Estudo da imobilização de proteases para a síntese de oligolisinas

Enzymatic synthesis of peptides using proteases has attracted a great deal of attention in recent years. One key challenge in peptide synthesis is to find supports for protease immobilization capable of working in aqueous medium at high performance, producing watersoluble oligopeptides. At present, few reports have been described using this strategy. Therefore, the aim of this thesis was to immobilize proteases applying different methods (Immobilization by covalent bound, entrapment onto polymeric gels of PVA and immobilization on glycidil metacrylate magnetic nanoparticles) in order to produce water-soluble oligopeptides derived from lysine. Three different proteases were used: trypsin, α-chymotrypsin and bromelain. According to immobilization strategies associated to the type of protease employed, trypsin-resin systems showed the best performance in terms of hydrolytic activity and oligopeptides synthesis. Hydrolytic activities of the free and immobilized enzymes were determined spectrophotometrically based on the absorbance change at 660 nm at 25 °C (Casein method). Calculations of oligolysine yield and average degree of polymerization (DPavg) were monitored by 1H-NMR analysis. Trypsin was covalently immobilized onto four different resins (Amberzyme, Eupergit C, Eupergit CM and Grace 192). Maximum yield of bound protein was 92 mg/g, 82 mg/g and 60 mg/g support for each resin respectively. The effectiveness of these systems (Trypsin-resins) was evaluated by hydrolysis of casein and synthesis of water-soluble oligolysine. Most systems were capable of catalyzing oligopeptide synthesis in aqueous medium, albeit at different efficiencies, namely: 40, 37 and 35% for Amberzyme, Eupergit C and Eupergit CM, respectively, in comparison with free enzyme. These systems produced oligomers in only 1 hour with DPavg higher than free enzyme. Among these systems, the Eupergit C-Trypsin system showed greater efficiency than others in terms of hydrolytic activity and thermal stability. However, this did not occur for oligolysine synthesis. Trypsin-Amberzyme proved to be more successful in oligopeptide synthesis, and exhibited excellent reusability, since it retained 90% of its initial hydrolytic and synthetic activity after 7 reuses. Trypsin hydrophobic interactions with Amberzyme support are responsible for protecting against strong enzyme conformational changes in the medium. In addition, the high concentration of oxirane groups on the surface promoted multi-covalent linking and, consequently, prevented the immobilized enzyme from leaching. The aforementioned results suggest that immobilized Trypsin on the supports evaluated can be efficiently used for oligopeptides synthesis in aqueous media

Magneto Immunoassays for Plasmodium falciparum Histidine-Rich Protein 2 Related to Malaria based on Magnetic Nanoparticles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Production of xylo-oligosaccharides by immobilized-stabilized derivatives of endo-xylanase from Streptomyces halstedii

An endoxylanase from Streptomyces halstedii was stabilized by multipoint covalent immobilization on glyoxyl-agarose supports. The immobilized enzyme derivatives preserved 65% of the catalytic activity corresponding to the one of soluble enzyme that had been immobilized. These immobilized derivatives were 200 times more stable 200 times more stable than the one-point covalently immobilized derivative in experiments involving thermal inactivation at 60 °C. The activity and stability of the immobilized enzyme was higher at pH 5.0 than at pH 7.0. The optimal temperature for xylan hydrolysis was 10 °C higher for the stabilized derivative than for the non-stabilized derivative. On the other hand, the highest loading capacity of activated 10% agarose gels was 75 mg of enzyme per mL of support. To prevent diffusional limitations, low loaded derivatives (containing 0.2 mg of enzyme per mL of support) were used to study the hydrolysis of xylan at high concentration (close to 1% (w/v)). 80% of the reducing sugars were released after 3 h at 55 °C. After 80% of enzymatic hydrolysis, a mixture of small xylo-oligosaccharides was obtained (from xylobiose to xylohexose) with a high percentage of xylobiose and minimal amounts of xylose. The immobilized-stabilized derivatives were used for 10 reaction cycles with no loss of catalytic activity. © 2013 Elsevier Ltd. All rights reserved.

