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1
David, Allan E. "Immobilization of enzymes on nanoporous, silica composites." College Park, Md. : University of Maryland, 2004. http://hdl.handle.net/1903/2055.
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Thesis (Ph. D.) -- University of Maryland, College Park, 2004.<br>Thesis research directed by: Chemical Engineering. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Dong, Liang-Chang. "Thermally reversible hydrogels for controlled drug delivery and enzyme immobilization /." Thesis, Connect to this title online; UW restricted, 1990. http://hdl.handle.net/1773/8009.
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Meneghello, Marta. "A modular approach for a controlled immobilization of enzymes." Thesis, University of Southampton, 2018. https://eprints.soton.ac.uk/422233/.
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Stable, site-specific immobilization of redox proteins and enzymes is of great interest for the development of biosensors and biofuel cells, where the long-term stability of enzymatic electrodes as well as the possibility of controlling the orientation of the biomolecules at the electrode surface have a great importance. For such applications, it would be desirable to immobilise redox proteins and enzymes in a specific orientation on the electrode in order to improve direct electron transfer. In this work, we describe such an approach using site directed mutagenesis to introduce cysteine residues at specific locations on the enzyme surface, and the reaction between the free thiol of the cysteine and maleimide groups attached on the electrode surface to immobilise the mutated enzymes. Using cellobiose dehydrogenase (CDH) as a model system, different types of electrodes (carbon and gold-based, flat and nanostructured) were firstly modified with maleimide groups using a modular approach based on electrografting and solid-phase synthesis. Therefore, the electrodes were used to covalently immobilise CDH variants bearing the free cysteine in different locations at their surface. The key point of this method is that the main elements of the modification can be independently varied to tune the architecture of the electrode surface as required, by simply changing the “bricks” of the structure. The CDH-modified electrodes were tested for direct and mediated electron transfer, showing excellent long-term storage stability as well as good catalytic responses. The mechanisms of the direct and mediated electron transfer were fully investigated, as well as the kinetics of the CDH electrodic reactions, also employing computer simulations.
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Li, Dan 1971. "Immobilization, characterization and use of fish protease." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=102996.
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Enzyme immobilization as a technique attaches free forms of enzyme molecules to stationary support materials to permit enzymes to be reused several times. Bovine trypsin as a model enzyme was immobilized onto controlled pore glass (CPG) beads to investigate the optimum conditions for immobilization, as well as the physico-chemical properties of the immobilized enzyme versus the free form of the enzyme. At pH 9, about 60% of the enzyme protein incubated with CPG was immobilized onto the CPG, and immobilized bovine trypsin activity was determined as 0.265 BAPNA U/g CPG beads. The immobilized bovine trypsin showed lower affinity for its substrate, lower susceptibility to inhibition by soybean trypsin inhibitor and higher thermal stability, while the optimum pH and optimum temperature values were shifted to higher values compared to those of the free enzyme. The immobilized enzyme was evaluated for its capacity to extract carotenoproteins from shrimp shell. After 11 re-uses, the immobilized enzyme retained about 77% of its initial activity, and the total yield of the product from the same immobilized trypsin was 4.3 times higher than a single use of the same amount of the free enzyme. Cunner fish is a cold water adapted, stomachless teleost fish. Cunner fish trypsin possesses some unique properties compared with homologous trypsins from (i) species acclimated to warm temperature regimes, and (ii) species with functionally distinct-stomachs. Cunner fish trypsin was extracted from pancreatic tissue, and immobilized onto CPG beads using glutaraldehyde as cross-linking reagent. The influence of enzyme loading, the properties of the immobilized enzyme in terms of specific activities, and responses to pH and temperature were investigated. The kinetic properties and operational stability of the immobilized cunner trypsin were studied as well. The pH optimum of the immobilized fish trypsin shifted from pH 8.5 to pH 9, and the temperature optimum also increased from 45ºC to 50ºC versus the free form of the cunner enzyme. The catalytic efficiencies (Vmax/Km) of the immobilized fish trypsin were determined for both amidase and esterase reactions, using BAPNA and TAME as substrates and were found to be greater than those of immobilized bovine trypsin. Thus, the immobilized cunner fish trypsin had a higher catalytic capacity for the hydrolysis of both the amide and ester substrates. The operational stability of immobilized fish trypsin was studied by extracting carotenoprotein from shrimp shell. The immobilized fish trypsin retained 75% of its initial hydrolytic capacity after 11 re-uses, and the yield obtained was over 20% higher than that of immobilized bovine trypsin. When the immobilized cunner fish trypsin was applied to digest native pectin methylesterase (PME), it exhibited greater capacity to inactivate the PME than immobilized bovine trypsin. The inactivation efficiency of the immobilized fish trypsin was 20% higher than that of the immobilized bovine trypsin. The inactivation of PME was influenced by PME concentration, incubation time and temperature. In general, higher temperature, longer incubation period, and lower initial PME concentration produced more PME inactivation. PME inactivated by immobilized fish trypsin and bovine trypsin regained part of its activity during storage at 4ºC. The initial PME concentration affected the reactivation period. The kinetic studies indicated that the inactivation rate constants increased and D-values (time to inactivate 90% of the enzyme) decreased with increasing temperature for both immobilized fish trypsin and bovine trypsin. The activation energy (Ea) of PME inactivation by the immobilized fish trypsin was lower than that by the immobilized bovine trypsin, which explains why the immobilized fish trypsin had higher catalytic capacity at various temperatures than immobilized bovine trypsin.
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Ampon, Kamaruzaman. "Immobilization of proteins on porous polymer beads /." The Ohio State University, 1987. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487324944212704.
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Chen, Xi. "Infrared and Uv-Vis Spectroscopic Studies of Catalytic Reaction of Enzymes and Immobilization Enzyme on Porous Polymers." University of Akron / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=akron1428327122.
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Reichstädter, Marek. "Imobilizace vybraných glykanohydroláz." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2015. http://www.nusl.cz/ntk/nusl-217152.
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The theoretical part of this thesis deals with cellulolytic enzymes, their microbial producers, the possibilities of using such enzymes in the industry and how can be enzymes - not only cellulolytic - immobilized. Experimental part examines the preparations created by immobilizing various amounts of the commercially used cellulolytic complex Cellulast 1.5L onto various synthetic carriers made of polyethylene terephthalate - commercially used Sorsilen, PET carrier and glutaraldehyde-treated PET carrier. Enzyme activity of these preparations was determined by Somogyi - Nelson method by spectrophotometry. For the highest activity immobilized preparation was determined the temperature- and the pH-optimum. The difference in effects change between the free and immobilized enzyme by measuring viscosity decrease of the substrate depending on the degradation of glycosidic bonds was also studied.
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Solé, Ferré Jordi. "Oxidoreductive bioprocess intensification through reaction engineering and enzyme immobilization." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/669346.
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La investigació plasmada en aquesta tesi doctoral tracta l’aplicació dels principis d’enginyeria de
reacció i immobilització enzimàtica per a la millora de reaccions d’oxidoreducció biocatalitzades.
En una primera part de la tesi, es va estudiar la co-immobilització de la monooxigenasa P450
BM3 juntament amb un enzim regenerador del cofactor NADPH, la glucosa deshidrogenasa
(GDH-Tac). Els millors derivats es van obtenir utilitzant dos suports d’agarosa, un funcionalitzat
amb grups epoxy (83% i 20% activitats retingudes respectivament) i l’altre amb grups amino
(28% i 25% activitats retingudes respectivament). Posteriorment es va provar de re-utilitzar
aquests enzims immobilitzats en diferents cicles de reacció utilitzant un dels substrats naturals
de la P450 BM3, el laurat de sodi.
Un cop demostrat que ambdós enzims immobilitzats podien ser reciclats, es va estudiar
l’aplicació d’aquests en dues reaccions d’interès industrial, la hidroxilació de la α-isoforona i la
hidroxilació del diclofenac. En el primer cas, va fer falta optimitzar certs paràmetres de la reacció
abans d’aplicar els derivats. Un cop es van tenir unes condicions acceptables, es va comprovar
que els resultats obtinguts anteriorment amb el laurat de sodi, no podien ser extrapolats. La
reutilització no va ser possible. En quant al diclofenac, es van obtenir resultats similars. En
ambdós casos però, l’aplicació dels enzims en la seva forma soluble, va permetre obtenir
conversions altes: 86.2% per a la α-isoforona (50 mM inicial) i 100% per al diclofenac (3.5 mM
inicial).
El producte hidroxilat de l’α-isoforona, la 4-hidroxi-isoforona, ha de ser oxidat en un segon pas
per arribar a l’intermediari desitjat, la keto-isoforona. Aquesta segona reacció es duu a terme
amb una alcohol deshidrogenasa, la qual també necessita un regenerador de cofactor, la NADPH
oxidasa. Al aplicar els dos enzims en la seva forma soluble, al cap de 24h, es va aconseguir un
95.7% de rendiment i una productivitat de 6.52 g L-1 dia-1. A part, l’enzim diana, l’alcohol
deshidrogenasa, es va poder immobilitzar en epoxy-agarosa obtenint una activitat retinguda del
58.2%. Al intentar re-utilitzar-lo, es va poder operar durant 96h (4 cicles) millorant el rendiment
del biocatalitzador fins a 2.5 vegades comparat amb la reacció en soluble.
La reacció d’hidrogenació de la α-isoforona, per altre banda, resulta en la 3,3,5-
trimetilciclohexanona, un substrat amb interès industrial per a l’obtenció de polímers. En aquest
cas, es va utilitzar la Baeyer-Villiger ciclohexanona monooxigenasa juntament amb una glucosa
deshidrogenasa comercial (GDH-01) per a realitzar la reacció d’inserció d’un àtom d’oxigen en
l’anell de carbonis. Es van optimitzar diferents paràmetres de la reacció com la formulació del
biocatalitzador, la velocitat d’adició de substrat i la quantitat d’enzim afegida. Un cop
optimitzada la reacció es va escalar primer a 1 litre i finalment a 100 litres. En aquesta última
reacció a escala pre-industrial, es va obtenir una conversió del 85%, una productivitat de 2.7 g L-
1 h-1 i un rendiment del biocatalitzador de 0.83 g g-1
cww.
Finalment aquesta mateixa reacció es va realitzar utilitzant els enzims immobilitzats i reciclantos.
Tant la ciclohexanona monooxigenasa com la glucosa deshidrogenasa (GDH-01) es van poder
immobilitzar en agarosa funcionalitzada amb grups amino. En el primer cas es va obtenir una
activitat retinguda del 62.4% (mètode obtingut de la bibliografia) i en el segon cas del 62.6%. En
reacció, els dos enzims immobilitzats van ser utilitzats tant per separat com conjuntament. Al
cap de sis cicles de reacció (132.5 mM de substrat inicial), es va assolir un rendiment de
biocatalitzador 3.6 vegades superior per a la monooxigenasa i 1.9 vegades superior per a la GDH-
01 comparat amb la reacció en soluble.<br>The research performed and disclosed in this thesis deals with the reaction engineering and the
enzyme immobilization principles as tools to improve biocatalyzed oxidoreductive reactions.
On a first stage, the co-immobilization of the P450 BM3 monooxygenase together with a NADPH
cofactor regeneration enzyme, the glucose dehydrogenase (GDH-Tac), was studied. The best
derivates were obtained when using two agarose supports, an epoxy functionalized (83% and
20% retained activity respectively) and an amino functionalized (28% and 25% retained activity
respectively). Later on, the re-cycling of the immobilized enzymes was tested in reaction cycles
using one of the natural substrates of the P450 BM3, the sodium laurate.
Once it could be demonstrated that re-cycling of both P450 BM3 and GDH-Tac was possible,
both enzymes were studied in two of the project’s target reactions, the hydroxylation of α-
isophorone and the hydroxylation of diclofenac. In the first case, the optimization of the reaction
conditions had to be performed prior to the reaction cycles. The reactor configuration, the
oxygen income or the glucose concentration were adjusted. However, when the reaction was
performed using the co-immobilized enzymes, the P450 BM3 was deactivated and it could not
be re-used. The same happened with the hydroxylation of diclofenac. On the other hand, the
reaction using soluble enzymes, resulted in 86.2% conversion for the α-isophorone (50 mM
initial concentration) and 100% for the diclofenac (3.5 mM initial concentration).
The product resulting from the hydroxylation of α-isophorone, the 4-hydroxy-isophorone, can
be further oxidized to keto-isophorone, an intermediary for the synthesis of carotenoids and
vitamin E. In order to enzymatically perform this step, an alcohol dehydrogenase and a NADPH
oxidase, as a cofactor regenerator, were employed. When used in their soluble form, after 24
hours, 95.7% yield and a space time yield of 6.52 g L-1 day-1 were achieved. Moreover, the alcohol
dehydrogenase was immobilized on epoxy-agarose and 58.2% retained activity was obtained.
When re-used, the derivate could operate for 96h (4 cycles) improving the biocatalyst yield 2.5-
fold compared with the reaction with soluble enzymes.
The hydrogenation of α-isophorone results in 3,3,5-trimethylcyclohexanone, an industrial
interesting substrate due to the polymers that can be obtained from its oxidized product, the
trimethyl-ε-caprolactone. This compound is obtained by the Baeyer-Villiger insertion of an
oxygen atom into the carbon ring. For this purpose, a cyclohexanone monooxygenase together
with a commercial glucose dehydrogenase (GDH-01) were used. Different parameters of the
reaction were optimized such as the biocatalyst formulation, the substrate addition rate or the
biocatalyst loading. Afterwards, the reaction was scaled up to 1 liter first and then up to 100
liters. In this last pre-industrial reaction, 85% conversion, a space time yield of 2.7 g L-1 h-1 and a
biocatalyst yield of 0.83 g g-1
cww could be obtained.
