Uso de invertase imobilizada em pó de sabugo de milho para produção de açúcar invertido

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Nanocompósitos multifuncionas de fibroína reforçados com biocelulose

This work shows the preparation and characterization of the new nanocomposites based on fibroin and biocellulose. Bacterial cellulose (BC) is an exopolysaccharide produced by bacteria of the genus Gluconacetobacter, which it has identical chemical structure of the cellulose from plants and it has gained attention in the field of research for its unique properties as excellent mechanical properties when dry and hydrated , higher capacity of water retention, moldability , biodegradability and excellent biological affinity . Silk fibroin (SF) is a structural protein present in the cocoon of the silkworm, Bombyx mori, has been identified as suitable for developing optical devices, tissue engineering application, enzyme immobilization, controlled release drug agent biopolymer. Silk fibroin/bacterial cellulose nanocomposite films were prepared impregnating different cellulose charges (0.5 %, 1.0 %, 1.5 %, 2.5 %, 5.0 % and 10.0 %) weight/weight. According mechanical tests and water and Paynes's cup permeability showed that SF/BC 1% nanocomposite has the most relevant results. Poliethylenoglicol (PEG) containing SF films improved optical and mechanical properties when compared to pristine SF film. New SF/BC nanocomposites could be applied in Medicine, as biodegradable packaging and flexible substrates for OLEDs.

Capillary bioreactors based on human purine nucleoside phosphorylase: A new approach for ligands identification and characterization

The enzyme purine nucleoside phosphorylase (PNP) is a target for the discovery of new lead compounds employed on the treatment severe T-cell mediated disorders. Within this context, the development of new, direct, and reliable methods for ligands screening is an important task. This paper describes the preparation of fused silica capillaries human PNP (HsPNP) immobilized enzyme reactor (IMER). The activity of the obtained IMER is monitored on line in a multidimensional liquid chromatography system, by the quantification of the product formed throughout the enzymatic reaction. The Km value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255 +/- 29.2 mu M and 133 +/- 114.9 mu M, respectively). A new fourth-generation immucillin derivative (DI4G: IC50 = 40.6 +/- 0.36 nM), previously identified and characterized in HsPNP free enzyme assays, was used to validate the IMER as a screening method for HsPNP ligands. The validated method was also used for mechanistic studies with this inhibitor. This new approach is a valuable tool to PNP ligand screening, since it directly measures the hypoxanthine released by inosine phosphorolysis, thus furnishing more reliable results than those one used in a coupled enzymatic spectrophotometric assay. (C) 2011 Elsevier B.V. All rights reserved.

Multi-Walled Carbon Nanotubes and Poly(lactic acid) Nanocomposite Fibrous Membranes Prepared by Solution Blow Spinning

Nanocomposite fibers based on multi-walled carbon nanotubes (MWCNT) and poly(lactic acid) (PLA) were prepared by solution blow spinning (SBS). Fiber morphology was characterized by scanning electron microscopy (SEM) and optical microscopy (OM). Electrical, thermal, surface and crystalline properties of the spun fibers were evaluated, respectively, by conductivity measurements (4-point probe), thermogravimetric analyses (TGA), differential scanning calorimetry (DSC), contact angle and X-ray diffraction (XRD). OM analysis of the spun mats showed a poor dispersion of MWCNT in the matrix, however dispersion in solution was increased during spinning where droplets of PLA in solution loaded with MWCNT were pulled by the pressure drop at the nozzle, producing PLA fibers filled with MWCNT. Good electrical conductivity and hydrophobicity can be achieved at low carbon nanotube contents. When only 1 wt% MWCNT was added to low-crystalline PLA, surface conductivity of the composites increased from 5 x 10(-8) to 0.46 S/cm. Addition of MWCNT can slightly influence the degree of crystallinity of PLA fibers as studied by XRD and DSC. Thermogravimetric analyses showed that MWCNT loading can decrease the onset degradation temperature of the composites which was attributed to the catalytic effect of metallic residues in MWCNT. Moreover, it was demonstrated that hydrophilicity slightly increased with an increase in MWCNT content. These results show that solution blow spinning can also be used to produce nanocomposite fibers with many potential applications such as in sensors and biosensors.