Continuous production of xylooligosaccharides in a packed bed reactor with immobilized-stabilized biocatalysts of xylanase from Aspergillus versicolor

The production of xylooligosaccharides (XOS) using a packed-bed enzymatic reactor was studied at lab-scale. For this, a xylanase from Aspergillus versicolor was immobilized on different supports. The optimal derivative was xylanase immobilized on glyoxyl-agarose supports. This derivative preserved 85% of its catalytic activity; it was around 700-fold more stable than the soluble enzyme after incubation at 60. °C and was able to be reused for at least 10 1. h-cycles retaining full catalytic activity. About 18% of oligosaccharides with prebiotic interest (X2-X6) were produced by the glyoxyl derivative in batch hydrolysis. The production of xylobiose was 2.5-fold higher using the immobilized preparation than with soluble enzyme and small concentrations of xylose (<0.1%) were observed only at the end of the reaction. The derivative was employed on a packed bed reactor, and the continuous operation with no recirculation reached 56% and 70% of the end of reaction with flow rates of 60. mL/h and 12. mL/h, respectively. In continuous operation with recirculation at a flow rate of 60. mL/h, the reaction was completed after four hours. © 2013 Elsevier B.V.

Imobilização multipontual das peroxidases da casca da soja e chuchu em suportes alternativos de pó de sabugo de milho e celulose bacteriana

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Bromophenol blue discoloration using peroxidase immobilized on highly activated corncob powder

The aim of the present study was to evaluate the efficacy of peroxidase immobilized on corncob powder for the discoloration of dye. Peroxidase was extracted from soybean seed coat, followed by amination of the surface of the tertiary structure. The aminated peroxidase was immobilized on highly activated corncob powder and employed for the discoloration of bromophenol blue. Amination was performed with 10 or 50 mmol.L-1carbodiimide and 1 mol.L-1ethylenediamine. The amount of protein in the extract was 0.235 ± 0.011 mg.mL-1and specific peroxidase activity was 86.06 ± 1.52 µmol min-1.mg-1, using 1 mmol.L-1ABTS as substrate. Ten mmol.L-1and 50 mmol.L-1 aminated peroxidase retained 88 and 100% of the initial activity. Following covalent immobilization on a corncob powder-glyoxyl support, 10 and 50 mmol.L-1aminated peroxidase retained 74 and 86% of activity, respectively. Derivatives were used for the discoloration of 0.02 mmol.L-1bromophenol blue solution. After 30 min, 93 and 89% discoloration was achieved with the 10 mmol.L-1and 50 mmol.L-1derivatives, respectively. Moreover, these derivatives retained 60% of the catalytic properties when used three times. Peroxidase extracted from soybean seed coat immobilized on a low-cost corncob powder support exhibited improved thermal stability. Keywords: Peroxidases. Multipoint immobilization of enzymes. Aminated enzymes. Corncob powder. RESUMO Descoloração de azul de bromofenol utilizando peroxidase imobilizada em pó de sabugo de milho altamente ativado Nesta pesquisa a enzima peroxidase foi extraída do tegumento de sementes de soja, e a superfície da estrutura terciária foi aminada. A peroxidase aminada foi imobilizada em suporte pó de sabugo de milho altamente ativado e utilizado na descoloração de azul de bromofenol. A aminação da peroxidase foi realizada com carbodiimida em concentrações de 10 e 50 mmol.L-1, e 1 mol.L-1de etilenodiamina. A quantidade de proteínas no extrato foi de 0,235 ± 0,011 mg.mL-1, e a atividade específica da peroxidase foi 86,06 ± 1,52 µmol min-1.mg-1, usando 1 mmol.L-1de ABTS como substrato. A peroxidase aminada a 10 mmol.L-1reteve 88% e a aminada a 50 mmol.L-1reteve 100% da atividade inicial. As peroxidases aminadas a 10 ou 50 mmol.L-1foram covalentemente imobilizadas em suporte glioxil-pó de sabugo de milho com atividade recuperada de 74% e 86%, respectivamente. Os derivados obtidos foram utilizados na descoloração de solução de azul de bromofenol 0,02 mmol.L-1. Após 30 min 93% de descoloração foram alcançados com o derivado glioxil-pó de sabugo de milho com a peroxidase aminada 10 mmol.L-1e 89% com a aminada 50 mmol.L-1. Estes derivados mantiveram 60% das propriedades catalíticas, quando utilizado por três vezes. A peroxidase extraída do tegumento da semente de soja imobilizada em suporte de baixo custo pó de sabugo de milho apresentou melhoria na estabilidade térmica da enzima. Palavras-chave: Peroxidases. Imobilização multipontual de enzimas. Aminação de enzimas. Pó de sabugo de milho.

