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.

Amination of enzymes to improve biocatalyst performance: coupling genetic modification and physicochemical tools

Improvement of the features of an enzyme is in many instances a pre-requisite for the industrial implementation of these exceedingly interesting biocatalysts. To reach this goal, the researcher may utilize different tools. For example, amination of the enzyme surface produces an alteration of the isoelectric point of the protein along with its chemical reactivity (primary amino groups are the most widely used to obtain the reaction of the enzyme with surfaces, chemical modifiers, etc.) and even its “in vivo” behavior. This review will show some examples of chemical (mainly modifying the carboxylic groups using the carbodiimide route), physical (using polycationic polymers like polyethyleneimine) and genetic amination of the enzyme surface. Special emphasis will be put on cases where the amination is performed to improve subsequent protein modifications. Thus, amination has been used to increase the intensity of the enzyme/support multipoint covalent attachment, to improve the interaction with cation exchanger supports or polymers, or to promote the formation of crosslinkings (both intra-molecular and in the production of crosslinked enzyme aggregates). In other cases, amination has been used to directly modulate the enzyme properties (both in immobilized or free form). Amination of the enzyme surface may also pursue other goals not related to biocatalysis. For example, it has been used to improve the raising of antibodies against different compounds (both increasing the number of haptamers per enzyme and the immunogenicity of the composite) or the ability to penetrate cell membranes. Thus, amination may be a very powerful tool to improve the use of enzymes and proteins in many different areas and a great expansion of its usage may be expected in the near future.

Co-encapsulation of enzymes and antibodies for chemical deactivation of pathogens on paper

Le papier bioactif est obtenu par la modification de substrat du papier avec des biomolécules et des réactifs. Ce type de papier est utilisé dans le développement de nouveaux biocapteurs qui sont portables, jetables et économiques visant à capturer, détecter et dans certains cas, désactiver les agents pathogènes. Généralement les papiers bioactifs sont fabriqués par l’incorporation de biomolécules telles que les enzymes et les anticorps sur la surface du papier. L’immobilisation de ces biomolécules sur les surfaces solides est largement utilisée pour différentes applications de diagnostic comme dans immunocapteurs et immunoessais mais en raison de la nature sensible des enzymes, leur intégration au papier à grande échelle a rencontré plusieurs difficultés surtout dans les conditions industrielles. Pendant ce temps, les microcapsules sont une plate-forme intéressante pour l’immobilisation des enzymes et aussi assez efficace pour permettre à la fonctionnalisation du papier à grande échelle car le papier peut être facilement recouvert avec une couche de telles microcapsules. Dans cette étude, nous avons développé une plate-forme générique utilisant des microcapsules à base d’alginate qui peuvent être appliquées aux procédés usuels de production de papier bioactif et antibactérien avec la capacité de capturer des pathogènes à sa surface et de les désactiver grâce à la production d’un réactif anti-pathogène. La conception de cette plate-forme antibactérienne est basée sur la production constante de peroxyde d’hydrogène en tant qu’agent antibactérien à l’intérieur des microcapsules d’alginate. Cette production de peroxyde d’hydrogène est obtenue par oxydation du glucose catalysée par la glucose oxydase encapsulée à l’intérieur des billes d’alginate. Les différentes étapes de cette étude comprennent le piégeage de la glucose oxydase à l’intérieur des microcapsules d’alginate, l’activation et le renforcement de la surface des microcapsules par ajout d’une couche supplémentaire de chitosan, la vérification de la possibilité d’immobilisation des anticorps (immunoglobulines G humaine comme une modèle d’anticorps) sur la surface des microcapsules et enfin, l’évaluation des propriétés antibactériennes de cette plate-forme vis-à-vis l’Escherichia coli K-12 (E. coli K-12) en tant qu’un représentant des agents pathogènes. Après avoir effectué chaque étape, certaines mesures et observations ont été faites en utilisant diverses méthodes et techniques analytiques telles que la méthode de Bradford pour dosage des protéines, l’électroanalyse d’oxygène, la microscopie optique et confocale à balayage laser (CLSM), la spectrométrie de masse avec désorption laser assistée par matrice- temps de vol (MALDI-TOF-MS), etc. Les essais appropriés ont été effectués pour valider la réussite de modification des microcapsules et pour confirmer à ce fait que la glucose oxydase est toujours active après chaque étape de modification. L’activité enzymatique spécifique de la glucose oxydase après l’encapsulation a été évaluée à 120±30 U/g. Aussi, des efforts ont été faits pour immobiliser la glucose oxydase sur des nanoparticules d’or avec deux tailles différentes de diamètre (10,9 nm et 50 nm) afin d’améliorer l’activité enzymatique et augmenter l’efficacité d’encapsulation. Les résultats obtenus lors de cette étude démontrent les modifications réussies sur les microcapsules d’alginate et aussi une réponse favorable de cette plate-forme antibactérienne concernant la désactivation de E. coli K-12. La concentration efficace de l’activité enzymatique afin de désactivation de cet agent pathogénique modèle a été déterminée à 1.3×10-2 U/ml pour une concentration de 6.7×108 cellules/ml de bactéries. D’autres études sont nécessaires pour évaluer l’efficacité de l’anticorps immobilisé dans la désactivation des agents pathogènes et également intégrer la plate-forme sur le papier et valider l’efficacité du système une fois qu’il est déposé sur papier.