Estudo da cinética da tirosinase imobilizada em nanopartícula de sílica com obtenção de revestimento de eumelanina

Melanina é um polímero constituído por uma grande heterogeneidade de monômeros tendo como característica comum a presença de grupos indóis. Por outro lado, a eumelanina produzida pela oxidação enzimática da tirosina é um polímero mais simples constituído principalmente de monômeros 5,6-dihidroxindol (DHI) e de indol-5,6-quinona (IQ). Tirosinase é a enzima chave na produção de melanina, sendo que a sua atividade cinética é medida em função da formação do intermediário dopacroma. Nanopartículas (NPs) de sílica são partículas nanométricas compostas de oxido de silício e são obtidas pelo processo sol-gel desenvolvido por Stöber de hidrólise e condensação de tetraetilortosilicato (TEOS), usando etanol como solvente em meio alcalino. As NPs foram funcionalizadas com 3-Aminopropiltrietoxissilano (ATPES) e depois com glutaraldeído. Este último permitiu a imobilização da tirosinase na superfície da sílica. Caracterizamos as NPs antes e após a reação da enzima, a atividade catalítica da enzima ligada à NP e o mecanismos de formação de melanina na superfície da sílica. As NPs foram caracterizadas por espectrofotometria de absorção e de reflectância, termogravimetria e microscopia eletrônica. A síntese da NP de sílica retornou partículas esféricas com 55nm de diâmetro e a funcionalização da partícula mostrou modificar eficientemente a sua superfície. A imobilização da tirosinase por ligação covalente foi de 99,5% contra 0,5% da adsorção física. A atividade da tirosinase foi caracterizada pela formação de dopacroma. O Km da enzima imobilizada não sofreu alteração em comparação com a tirosinase livre, mas a eficiência catalítica - que considera a eficiência recuperada - foi de apenas 1/3 para a enzima ligada covalentemente, significando que 2/3 das enzimas ligadas não estão ativas. Obtivemos NPs revestidas com melanina a partir de oxidação de tirosina solubilizada em duas preparações: NP com tirosinase ligada covalentemente na superfície e NP funcionalizada com glutaraldeido dispersa em solução de DHI e IQ. O revestimento de melanina foi na forma de um filme fino com espessura ~1,9nm, conferindo perfil de absorção luminosa equivalente ao da própria melanina. Mostramos que o mecanismo de polimerização passa pela oxidação da tirosina pela tirosinase, que gera intermediários oxidados (principalmente DHI e IQ) que vão para solução (mesmo quando a tirosinase está ligada covalentemente na sílica). Estes intermediários ligam-se ao glutaraldeido e a superfície da sílica passa a funcionar como ambiente de polimerização da melanina.

Biotransformações na obtenção de hidróxi-selenetos e hidróxi-teluretos quirais

Neste trabalho foi estudado o comportamento de hidróxi-calcogenetos (Se e Te) frente a biotransformações, empregando enzimas isoladas em meio orgânico ou aquoso e empregando microorganismos (fungos). Estudos comparativos sobre a influência de diversas variáveis, como solvente, temperatura, imobilização enzimática e estrutura do hidróxi-calcogeneto, foram realizados. Inicialmente os compostos foram sintetizados utilizando métodos descritos na literatura, em seguida foi estudada a resolução de hidróxiselenetos em meio orgânico empregando lipases isoladas (Esquema 1), (ver arquivo), incluindo um estudo de imobilização da PSL em diversos suportes, além do estudo da influência da variação do solvente, da temperatura, da lipase, etc. Na resolução em meio aquoso empregando enzimas isoladas, primeiramente os hidróxi-selenetos foram acetilados quimicamente e depois realizado uma triagem (com dez enzimas de diferentes fontes) empregando indicador de pH colori métrico. Posteriormente os acetatos dos hidróxi-selenetos (Esquema 2) (ver arquivo) foram submetidos à resolução enzimática em meio aquoso empregando as enzimas que foram selecionadas na triagem enzimática. As biotransformações utilizando fungos foram realizadas empregando células inteiras de algumas linhagens de Aspergillus terreus. Na seqüência foi realizada a resolução de hidróxi-teluretos em meio orgânico utilizando lipases isoladas (Esquema 3)(ver arquivo). Nessas resoluções também foi estudada a influência da variação do solvente, da lipase, do tempo, etc. De forma a demonstrar a importância dos compostos resolvidos, um hidróxi-seleneto quiral e dois hidróxi-teluretos quirais foram usados para preparar compostos pertencentes a classes de unidades estruturais de vasta ocorrência em produtos naturais: um álcool alílico e duas lactonas (Esquema 4)(ver arquivo).

