel for EPI derivatives, the latter presenting the maximum apparent hydrolytic activity (364.8 IU g(-1) of gel). The three derivatives catalyzed conversion of palm oil to biodiesel, but chitosan-alginate-TNBS activated via GLY and EPI led to higher recovered activities of the enzyme. Thus, this is a more attractive option for both hydrolysis and transesterification of vegetable oils using immobilized TLL, although industrial application of this biocatalyst still demands further improvements in its half-life to make the enzymatic process economically attractive.

Immobilization and stabilization of microbial lipases by multipoint covalent attachment on aldehyde-resin affinity: Application of the biocatalysts in biodiesel synthesis

Microbial lipase preparations from Thermomyces lanuginosus (TLL) and Pseudomonas fluorescens (PFL) were immobilized by multipoint covalent attachment on Toyopearl AF-amino-650M resin and the most active and thermal stable derivatives used to catalyze the transesterificanon reaction of babassu and palm oils with ethanol in solvent-free media For this different activating agents mainly glutaraldehyde glycidol and epichlorohydrin were used and immobilization parameters were estimated based on the hydrolysis of olive oil emulsion and butyl butyrate synthesis ILL immobilized on glyoxyl-resin allowed obtaining derivatives with the highest hydrolytic activity (HA(der)) and thermal stability between 27 and 31 times more stable than the soluble lipase Although PFL derivatives were found to be less active and thermally stables similar formation of butyl butyrate concentrations were found for both ILL and PFL derivatives The highest conversion into biodiesel was found in the transesterification of palm oil catalyzed by both ILL and PFL glyoxyl-derivatives (c) 2010 Elsevier B V All rights reserved

Covalent attachment of Candida rugosa lipase on chemically modified hybrid matrix of polysiloxane-polyvinyl alcohol with different activating compounds

Candida rugosa lipase was immobilized by covalent binding on hybrid matrix of polysiloxane-polyvinyl alcohol chemically modified with different activating agents as glutaraldehyde, sodium metaperiodate and carbonyldiimidazole. The experimental results suggested that functional activating agents render different interactions between enzyme and support, producing consequently alterations in the optimal reaction conditions. Properties of the immobilized systems were assessed and their performance on hydrolytic and synthetic reactions were evaluated and compared with the free enzyme. In hydrolytic reactions using p-nitrophenyl palmitate as substrate all immobilized systems showed higher thermal stability and optima pH and temperature values in relation to the free lipase. Among the activating compounds, carbonyldiimidazole resulted in a total recovery of activity on the support and the highest thermal stability. For the butyl butyrate synthesis, the best performance (molar conversion of 95% and volumetric productivity of 2.33 g L-1 h(-1)) was attained with the lipase immobilized on POS-PVA activated with sodium metaperiodate. The properties of the support and immobilized derivatives were also evaluated by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopies and chemical composition (FTIR). (c) 2007 Elsevier B.V. All rights reserved.

Development of novel bioanodes for ethanol biofuel cell using PAMAM dendrimers as matrix for enzyme immobilization

This paper describes the preparation and application of a novel bioanode for use in ethanol/O(2) biofuel cells based upon immobilization of alcohol dehydrogenase (ADH) and polyamidoamine (PAMAM) dendrimers onto carbon cloth platforms. The power density measurements indicated a direct relationship between the amount of anchored ADH and the anode power values, which increased upon enzyme loading. The power density values ranged from 0.04 to 0.28 mW cm(-2), and the highest power density was achieved with the bioanode prepared with 28 U of ADH, which provided a power density of 0.28 mW cm(-2) at 0.3 V. The latter power output values were the maximum observed, even for higher enzyme concentrations. Stability of the bioanodes was quite satisfactory, since there was no appreciable reduction of enzymatic activity during the measurements. The method of bioanode preparation described here has proven to be very effective. The PAMAM dendrimer represents a friendly environment for the immobilization of enzymes, and it is stable and capable of generating high power density compared to other immobilization methods. (C) 2010 Elsevier B.V. All rights reserved.