Finally, this same reaction was performed using both enzymes immobilized and re-cycling was
intended. The cyclohexanone monooxygenase could be immobilized following a previously
described method and 62.4% retained activity was achieved. In the GDH-01 case, different
supports were screened albeit at the end, it was also the amino functionalized agarose that
resulted successful. A retained activity of 62.6% was obtained. In the reaction cycles, the
immobilized enzymes were used either separately or both together. In the best case scenario,
after six cycles of reaction (132.5 mM initial substrate) 3.6-fold and 1.9-fold higher biocatalysts
yields were obtained for the monooxygenase and the GDH-01, respectively.
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Myung, Suwan. "Cell-Free Biosystems Comprised of Synthetic Enzymatic Pathways: Development of Building Blocks, Immobilization of Enzymes, Stabilization of Cascade Enzymes, and Generation of Hydrogen." Diss., Virginia Tech, 2013. http://hdl.handle.net/10919/50645.
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The production of hydrogen from low-cost abundant renewable biomass would be vital to sustainable development. Cell-free (in vitro) biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it is essential to produce (purified) enzymes at low costs and stabilize them for long periods to decrease biocatalyst costs.<br /><br />Thermophilic recombinant enzymes as building blocks were discovered and developed: fructose 1,6-bisphosphatase (FBP) from Thermotoga maritime, phosphoglucose isomerase (PGI) from Clostridium thermocellum, triose phosphate isomerase (TIM) from Thermus thermophiles and fructose bisphosphate aldolase (ALD) from T. maritima and T. thermophilus. The recombinant proteins were over-expressed in E. coli, purified and characterized. <br /><br />For purification and stabilization of enzymes, one-step, simple, low-cost purification and immobilization methods were developed based on simple adsorption of cellulose-binding module (CBM)-tagged protein on the external surface of high-capacity regenerated amorphous cellulose. Also, a simple, low-cost purification method of thermophilic enzymes was developed utilizing a combination of heat and ammonium sulfate precipitation.<br /><br />For development of cascade enzymes as building modules (biocatalyst modules), it was discovered that the presence of other enzymes/proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme (e.g., PGI) due to the in vitro macromolecular crowding effect. And substrate channeling among CBM-tagged self-assembled three-enzyme complex (synthetic matabolon) immobilized on the easily-recycled cellulose-containing magnetic nanoparticles can not only increase cascade reaction rates greatly, but also decrease enzyme cost in cell-free biosystems.<br /><br />The high product yield and fast reaction rate of dihydrogen from sucrose was validated in a batch reaction containing fifteen enzymes comprising a non-natural synthetic pathway. The yield of dihydrogen production from 2 mM of sucrose was 96.7 % compared to theoretical yield at 37 oC. The maximum rate was increased 3.1 fold when the substrate concentration was increased from 2 to 50 mM in a fed-batch reaction.<br /><br />The research and development of cell-free biosystems for biomanufacturing require more efforts, especially in low-cost recombinant thermostable enzymes as building blocks, efficient cofactor recycling, enzyme and cofactor stabilization, and fast reaction rates.<br>Ph. D.
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Gao, Xiaojian. "Immobilization of lipases via sol-gel procedures and application of the immobilized lipases in oleochemicial [sic] reactions." [Lincoln, Neb. : University of Nebraska-Lincoln], 2004. http://www.unl.edu/libr/Dissertations/2004/GaoDis.pdf.
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Thesis (Ph. D.)--University of Nebraska--Lincoln, 2004.<br>PDF text: [2] leaves abstract, vii, 156 leaves dissertation : ill. (some col.). Site viewed on Jan. 25, 2005. Includes bibliographical references.
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Techer, Vincent. "Electrodes catalytiques à base d’enzymes pour le développement de biopiles alcool/oxygène microfluidiques." Thesis, Montpellier 2, 2013. http://www.theses.fr/2013MON20166/document.
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Les biopiles enzymatiques sont considérés comme des systèmes potentiellement utilisables pour la production d'énergie renouvelable dans des marchés niches. Une biopile est constituée de deux électrodes associées à des enzymes, catalyseurs biologiques, qui permettent la production d'énergie électrique à partir de réactions chimiques d'oxydoréduction. Ce travail présente la réalisation d'une biopile alcool/oxygène, au sein de laquelle l'alcool est oxydé à l'anode par l'alcool déshydrogénase alors que l'oxygène moléculaire est réduit en eau à la cathode par l'enzyme laccase, en présence de médiateurs spécifiques. L'objectif de ce travail a été tout d'abord de développer des bioélectrodes avec des enzymes immobilisées de manière à minimiser la quantité de biocatalyseur et augmenter sa stabilité. Dans un second temps, l'assemblage de biocathodes et de bioanodes a permis de fabriquer des biopiles à alcool macroscopique et microfluidique. Différentes poudres de carbone combinées à des polymères ont été utilisées pour immobiliser les enzymes et les médiateurs par encapsulation selon diverses configurations. Des analyses électrochimiques ont permis de mettre en évidence l'influence importante de certains paramètres comme la nature du carbone et du polymère, le pH et la température sur les performances des bioélectrodes. Une fois assemblées dans les configurations classique ou microfluidique, ces bioélectrodes ont conduit à des systèmes électrochimiques de génération d'énergie délivrant une densité de puissance maximale de 300μW/cm2 à 0,61V pour la biopile macroscopique et de 45μW/cm2 à 0,5V pour le système microfluidique<br>Enzymatic biofuel cells (BFC) are systems of great interest for the production of renewable energy in niche markets. A BFC consists of two electrodes associated with enzymes as catalysts allowing energy production from oxydoreduction reactions. This work is devoted to the development of an alcohol/oxygen BFC for which alcohol is oxidized at the anode by alcohol dehydrogenase while molecular oxygen is reduced to water at the cathode by laccase, in the presence of specific mediators. The objective of this work was first to develop bioelectrodes with immobilized enzymes in order to minimize the amount of biocatalyst and increase its stability. In a second step, biocathodes and bioanodes were assembled to make macroscopic and microfluidic alcohol BFCs. Various carbon powders combined to polymers were used to immobilize enzymes and mediators in various configurations by entrapment. Electrochemical analysis have highlighted the significant influence of certain parameters like the nature of polymer and carbon, the pH or the temperature on the bioelectrodes performances. Once assembled in classical or microfluidic configurations, these bioelectrode led to electrochemical energy generation systems delivering a maximum power density of 300μW/cm2 at 0,61V for the macroscopic BFC and 45μW/cm2 at 0,5V for the microfluidic system
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Döbber, Johannes [Verfasser], Martina [Gutachter] Pohl, and Vlada B. [Gutachter] Urlacher. "Fusion tag-based immobilization methods for the one-step purification and immobilization of enzymes / Johannes Döbber ; Gutachter: Martina Pohl, Vlada B. Urlacher." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2018. http://d-nb.info/1163449865/34.
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Gkaniatsou, Effrosyni. "Elaboration of novel enzymatic immobilization matrices, based on Metal-Organic Frameworks for the catalytic degradation of environmental pollutants." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLV005.
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Les enzymes sont des biocatalyseurs de plus en plus utilisés pour la transformation de molécules organiques (chimie fine, bioconversions, dépollution, chimie du pétrole) car elles possèdent de très bonnes sélectivité et réactivité, générant rapidement de larges quantités de produit. Cependant, la fragilité des enzymes, notamment en solution, limite souvent leur utilisation. Il est donc crucial de les immobiliser et de les stabiliser dans des supports adaptés. Une grande variété de matrices d’immobilisation (organiques ou inorganiques) a déjà étudiée, mais aucune ne satisfait pleinement aux critères nécessaires pour le développement de bio-réacteurs (accessibilité au site actif de l’enzyme, relargage de l’enzyme, diffusion des réactifs, recyclabilité, stabilité..). En outre, la majorité de ces matrices présente une porosité désordonnée, inadaptée pour une immobilisation homogène. L’utilisation de matériaux hybrides, cristallins et poreux de type Metal-Organic Frameworks (MOFs) a été récemment proposée comme alternative avec des applications en biocatalyse et en biodétection.Le travail de cette thèse a consisté à associer des matériaux de type Metal-Organic Frameworks à une mini-enzyme, la microperoxidase 8 (MP8), afin d’obtenir des matériaux multifonctionnels. Dans une première partie, le MOF mésoporeux, MIL-101(Cr), a été utilisé pour encapsuler la MP8, ce qui a conduit à une amélioration de son activité catalytique dans des conditions qui ne sont pas adéquates pour l’activité enzymatique (conditions acides, forte concentration en H2O2), démontrant ainsi le rôle protecteur du MOF vis-à-vis de l’enzyme. De plus, il a été possible de recycler le biocatalyseur. Cette approche a également permis d’améliorer considérablement la sélectivité de la MP8 pour la dégradation d’un colorant organique toxique négativement chargé, le méthyl orange, grâce à son adsorption sélective par interaction électrostatique avec les particules de MIL-101(Cr). La seconde partie a été consacrée à l’utilisation de matériaux MIL-101(Cr) fonctionnalisés. Tout d’abord, l’influence de la fonctionnalisation du ligand (avec un groupement –NH2 ou –SO3H) sur l’encapsulation de la MP8 ainsi que sur son activité catalytique pour des réactions de sulfoxydation a été étudiée. Il a été montré que l’activité catalytique et la réactivité de la MP8 sont affectées par le microenvironnement spécifique des pores du MOF, notamment pour des réactions de sulfoxydation mettant en jeu des dérivés thioanisole. Ensuite, un MOF à métal mixte (MIL-101(Cr/Fe)) choisi pour ses propriétés catalytiques stables, a été synthétisé et caractérisé. Enfin, la dernière partie de cette thèse a été consacrée à la synthèse in-situ d’un MOF (le microporeux MIL-53(Al)-FA) en présence de biomolécules (BSA) dans des conditions compatibles avec la préservation de la structure protéique (en solution aqueuse à température ambiante). Les matériaux hybrides obtenus ont été caractérisés en couplant de nombreuses techniques. Cette méthode d’encapsulation a conduit à des taux d’immobilisation extrêmement élevés. Une étude préliminaire a été initiée avec l’enzyme, Horseradish Peroxidase , qui conserve son activité catalytique après immobilisation<br>The use of enzymes in biocatalytic processes has been a challenging goal over the years. While enzymes present exceptional catalytic properties, their fragility hinders their industrial application. Their stabilization and protection are therefore of paramount importance. This can be effectively addressed through their immobilization within host solid matrices. Traditional materials (silica, clays, polymers, biopolymers, porous carbons…) have been widely studied as supports. Their pure organic or inorganic nature often requires a compromise between affinity with enzymes and robustness of the matrix. Besides, most of them have non-ordered porosity, with non-homogenous pore size distributions, unsuitable for homogeneous immobilization. Metal-Organic Frameworks (MOFs) have been recently introduced as alternative supports, thanks to their hybrid nature and their crystalline and highly porous structures.The aim of this PhD was to combine Metal-Organic Frameworks (highly porous and chemically stable polycarboxylate MOFs) and a mini-enzyme, microperoxidase 8 (MP8) to obtain multifunctional biocatalysts. In a first part, the mesoporous MIL-101(Cr) was used as a host matrix to encapsulate MP8. The encapsulation led to an increased catalytic activity under conditions (acidic conditions, high concentration of H2O2) detrimental to the catalytic activity of MP8, thereby demonstrating the protecting effect of MIL-101(Cr) matrix. The biocatalyst was also efficiently recycled. The selectivity of MP8 for the degradation of the harmful negatively charged organic dye methyl orange was also enhanced, thanks to the charged-based selective adsorption of the dye in MIL-101(Cr) porosity. A second part of the work was devoted to the use of functionalized MIL-101(Cr) analogs. First, functionalized ligands (bearing –NH2 and –SO3H groups) were used, and their influence on MP8 encapsulation was evaluated. The catalytic activity toward sulfoxidation reactions was also studied. The successful encapsulation of MP8 was strongly dependent on charge matching between the enzyme and the MOFs particles, while its catalytic activity was affected by the specific microenvironment of the pores. The MOF frameworks also modified the reactivity of MP8 toward different thioanisole derivatives. Then, a mixed metal MOF (MIL-101(Cr/Fe)), selected for its stable catalytic properties, was synthesized and characterized. Finally, the last part was devoted to the in-situ synthesis of MOFs (microporous MIL-53(Al)-FA) in presence of biomolecules (BSA) under compatible conditions with the preservation of the protein’s quaternary structure (aqueous media and room temperature). The resulting hybrid materials were thoroughly characterized and presented high loadings of BSA. A preliminarily study was performed with the enzyme, Horseradish Peroxidase, which retained its catalytic activity after immobilization
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Mallin, Hendrik [Verfasser]. "Development of Enzymes for Biocatalytic Applications : Protein Engineering, Immobilization and Reactor Concepts / Hendrik Mallin." Greifswald : Universitätsbibliothek Greifswald, 2014. http://d-nb.info/1060815486/34.
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Ménard, Amélie. "P450 enzymes in biocatalysis: exploration of chemical auxiliaries, macromolecular crowding, bioconjugation and oriented-immobilization." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114213.