Desenvolvimento de biossensores enzimáticos amperométricos para a determinação de compostos de importância clínica

Este trabalho descreve a preparação e caracterização de eletrodos modificados com azul da Prússia e materiais relacionados e a sua aplicação na construção de biossensores enzimáticos amperométricos para a detecção de oxalato e de glicose. Os materiais utilizados na modificação dos eletrodos foram azul da Prússia e compostos híbridos formados por hexacianoferrato de níquel e polipirrol ou hexacianoferrato de cobre e polipirrol. Os materiais lubridos mostraram-se capazes de mediar na eletroredução de peróxido de hidrogênio, mesmo em eletrólitos contendo Na+, apresentando melhor desempenho analítico quando comparados aos respectivos hexacianoferratos sem a presença do polímero condutor. Estes materiais foram utilizados com êxito na construção de biossensores para oxalato e para glicose, imobilizando as enzimas Oxalato Oxidase e Glicose Oxidase, respectivamente. Também foi estudada a preparação de um biossensor para a detecção de glicose utilizando a técnica de automontagem eletrostática camada por camada. Esta técnica permite otimizar o processo de immobilização da enzima, obtendo excelente desempenho analítico com pouca quantidade de enzima. Finalmente, são apresentadas a síntese, caracterização e aplicação de nanopartículas de azul da Prússia na determinação de peróxido de hidrogênio. Foi possível preparar nanopartículas com um diâmetro médio de 5 nm, as quais foram imobilizadas em eletrodos mediante a técnica de automontagem eletrostática camada por camada, a fim de estudar seu comportamento eletroquímico.

C-MEMS Based Micro Enzymatic Biofuel Cells

Miniaturized, self-sufficient bioelectronics powered by unconventional micropower may lead to a new generation of implantable, wireless, minimally invasive medical devices, such as pacemakers, defibrillators, drug-delivering pumps, sensor transmitters, and neurostimulators. Studies have shown that micro-enzymatic biofuel cells (EBFCs) are among the most intuitive candidates for in vivo micropower. In the fisrt part of this thesis, the prototype design of an EBFC chip, having 3D intedigitated microelectrode arrays was proposed to obtain an optimum design of 3D microelectrode arrays for carbon microelectromechanical systems (C-MEMS) based EBFCs. A detailed modeling solving partial differential equations (PDEs) by finite element techniques has been developed on the effect of 1) dimensions of microelectrodes, 2) spatial arrangement of 3D microelectrode arrays, 3) geometry of microelectrode on the EBFC performance based on COMSOL Multiphysics. In the second part of this thesis, in order to investigate the performance of an EBFC, behavior of an EBFC chip performance inside an artery has been studied. COMSOL Multiphysics software has also been applied to analyze mass transport for different orientations of an EBFC chip inside a blood artery. Two orientations: horizontal position (HP) and vertical position (VP) have been analyzed. The third part of this thesis has been focused on experimental work towards high performance EBFC. This work has integrated graphene/enzyme onto three-dimensional (3D) micropillar arrays in order to obtain efficient enzyme immobilization, enhanced enzyme loading and facilitate direct electron transfer. The developed 3D graphene/enzyme network based EBFC generated a maximum power density of 136.3 μWcm-2 at 0.59 V, which is almost 7 times of the maximum power density of the bare 3D carbon micropillar arrays based EBFC. To further improve the EBFC performance, reduced graphene oxide (rGO)/carbon nanotubes (CNTs) has been integrated onto 3D mciropillar arrays to further increase EBFC performance in the fourth part of this thesisThe developed rGO/CNTs based EBFC generated twice the maximum power density of rGO based EBFC. Through a comparison of experimental and theoretical results, the cell performance efficiency is noted to be 67%.

Hydrazide-functionalized hollow porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) spheres for immobilization of enzyme

Hollow porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate)(HEMA-co-EDMA) spheres were prepared by emulsifier-free emulsion polymerization, swelling, seed emulsion polymerization and extraction. Then the spheres activated with 2,4,6-trichloro-1,3,5-triazine were functioned with adipohydrazide (AH). After periodate oxidation of its carbohydrate moieties, horseradish peroxidase was immobilized on the hydrazide-functionalized hollow porous poly(HEMA-co-EDMA) spheres. The amount of immobilized enzyme was up to 43.4 mu g of enzyme/g of support. Moreover, the immobilized horseradish peroxidase exhibited high activity and good stability.

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.

Immobilization of papain on chitin and chitosan and recycling of soluble enzyme for deflocculation of saccharomyces cerevisiae from bioethanol distilleries

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

Immobilization and characterization of recombinant esterase enzyme on chitosan nanoparticles