Co-encapsulation of enzymes and antibodies for chemical deactivation of pathogens on paper

Le papier bioactif est obtenu par la modification de substrat du papier avec des biomolécules et des réactifs. Ce type de papier est utilisé dans le développement de nouveaux biocapteurs qui sont portables, jetables et économiques visant à capturer, détecter et dans certains cas, désactiver les agents pathogènes. Généralement les papiers bioactifs sont fabriqués par l’incorporation de biomolécules telles que les enzymes et les anticorps sur la surface du papier. L’immobilisation de ces biomolécules sur les surfaces solides est largement utilisée pour différentes applications de diagnostic comme dans immunocapteurs et immunoessais mais en raison de la nature sensible des enzymes, leur intégration au papier à grande échelle a rencontré plusieurs difficultés surtout dans les conditions industrielles. Pendant ce temps, les microcapsules sont une plate-forme intéressante pour l’immobilisation des enzymes et aussi assez efficace pour permettre à la fonctionnalisation du papier à grande échelle car le papier peut être facilement recouvert avec une couche de telles microcapsules. Dans cette étude, nous avons développé une plate-forme générique utilisant des microcapsules à base d’alginate qui peuvent être appliquées aux procédés usuels de production de papier bioactif et antibactérien avec la capacité de capturer des pathogènes à sa surface et de les désactiver grâce à la production d’un réactif anti-pathogène. La conception de cette plate-forme antibactérienne est basée sur la production constante de peroxyde d’hydrogène en tant qu’agent antibactérien à l’intérieur des microcapsules d’alginate. Cette production de peroxyde d’hydrogène est obtenue par oxydation du glucose catalysée par la glucose oxydase encapsulée à l’intérieur des billes d’alginate. Les différentes étapes de cette étude comprennent le piégeage de la glucose oxydase à l’intérieur des microcapsules d’alginate, l’activation et le renforcement de la surface des microcapsules par ajout d’une couche supplémentaire de chitosan, la vérification de la possibilité d’immobilisation des anticorps (immunoglobulines G humaine comme une modèle d’anticorps) sur la surface des microcapsules et enfin, l’évaluation des propriétés antibactériennes de cette plate-forme vis-à-vis l’Escherichia coli K-12 (E. coli K-12) en tant qu’un représentant des agents pathogènes. Après avoir effectué chaque étape, certaines mesures et observations ont été faites en utilisant diverses méthodes et techniques analytiques telles que la méthode de Bradford pour dosage des protéines, l’électroanalyse d’oxygène, la microscopie optique et confocale à balayage laser (CLSM), la spectrométrie de masse avec désorption laser assistée par matrice- temps de vol (MALDI-TOF-MS), etc. Les essais appropriés ont été effectués pour valider la réussite de modification des microcapsules et pour confirmer à ce fait que la glucose oxydase est toujours active après chaque étape de modification. L’activité enzymatique spécifique de la glucose oxydase après l’encapsulation a été évaluée à 120±30 U/g. Aussi, des efforts ont été faits pour immobiliser la glucose oxydase sur des nanoparticules d’or avec deux tailles différentes de diamètre (10,9 nm et 50 nm) afin d’améliorer l’activité enzymatique et augmenter l’efficacité d’encapsulation. Les résultats obtenus lors de cette étude démontrent les modifications réussies sur les microcapsules d’alginate et aussi une réponse favorable de cette plate-forme antibactérienne concernant la désactivation de E. coli K-12. La concentration efficace de l’activité enzymatique afin de désactivation de cet agent pathogénique modèle a été déterminée à 1.3×10-2 U/ml pour une concentration de 6.7×108 cellules/ml de bactéries. D’autres études sont nécessaires pour évaluer l’efficacité de l’anticorps immobilisé dans la désactivation des agents pathogènes et également intégrer la plate-forme sur le papier et valider l’efficacité du système une fois qu’il est déposé sur papier.