Bromofhenol blue discoloration using peroxidase immobilized on highly activated corncob powder

The aim of the present study was to evaluate the efficacy of peroxidase immobilized on corncob powder for the discoloration of dye. Peroxidase was extracted from soybean seed coat, followed by amination of the surface of the tertiary structure. The aminated peroxidase was immobilized on highly activated corncob powder and employed for the discoloration of bromophenol blue. Amination was performed with 10 or 50 mmol.L-1 carbodiimide and 1 mol.L-1 ethylenediamine. The amount of protein in the extract was 0.235 ± 0.011 mg.mL-1 and specific peroxidase activity was 86.06 ± 1.52 µmol min-1 . mg-1, using 1 mmol.L-1 ABTS as substrate. Ten mmol.L-1 and 50 mmol.L-1 aminated peroxidase retained 88 and 100% of the initial activity. Following covalent immobilization on a corncob powder-glyoxyl support, 10 and 50 mmol.L-1 aminated peroxidase retained 74 and 86% of activity, respectively. Derivatives were used for the discoloration of 0.02 mmol.L-1 bromophenol blue solution. After 30 min, 93 and 89% discoloration was achieved with the 10 mmol.L-1 and 50 mmol.L-1 derivatives, respectively. Moreover, these derivatives retained 60% of the catalytic properties when used three times. Peroxidase extracted from soybean seed coat immobilized on a low-cost corncob powder support exhibited improved thermal stability.

PHYSICO-CHEMICAL, SPECTROSCOPICAL AND THERMAL CHARACTERIZATION OF BIODIESEL OBTAINED BY ENZYMATIC ROUTE AS A TOOL TO SELECT THE MOST EFFICIENT IMMOBILIZED LIPASE

Two microbial lipases from Burkholderia cepacia and Pseudomonas fluorescens were evaluated as catalysts for the enzymatic transesterification of beef tallow with ethanol and the most efficient lipase source was selected by taking into account the properties of the product to be used as fuel. Both lipases were immobilized on an epoxy silica-polyvinyl alcohol composite by covalent immobilization and used to perform the reactions under the following operational conditions: beef tallow-to-ethanol molar ratio of 1:9, 45 degrees C and 400 units of enzymatic activity per gram of fat. Products, characterized using Fourier Transform Infrared spectroscopy (FTIR), viscosimetry, thermogravimetry and H-1 NMR spectroscopy, suggested that the biodiesel sample obtained in the reaction catalyzed by Burkholderia cepacia lipase has the best set of properties for fuel usage.