A biosensor based on immobilization of lactate oxidase in a PB-CTAB film for FIA determination of lactate in beer samples

An amperometric lactate biosensor with lactate oxidase immobilized into a Prussian Blue (PB) modified electrode was fabricated. The advantage of using cetyltrimethylammonium bromide (CTAB) in the electrodeposition step of PB films onto glassy carbon surfaces was confirmed taking into account both the stability and sensitivity of the measurements. The biosensor was used in the development of a FIA amperometric method for the determination of lactate. Under optimal operating conditions (pH = 6.9, E = -0.1 V), the linear response of the method was extended up to 0.28 µmol L-1 lactate with a limit of detection of 0.84 mmol L-1. The repeatability of the method for injections of a 0.28 mmol L-1 lactate solution was 2.2 % (n = 18). The usefulness of the method was demonstrated by determining lactate in beer samples and the results were in good agreement with those obtained by using a reference spectrophotometric enzyme method.

Influence of support material on the immobilization of biomass for the degradation of linear alkylbenzene sulfonate in anaerobic reactors

Two horizontal-flow anaerobic immobilized biomass reactors (HAIB) were used to study the degradation of the LAS surfactant: one filled with charcoal (HAIB1) and the other with a mixed bed of expanded clay and polyurethane foam (HAIB2). The reactors were fed with synthetic substrate supplemented with 14 mg l(-1) of LAS, kept at 30 +/- 2 degrees C and operated with a hydraulic retention time (HRT) of 12 h. The surfactant was quantified by HPLC. Spatial variation analyses were done to quantify organic matter and LAS consumption along the reactor length. The presence of the surfactant in the load did not affect the removal of organic matter (COD), which was close to 90% in both reactors for an influent COD of 550 ring l(-1). The results of a mass balance indicated that 28% of all LAS added to HAIB1 was removed by degradation. HAIB2 presented 27% degradation. Molecular biology techniques revealed microorgan isms belonging the uncultured Holophaga sp., uncultured delta Proteobacterium, uncultured Verrucomicrobium sp., Bacteroides sp. and uncultured gamma Proteobacterium sp. The reactor with biomass immobilized on charcoal presented lower adsorption and a higher kinetic degradation coefficient. So, it was the most suitable support for LAS anaerobic treatment. (c) 2008 Elsevier Ltd. All rights reserved.

Lysine biosynthesis and nitrogen metabolism in quinoa (Chenopodium quinoa): Study of enzymes and nitrogen-containing compounds

Aspartate kinase (AK, EC 2.7.2.4), homoserine dehydrogenase (HSDH, EC 1.1.1.3) and dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) were isolated and partially purified from immature Chenopodium quinoa Willd seeds. Enzyme activities were studied in the presence of the aspartate-derived amino acids lysine, threonine and methionine and also the lysine analogue S-2-aminoethyl-L-cysteine (AEC), at 1 mM and 5 mM. The results confirmed the existence of, at least, two AK isoenzymes, one inhibited by lysine and the other inhibited by threonine, the latter being predominant in quinoa seeds. HSDH activity was also shown to be partially inhibited by threonine, whereas some of the activity was resistant to the inhibitory effect, indicating the presence of two isoenzymes, one resistant and another sensitive to threonine inhibition. Only one DHDPS isoenzyme highly sensitive to lysine inhibition was detected. The results suggest that the high concentration of lysine observed in quinoa seeds is possibly due to a combined effect of increased lysine, synthesis and accumulation in the soluble form and/or as protein lysine. Nitrogen assimilation was also investigated and based on nitrate content, nitrate reductase activity, amino acid distribution and ureide content, the leaves were identified as the predominant site of nitrate reduction in this plant species. The amino acid profile analysis in leaves and roots also indicated an important role of soluble glutamine as a nitrogen transporting compound. (c) 2007 Elsevier Masson SAS. All rights reserved.

Ascorbic acid metabolism in fruits: activity of enzymes involved in synthesis and degradation during ripening in mango and guava