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Cytochrome P450 enzymes (CYPs or P450s) form a ubiquitous family of heme-dependent monooxygenases known mainly for their role in xenobiotic metabolism and their remarkable ability to regio- and stereoselectively oxidize inactivated C-H bonds, a feat that is difficult to achieve by chemical methods. Unfortunately, our ability to study and exploit these enzymes as in vitro biocatalysts has been limited by their low activity, low stability and poor product predictability. This thesis focuses on the study of human drug metabolizing P450 isoforms, namely CYP2E1, CYP3A4 and CYP2D6 because of their exceptional ability to accept a large variety of substrates. In Chapter 2, we demonstrate the utility of "type II ligands" as chemical auxiliaries for biocatalysis with human CYP2E1. We show that linking the chemical auxiliary nicotinate to a variety of short hydrocarbon substrates can promote their oxidation with predictable regioselectivity at the secondary aliphatic or alkenyl C-H bond furthest from the auxiliary. The origin of this selectivity was rationalized through docking studies of our auxiliary-substrate compounds with reported X-ray crystals structures of CYP2E1. These results not only confirm the general utility of the chemical auxiliary approach pioneered by our lab to direct the predictable oxidation of inactivated C-H bonds by P450 enzymes, but also provide a system with complementary regioselectivity.A short study of the effects of macromolecular crowding on the activity of human CYP3A4 and CYP2D6 is presented in Chapter 3. We found that certain crowding agents were not detrimental to enzyme activity while others had a negative effect. Moreover, certain conditions (initially tested as controls) that improved enzymatic activity were uncovered. In Chapter 4, the non-covalent oriented-immobilization of CYP3A4 via its C-terminal histidine-tag is described. We show that immobilization on Ni-NTA agarose resin via this strategy has no detrimental effect on enzyme activity or stability. The lyoprotectant properties of Ni-NTA were also investigated. In Chapter 5, we designed and characterized a mutant of CYP3A4 that retains its enzymatic activity upon modification with a variety of fluorescent maleimide dyes via a single cysteine residue on its surface, namely C64. We also show that the activity of this mutant is preserved upon immobilization onto solid supports via this same cysteine residue. Finally, results of a preliminary feasibility study towards applying this immobilization strategy to eventual single-molecule fluorescence microscopy studies are presented.<br>Les enzymes cytochrome P450 (CYPs ou P450) forment une famille omniprésente de mono-oxygénases possédant un noyau hème au site-actif. Ces enzymes sont surtout connues pour leur rôle dans le métabolisme de produits pharmaceutiques et pour leur capacité remarquable à oxyder les liens C-H non-activés de façon régio- et stéréosélective. Malheureusement, notre capacité d'étudier et d'utiliser ces enzymes comme biocatalyseurs in vitro est limitée par leur faible activité, instabilité et une incapacité des connaissances actuelles à prédire leurs produits.Dans le chapitre 2, nous démontrons l'utilité des ligands de type II comme auxiliaires chimiques pour la biocatalyse avec CYP2E1 humaine. Nous démontrons que l'auxiliaire chimique nicotinate, lorsque lié à une variété d'hydrocarbures courts, peux promouvoir leur oxydation avec une régiosélectivité prévisible pour le lien C-H secondaire aliphatique ou alcényle le plus éloigné de l'auxiliaire. L'origine de cette sélectivité a été rationalisée à l'aide de «docking» moléculaire de nos composés auxiliaire-substrats à l'intérieure de structures cristallines de CYP2E1 publiées par d'autres chercheurs. L'utilité d'auxiliaires chimiques pour contrôler la régiosélectivité des enzymes P450 avait déjà été démontrée par notre groupe de recherche. Les résultats présentés dans ce chapitre offrent non seulement une confirmation du potentiel de cette stratégie, mais aussi un système complémentaire pour l'oxydation prévisible de liens C-H non-activés par les enzymes P450. Ces résultats confirment également la généralité de l'approche mis au point dans notre laboratoire qui décrit l'utilisation d'une auxiliaire chimique pour diriger l'oxydation prévisible de liens C-H non-activés par les enzymes P450.Une étude des effets de l'encombrement macromoléculaire sur l'activité enzymatique des CYP3A4 et CYP2D6 humaines est présentée dans le chapitre 3. Nous avons trouvé que leur activité demeure inchangée par la présence de certains agents encombrants alors que d'autres ont un effet négatif. De plus, certaines conditions (testées initialement comme contrôle) qui améliorent l'activité enzymatique ont été découvertes.Dans le chapitre 4, l'immobilisation orienté non-covalente de CYP3A4 par son étiquette de type his-tag C-terminale est décrite. Nous démontrons que son immobilisation sur une résine Ni-NTA à base d'agarose via cette stratégie n'a aucun effet négatif sur l'activité ou la stabilité de l'enzyme. Les propriétés lyoprotectrices de cette résine ont aussi été investiguées.Dans le chapitre 5, nous concevons et caractérisons un mutant actif de CYP3A4 lors de modifications avec une variété de maléimides fluorescentes à l'endroit d'un unique résidu cystéinique à sa surface, soit le C64. Nous démontrons aussi que ce mutant préserve son activité lorsqu'immobilisé sur des supports solides par ce même résidu cystéinique. Finalement, les résultats d'études préliminaires sont présentés qui envisagent l'application de cette stratégie d'immobilisation envers des études éventuelles de spectroscopie de fluorescence à la résolution d'une seule molécule.
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Schafhauser, Doris Yvonne Carleton University Dissertation Biology. "Chicken bone as a support matrix for the immobilization of some carbohydrate degrading enzymes." Ottawa, 1990.
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Fischback, Michael Bryant. "Introduction and characterization of an innovative biofuel cell platform with improved stability through novel enzyme immobilization techniques." Online access for everyone, 2006. http://www.dissertations.wsu.edu/Thesis/Fall2006/m_fischback_121206.pdf.
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Kurt, Elif [Verfasser], Wilhelm [Akademischer Betreuer] Schwieger, and Martin [Gutachter] Hartmann. "Immobilization of Enzymes on Hierarchically-Structured Supports / Elif Kurt ; Gutachter: Martin Hartmann ; Betreuer: Wilhelm Schwieger." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2020. http://d-nb.info/121224267X/34.
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Zichová, Miroslava. "Příprava mikrobiálních metabolitů z odpadních surovin." Doctoral thesis, Vysoké učení technické v Brně. Fakulta chemická, 2017. http://www.nusl.cz/ntk/nusl-295715.
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In this thesis the use of waste materials for the microbial production of important metabolites is reported. The first part is focused on the use of waste paper (a lignocellulosic material) as a non-traditional source for the production of bioethanol. The second part is focused on the immobilization of cellulolytic enzymes, which are used for the hydrolysis of lignocellulosic materials. First, the waste paper (cardboard) was pre-treated using a blender and a vibratory mill. The pre-treated cardboard was used for the production of ethanol by the method of simultaneous saccharification and fermentation. This method was optimized with free cells of Saccharomyces cerevisiae. Then strains suitable for the immobilization were selected. Strains of S. cerevisiae and Pichia kudriavzevii were immobilized by encapsulation into the polyvinyl alcohol carrier and tested again for the ethanol production by simultaneous saccharification and fermentation. In the second part of the work a carrier from waste polyethylene terephthalate bottles was prepared and used for the immobilization of the cellulolytic complex. The basic characteristics were determined, such as optimal pH and optimal temperature, storage, operational and thermal stability, enzyme kinetics and the mode of action of the enzyme. Compared to two other commercial carriers this carrier showed to be suitable for the immobilization of the cellulolytic complex.
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WANG, CUI. "Enhanced Activity And Stability Of Enzymes Associated With Delayed Fruit Ripening In Rhodococcus rhodochrous DAP 96253." Digital Archive @ GSU, 2013. http://digitalarchive.gsu.edu/biology_diss/131.
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Rhodococcus has diverse metabolic capabilities, such as delaying ripening of certain climacteric fruit. Nitrile hydratase (NHase), amidase, 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase), cyanidase, and β-cyanoalanine synthase-like enzyme (βCAS-like) are possibly involved in fruit ripening. The activity of these enzymes in Rhodococcus rhodochrous DAP 96253 cells were induced with selected multiple inducers (i.e. cobalt and urea).
This research showed that the supplementation of selected sugars, i.e. trehalose and maltodextrin in growth media and storage buffers of R. rhodochrous DAP 96253 affected activity and stability of the enzymes mentioned above. Thermostability and osmostability of the five enzymes in whole cells (plate grown and fermented) were evaluated in this study, i.e. βCAS-like was more stable than the other four enzymes in storage conditions.
Immobilized biocatalysts have practical advantages over the use of “free” whole cells. Immobilization of whole rhodococcal cells (plate grown and fermented) was employed, using techniques such as glutaraldehyde-polyethylenimine (GA-PEI) cross-linking, waxing and calcium-alginate entrapment. The GA-PEI immobilized catalysts were non-replicating and more stable in storage conditions than the catalysts produced by the other two methods. Wax or calcium-alginate immobilized catalysts (live catalysts) showed higher enzyme activity than the GA-PEI catalyst.
The effects of whole and immobilized catalysts were evaluated on delayed ripening of fruit. Both free whole cells and immobilized catalysts delayed the ripening of bananas and peaches. Delayed ripening experiments showed that the catalysts were effective in direct contact and not in contact with fruit. Moreover, both free whole cells and immobilized catalysts showed antifungal activity against Aspergillus niger and Penicillium spp.
Gas chromatography was performed to analyze volatile interactions between the biocatalysts and fruit. This analysis revealed that cyanide in an atmosphere with ethylene was utilized by the biocatalysts. There was also less volatile production by exposed fruit (bananas) than fruit unexposed to biocatalysts, either rhodococcal immobilized catalysts or live whole cells (plate grown and fermented).
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Kiralp, Senem. "Synthesis Of Conducting Block Copolymers And Their Use In The Immobilization Of Invertase And Polyphenol Oxidase Enzymes." Phd thesis, METU, 2004. http://etd.lib.metu.edu.tr/upload/12604912/index.pdf.
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A new thiophene derivative containing menthyl group (MM) was synthesized and polymerized via chemical and electrochemical methods. Polymers obtained and MM itself were used to synthesize copolymers with pyrrole under conditions of constant potential electrolysis. Cyclic Voltammetry, thermal analysis and scanning electron microscopy analyses were performed for the characterization of samples.
Immobilization of invertase and polyphenol oxidase enzymes was performed in the matrices obtained via copolymerization of MM with pyrrole. Immobilization was carried out via entrapment of enzyme in matrices during the polymerization of pyrrole. Temperature optimization, operational stability and shelf-life of the enzyme electrodes were investigated. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined.
It is known that wine includes phenolic groups that give astringency in high concentrations. Polyphenol oxidase (PPO) converts mono and diphenols to quinone. By analyzing the product, one can find out the amount of phenolic groups. By using obtained enzyme electrodes via immobilization of PPO, amount of phenolics in different wines were analyzed.
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Dey, Pradip [Verfasser]. "Polyglycerol Based Hydrogels for the Immobilization of Catalytically Active Enzymes and as Scaffolds for Cells / Pradip Dey." Berlin : Freie Universität Berlin, 2015. http://d-nb.info/1079525408/34.
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Morris, R. A. "The preparation and use of graft copolymers as supports for immobilization of enzymes, antibodies and affinity absorbants." Thesis, Leeds Beckett University, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.372845.
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Malmstadt, Noah. "Temperature-dependant [sic] smart bead adhesion : a versatile platform for biomolecular immobilization in microfluidic devices /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/8019.
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Jung, Dirk [Verfasser], and Martin [Akademischer Betreuer] Hartmann. "Immobilization of Enzymes on mesoporous Supports and their Application in continuous-flow Biotransformations / Dirk Jung. Betreuer: Martin Hartmann." Erlangen : Universitätsbibliothek der Universität Erlangen-Nürnberg, 2012. http://d-nb.info/1028392664/34.
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Parra, Guardado Ana Luisa. "Dégradation enzymatique de micropolluants récalcitrants d'origine pharmaceutique." Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTG013/document.
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Ce travail concerne l'étude de la dégradation enzymatique de micropolluants pharmaceutiques récalcitrants présents dans l'eau. Tout d’abord, les efficacités de trois laccases différentes issues respectivement de : Pycnoporus sanguineus CS43, Trametes versicolor (Tv) et Myceliophtora thermophila ont été comparés lors d’essais de dépollution de solutions modèles renfermant trois antibiotiques (amoxicilline, ciprofloxacine et sulfaméthoxazole) et un antiépileptique (carbamazépine). Les essais ont été réalisés avec les laccases libres en présence ou non de médiateurs redox. L'impact de plusieurs paramètres opératoires sur les performances des enzymes a également été étudié. Puis, une nouvelle méthode d’immobilisation des laccases impliquant l’activation du support (microparticules à base de silice commerciales) par du glutaraldéhyde en phase vapeur a été mise au point et optimisée en utilisant la méthodologie de plans d’expériences. Après immobilisation, la laccase Tv s’est avérée être la plus active. Des essais de dégradation en présence de médiateurs redox ont confirmé l’efficacité de l’enzyme immobilisée et sa possible réutilisation lors de cycles successifs. La toxicité des solutions après traitement a été évaluée par des tests Microtox®. La laccase Tv a également été immobilisée sur des nanoparticules non commerciales à base de silice ou d’argile ainsi que sur des composites à base de silice et d’argile. La laccase Tv immobilisée sur les supports composites riches en silice a montré une plus grande réactivité et de meilleures performances pour l'élimination des composés cibles<br>This work is focused on the study of the enzymatic depletion of recalcitrant pharmaceutical micropollutants in water. The potential degradation of three antibiotics (amoxicillin, ciprofloxacin and sulfamethoxazole) and one anti-epileptic (carbamazepine) was studied with three laccases: Pycnoporus sanguineus CS43, Trametes versicolor (Tv) and Myceliophtora thermophila. Free laccase systems were evaluated for pharmaceuticals depletion on model solutions in the presence or absence of redox mediators and the impact of several parameters on the performance of laccases for degradation were studied. The enzymes were then immobilized on different solid supports: commercial silica, laboratory synthetized nano-silica and clay based composite nanomaterials and used for degradation tests. A novel methodology for the covalent binding of laccases onto carriers was developed by using glutaraldehyde in vapour phase and the best immobilization conditions were determined through a 23 full factorial design. The immobilized Tv shown the highest activity and was tested in presence of redox mediators. Moreover, the reusability was evaluated in several degradation cycles and the toxicity of the solutions after treatment was assessed with the Microtox® test. In comparison to laccase immobilized on commercial silica, the Tv supported on laboratory synthetized materials showed higher activity and a better performance for the removal of target compounds
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Hitaishi, Vivek Pratap. "Enzymatic oxygen electroreduction : from molecular basis of effective enzymes immobilization on planar electrodes to the electrocatalysis on nanostructured electrodes." Electronic Thesis or Diss., Aix-Marseille, 2020. http://theses.univ-amu.fr.lama.univ-amu.fr/200121_HITAISHI_431ad491kqfawo954pehyij849p_TH.pdf.