Iron(III) Porphyrin Covalently Supported onto Magnetic Amino-Functionalized Nanospheres as Catalyst for Hydrocarbon and Herbicide Oxidations

This work describes the covalent immobilization of an ironporphyrin, 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin iron(III) chloride (FeTFPP), onto maghemite/silica magnetic nanospheres covered with aminofunctionalized silica. The resulting material (gamma-Fe2O3/SiO2-NHFeP) was characterized by diffuse reflectance infrared spectroscopy (DRIFTS) and UV-Vis absorption spectroscopy. The catalytic activity of this magnetic ironporphyrin was investigated in the oxidation of hydrocarbons (styrene, (Z)-cyclooctene and R-(+)-limonene) and an herbicide (simazine) by hydrogen peroxide or 3-chloroperoxybenzoic acid. Hydrocarbon and simazine oxidation reaction products were analyzed by gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively. This catalytic system proved to be efficient and selective for hydrocarbon oxidation, leading to high product yields from styrene (89%), cyclooctene (71%) and R-(+) -limonene (86%). Simazine oxidation was attained with 100% selectivity for a dechlorinated product (OEAT), while several oxidation products were obtained for the same catalyst in homogeneous media. The catalyst can be easily recovered through application of an external magnetic field and washed after reaction. Catalyst reuse experiments for R-(+)-limonene oxidation have shown that the catalytic activity is kept at 90% after 10 consecutive reactions.

Iron(III) porphyrin covalently supported onto magnetic amino-functionalized nanospheres as catalyst for hydrocarbon and herbicide oxidations

This work describes the covalent immobilization of an ironporphyrin, 5,10,15,20- tetrakis(pentafluorophenyl)porphyrin iron(III) chloride (FeTFPP), onto maghemite/silica magnetic nanospheres covered with aminofunctionalized silica. The resulting material (γ-Fe2O3/SiO2-NHFeP) was characterized by diffuse reflectance infrared spectroscopy (DRIFTS) and UV-Vis absorption spectroscopy. The catalytic activity of this magnetic ironporphyrin was investigated in the oxidation of hydrocarbons (styrene, (Z)-cyclooctene and R-(+)-limonene) and an herbicide (simazine) by hydrogen peroxide or 3-chloroperoxybenzoic acid. Hydrocarbon and simazine oxidation reaction products were analyzed by gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively. This catalytic system proved to be efficient and selective for hydrocarbon oxidation, leading to high product yields from styrene (89%), cyclooctene (71%) and R-(+)-limonene (86%). Simazine oxidation was attained with 100% selectivity for a dechlorinated product (OEAT), while several oxidation products were obtained for the same catalyst in homogeneous media. The catalyst can be easily recovered through application of an external magnetic field and washed after reaction. Catalyst reuse experiments for R-(+)-limonene oxidation have shown that the catalytic activity is kept at 90% after 10 consecutive reactions.

Physico-chemical, spectroscopical and thermal characterization of biodiesel obtained by enzymatic route as a tool to select the most efficient immobilized lipase

Two microbial lipases from Burkholderia cepacia and Pseudomonas fluorescens were evaluated as catalysts for the enzymatic transesterification of beef tallow with ethanol and the most efficient lipase source was selected by taking into account the properties of the product to be used as fuel. Both lipases were immobilized on an epoxy silica-polyvinyl alcohol composite by covalent immobilization and used to perform the reactions under the following operational conditions: beef tallow-to-ethanol molar ratio of 1:9, 45ºC and 400 units of enzymatic activity per gram of fat. Products, characterized using Fourier Transform Infrared spectroscopy (FTIR), viscosimetry, thermogravimetry and ¹H NMR spectroscopy, suggested that the biodiesel sample obtained in the reaction catalyzed by Burkholderia cepacia lipase has the best set of properties for fuel usage.

Biomimese biologischer Membranen: Konzeptionierung, Synthese und biophysikalische Charakterisierung einer Hierarchie festkörpergestützter Mimikry biologischer Membranen