BACKGROUND: Ascorbic acid is a very important compound for plants. It has essential functions, mainly as an antioxidant and growth regulator. Ascorbic acid biosynthesis has been extensively studied, but studies in fruits are very limited. In this work we studied the influence of five enzymes involved in synthesis (L-galactono-1,4-lactone dehydrogenase, GalLDH, EC 1.3.2.3), oxidation (ascorbate oxidase, EC 1.10.3.3, and ascorbate peroxidase, APX, EC and recycling (monodehydroascorbate reductase, EC 1.6.5.4, and dehydroascorbate reductase, DHAR, EC 1.8.5.1) on changes in ascorbic acid content during development and ripening of mangoes (Mangifera indica L. cv. Keitt) and during the ripening of white pulp guavas (Psidium guayava L. cv. Paloma). RESULTS: It was found that there was a balance between the activities of GalLDH, APX and DHAR, both in mangoes and guavas. CONCLUSIONS: Equilibrium between the enzymatic activities of synthesis, catabolism and recycling is important for the regulation of ascorbic acid content in mango and guava. These results have contributed to understanding some of the changes that occur in ascorbic acid levels during fruit ripening. (C) 2008 Society of Chemical Industry.

Phase diagrams of a CTAB/organic solvent/buffer system applied to extraction of enzymes by reverse micelles

A partial pseudo-ternary phase diagram has been studied for the cethyltrimethylammonium bromide/isooctane:hexanol:butanol/potassium phosphate buffer system, where the two-phase diagram consisting of the reverse micelle phase (L-2) in equilibrium with the solvent is indicated. Based on these diagrams two-phase systems of reverse micelles were prepared with different compositions of the compounds and used for extraction and recovery of two enzymes, and the percentage of enzyme recovery yield monitored. The enzymes glucose-6-phosphate dehydrogenase (G6PD) and xylose redutase (XR) obtained from Candida guilliermondii yeast were used in the extraction procedures. The recovery yield data indicate that micelles having different composition give selective extraction of enzymes. The method can thus be used to optimize enzyme extraction processes. (c) 2007 Elsevier B.V. All rights reserved.

Lectins and/or xyloglucans/alginate layers as supports for immobilization of dengue virus particles

Formation of stable thin films of mixed xyloglucan (XG) and alginate (ALG) onto Si/SiO2 wafers was achieved under pH 11.6, 50 mM CaCl2, and at 70 degrees C. XG-ALG films presented mean thickness of (16 +/- 2) nun and globules rich surface, as evidenced by means of ellipsometry and atomic force microscopy (AFM), respectively. The adsorption of two glucose/mannose-binding seed (Canavalia ensiformis and Dioclea altissima) lectins, coded here as ConA and DAlt, onto XG-ALG surfaces took place under pH 5. Under this condition both lectins present positive net charge. ConA and DAIt adsorbed irreversibly onto XG-ALG forming homogenous monolayers similar to(4 +/- 1)nm thick. Lectins adsorption was mainly driven by electrostatic interaction between lectins positively charged residues and carboxylated (negatively charged) ALG groups. Adhesion of four serotypes of dengue virus, DENV (1-4), particles to XG-ALG surfaces were observed by ellipsometry and AFM. The attachment of dengue particles onto XG-ALG films might be mediated by (i) H bonding between E protein (located at virus particle surface) polar residues and hydroxyl groups present on XG-ALG surfaces and (ii) electrostatic interaction between E protein positively charged residues and ALG carboxylic groups. DENV-4 serotype presented the weakest adsorption onto XG-ALG surfaces, indicating that E protein on DENV-4 surface presents net charge (amino acid sequence) different from E proteins of other serotypes. All four DENV particles serotypes adsorbed similarly onto lectin films adsorbed. Nevertheless, the addition of 0.005 mol/L of mannose prevented dengue particles from adsorbing onto lectin films. XG-ALG and lectin layers serve as potential materials for the development of diagnostic methods for dengue. (c) 2008 Elsevier B.V. All rights reserved.

Immobilization of glucose oxidase enzyme (GOD) in large pore ordered mesoporous cage-like FDU-1 silica

Large pore ordered mesoporous silica FDU-1 with three-dimensional (3D) face-centered cubic, Fm3m arrangement of rnesopores, was synthesized under strong acid media using B-50-6600 poly(ethylene oxide)-poly(butylene oxide)-poly(ethylene oxide) triblock copolymer (EO(39)BO(47)EO(39)), tetraethyl orthosilicate (TEOS) and trimethyl-benzene (TMB). Large pore FDU-1 silica was obtained by using the following gel composition 1TEOS:0.00735B50-6600:0.00735TMB:6HCl:155H(2)O. The pristine material exhibited a BET specific surface area of 684 m(2) g(-1), total pore volume of 0.89 cm(3) g(-1), external surface area of 49 m(2) g(-1) and microporous volume of 0.09 cm(3) g(-1). The enzyme activity was determined by the Flow Injection Analysis-Chemiluminescence (FIA-CL) method. For GOD immobilized on the FDU-1 silica, GOD supernatant and GOD solution, the FIA-CL results were 9.0, 18.6 and 34.0 U, respectively. The value obtained for the activity of the GOD solution with FIA-CL method is in agreement with the 35 U, obtained by spectrophotometry. (C) 2011 Elsevier B.V. All rights reserved.