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L’immobilisation fonctionnelle des enzymes redox sur un support solide conducteur, en termes de densités de courant catalytique et de stabilité, est l’un des défis les plus importants à relever avant la commercialisation de systèmes tels que les piles à combustible enzymatiques et les biocapteurs. Cette thèse vise à la compréhension des facteurs moléculaires qui contrôlent le processus de transfert d'électrons interfacial et l'efficacité catalytique des enzymes redox immobilisées, en considérant l'effet de la quantité, de l'orientation et de la conformation des biomolécules à l’interface électrochimique. L'objectif ultime est d'obtenir une rationalisation des bioélectrodes. Des enzymes redox d’origine et de propriétés différentes, et des surfaces d’électrodes de chimie et structuration variées sont explorées pour moduler la connexion électrique de l’enzyme, et donc le processus de transfert d'électrons. Les propriétés requises de surface de l'électrode en tant que matrice hôte pour les enzymes sont tout d’abord déterminées sur électrodes planes puis étendues à des électrodes nanostructurées. Afin de déduire un modèle d'adsorption, un couplage sans précédent de l'électrochimie à des techniques de surface telles que la résonance plasmonique de surface (SPR), la spectroscopie d'absorption par réflectance infrarouge à modulation de polarisation (PMIRRAS) et l'ellipsométrie. Enfin, de nouvelles électrodes nanostructurées obtenues par ablation au laser sont explorées comme de nouvelles plates-formes stables capables de contrôler le taux de recouvrement surfacique enzymatique, ouvrant la voie à l'électrochimie d'une molécule unique<br>Functional immobilization of redox enzymes on conductive solid support, which must result in high current densities and operation stabilities, is one of the most significant challenge before the commercialization of biodevices like biofuel cells and biosensors. In order to get and to maintain the enzymatic activity in the immobilized state, this thesis aims to develop the molecular understanding that controls the efficiency of immobilized redox enzymes while considering the effect of loading, orientation and conformation as a function of various parameters like pH, electric field, ionic strength and covalent connections. The overall goal is to get a full rationalization of bioelectrodes.In this thesis, varieties of combinations that include properties of redox enzymes and/or electrode surfaces are explored to optimize immobilization, electrical wiring and thereby the ET process and its viability for bioelectronics. In order to deduce an adsorption model based on electrostatic interaction, an unprecedented coupling of electrochemistry to surface sensitive techniques like surface plasmon resonance (SPR), Polarization Modulation Infrared Reflectance Absorption Spectroscopy (PMIRRAS) and Ellipsometry is discussed which will ultimately help in understanding the combined effect of loading, orientation, and conformation on bioelectrocatalysis. Additionally, role of nanomaterials for the optimization of bioelectrocatalytic process is also explored. The desired properties of the electrode surface as a host matrix for enzymes are put forward accordingly while comparing the merit of planar and nanostructured electrodes
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Rocha, Caroline Oliveira da [UNESP]. "Nanopartículas magnéticas como suporte para imobilização de lipases." Universidade Estadual Paulista (UNESP), 2016. http://hdl.handle.net/11449/137809.
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Previous issue date: 2016-03-14<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)<br>As enzimas são catalisadores de alto custo, sendo necessário a imobilização para que haja a recuperação e a reutilização tornando o processo viável economicamente. Além disso, a utilização de enzimas imobilizadas permite simplificar o modelo de reatores e o controle da reação. Assim, a imobilização é geralmente um requisito para a utilização de enzimas como biocatalisadores industriais. A escolha do suporte para imobilização depende das propriedades da enzima a ser imobilizada. Suportes sólidos podem interagir com a enzima por diferentes vias: por adsorção, ligação covalente ou encapsulamento. Um importante fator para imobilizar a enzima é que o suporte deve ser inerte e biocompatível ao ambiente, ou seja, não deve interferir na estrutura nativa da proteína e nem comprometer sua atividade biológica. Dentre as principais enzimas, as lipases hidrolisam triglicerídeos (TAG) em glicerol e ácidos graxos e por este motivo estão na classe das hidrolases. Uma proposta de imobilização destas enzimas consiste na utilização de nanoestruturas magnéticas como biocatalisadores da reação de transesterificação para a produção de biodiesel, devido à facilidade e a rápida separação das enzimas imobilizadas, a partir da mistura reacional, usando um campo magnético externo. As vantagens das enzimas imobilizadas em relação às enzimas livres surgem da sua maior estabilidade e facilidade de separação, o que acarreta economia significativa no custo global do processo, desde que o procedimento de imobilização não seja muito caro, haja boa recuperação da atividade enzimática e que a estabilidade operacional da enzima imobilizada seja suficientemente longa. O uso de enzimas imobilizadas permite a retenção do biocatalisador no reator; elevada concentração de catalisador no reator permitindo intensificar o processo; controle do microambiente da enzima; facilidade de recuperação e reutilização do catalisador, o que reduz os custos das enzimas; possibilidade de ser utilizado em sistemas contínuos. Utilizando a técnica de difração de raios X foi possível confirmar a fase magnetita nas sínteses propostas: método de coprecipitação e em meio orgânico. A funcionalização da superfície da NP e SP com APTS, foi comprovado por espectroscopia na região de infravermelho, apresentando bandas de –NH2. A técnica de DLS comprovou a funcionalização, pelo aumento dos diâmetros hidrodinâmicos das amostras NP-APTS e SP-APTS comparada a NP e SP. O ponto isoelétrico das amostras SP e SP-APTS apresentou aumento de 2,33 para 6,44. O derivado imobilizado apresentou bandas típicas de amidas. As lipases imobilizadas apresentaram diâmetros hidrodinâmicos maiores que NP-APTS e SP-APTS. Os resultados da atividade hidrolítica das enzimas suportadas foram satisfatórios, sendo que SP-APTS-LPP apresentou maior atividade. Pela análise termogravimétrica comprovou-se rendimento de imobilização de 22,86%. Determinou-se o pH ótimo da lipase imobilizada que mostrou maior atividade em pH 8 enquanto a LPP livre em pH 6,5. As medidas de temperatura ótima demostrou que o derivado imobilizado possui maior atividade que a LPP livre a 50 °C, favorecendo a utilização deste suporte em processos industriais de biodiesel que opera em altas temperaturas. Neste contexto, a síntese de suportes magnéticos porosos e a imobilização de lipases com este suporte, apresentou excelentes resultados para a aplicação em biocatálise na reação de transesterificação para a síntese de biodiesel.<br>Enzymes are expensive catalysts, immobilization is necessary for recovery and reuse making the process economically viable. Furthermore, use of immobilized enzymes can simplify model reactor and control reaction. Thus, immobilization is generally a requirement for use of the enzyme as an industrial biocatalyst. The choice of support for biocatalysts immobilization depends on properties of enzyme to be immobilized. Solid supports can interact with enzyme in different ways: by adsorption, covalent bonding or encapsulation. An important factor to immobilize the enzyme is that support must be inert and biocompatible to environment; it should not interfere in native structure of protein and not compromising their biological activity. The main enzymes, lipases hydrolyze triglycerides (TAG) to glycerol and fatty acids and for this reason; they are in the class of hydrolases. These enzymes are carboxylesterases that catalyze hydrolysis in glycerides synthesis. A proposal for immobilization of these enzymes is use of magnetic nanostructures in biocatalysts transesterification reaction for producing biodiesel due to ease and rapid separation of immobilized enzyme, from a mixture reaction using an external magnetic field. The benefits of immobilized enzymes compared to free enzymes arise from their greater stability and ease of separation, which leads to significant savings in the overall cost of the process, provided that immobilization procedure is not very expensive, there is good recovery of enzyme activity, and operational stability of immobilized enzyme is sufficiently long. The use of immobilized enzymes allow retention of biocatalyst in reactor; high concentration of catalyst in reactor to intensify the process; control of microenvironment of enzyme; ease of recovery and reuse of catalyst, which reduces the costs of enzymes; possibility of being used in continuous systems. Using the technique of X-ray diffraction was confirmed magnetite phase in syntheses proposed: co-precipitation method and organic solvent. The functionalization of surface NP and SP with APTS confirmed by spectroscopy in infrared region, with bands of -NH2. The DLS technique proved the functionalization, the increase of hydrodynamic diameters NP-APTS samples and SP-APTS compared to NP and SP. The isoelectric point of SP and SP-APTS samples increased by 2.33 to 6.44. The immobilized derivative showed typical bands of amides. Immobilized lipases showed higher hydrodynamic diameters NP-APTS and SP-APTS. The results of hydrolytic activity of supported enzymes were satisfactory and SP-APTS-LPP showed higher activity. By thermogravimetric analysis, it was shown immobilization yield 22.86%. It was determined the pH optimum of immobilized lipase showed highest activity at pH 8 while the LPP free at pH 6.5. The optimum temperature measurements demonstrated that immobilized derivative is more active than free LPP at 50 ° C, favoring the use of support in industrial processes of biodiesel, which operates at high temperatures. In this context, the synthesis of porous magnetic support and immobilization of lipases showed excellent results for use in biocatalysis in transesterification reaction for biodiesel synthesis.
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Bahrami, Atieh. "Immobilization of cytochrome P450 BM3 from Bacillus megaterium on magnetic nanoparticles to develop an effective biocatalyst for hydroxylation reactions." Doctoral thesis, Université Laval, 2018. http://hdl.handle.net/20.500.11794/34494.