Im Rahmen dieser Arbeit wurden drei neue Modelle zur funktionellen Mimiese biologischer Membranen im Bereich der Bionanotechnologie entwickelt. Um den Rahmen der notwendigen Faktoren und Komponenten für biomimetische Membranmodelle abzustecken, wurde das biologische Vorbild im Bezug auf Zusammensetzung, Organisation und Funktion analysiert. Die daraus abgeleiteten Erkenntnisse erlauben das Erreichen von biologisch relevanten Membranwiderständen im Bereich von mehreren MOhm cm2 und eine gute lokale Fluidität. Ein weiteres Ziel dieser Arbeit war die Entwicklung einer Hierachie unterschiedlich stark von der Festkörperoberfläche entkoppelter Membranen zur Vergrößerung des submembranen Raumes. Diese Ziele konnten realisiert werden. Das auf archaealen Etherlipiden basierende DPTL-System wurde analog dem biologischen Vorbild stereoselektiv synthetisiert und ist in der Lage die Membran bei maximaler Elongation des TEG-Spacers mit mehr als 2 nm von der Oberfläche zu entkoppeln. Die erzielten Wiederstände liegen im hohen ein- bis zweistelligen MOhm-Bereich, die Kapazität entspricht mit 0,5 µF cm-2 ebenfalls dem Wert biologischer Membranen. Die Membraneigenschaften wurden mit Hilfe von SPS, EIS, IR-Spektroskopie, QCM, AFM und Kontaktwinkelmessungen charakterisiert. Die Funktionalität und lokale Fluidität der DPTL-Membran konnte anhand des Valinomycin vermittelten K+-Transports über die Membran gezeigt werden. Fluide Elektroden oder laterale Verdünnung mit TEGL erlauben den Einbau größerer Ionenkanäle. Lipo-Glycopolymere (LGP) mit unterschiedlichen Kettenlängen wurden mit Hilfe der kontrollierten radikalischen Polymerisation mit einer PD < 1.2 synthetisiert. Es zeigte sich, daß die Vororientierung der LGPs auf dem LB-Trog, gefolgt von einem LB-Übertrag auf einen funktionalisierten Träger mit photoreaktivem SAM, nach Belichten des Systems zu einer verlässlichen kovalenten Anbindung der supramolekularen LGP-Architektur führt. Da die Lipo-Glycopolymerketten am Glycopolymerterminus nur mit oberflächennahen Repetiereinheiten an die photoaktivierte Oberfläche binden, sind sie in der Lage Oberflächenrauhigkeiten des Festkörpersubstrates auszugleichen. Die photochemische Immobilisierung von funktionell orientierten supramolekularen LGP-Architekturen auf Goldoberflächen resultiert in tBLMs mit großen vertikalen Enkopplungen der Membran von der Festkörperoberfläche (>8 nm). Der funktionelle Ionentransport von Kaliumionen durch Valinomycin zeigt eine ausreichende lokale Fluidität der Membran die mit einem guten Membranwiderstand (mehrere MOhm) kombiniert ist. Große Membran-Oberflächenentkopplungen konnten mit Hilfe plasmapolymerisierter elektrophiler Polymere erreicht werden. Filmdicken von 50 nm sind mit homogener Oberfläche und Rauhigkeiten im Bereich von Nanometern möglich. Das System zeigt interessante fluide Eigenschaften mit guten Erholungsraten bei FRAP-Experimenten (Diffusionskonstanten von etwa 17 mikro m2 s-1). Die elektrischen Eigenschaften liegen mit Widerständen von wenigen kOhm unterhalb der für gute Membranmimikrie notwendigen Werte. Erstmalig konnte gezeigt werden, daß mit Hilfe dieser Methode inerte Polymere/Plastikträger (zum Beispiel Polypropylen und TOPAS) in effizienter Weise kovalent mit reaktiven Polymeroberflächen modifiziert werden können (Anwendung als DNA-Chip ist beschrieben).