On the steady-state assumption and its application to the rotating disk voltammetry of adsorbed enzymes

Rotating disk voltammetry is routinely used to study electrochemically driven enzyme catalysis because of the assumption that the method produces a steady-state system. This assumption is based on the sigmoidal shape of the voltammograms. We have introduced an electrochemical adaptation of the King-Altman method to simulate voltammograms in which the enzyme catalysis, within an immobilized enzyme layer, is steadystate. This method is readily adaptable to any mechanism and provides a readily programmable means of obtaining closed form analytical equations for a steady-state system. The steady-state simulations are compared to fully implicit finite difference (FIFD) simulations carried out without any steady-state assumptions. On the basis of our simulations, we conclude that, under typical experimental conditions, steady-state enzyme catalysis is unlikely to occur within electrode-immobilized enzyme layers and that typically sigmoidal rotating disk voltammograms merely reflect a mass transfer steady state as opposed to a true steady state of enzyme intermediates at each potential.

Preparation and assessment of immobilized laccase-based micro- and nanocatalysts for the degradation of anthropogenic compounds

Since the dawn of its presence on earth, the human being has been able to exploit the enzymes for its subsistence. More recent is the meeting between the enzymatic processes and the urgent need for technologies that aim to preserve our planet. In this field nowadays enzymatic catalysis is tested either to depollution/remediation as well as waste disposal. The work presented in this thesis, regarding both these two topics, is tailored on two European projects (EU 2020), MADFORWATER and TERMINUS respectively. Firstly, production of micro- and nanocatalysts via immobilization of laccases (a lignin-degrader enzyme) is performed. In the second part of the thesis laccase is applied to a tertiary treatment of wastewater with the aim to degrade 9 pharmaceutical active compounds in batch reactors. Despite several optimizations, poor degradation is reached and we did not proceed with the study of different bioreactor setups. Therefore, the focus is moved to a project concerning the production of smart multi-layer plastic packaging containing enzymes to improve the possibilities of recycling. In this field shielded nanocatalysts produced via coating techniques able to interact with redox mediators are investigated. The target substrate in this second project is produced in laboratory (i.e. polyurethane like compounds), starting from monomers whose degradation had already been tested, as a proof of concept. The first enzyme studied is still the laccase.

Digestive Peptidase Evolution In Holometabolous Insects Led To A Divergent Group Of Enzymes In Lepidoptera.

Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic l-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.

Immobilization Of Papain On Chitin And Chitosan And Recycling Of Soluble Enzyme For Deflocculation Of Saccharomyces Cerevisiae From Bioethanol Distilleries.