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Les catalyseurs chimiques sont utilisés dans différents procédés de synthèse. Cependant, la pollution qu'ils causent sur l'environnement n’est pas prise en considération. Les procédés de synthèse chimique nécessitent généralement un grand volume de solvants organiques, produisant d’énormes quantités de déchets chimiques, souvent toxiques et non dégradables. Le remplacement des catalyseurs chimiques par des biocatalyseurs (enzymes) pourrait donc bénéficier de leur nature écologique et de leur grande sélectivité envers les produits désirés. Néanmoins, la faible activité et stabilité des enzymes ainsi que leurs coûts élevés sont des obstacles majeurs au développement des systèmes enzymatiques. Par conséquent, des études axées sur le développement de systèmes biocatalytiques plus actifs, stables et rentables, pouvant ouvrir les portes vers un environnement plus vert, sont très souhaitables. Parmi les enzymes qui catalysent des réactions d’importance dans de nombreux procédés de synthèse, le cytochrome P450 BM3 issu de Bacillus megaterium fait l'objet de cette thèse. L'enzyme est capable d’hydroxyler les liaisons C–H des acides gras (C₁₂-C₂) à température ambiante et pH physiologique. Pour cette réaction, BM3 n'a besoin que d’oxygène et de deux électrons habituellement obtenus de son cofacteur naturel, le NADPH. Cependant, pour engager cette enzyme dans les réactions d'hydroxylation, quelques obstacles importants doivent être surmontés : (i) le cofacteur coûteux (NADPH), devrait être remplacé par une source d'électrons moins chère ou régénérée, (ii) la stabilité enzymatique devrait être améliorée et (iii) l'enzyme devrait être facilement récupérable du milieu de réaction pour être réutilisée. Dans ce contexte, cette étude propose pour la première fois l'immobilisation d'un BM3 sur des nanoparticules magnétiques (NMP) d’oxyde de fer. Ce système enzymatique bénéficie (i) de la préférence de l'enzyme pour les cofacteurs NADH et BNAH (moins chers que le NADPH), (ii) de la réutilisation facile du biocatalyseur et (iii) d’une stabilité significative de l’enzyme lors du stockage. Les NMP synthétisées ont été fonctionnalisées pour permettre l’immobilisation de l'enzyme par adsorption ou liaison covalente. Par conséquent, les BM3-NMP adsorbées / réticulées ou liées de façon covalente ont été obtenues en immobilisant P450 BM3 (R966D / W1046S) sur Ni²⁺-PMIDA-NMP ou sur des NMP activés par glutaraldéhyde, respectivement.<br>L'activité de l’enzyme immobilisée a été comparée avec celle de l’enzyme libre dans la réaction d'hydroxylation du 10-pNCA comme substrat modèle. L'acide myristique a également été utilisé comme substrat modèle pour confirmer la capacité d’hydroxylation sélective de l’enzyme sur les atomes de carbone ω-1, -2 ou -3. Pour les mêmes conditions opératoires, le BM3 adsorbé / réticulé a montré plus de 85% de l'activité de l’enzyme libre, alors que pour les BM3-NMP liées de manière covalente cela représente 60%. La séparation facile des NMP du milieu réactionnel à l’aide d’un aimant a permis de réutiliser le système enzymatique cinq fois consécutives. Après 5 cycles de réaction, l'enzyme réticulée a conservé 100% de son activité initiale. Compte tenu que le recyclage de l’enzyme libre n’est pas faisable, ce résultat est d’une importance considérable dans les applications pratiques. De plus, la stabilité de l’enzyme pendant un mois de stockage à 4 ºC a été évaluée pour chaque système de BM3. Les résultats ont montré que l’enzyme libre n’était plus active après seulement une semaine de stockage dans ces conditions. L'enzyme réticulée n'a montré qu'une activité relative de 41% après un mois de stockage, mais pour le BM3 fixée de façon covalente, la valeur correspondante a été de 80%. La cinétique de l'hydroxylation du 10-pNCA en présence de l’enzyme libre ou immobilisée a été également étudiée. Sur la base des données expérimentales, un modèle de Hill (coefficient de Hill égal à 2) a été obtenu pour l'enzyme libre. Il a été démontré que les mêmes paramètres cinétiques sont capables de prédire le comportement du système BM3-adsorbé et BM3-réticulé dans la réaction d’hydroxylation, étant donné sa similarité avec celui de l’enzyme libre. En conclusion, les résultats de cette thèse ont montré qu'un système enzymatique actif, stable et rentable peut être obtenu en immobilisant le BM3 sur des NMP fonctionnalisées. Il bénéficie autant des avantages de l'enzyme que du support. Ainsi, l'immobilisation sur des NMP d’une enzyme spécialement conçue pour remplacer le couteux NADPH par des cofacteurs moins chers mais efficaces (NADH et BNAH) offre en même temps une amélioration significative de sa stabilité et facilite son recyclage.<br>MNPs have been synthesized and surface functionalized to attach the enzyme via two different methods, adsorption and covalent binding. Moreover, glutaraldehyde was used to treat the adsorbed enzyme molecules on MNPs (crosslinking-adsorption). Therefore, adsorbed, crosslinked-adsorbed, or covalently bound BM3-MNPs were obtained by immobilizing P450 BM3 on synthesized Ni²⁺-functionalized MNPs or glutaraldehyde pre-activated MNPs, respectively. The immobilized enzyme activity was compared to its free counterpart in hydroxylation reaction of 10-pNCA (10-(4-Nitrophenoxy) decanoic acid) as a substrate model. Myristic acid was also used as a substrate model to confirm the enzyme selective hydroxylation at ω-1, -2, or -3 carbon positions. The effect of cofactor (NADH and its analogue, BNAH) on the enzyme activity was also investigated. The adsorbed/crosslinked-adsorbed BM3 showed more than 85% of the free enzyme activity while the covalently bound BM3-MNPs presented 60% of the free enzyme activity under the same reaction conditions. An important feature of BM3-MNPs system is the possibility of recycling the biocatalyst. Facile separation of the magnetic nanoparticles from the reaction medium by applying a magnet provided the opportunity of reusing the enzymatic system for five times. After 5 cycles of reaction, the crosslinked-adsorbed enzyme retained 100% of its initial activity. Although the covalently bound enzyme showed, only half of the crosslinked-adsorbed enzyme activity, its storage stability was more significant. Taking into account that the enzyme reuse is an essential concern in many large-scale applications and the free BM3 cannot be recovered and reused, this result is noteworthy. Storage stability tests revealed that the free enzyme became inactive after one-week while the crosslinked-adsorbed enzyme and the covalently attached BM3 on MNPs showed 41% and 80% relative activity after one month, respectively. Finally, the steady-state kinetics of 10-pNCA hydroxylation by free and immobilized BM3 was investigated. Based on the experimental data, a non-Michaelis-Menten, Hill model (Hill coefficient of 2) was obtained for the free enzyme which could also predict the adsorbed and crosslinked-adsorbed BM3-MNPs system performance. This sigmoidal behavior was found to be independent of enzyme concentration and type of cofactor. However, since the enzyme activity was only 60% of the free enzyme for covalently bound BM3, further studies are necessary for a better understanding of this system. In summary, the results of this thesis show that an active, stable, and cost-effective BM3-MNPs system can be obtained by immobilizing an engineered BM3 on functionalized MNPs. Such systems benefit from the advantages of both enzyme and support. An engineered enzyme can fulfill the desired targets including the replacement of costly NADPH by less-expensive, yet effective cofactors namely NADH and BNAH. Furthermore, immobilization of this enzyme on MNPs improves its stability and facilitates the recycling process.<br>Chemical catalysts are used in different synthetic processes from lab to industrial scales. High reaction yields usually achieved by this type of processes favor their application in many industries without considering the pollution they cause to the environment. Chemical synthesis processes usually require a high volume of organic solvents and produce tons of chemical wastes which are often toxic and not degradable. Replacing conventional catalysts by biocatalysts (enzymes) can benefit from their environmentally friendly nature and high selectivity toward the desired products. Although the advantages of biocatalysts over chemical catalysts have been proven, the application of enzymes in an industrial level is still not considerable. The enzyme low activity, stability, and high cost are the main concerns in developing large-scale enzymatic systems. Therefore, in the context of a greener environment, studies focusing on the development of more active, stable, and cost-effective enzymatic systems are in great demand. Among several enzymes that can catalyze essential synthesis reactions, cytochrome P450 BM3 from Bacillus megaterium is the subject of this thesis. This enzyme hydroxylates the saturated and unsaturated C–H bonds of medium to long chain fatty acids at room temperature and physiological pH. For this reaction, BM3 only needs molecular oxygen and two electrons usually obtained from its natural cofactor, NADPH. However, to engage this enzyme in hydroxylation reactions, some important obstacles should be overcome: (i) the costly cofactor (NADPH) should be replaced by a cheaper source of electrons or regenerated, (ii) the enzyme stability should be improved, and (iii) the enzyme should be easily recovered from the reaction medium to be reused. In this context, this study proposes for the first time the immobilization of an optimized BM3 mutant on functionalized iron oxide magnetic nanoparticles (MNPs). This enzymatic system benefits from (i) the enzyme preference towards cofactors like the reasonably priced NADH and the very cheap BNAH, (ii) facile recovery and reuse of the biocatalyst (enzyme-MNPs), and (iii) the enzyme significant storage stability.
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Boyukbayram, Ayse Elif. "Immobilization Of Invertase, Polyphenol Oxidase And Glucose Oxidase In Conducting Copolymers Of Thiophene-capped Polytetrahydrofuran And Pyrrole." Phd thesis, METU, 2005. http://etd.lib.metu.edu.tr/upload/12605935/index.pdf.
Full textAbstract:
ABSTRACT
IMMOBILIZATION OF INVERTASE, POLYPHENOL OXIDASE AND GLUCOSE OXIDASE IN CONDUCTING COPOLYMERS OF
THIOPHENE-CAPPED POLYTETRAHYDROFURAN AND PYRROLE
Bö<br>yü<br>kbayram, AySe Elif
Ph.D., Department of Chemistry
Supervisor: Prof. Dr. Levent Toppare
January 2005, 123 pages
Immobilization of invertase, polyphenol oxidase (PPO) and glucose oxidase (GOD) enzymes were performed in electrochemically synthesized two types of conducting copolymers. One end and two end thiophene-capped polytetrahydrofuran (TPTHF-1 and TPTHF-2) were copolymerized with pyrrole under conditions of constant potential electrolysis. The copolymers were characterized by thermal, spectroscopic and scanning electron microscopy analyses.
Immobilization was carried out via entrapment of enzymes in two types of matrices during the copolymerization of pyrrole with the insulating polymers in the presence of sodium dodecyl sulphate (SDS). Kinetic parameters: Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for the enzyme electrodes. Temperature optimization, pH optimization, operational stability and shelf-life of the enzyme electrodes were investigated.
Enzyme electrodes of polyphenol oxidase and glucose oxidase were used to determine the amount of their substrates in samples. Polyphenol oxidase converts mono and diphenols to quinone. Amount of phenolic compounds in two kinds of wines were determined by analyzing the quinone amount. Glucose oxidase converts &<br>#61538<br>-D-glucose to D-glucono-1,5-lactone. Glucose amount was determined in two kind of factory-produced orange juices by analyzing D-glucono-1,5-lactone.
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Ekinci, Olcun. "Immobilization Of Glucose Oxidase And Polyphenol Oxidase In Conducting Copolymer Of Pyrrole Functionalized Polystyrene With Pyrrole." Master's thesis, METU, 2006. http://etd.lib.metu.edu.tr/upload/2/12607373/index.pdf.
Full textAbstract:
Electrochemical polymerization of pyrrole functionalized polystyrene (PStPy) with pyrrole was carried out in water-sodium dodecyl sulfate solvent-electrolyte couple. Characterization of the resulting copolymer was performed via Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and four probe conductivity measurements. Glucose oxidase and polyphenol oxidase enzymes were immobilized in polypyrrole (PPy) and conducting copolymer of pyrrole functionalized polystyrene with pyrrole (P(PStPy-co-Py). Resulting enzyme electrodes were characterized by kinetic parameters<br>Vmax and Km. Behavior of enzyme electrodes upon temperature and pH changes were investigated. Glucose oxidase electrode was used for the determination of glucose in orange juice and polyphenol oxidase electrode was used for the determination of polyphenolic compounds in red wine.
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Periyasamy, Karthik. "Production de bioéthanol à partir de biomasse lignocellulosique en utilisant des enzymes cellulolytiques immobilisées." Thesis, Université Grenoble Alpes (ComUE), 2018. http://www.theses.fr/2018GREAI024/document.
Full textAbstract:
L'objectif global de cette étude était de produire du bioéthanol à partir de biomasse lignocellulosique en utilisant des enzymes libres ou immobilisées de type xylanase, cellulase et β-1,3-glucanase. L'isolement de la souche AUKAR04 de Trichoderma citrinoviride a permis de produire par fermentation solide ces trois enzymes à un taux de 55 000, 385 et 695 UI / gd, respectivement. L’activité biochimique des enzymes libres a été caractérisée en faisant varier différents paramètres : pH, température et concentration en cations métalliques, et les paramètres cinétiques correspondants ont été identifiés. Par la suite, les enzymes ont été immobilisées en phase solide, soit sous forme d’agrégats sans support de type (combi-CLEA), soit par association avec des nanoparticules magnétiques bifonctionnalisées (ISN-CLEA). Ces dernières ont fourni de meilleures performances en termes de stabilité thermique, d’activité et d’aptitude à réutilisation après un temps de conservation prolongé. Le substrat végétal utilisé (SCB : bagasse de canne à sucre) a été prétraité chimiquement par cuisson à l'ammoniac, permettant d’éliminer 40% de la lignine initiale tout en préservant 95% de glucane, 65% de xylane et 41% d'arabinane. L’hydrolyse enzymatique du substrat prétraité a permis une conversion de la cellulose en 87% de glucose, et une conversion des hémicelluloses (arabinoxylanes) en 74% de xylose et 64% d'arabinose, chiffres notoirement supérieurs à l'activité des enzymes libres. L'analyse chimique et structurale du substrat a été faite par spectrométrie ATR-FTIR et DRX, et par analyse TGA. L’étude FTIR a prouvé l’efficacité du traitement enzymatique en montrant que les hémicelluloses et la cellulose subissent une dépolymérisation partielle par l’action simultanée des trois enzymes immobilisées dans les ISN-CLEA. L’étude TGA a montré que la stabilité thermique des échantillons prétraités à l'ammoniac puis traités par des enzymes est notoirement améliorée. L’analyse DRX a montré que l'indice de cristallinité du substrat prétraité à l’ammoniac puis traité par l'ISN-CLEA a augmenté de 61,3 ± 1%, par rapport au substrat avant traitement enzymatique. La fermentation par la levure Saccharomyces cerevisiae LGP2Y1 utilisée en monoculture, à partir d’un hydrolysat enzymatique contenant 103,8 g / L de glucose, a produit 42 g / L d'éthanol en 36 h de fermentation. Le rendement métabolique global atteint ainsi environ 79% du rendement théorique. La fermentation en co-culture avec Saccharomyces cerevisiae LGP2Y1 et Candida utilis ATCC 22023 d’un hydrolysat à 107,6 g / L de glucose et 41,5 g / L de xylose a produit 65 g / L d'éthanol en 42 h de fermentation. Ainsi, en co-culture fermentaire, le rendement métabolique global atteint environ 88 % du rendement théorique<br>The overall objective of the study was to produce bioethanol from lignocellulosic biomass by using free and immobilized xylanase, cellulase and β-1, 3-glucanase. Specifically, this study was focused on the isolation of Trichoderma citrinoviride strain AUKAR04 and it produces xylanase (55,000 IU/gds), Cellulase (385 IU/gds) and β-1, 3-glucanase (695 IU/gds) in solid state fermentation. Then the free enzymes were biochemically characterized such as effect of pH, temperature and metal ion concentration and kinetics parameters. Then the enzymes were subjected to two types of immobilization using carrier-free co-immobilization (combi-CLEAs) method and immobilized on bifunctionalized magnetic nanoparticles (ISN-CLEAs) with higher thermal stability, extended reusability and good storage stability. Liquid ammonia pretreatment removed 40% lignin from the biomass and retained 95% of glucan, 65% of xylan and 41% of arabinan in sugarcane bagasse (SCB). SCB was enzymatically hydrolyzed and converted to 87% glucose from cellulose and 74% of xylose, 64% of arabinose from the hemicelluloses which is remarkably higher than the activity of the free enzymes. Chemical and structural analysis of SCB was done by ATR-FTIR, TGA and XRD. FTIR result showed a successful pretreatment of the SCB raw material. It showed that hemicelluloses and cellulose are partially depolymerized by the action of xylanase, cellulase and β-1,3-glucanase in ISN-CLEAs. TGA studies showed that the thermal stability of the ammonia pretreated and enzymatically treated samples have improved remarkably. XRD results showed that the crystallinity index of the ISN-CLEAs treated SCB increased to 61.3±1% when compared to the ammonia-treated SCB. Mono-culture fermentation using Saccharomyces cerevisiae LGP2Y1 utilized SCB hydrolysate containing 103.8 g/L of glucose and produced 42 g/L ethanol in 36 h of fermentation. The overall metabolic yield achieved was about 79% of theoretical yield. Co-culture fermentation using Saccharomyces cerevisiae LGP2Y1 and Candida utilis ATCC 22023 utilized SCB hydrolysate containing 107.6 g/L of glucose and 41.5 g/L xylose and produced 65 g/L ethanol in 42 h of fermentation. The overall metabolic yield in co-culture fermentation achieved was about 88 % of the theoretical yield
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Cotignoli, Matteo. "Preparation and assessment of immobilized laccase-based micro- and nanocatalysts for the degradation of anthropogenic compounds." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2019.