Development and characterization of fluorescent pH sensors based on porous silica and hydrogel support matrices

The hydrogen ion activity (pH) is a very important parameter in environment monitoring, biomedical research and other applications. Optical pH sensors have several advantages over traditional potentiometric pH measurement, such as high sensitivity, no need of constant calibration, easy for miniaturization and possibility for remote sensing. Several pH indicators has been successfully immobilized in three different solid porous materials to use as pH sensing probes. The fluorescent pH indicator fluorescein-5-isothiocyanate (FITC) was covalently bound onto the internal surface of porous silica (pore size ~10 nm) and retained its pH sensitivity. The excited state pK* a of FITC in porous silica (5.58) was slightly smaller than in solution (5.68) due to the free silanol groups (Si-OH) on the silica surface. The pH sensitive range for this probe is pH 4.5 - 7.0 with an error less than 0.1 pH units. The probe response was reproducible and stable for at least four month, stored in DI water, but exhibit a long equilibrium of up to 100 minutes. Sol-gel based pH sensors were developed with immobilization of two fluorescent pH indicators fluorescein-5-(and-6)-sulfonic acid, trisodium salt (FS) and 8-hydroxypyrene- 1,3,6-trisulfonic acid (HPTS) through physical entrapment. Prior to immobilization, the indicators were ion-paired with a common surfactant hexadecyltrimethylammonium bromide (CTAB) in order to prevent leaching. The sol-gel films were synthesized through the hydrolysis of two different precursors, ethyltriethoxysilane (ETEOS) and 3- glycidoxypropyltrimethoxysilane (GPTMS) and deposited on a quartz slide through spin coating. The pK a of the indicators immobilized in sol-gel films was much smaller than in solutions due to silanol groups on the inner surface of the sol-gel films and ammonium groups from the surrounding surfactants. Unlike in solution, the apparent pK a of the indicators in sol-gel films increased with increasing ionic strength. The equilibrium time for these sensors was within 5 minutes (with film thickness of ~470 nm). Polyethylene glycol (PEG) hydrogel was of interest for optical pH sensor development because it is highly proton permeable, transparent and easy to synthesize. pH indicators can be immobilized in hydrogel through physical entrapment and copolymerization. FS and HPTS ion-pairs were physically entrapped in hydrogel matrix synthesized via free radical initiation. For covalent immobilization, three indicators, 6,8-dihydroxypyrene-1,3- disulfonic acid (DHPDS), 2,7-dihydroxynaphthalene-3,6-disulfonic acid (DHNDS) and cresol red were first reacted with methacrylic anhydride (MA) to form methacryloylanalogs for copolymerization. These hydrogels were synthesized in aqueous solution with a redox initiation system. The thickness of the hydrogel film is controlled as ~ 0.5 cm and the porosity can be adjusted with the percentage of polyethylene glycol in the precursor solutions. The pK a of the indicators immobilized in the hydrogel both physically and covalently were higher than in solution due to the medium effect. The sensors are stable and reproducible with a short equilibrium time (less than 4 minutes). In addition, the color change of cresol red immobilized hydrogel is vivid from yellow (acidic condition) to purple (basic condition). Due to covalently binding, cresol red was not leaching out from the hydrogel, making it a good candidate of reusable "pH paper".

Antigen binding forces of individually addressed single-chain Fv antibody molecules

Antibody single-chain Fv fragment (scFv) molecules that are specific for fluorescein have been engineered with a C-terminal cysteine for a directed immobilization on a flat gold surface. Individual scFv molecules can be identified by atomic force microscopy. For selected molecules the antigen binding forces are then determined by using a tip modified with covalently immobilized antigen. An scFv mutant of 12% lower free energy for ligand binding exhibits a statistically significant 20% lower binding force. This strategy of covalent immobilization and measuring well separated single molecules allows the characterization of ligand binding forces in molecular repertoires at the single molecule level and will provide a deeper insight into biorecognition processes.

Real-time detection of DNA interactions with long-period fiber-grating-based biosensor

Using an optical biosensor based on a dual-peak long-period fiber grating, we have demonstrated the detection of interactions between biomolecules in real time. Silanization of the grating surface was successfully realized for the covalent immobilization of probe DNA, which was subsequently hybridized with the complementary target DNA sequence. It is interesting to note that the DNA biosensor was reusable after being stripped off the hybridized target DNA from the grating surface, demonstrating a function of multiple usability.