Yeast flocculation (Saccharomyces cerevisiae) is one of the most important problems in fuel ethanol production. Yeast flocculation causes operational difficulties and increase in the ethanol cost. Proteolytic enzymes can solve this problem since it does not depend on these changes. The recycling of soluble papain and the immobilization of this enzyme on chitin or chitosan were studied. Some cross-linking agents were evaluated in the action of proteolytic activity of papain. The glutaraldehyde (0.1-10% w·v(-1)), polyethyleneimine (0.5% v·v(-1)), and tripolyphosphate (1-10% w·v(-1)) inactivated the enzyme in this range, respectively. Glutaraldehyde inhibited all treatments of papain immobilization. The chitosan cross-linked with TPP in 5 h of reaction showed the yield of active immobilized enzyme of 15.7% and 6.07% in chitosan treated with 0.1% PEI. Although these immobilizations have been possible, these levels have not been enough to cause deflocculation of yeast cells. Free enzyme was efficient for yeast deflocculation in dosages of 3 to 4 g·L(-1). Recycling of soluble papain by centrifugation was effective for 14 cycles with yeast suspension in time perfectly compatible to industrial conditions. The reuse of proteases applied after yeast suspension by additional yeast centrifugation could be an alternative to cost reduction of these enzymes.

Enzymatic Biodiesel Synthesis From Yeast Oil Using Immobilized Recombinant Rhizopus Oryzae Lipase.

The recombinant Rhizopus oryzae lipase (1-3 positional selective), immobilized on Relizyme OD403, has been applied to the production of biodiesel using single cell oil from Candida sp. LEB-M3 growing on glycerol from biodiesel process. The composition of microbial oil is quite similar in terms of saponifiable lipids than olive oil, although with a higher amount of saturated fatty acids. The reaction was carried out in a solvent system, and n-hexane showed the best performance in terms of yield and easy recovery. The strategy selected for acyl acceptor addition was a stepwise methanol addition using crude and neutralized single cell oil, olive oil and oleic acid as substrates. A FAMEs yield of 40.6% was obtained with microbial oils lower than olive oil 54.3%. Finally in terms of stability, only a lost about 30% after 6 reutilizations were achieved.

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

Herein, the immobilization of some Schiff base-copper(II) complexes in smectite clays is described as a strategy for the heterogenization of homogeneous catalysts. The obtained materials were characterized by spectroscopic techniques, mostly UV/Vis, EPR, XANES and luminescence spectroscopy. SWy-2 and synthetic Laponite clays were used for the immobilization of two different complexes that have previously shown catalytic activity in the dismutation of superoxide radicals, and disproportionation of hydrogen peroxide. The obtained results indicated the occurrence of an intriguing intramolecular redox process involving copper and the imine ligand at the surface of the clays. These studies are supported by computational calculations.

Enzymatic kinetic resolution of (RS)-1-(Phenyl)ethanols by Burkholderia cepacia lipase immobilized on magnetic nanoparticles

Lipase from Burkholderia cepacia immobilized on superparamagnetic nanoparticles using adsorption and chemisorption methodologies was efficiently applied as recyclable biocatalyst in the enzymatic kinetic resolution of (RS)-1-(phenyl)ethanols via transesterification reactions. (R)-Esters and the remaining (S)-alcohols were obtained with excellent enantiomeric excess (> 99%), which corresponds to a perfect process of enzymatic kinetic resolution (conversion 50%, E > 200). The transesterification reactions catalysed with B. cepacia lipase immobilized by the glutaraldehyde method showed the best results in terms of reusability, preserving the enzyme activity (conversion 50%, E > 200) for at least 8 successive cycles.