Find full textAbstract:
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.
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Artigues, Cladera Margalida Esmeralda. "Estudio de biosensores electroquímicos basados en inmovilización enzimática." Doctoral thesis, Universitat Ramon Llull, 2019. http://hdl.handle.net/10803/667847.
Full textAbstract:
Els biosensors electroquímics són dispositius d'anàlisi que combinen l'especificitat de les reaccions bioquímiques amb la capacitat analítica de les tècniques electroquímiques. Gràcies a aquesta combinació, és possible determinar de forma ràpida, sensible i fiable diferents analits en mostres amb matrius complexes. Per tot això, l'ús de biosensors és una alternativa als mètodes clàssics d'anàlisi per a realitzar processos de control de qualitat en diferents sectors industrials.
En la present Tesi, s'han desenvolupat biosensors amperomètrics enzimàtics basats en la immobilització d’oxidases sobre una interfase electroquímica de nanotubs d'òxid de titani (IV) altament ordenats (TiO2NTAs). Per a això, s'han estudiat diferents processos d'immobilització d'enzims basats en captura polimèrica i en immobilització covalent, i s'han avaluat els paràmetres analítics dels biosensors desenvolupats.
En als processos de captura polimèrica, s'ha estudiat l'ús de kappa-carragenina, 2-hidroxietilmetacrilat (HEMA) i quitosà. Aquests hidrogels s'han utilitzat per a la immobilització de l'enzim glucosa oxidasa (GOx) i s'ha observat que tant el HEMA com el quitosà generen un microentorn favorable per a la conservació de l'activitat biològica de l'enzim.
banda Per a la immobilització covalent, s'ha utilitzat pentafluorofenilmetacrilat (PFM) generació amb l’objectiu de generar enllaços entre les molècules d'enzim i la superfície del transductor. Per a això, s'ha modificat la superfície de la interfase electroquímica TiO2NTAs mitjançant dues tècniques de plasma: polimerització de PFM i sembrat del mateix polímer. S'ha observat que la superfície polimeritzada de PFM (ppPFM) presenta major hidrofobicitat que la superfície en la qual s'ha realitzat el sembrat de PFM (pgPFM) i que això té influència en la conformació que adopten les molècules d'enzim. Mentre que en la superfície ppPFM predominen conformacions amb baixa activitat, en la superfície pgPFM la major part de la població de les molècules de GOx adopten conformacions amb activitat catalítica. Per aquests motius, els biosensors amb sembrat per plasma de PFM presenten major sensibilitat enfront de la presència de glucosa que els biosensors basats en la polimerització de PFM.
Finalment, s'han desenvolupat biosensors amperomètrics de glucosa i de glutamat amb matrius d'immobilització polimèriques i covalents: Ti/TiO2NTAs/GOx/Quitosà, Ti/TiO2NTAs/HEMA-co-EGDA/pgPFM/GOx/Quitosà i Ti/TiO2NTAs/GmOx/Quitosà. S'han realitzat determinacions sobre mostres alimentàries emprant aquests biosensors i els resultats s'han comparat amb els obtinguts amb tècniques de referència.<br>Los biosensores electroquímicos son dispositivos de análisis que combinan la especificidad de las reacciones bioquímicas con la capacidad analítica de las técnicas electroquímicas. Gracias a esta combinación, es posible determinar de forma rápida, sensible y fiable distintos analitos en muestras con matrices complejas. Por ello, el uso de biosensores es una alternativa a los métodos clásicos de análisis para realizar procesos de control de calidad en distintos sectores industriales.
En la presente Tesis, se han desarrollado biosensores amperométricos enzimáticos basados en la inmovilización de oxidasas sobre una interfase electroquímica de nanotubos de óxido de titanio (IV) altamente ordenados (TiO2NTAs). Para ello, se han estudiado diferentes procesos de inmovilización de enzimas basados en captura polimérica y en inmovilización covalente, y se han evaluado los parámetros analíticos de los biosensores desarrollados.
En los procesos de captura polimérica, se ha estudiado el uso de kappa-carragenina, 2-hidroxietilmetacrilato (HEMA) y quitosano. Estos hidrogeles se han utilizado para la inmovilización del enzima glucosa oxidasa (GOx) y se ha observado que tanto HEMA como quitosano generan un microentorno favorable para la conservación de la actividad del enzima.
Para la inmovilización covalente, se ha utilizado pentafluorofenilmetacrilato (PFM) con el objetivo de generar enlaces entre las moléculas de enzima y la superficie del transductor. Para ello, se ha modificado la superficie de la interfase electroquímica TiO2NTAs mediante dos técnicas de plasma: polimerización de PFM y sembrado del mismo polímero. Se ha observado que la superficie polimerizada de PFM (ppPFM) presenta mayor hidrofobicidad que la superficie en la que se ha realizado el sembrado de PFM (pgPFM) y que ello tiene influencia en la conformación que adoptan las moléculas de enzima. Mientras que en la superficie ppPFM predominan conformaciones con baja actividad, en la superficie pgPFM la mayor parte de la población de las moléculas de GOx adoptan conformaciones con actividad catalítica. Por estos motivos, los biosensores con sembrado por plasma de PFM presentan mayor sensibilidad frente a la presencia de glucosa que los biosensores basados en la polimerización de PFM.
Finalmente, se han desarrollado biosensores amperométricos de glucosa y de glutamato con matrices de inmovilización poliméricas y covalentes: Ti/TiO2NTAs/GOx/Quitosano, Ti/TiO2NTAs/HEMA-co-EGDA/pgPFM/GOx/Quitosano y Ti/TiO2NTAs/GmOx/Quitosano. Se han realizado determinaciones sobre muestras alimentarias empleando estos biosensores y los resultados se han comparado con los obtenidos con técnicas de referencia.<br>Electrochemical biosensors are analytical devices that combine the specificity of biochemical recognition processes with the analytical power of electrochemical techniques. Consequently, it is possible to perform rapid, sensitive and reliable determinations of different analytes present in complex samples. For this reason, the use of biosensors is an alternative to classical analytical methods to perform quality control processes in different industrial sectors.
In this work, we have developed enzymatic amperometric biosensors based on the immobilization of oxidases on an electrochemical interface of highly ordered titanium dioxide nanotubes array (TiO2NTAs). Thus, processes of enzyme immobilization based on polymeric entrapment and covalent immobilization have been studied. The analytical parameters of these biosensors have been evaluated.
For polymeric entrapment processes, kappa-carrageenan, 2-hydroxyethyl methacrylate (HEMA) and chitosan have been studied as immobilization matrices. These hydrogels have been used for the immobilization of the enzyme glucose oxidase (GOx) and it has been observed that both, HEMA and chitosan, generate a favorable microenvironment for the conservation of the activity of the enzyme.
For covalent immobilization, pentafluorophenylmethacrylate (PFM) has been used in order to generate bonds between the enzyme molecules and the surface of the transducer. Thus, the electrochemical interface TiO2NTAs has been modified by two plasma techniques: polymerization of PFM and grafting of the same polymer. It has been observed that the polymerized surface of PFM (ppPFM) has a higher hydrophobicity than the surface in which the PFM has been grafted (pgPFM). Hydrophobicity has influence on the adopted enzyme molecules conformation. On the ppPFM surface, conformations with low activity predominate, and on the pgPFM surface most of the population of GOx molecules adopt conformations with catalytic activity. For these reasons, the biosensors with plasma grafted PFM show higher sensitivity in presence of glucose than the biosensors based on the PFM polymerization.
Finally, amperometric glucose and glutamate biosensors with polymeric and covalent immobilization matrices have been developed: Ti/TiO2NTAs/GOx/Chitosan, Ti/TiO2NTAs/HEMA-co-EGDA/pgPFM/GOx/Chitosan and Ti/TiO2NTAs/GmOx/Chitosan. These biosensors have been used to determine the glucose and glutamate content in different food samples. The results have been compared with those obtained with reference techniques.
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Nunes, Gisele Fátima Morais. "Avaliação da interesterificação enzimática de misturas binárias e ternárias de gordura de leite com óleos de canola e castanha-do-pará nas propriedades do produto obtido." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/97/97132/tde-22082013-164341/.
Full textAbstract:
Este trabalho teve como objetivo avaliar o efeito da interesterificação enzimática da gordura de leite com óleos de canola e castanha-do-pará nas propriedades do produto alimentício obtido, empregando lipase de Rhizopus oryzae imobilizada em sílica-álcool polivinílico (SiO2-PVA) como catalisador. Considerou-se desejável a obtenção de um produto que, ao incorporar parte dos ácidos graxos insaturados e essenciais presentes nos óleos, apresentasse boa espalhabilidade sob temperatura de refrigeração. Na primeira etapa as propriedades das matérias-primas foram determinadas aplicando técnicas oficiais de análise e verificou-se que todas apresentaram características de acordo com a legislação brasileira para uso em produtos alimentícios. Em seguida, foram testados dois métodos (adsorção física e ligação covalente) para efetuar a imobilização da lipase selecionada em SiO2-PVA e os resultados obtidos indicaram a adequação do procedimento de adsorção física. As condições otimizadas para conduzir as reações de interesterificação enzimática de blendas binárias de gordura de leite e óleo de canola, e de gordura de leite e óleo de castanha-do-pará, foram determinadas por planejamento composto central (CCD) constituído de 11 experimentos. A influência das variáveis temperatura (45-65?C) e teor de gordura no meio reacional (50-80%) foi avaliada simultaneamente, considerando como variáveis-resposta o grau de interesterificação (GI) e a consistência dos produtos. Modelos empíricos que possibilitaram a seleção de condições para obtenção de produtos interesterificados com satisfatória espalhabilidade (consistência entre 200 e 800 gf/cm²) foram compostos e confirmados para cada caso. Para a blenda gordura de leite e óleo de canola, as condições selecionadas corresponderam a um meio contendo 65% de gordura e 35% de óleo e incubado a 45°C por 12 h. Nessas condições foram obtidos produtos com consistência de 700 gf/cm². No caso da blenda gordura de leite e óleo de castanha-dopará, produtos interesterificados que atendem o parâmetro desejado (200-800 gf/cm²) foram obtidos empregando-se um meio contendo 50% de gordura e 50% do óleo, incubados a 45°C por 24 h. Reações de interesterificação de blendas ternárias de gordura de leite, óleo de canola e óleo de castanha-do-pará foram também efetuadas de acordo com um planejamento de misturas, constituído de 17 experimentos visando avaliar a influência da proporção de cada componente da blenda na consistência do produto interesterificado. Na faixa de variação investigada, o uso de blendas contendo 56% de gordura de leite, 22% de óleo de canola e 22% de óleo de castanha-do-pará incubadas por 3 h a 45°C resultou em produtos interesterificados com satisfatória consistência e plasticidade (634 gf/cm²). O desempenho das reações enzimáticas conduzidas sob aquecimento convencional e não convencional (irradiação de micro-ondas) foi ainda avaliado para as blendas binárias nas condições preditas pelo planejamento composto central, não sendo observada interferência das micro-ondas na atuação da enzima, obtendo-se produtos interesterificados com valores similares de consistência. Os dados obtidos sugerem que o processo de interesterificação enzimática catalisado pela lipase de Rhizopus oryzae imobilizada em SiO2-PVA foi eficaz para a modulação das características de plasticidade dos produtos obtidos empregando tanto misturas binárias como ternárias.<br>The objective of this work was to assess the effect of the enzymatic interesterification of milkfat with canola oil and Brazil nut oil on the properties of the resulting food product, using Rhizopus oryzae lipase immobilized on silica-polyvinyl alcohol (SiO2-PVA) as catalyst. The work was carried out considering as desirable to obtain a more spreadable product under domestic refrigerated conditions as well as enriched with unsaturated and essential fatty acids. Firstly, the properties of the raw materials were determined by applying official analysis techniques and results indicated that all raw materials were in agreement with the Brazilian legislation to food products. Then, two methodologies (physical adsorption and covalent binding) were tested for immobilizing the selected lipase on SiO2-PVA and physical adsorption was found to be the most suitable procedure. The optimized conditions to perform the enzymatic interesterification reactions of binary blends (milkfat and canola oil and milkfat and Brazil nut oil) were determined by central composite design (CCD), leading to a set of 11 runs. The influence of the variables, temperature (45-65°C) and the content of milk fat in the reaction medium (50-80%), was assessed simultaneously, taking the interesterification degree (ID) and consistency (10°C) as response variables. Empiric models were composed and confirmed for each case to establish conditions at which products with satisfactory spreadability (consistency in the range from 200 and 800 gf/cm²) can be obtained. For the milkfat and canola oil blend, the established conditions corresponded to a medium containing 65% of milk fat and 35% of oil, and lipase incubated at 45°C for 12 h. In these conditions, products with consistency of 700 gf/cm² were obtained. In the case of milkfat and Brazil nut oil blend, interesterified products with desirable parameter (200 and 800 gf/cm²) were obtained from reactions carried out with medium containing 50% of milk fat and 50% of oil, and lipase incubated at 45°C for 24 h. Interesterification reactions of ternary blends of milkfat, canola oil and Brazil nut oil were also carried out according to a mixture design with 17 runs to assess the influence of the mass proportion of each compound in the blend on the consistency of the interesterified products. In the range studied, the use of blends with 56% of milkfat, 22% of canola oil and 22% of Brazil nut oil and incubation at 45°C for 3 h resulted in products with satisfactory consistency and plasticity (634 gf/cm²). The performance of enzymatic reactions carried out under conventional and non-conventional (microwave irradiation) heating was also assessed for binary blends under the conditions predicted by the central composite design, and no interference of the microwave in the enzyme action was observed, resulting in interesterified products with similar values of consistency. The results obtained suggested that the process of enzymatic interesterification catalyzed by Rhizopus oryzae immobilized on SiO2-PVA was effective in modulating the plasticity properties of the products obtained using both binary and ternary blends.