Immobilization of biologically important molecules on myriad nano-sized materials has attracted great attention. Through this study, thermophilic esterase enzyme was obtained using recombinant DNA technology and purified applying one-step His-Select HF nickel affinity gel. The synthesis of chitosan was achieved from chitin by deacetylation process and degree of deacetylation was calculated as 89% by elemental analysis. Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions. The physicochemical properties of the chitosan and chitosan nanoparticles were determined by several methods including SEM (Scanning Electron Microscopy), FT-IR (Fourier Transform Infrared Spectroscopy) and DLS (Dynamic Light Scattering). The morphology of chitosan nanoparticles was spherical and the nanospheres’ average diameter was 75.3 nm. The purified recombinant esterase was immobilized efficiently by physical adsorption onto chitosan nanoparticles and effects of various immobilization conditions were investigated in details to develope highly cost-effective esterase as a biocatalyst to be utilized in biotechnological purposes. The optimal conditions of immobilization were determined as follows; 1.0 mg/mL of recombinant esterase was immobilized on 1.5 mg chitosan nanoparticles for 30 min at 60°C, pH 7.0 under 100 rpm stirring speed. Under optimized conditions, immobilized recombinant esterase activity yield was 88.5%. The physicochemical characterization of enzyme immobilized chitosan nanoparticles was analyzed by SEM, FT-IR and AFM (Atomic Force Microscopy).

Modifying enzyme activity and selectivity by immobilization

Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will be positive. For example, they may be related to the stabilization of a hyperactivated form of the enzyme, like in the case of lipases immobilized on hydrophobic supports via interfacial activation. In some other instances, these improvements will be just a consequence of random modifications in the enzyme properties that in some reactions will be positive while in others may be negative. For this reason, the preparation of a library of biocatalysts as broad as possible may be a key turning point to find an immobilized biocatalyst with improved properties when compared to the free enzyme. Immobilized enzymes will be dispersed on the support surface and aggregation will no longer be possible, while the free enzyme may suffer aggregation, which greatly decreases enzyme activity. Moreover, enzyme rigidification may lead to preservation of the enzyme properties under drastic conditions in which the enzyme tends to become distorted thus decreasing its activity. Furthermore, immobilization of enzymes on a support, mainly on a porous support, may in many cases also have a positive impact on the observed enzyme behavior, not really related to structural changes. For example, the promotion of diffusional problems (e.g., pH gradients, substrate or product gradients), partition (towards or away from the enzyme environment, for substrate or products), or the blocking of some areas (e.g., reducing inhibitions) may greatly improve enzyme performance. Thus, in this tutorial review, we will try to list and explain some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization.

Immobilization of Aspergillus oryzae alpha-galactosidase in gelatin and its application in removal of flatulence-inducing sugars in soymilk

Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean-derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. In this study, alpha-galactosidase from Aspergillus oryzae was entrapped in gelatin using formaldehyde as the hardener. The immobilization yield was 64.3% under the optimum conditions of immobilization. The immobilized alpha-galactosidase showed a shift in optimum pH from 4.8 to 5.4 in acetate buffer. The optimum temperature also shifted from 50 degrees C to 57 degrees C compared with soluble enzyme. Immobilized alpha-galactosidase was used in batch, repeated batch and continuous mode to degrade RO present in soymilk. In the repeated batch, 45% reduction of RO was obtained in the fourth cycle. The performance of immobilized alpha-galactosidase was tested in a fluidized bed reactor at different flow rates and 86% reduction of RO in soymilk was obtained at 25 ml h(-1) flow rate. The study revealed that immobilized alpha-galactosidase in continuous mode is efficient in reduction of RO present in soymilk.

Magnetic Cross-Linked Enzyme Aggregates (mCLEAs) of Candida antarctica Lipase: An Efficient and Stable Biocatalyst for Biodiesel Synthesis

Enzyme-catalyzed production of biodiesel is the object of extensive research due to the global shortage of fossil fuels and increased environmental concerns. Herein we report the preparation and main characteristics of a novel biocatalyst consisting of Cross-Linked Enzyme Aggregates (CLEAs) of Candida antarctica lipase B (CALB) which are covalently bound to magnetic nanoparticles, and tackle its use for the synthesis of biodiesel from non-edible vegetable and waste frying oils. For this purpose, insolubilized CALB was covalently cross-linked to magnetic nanoparticles of magnetite which the surface was functionalized with –NH2 groups. The resulting biocatalyst combines the relevant catalytic properties of CLEAs (as great stability and feasibility for their reutilization) and the magnetic character, and thus the final product (mCLEAs) are superparamagnetic particles of a robust catalyst which is more stable than the free enzyme, easily recoverable from the reaction medium and reusable for new catalytic cycles. We have studied the main properties of this biocatalyst and we have assessed its utility to catalyze transesterification reactions to obtain biodiesel from non-edible vegetable oils including unrefined soybean, jatropha and cameline, as well as waste frying oil. Using 1% mCLEAs (w/w of oil) conversions near 80% were routinely obtained at 30°C after 24 h of reaction, this value rising to 92% after 72 h. Moreover, the magnetic biocatalyst can be easily recovered from the reaction mixture and reused for at least ten consecutive cycles of 24 h without apparent loss of activity. The obtained results suggest that mCLEAs prepared from CALB can become a powerful biocatalyst for application at industrial scale with better performance than those currently available.