Optimization of the Enzymatic Interesterification of Milk Fat and Canola Oil Blends Using Immobilized Rhizopus oryzae Lipase by Response Surface Methodology

Blends of milk fat and canola oil (MF:CNO) were enzymatically interesterified (EIE) by Rhizopus oryzne lipase immobilized on polysiloxane-polyvinyl alcohol (SiO(2)-PVA) composite, in a solvent-free system. A central composite design (CCD) was used to optimize the reaction, considering the effects of different mass fractions of binary blends of MF:CNO (50:50, 65:35 and 80:20) and temperatures (45, 55 and 65 degrees C) on the composition and texture properties of the interesterified products, taking the interesterification degree (ID) and consistency (at 10 degrees C) as response variables. For the ID variable both mass fraction of milk fat in the blend and temperature were found to be significant, while for the consistency only mass fraction of milk fat was significant. Empiric models for ID and consistency were obtained that allowed establishing the best interesterification conditions: blend with 65 % of milk fat and 35 %, of canola oil, and temperature of 45 degrees C. Under these conditions, the ID was 19.77 %) and the consistency at 10 degrees C was 56 290 Pa. The potential of this eco-friendly process demonstrated that a product could be obtained with the desirable milk fat flavour and better spreadability under refrigerated conditions.

An Evaluation of Different Bioreactor Configurations with Immobilized Yeast for Bioethanol Production

The bioethanol industry expects a huge expansion and new technologies are being implemented with the aim of optimizing the fermentation process. The behavior of cells of Saccharomyces cerevisiae immobilized in PVA-LentiKats, during the production of bioethanol in two reactor systems, was studied. The entrapped cell in LentiKats lenses showed a different profile using stirred tank reactor (STR) and packed column reactor (PCR). Low free cells accumulation in the medium was observed for the STR after 72 h of fermentation. On the other hand, no free cells accumulation was observed, probably due to the absence of mechanical agitation in PCR configuration. Better fermentation results were obtained working with STR (final cellular concentration = 13 g.L-1, Pf = 28 g.L-1, Qp = 1.17 g.L-1.h-1,and Yp/s = 0.3 g.g-1) in comparison to PCR (final cellular concentration = 11.4 g.L-1, Pf = 20 g.L-1, Qp = 0.83 g.L-1.h-1,and Yp/s = 0.25 g.g-1). Such results are probably due to the mechanical agitation of the medium provided by STR configuration, which permitted a better heat and mass transference.

Production of Cellulolytic Enzymes by Anaerobic Fungi Cultivated in Different Conditions

The present paper studies the influence of different nutrients for the production of two cellulolytic enzymes: endo beta-1.4 glucanase and exo beta-1.4 glucanase by anaerobic fungi taken from cow rumen, that were fed a diet of corn silage and Brachiaria decumbens grass hay. During the enzymatic degradation assays, it was observed that the addition of some essential nutrients in the formulation of the culture medium contributed positively in the cellulolytic enzyme production, with exception of riboflavin. Such results contributed in the establishment of an effective method for the evaluation of enzymatic activities in anaerobic fibrolytic fungi. In this work, nutrients added to enrich the culture medium have successfully proven that they can be used as inoculating agents (inductors) in diets rich in ensilage with law nutritive value.

Fermentation of Groundnut Shell Enzymatic Hydrolysate for Fuel Ethanol Production by Free and Sorghum Stalks Immobilized Cells of Pichia stipitis NCIM 3498

Groundnut shell (GS), after separation of pod, is readily available as a potential feedstock for production of fermentable sugars. The substrate was delignified with sodium sulfite. The delignified substrate released 670 mg/g of sugars after enzymatic hydrolysis (50 degrees C, 120 rpm, 50 hrs) using commercial cellulases (Dyadic Xylanase PLUS, Dyadic Inc. USA). The groundnut shell enzymatic hydrolysate (45.6 g/L reducing sugars) was fermented for ethanol production with free and sorghum stalks immobilized cells of Pichia stipitis NCIM 3498 under submerged cultivation conditions. Immobilization of yeast cells on sorghum stalks were confirmed by scanning electron microscopy (SEM). A maximum of ethanol production (17.83 g/L, yield 0.44 g/g and 20.45 g/L, yield 0.47 g/g) was observed with free and immobilized cells of P. stipitis respectively in batch fermentation conditions. Recycling of immobilized cells showed a stable ethanol production (20.45 g/L, yield 0.47 g/g) up to 5 batches followed by a gradual downfall in subsequent cycles.