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Ferey, Justine. "Développement d'outils analytiques basés sur la spectrométrie de masse pour le suivi d'interactions enzyme-ligand dans le domaine de la santé." Thesis, Orléans, 2017. http://www.theses.fr/2017ORLE2051/document.
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Les enzymes et leur diversité d’actions sont appréciées dans des domaines d’applications variés allant del’agroalimentaire à la thérapeutique. Ainsi, une attention toute particulière est portée à leur étude afin d’améliorer uneaction (contre le vieillissement de la peau, antivirale, anticancéreuse…) ou un procédé de synthèse. Ce projet derecherche s’inscrit dans une démarche de développement d’outils analytiques basés sur la spectrométrie de masse,permettant le suivi rapide et sensible d’interactions enzyme-ligand.Dans une première étude, l’approche TLC couplée à une détection par UV a été évaluée pour la déterminationde constantes enzymatiques de l’enzyme invertase. Cette approche couplée à un MALDI/TOF MS a permis d’identifierdes substrats spécifiques de l’invertase au sein d’extraits de plantes. Pour preuve de concept, l’interactioncellobiohydrolase II–ligand est présentée dans le cadre de l’identification d’inhibiteur par TLC-MALDI/TOF et TLCENALDIMS.En seconde étude, nos travaux ont porté sur la caractérisation directe de différentes enzymes kinases, puis auxsuivis des réactions de phosphorylation de nucléosides /tides endogènes. Ces études, basées sur des approches « offline» (Flow Injection Analysis, FIA) et « on-line » (Frontal Affinity Chromatography, FAC) couplées à unspectromètre de masse haute résolution, ont été réalisées au moyen de ces kinases libres et immobilisées. Dans le cadrede la recherche de nouveaux candidats médicamenteux antiviraux, le suivi d’une phosphorylation spécifique desmolécules de synthèse, au regard de souches humaine ou virale de kinase, a également été évalué par ces deuxméthodologies<br>Enzymes are very appreciated and useful in various application fields from agri-business to therapeutic due to theirdiversity of actions. Therefore, their action mechanisms are widely studied in order to enhance an action (anti-aging ofskin, antiviral, antitumorous) or a synthesis process. This research project is part of the approach to propose analyticaltools based on mass spectrometry, allowing rapid and sensitive follow-up of enzyme-ligand interactions.In a first study, the Thin-Layer Chromatography (TLC) approach coupled with UV detection was evaluated forthe determination of invertase kinetic constants. This approach coupled with a MALDI / TOF-MS led to theidentification of invertase substrates in plant extracts. As a proof of concept, the cellobiohydrolase II - ligand interactionwas presented in the framework of the identification of inhibitor by TLC-MALDI / TOF and TLC-ENALDI MS.In the second study, our work aimed at developing a direct method for the determination of kinetic parametersof kinases and following-up the phosphorylation reactions of endogenous nucleosides / tides. These studies, based on“off-line” (Flow Injection Analysis, FIA) and “on-line” (Frontal Affinity Chromatography, FAC) approaches coupledwith a high-resolution mass spectrometer, were carried out using free and immobilized kinases. In the context of thesearch for new antiviral drug candidates, a specific phosphorylation of synthetic molecules regards to human or viralkinase was also evaluated by these both approaches
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Calil, Felipe Antunes. "Biorreatores capilares de NTPDase-1 de Trypanosoma cruzi: desenvolvimento e aplicação na triagem de inibidores seletivos." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/59/59138/tde-21072014-144130/.
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Uma das estratégias utilizadas no desenvolvimento de novas drogas envolve a descoberta de compostos que modulem a atividade de enzimas, importantes no processo infeccioso de patógenos. Uma abordagem interessante na triagem de novos ligantes é o uso de métodos baseados na imobilização de enzimas em suportes cromatográficos acoplados a sistemas de cromatografia líquida. O uso de IMERs (Immobilized Enzyme Reactors) como uma fase estacionária acoplado a sistemas de cromatografia líquida de alta eficiência consiste em uma estratégia para triagem de compostos rápida e eficiente e tem vantagens em relação ao uso de enzimas em solução. A enzima NTPDase-1 de Trypanosoma cruzi age como um facilitador da infecção do patógeno, inibindo assim a resposta imune do hospedeiro, permitindo uma infecção silenciosa, o que sugere seu uso como um bom alvo na busca por inibidores. Neste trabalho, a enzima NTPDase-1 foi imobilizada na parede interna de capilares de sílica fundida formando ICERs (Immobilized Capillary Enzyme Reactors). Estudos das condições de uso destes biorreatores juntamente com o desenvolvimento de um método cromatográfico multidimensional, foram realizados e validados. A otimização do método cromatográfico e sua validação, apresentaram ótimos resultados em relação aos valores obtidos para os parâmetros avaliados para métodos bioanalíticos. A imobilização da enzima foi realizada com sucesso, sendo possível a detecção da atividade catalítica no sistema cromatográfico (TcNTPDase1-ICER). Foi realizado também, o estudo cinético para ATP no TcNTPDase1-ICER, obtendo-se KM de 0,317 ± 0,044 mM, que comparado com estudos em solução, KM de 0,096 mM, ainda apresenta grande afinidade pelo substrato.<br>One of the strategies used in the development of new drugs involves the discovery of compounds that modulate the activity of enzymes, important in the infectious pathogens process. An interesting approach in the screening of new ligands is the use of methods based on immobilization of enzymes in chromatographic supports coupled to liquid chromatography systems. The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems consist in a strategy to a fast and efficient compounds screening and it has advantages comparing to the use of enzymes in solution. The enzyme NTPDase-1 Trypanosoma cruzi acts as a pathogen infection facilitator, thus inhibits the host immune response allowing a silent infection, suggesting its use as a good target in the search for inhibitors. In this paper, the enzyme NTPDase-1 was immobilized for the manufacturing of ICERs (Immobilized Capillary Enzyme Reactors). Studies of conditions to the use of these bioreactors in the ligands screening along with the development of a multidimensional chromatographic method were performed and validated. The chromatographic method optimization and validation, presented excellent results, relating to the obtained values, from evaluated parameters in bioanalytical methods. The enzyme immobilization was successfully performed, being possible to detect the catalytic activity in the chromatographic system (TcNTPDase1-ICER). The kinetic study for the substrate ATP was also performed in the TcNTPDase1-ICER, obtaining KM of 0.317 ± 0.044 mM, which in comparison with studies in solution KM of 0.096 mM, still presents high affinity for the substrate.
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Bourkaib, Mohamed Chafik. "Procédé enzymatique intensifié et durable de N-acylation d'acides aminés en milieux écocompatibles : caractérisation, cinétiques et immobilisation de nouveaux biocatalyseurs." Electronic Thesis or Diss., Université de Lorraine, 2020. http://www.theses.fr/2020LORR0114.
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Les acides aminés acylés constituent un important groupe de tensioactifs biosourcés présentant des propriétés techno-fonctionnelles et bioactives très attractifs pour des applications en industries alimentaires, cosmétiques, pharmaceutiques… La production de ces molécules à l’échelle industrielle se fait par voie chimique suivant la réaction de Schotten-Baumann, qui en dépit de son efficacité, a un effet négatif sur l’environnement et ne présente aucune spécificité de réaction. L’utilisation de biocatalyseurs enzymatiques est l’alternative la plus prometteuse à cette voie chimique. L’objectif de cette thèse était d’évaluer le potentiel catalytique de différentes enzymes pour la réaction de N-acylation des acides aminés en solvants verts. Dans un premier temps, le couple enzyme / substrat le plus adapté à la réaction a été identifié. Les aminoacylases de Streptomyces ambofaciens en milieu aqueux ont montré une bonne spécificité de substrat vis-à-vis de nombreux acides aminés contrairement à la lipase B de Candida antarctica en milieu non-aqueux. Une relation entre la régiosélectivité des aminoacylases et la longueur de la chaine du donneur d’acyle a été mise en évidence. Cela été lié à la présence de l’ε-lysine acylase dans le mélange enzymatique. L’ajout d’ions cobalt a permis une accélération importante de la vitesse initiale de la réaction de N-acylation et une augmentation du taux de conversion. Dans une deuxième phase, les enzymes ont été immobilisées sur différents supports afin de faciliter leur recyclage. Les enzymes immobilisées sur les supports mésoporeux (SBA15) fonctionnalisés avec de l’APTES ont été sélectionnés comme les meilleurs supports pour l’immobilisation des aminoacylases de S. ambofaciens permettant de maintenir une certaine activité de l’enzyme après immobilisation. Les performances de CALB après immobilisation ont été évalués sous scCO2 pour la réaction de synthèse de l’acétate de géraniol. L’immobilisation de CALB par chimisorption sur des supports siliceux macroporeux avec de l’isocyanate comme greffon a permis une augmentation importante de l’activité par rapport aux autres supports et à l’enzyme CALB commerciale immobilisée sur une résine acrylique. Enfin, l’impact des conditions réactionnelles sur la production de l’undécénoyl-phénylalanine (un surfactant d’intérêt en cosmétique) par les aminoacylases de S. ambofaciens a été évaluée. Le produit de la réaction a été purifié à >99% et l’activité de dépigmentation de la peau a été vérifié<br>Acylated amino acids are one of the most attractive biobased surfactants for food, cosmetic and pharmaceutic industry regarding their techno-functional and bioactive properties. Those molecules are produced at industrial scale by using Schotten-Baumann chemical method which is a very productive way but non-specific and have a negative impact on the environment. Enzymatic catalysis is one of the most promising alternatives. The aim of this work was to find the best enzymatic way for the N-acylation of amino acids in green solvent. The first step was to select the couple enzyme / reactional medium enabling the implementation of this reaction. Aminoacylases from Streptomyces ambofaciens in aqueous medium showed the best N-acylation specificity towards the amino acids and the acyl donors with some correlating between the structure of the substrates and the catalytic performances compared to CALB which doesn’t show any N-α-acylation activity. The addition of cobalt to the reaction medium allowed an acceleration of the reaction rate and an increase of the activity. In a second time, the activity of the different enzymes was evaluated after the immobilization on various supports. The aminoacylases immobilized on SBA-15 functionalized with APTES showed the best performances compared to the use of the others supports but a decrease of the activity compared to the free enzymes was observed. In the case of CALB, the covalent immobilization using isocyanate as grafting agent on the macroporous silica material allowed a significant improvement of the catalytic performance under scCO2 even better than the commercial immobilized CALB. Lastly, the operational condition affecting the aminoacylases activity for the synthesis of undecenoyl-phenylalanine, a product with cosmetic interest, was evaluated. The product of the reaction was purified to >99% and the skin depigmentation activity was checked
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Poojari, Yadagiri. "Enzyme Immobilization and Biocatalysis of Polysiloxanes." University of Cincinnati / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1265987790.
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Dakhmouche-Djekrif, Scheherazed. "Production et caractérisation de l'amylopullulanase de la levure Clavispora lusitaniae ABS7 isolée de blé cultivé et stocké en zones arides." Thesis, Compiègne, 2016. http://www.theses.fr/2016COMP2258/document.