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.

Immobilization of Alcohol Dehydrogenase in Phospholipid Langmuir-Blodgett Films To Detect Ethanol

Enzyme immobilization in nanostructured films may be useful for a number of biomimetic systems, particularly if suitable matrixes are identified. Here we show that alcohol dehydrogenase (ADH) has high affinity toward a negatively charged phospholipid, dimyristoylphosphatidic acid (DMPA), which forms a Langmuir monolayer at an air-water interface. Incorporation of ADH into the DMPA monolayer was monitored with Surface pressure measurements; and polarization-modulation infrared reflection absorption spectroscopy, with the alpha-helices from ADH being mainly oriented parallel to the water surface. ADH remained at the interface even at high surface pressures, thus allowing deposition of Langmuir-Blodgett (LB) films from the DMPA-ADH film. Indeed, interaction with DMPA enhances the transfer of ADH, where the mass transferred onto a solid support increased from 134 ng for ADH on a Gibbs monolayer to 178 ng for an LB film with DMPA. With fluorescence spectroscopy it was possible to confirm that the ADH structure was preserved even after one month of the LB deposition. ADH-containing films deposited onto gold-interdigitated electrodes were employed in a sensor array capable of detecting ethanol at concentrations down to 10 ppb (in volume), using impedance spectroscopy as the method of detection.

Dendrimer-assisted immobilization of alcohol dehydrogenase in nanostructured films for biosensing: Ethanol detection using electrical capacitance measurements

The selective determination of alcohol molecules either in aqueous solutions or in vapor phase is of great importance for several technological areas. In the last years, a number of researchers have reported the fabrication of highly sensitive sensors for ethanol detection, based upon specific enzymatic reactions occurring at the surface of enzyme-containing electrodes. In this study, the enzyme alcohol dehydrogenase (ADH) was immobilized in a layer-by-layer fashion onto Au-interdigitated electrodes (IDEs), in conjunction with layers of PAMAM dendrimers. The immobilization process was followed in Teal time using quartz crystal microbalance (QCM), indicating that an average mass of 52.1 ng of ADH was adsorbed at each deposition step. Detection was carried out using a novel strategy entirely based upon electrical capacitance measurements, through which ethanol could be detected at concentrations of 1 part per million by volume (ppmv). (C) 2007 Elsevier B.V. All rights reserved.

Enhanced activity of horseradish peroxidase in Langmuir-Blodgett films of phospholipids

The immobilization of enzymes in nanostructured films has potential applications, e.g. in biosensing, for which the activity may not only be preserved, but also enhanced if optimized conditions are identified. Optimization is not straightforward because several requirements must be fulfilled, including a suitable matrix and film-forming technique. In this study, we show that horseradish peroxidase (HRP) has its activity enhanced when immobilized in Langmuir-Blodgett (LB) films, in conjunction with dipalmitoylphosphaticlylglycerol (DPPG). Incorporation of HRP into a DPPG monolayer at the air-water interface was demonstrated with compression isotherms, and Polarization-Modulation Infrared Reflection Absorption Spectroscopy (PM-IRRAS). From the PM-IRRAS data, we inferred that HRP was not denatured when adsorbed on a pre-formed, low pressure DPPG monolayer. A change in orientation was induced by the phospholipid matrix, with the amide C=O and NH groups from HRP being oriented perpendicular to the surface, parallel to the DPPG acyl chains, i.e. the alpha-helix was inserted into the monolayer. The mixed DPPG-HRP monolayer could be transferred onto solid supports, to which HRP activity was ca. 23% higher than in solution. The control of molecular architecture and choice of a suitable phospholipid matrix allowed HRP-containing LB films to be used in sensing peroxide. (c) 2008 Elsevier B.V. All rights reserved.