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Cette étude vise à produire deux enzymes amylolytiques (α-amylase et pullulanase) thermostables par des levures contaminant le blé récolté dans des zones semi arides et arides (Biskra - Sahara, Sud Algérien) et capables d’hydrolyser à la fois les liaisons α-1-4 et α-1-6 de polysaccharides comme l’amidon et le pullulane, molécules d’intérêt industriel. Après isolement et caractérisation de colonies levuriennes, la méthode de plate-test-agar permet d’isoler des souches amylolytiques et de montrer que la souche L7 est la plus performante dans la production enzymatique parmi une douzaine de souches de levures productrices d’α-amylase et de pullulanase thermostables. L’identification des souches, basée sur les caractères morphologiques, les tests biochimiques et la biologie moléculaire a permis de répartir la population comme suit : 50% Clavispora lusitaniae (forme anamorph Candida lusitaniae), 25%, Pichia guilliermondii, 8% Pichia carribbicca, 8% Meyerozyma guilliermondii et 8% Rhodotorula rubra. Par sa richesse en amidon, le biotope du blé est favorable à la survie des levures amylolytiques. La majorité de ces souches dont la souche L7 est productrice de pseudo ou vrai mycélium et est tolérante à certains paramètres comme la température, la salinité, les stress osmotique et éthanolique. La souche de levure L7, Clavispora lusitaniae ABS7, semble être la plus performante dans la production d’enzymes thermostables. Son identification moléculaire a montré deux bandes avec l’endonucléase HAE III alors que les autres souches de la même espèce de Clavispora lusitaniae (L5, L9, L10, L11 et L12) présentent une seule bande. En conditions optimales (agitation 136,56 rpm, température 54,14°C, amidon 2,66g/l, extrait de levure 0,365g/l, sels 8, 75ml/l et oligo-éléments 4,3ml/l en erlenmeyers de 250 ml), la production maximale atteint les valeurs suivantes : 13456,36±300 UI pour l’ α-amylase et 12611, 6±154 UI pour la pullulanase. Ces performances sont en accord étroit avec la prédiction du modèle statistique évaluée à 13231UI pour l’α-amylase et 12825,5 UI pour la pullulanase. La production optimisée a pratiquement doublé par rapport à la production avant l’optimisation (6639,16 UI pour l’α-amylase et 6308,5 UI pour la pullulanase). En conditions optimales et en fermenteur de 2 L, la production maximale pour les deux enzymes de la levure Clavispora lusitaniaeABS7 est obtenue au bout de 28 h avec un optimum de croissance obtenu à 40 heures. La production des deux enzymes n’est donc pas associée à la croissance. La production maximale des deux enzymes s’effectue à pH 8. pic protéique. L’élution sur DEAE-cellulose confirme la présence des deux activités dans la même fraction. Les deux enzymes sont donc présentes sur la même molécule. L’α-amylase et la pullulanase sont purifiées avec un taux de purification de 50,5 et 44,6 respectivement et des rendements respectifs de 23,9% et 21,1%. L’extrait purifié montre une seule bande sur le gel de SDS-PAGE avec un poids moléculaire estimé à 75KDa et une activité amylolytique contenant à la fois les activités α-amylasique (indépendante de Ca2+) et pullulanasique (une métalloenzyme à calcium). La souche de la levure Clavispora lusitaniae ABS7 possède donc une enzyme amylolytique avec deux sites actifs. La CCM révèle une enzyme qui hydrolyse l’amidon en maltose et glucose et le pullulane en maltotriose, maltose et glucose, ce qui montre que l’enzyme est saccharifiante et correspond à une pullulanase de type II (amylopullulase). L’optimisation de l’immobilisation de l’enzyme a permis l’amélioration de l’activité: α-amylasique à 4907,75 UI (rendement 72,3 %) et celle de la pullulanase à 4491,83 UI (rendement 70,1%) avec un pH optimum de 8,5. Il ressort de notre étude que l’amylopullulanase type II libre de Clavispora lusitaniae ABS7 est thermostable puisqu’elle résiste à un traitement thermique de 75°C pendant 3 heures d’incubation et conserve 88% de son activité initiale<br>This study aims to produce two amylolytic enzymes (α-amylase and pullulanase) by thermostable wheat contaminant yeast harvested in semi arid and arid zones (Biskra, Sahara, Algeria SUD) and capable of hydrolyzing both the α links 1-4 and 1-6 of polysaccharides such as starch and pullulan, molecules of industrial interest. After isolation and characterization of levuriennes colonies, the test method of agar-plate allows to isolate amylolytic strains and show, that the L7 strain is the most effective, in the enzymatic production of the 12 yeast strains producing α-amylase and pullulanase the thermostable. The identification of strains, based on morphological, biochemical tests and molecular biology has helped spread the population as follows: 50% Clavispora lusitaniae (anamorph form Candida lusitaniae), 25%, Pichia guilliermondii, 8% carribbicca Pichia, 8% Meyerozyma guilliermondii and 8% Rhodotorula rubra. By its high starch, the wheat biotope is favorable to the survival of amylolytic yeasts. Most of these strains, including the strain L7, is producer, pseudo or true mycelium and is tolerant to certain parameters such as temperature, salinity, osmotic stress and ethanolic stress. The yeast strain Clavispora lusitaniae ABS7 (L7) seems to be the most efficient in the production of thermostable enzymes. Its molecular identification showed two bands with the endonuclease HAE III while other strains of the same species Clavispora lusitaniae (L5, L9, L10, L11 and L12) have a single band. In optimal conditions (agitation 136.56 rpm, temperature 54.14 ° C, starch 2,66g / l, yeast extract 0,365g / l, salts 8 75ml / l and trace elements 4,3ml / liter Erlenmeyer flasks into 250 ml), the maximum production reached: 13456.36 ± 300 IU for the α-amylase and 12611, 6 ± 154 IU for pullulanase. This performance is in close agreement with the prediction of the statistical model 13231UI evaluated for α-amylase and 12825.5 IU for pullulanase. The optimized production almost doubled compared to production before optimization (6639.16 IU for the α-amylase and pullulanase for 6308.5 IU). In optimal conditions, and 2 L fermenter, the maximum production for the two enzymes of Clavispora lusitaniae ABS7 obtained after 28 hours, with an optimum of growth obtained at 40 hours. The production of both enzymes is thus not associated with growth. The maximum production of both enzymes is obtained at pH 8. The kinetics are characterized by an increase in carbohydrate and a substance spooning the wall of the fermenter, probably an exo-polysaccharide. The chromatographic profile on Sephacryl S200 reveals two α-amylase and pullulanase activities eluted along with the protein peak. Elution DEAE cellulose confirms the presence of both activities in the same fraction. Both enzymes are present on the same molecule. The α-amylase and pullulanase were purified with a purification rate of 50.45 and 44.59 respectively and respective yields of 23.88% and 21.11%. The purified enzyme showed a single band on SDS-PAGE gel with a molecular weight estimated at 75 KDa and an amylolytic activity containing both the α-amylase activities (independent of Ca2+) and pullulanase (a calcium metalloenzyme). The strain of the yeast Clavispora lusitaniae ABS7 therefore has an amylolytic enzyme with two active sites. TLC reveals an enzyme which hydrolyzes starch into maltose and glucose and pullulan into maltotriose, maltose and glucose, which shows that the saccharifying enzyme, and corresponds to a pullulanase type II (amylopullulase). The optimization of the immobilization of the enzyme enabled the improvement of the activity: α-amylase to 4907.75 IU (yield 72.3%) and pullulanase to 4491.83 IU (yield 70, 1%) with a pH optimum of 8.5. It appears from our study that amylopullulanase type II free is thermostable to heat treatment of 75 ° C for 3 hours of incubation, and retains 88% of its original activity
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Laaroussi, Awatef. "Fabrication de biocathodes flexibles pour biopiles enzymatiques implantables par procédés d’impression." Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAI014/document.
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Les biopiles enzymatiques, capables de convertir le glucose présent dans le fluide physiologique en électricité, sont une source d’alimentation pour les dispositifs implantables. Cependant, les faibles puissances délivrées ne permettent pas d’alimenter actuellement des organes artificiels implantables. Une nouvelle architecture de biocathode tirant profit des technologies d’impression a été testée en vue d’améliorer les performances des Biopiles implantables. Ce travail démontre la pertinence des procédés d’impression tels que le spray ultrasonique et l’héliogravure dans l’élaboration de biocathodes homogènes, fines et flexibles. Ainsi, des encres fonctionnelles, dont la formulation à base de nanotubes de carbone et de surfactant a été optimisée, ont pu être déposées sur un substrat flexible hydrophobe (feuilles de carbone). Les problèmes d’imprimabilité du substrat ont été surmontés et des couches actives flexibles ont été obtenues (épaisseur entre 5 et 10 µm). Enfin, une technique d’immobilisation non-covalente des laccases (via le pyrène adamantane) a été testée et un courant catalytique de l’ordre de 130 mA.cm-2 a été obtenu<br>Enzymatic Biofuel Cells, capable of converting efficiently the glucose from extracellular fluid into electrical energy, are a power source for implantable devices. However, the power output generated by these cells is not sufficient to fulfill the energy required by implantable artificial organs. Therefore, a new packaging architecture design based on flexible materials derived from printing technologies has been explored in order to enhance the power output of this cell. This work demonstrates the relevance of printing processes such as ultrasonic spray and gravure to develop homogeneous, thin and flexible biocathodes. During this work, a carbon nanotubes / surfactant suspensions were deposited on a hydrophobic flexible substrate (carbon paper). Despite the poor printability of the substrate, flexible active layers were obtained (thickness between 5 and 10 µm). Finally, a non-covalent immobilization of laccases (via adamantane pyrene) was tested and a catalytic current of approximately 130 µA.cm-2 was obtained. mA.cm-2 was obtained
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Harguindeguy, Marine. "Développement de supports d’immobilisation biosourcés pour la dégradation enzymatique de micro polluants dans un réacteur à lit fluidisé." Thesis, Montpellier, 2020. http://www.theses.fr/2020MONTG023.
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Ce travail de thèse a pour objectif l’élaboration de supports d’immobilisation d’enzymes bio sourcés en vue de la dégradation de micropolluants dans l’eau. Des billes de gélatine de diamètre moyen 1,9 mm ont été formées dans ce but par un procédé de goutte à goutte n’employant aucun solvant organique. La réticulation des billes par le glutaraldéhyde permet d’une part de limiter le gonflement de ce polymère dans l’eau et d’améliorer sa tenue mécanique en immersion, et d’autre part de créer des liaisons covalentes entre la gélatine et la laccase. Un plan d’expérience 2^4 a été réalisé afin d’identifier les paramètres influant sur l’efficacité du greffage.La dégradation de deux micropolluants par les billes activées a été étudiée en réacteur agité et dans un réacteur à lit fluidisé conçu pour ce projet. La tétracycline s’est montrée sensible à l’oxydation par les laccases greffées à la surface des billes. Divers paramètres ont été étudiés comme le pH de la solution, le débit de recirculation, ou l’addition d’oxygène dans le système. Le glyphosate s’est montré plus récalcitrant à l’action de la laccase seule à cause d’une structure plus complexe. L’ajout d’un médiateur d’oxydoréduction, l’ABTS, a néanmoins permis de faciliter son oxydation. La co-immobilisation du médiateur et du biocatalyseur sur les supports à base de gélatine a été étudiée, afin de limiter l’impact économique et environnemental du projet<br>The aim of this thesis is to develop bio-based supports for immobilization of enzymes for micro pollutants degradation in water. Gelatin beads of around 1,9 mm diameter were formed by a dripping process using no organic solvent. A crosslinking step with glutaraldehyde was then carried out with two objectives: to improve the mechanical properties of the beads when immersed in water, and to create covalent bonds between gelatin and laccase. The parameters affecting the efficiency of the enzyme grafting were investigated using a full factorial design 2^4.The degradation of two micro pollutants by the activated beads was studied in a stirred tank reactor and in a fluidized bed reactor designed for this project. Tetracycline was efficiently oxidized by the grafted enzymes. The influences of pH, recirculation flowrate, and addition of supplementary oxygen on the TC degradation were studied. Glyphosate was not sensitive to laccase oxidation at first due to its complex structure, but the addition of ABTS as an oxidoreduction mediator considerably improved its removal. Mediator and bio-catalyst were co-immobilized on the gelatin beads to reduce the environmental and economical cost of the project
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Roucher, Armand. "Elaboration de matériaux biofonctionnels par chimie intégrative." Thesis, Bordeaux, 2018. http://www.theses.fr/2018BORD0303/document.
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Bien que les matériaux poreux soient nombreux dans la nature, la synthèse en laboratoirede matériaux présentant une porosité multi-échelle ou hiérarchisée est toujours délicate. Enutilisant la matière molle (émulsions concentrées, auto-assemblages, mésophases lyotropes, etc)et le procédé sol-gel, il est possible d’obtenir une grande variété de matériaux monolithiques, àporosité hiérarchisée, composés d’un squelette silicique. La porosité de ces matériaux peut êtreoptimisée en jouant avec la nature de l’émulsion, le tensioactif utilisé, ou avec l’ajout d’agentd’extérieur comme le sel. En combinant ces méthodes, des matériaux possédant une mésoporositéhexagonale ont été obtenus. Grâce à leur surface riche en silanols, ces matériaux poreux ont étéfonctionnalisés par greffage post-synthèse de molécules organiques. Dès lors, l’immobilisationd’entités biologiques comme les enzymes au sein de la structure poreuse a permis d’utiliser cesmatériaux pour des réactions d’hydrolyse, de synthèse ou de décoloration en milieu aqueux dansune approche de « chimie verte ». Enfin, des micro-organismes ont été piégés dans ces matériauxporeux qui ont été recouverts d’une coque en silice. Les micro-organismes peuvent s’y développersans restriction et leur croissance est très différente de celle observée dans les cultures classiques.La coque en silice, formée en surface, est donc imperméable au passage des bactéries (taillemicrométrique) mais perméable à la diffusion des substrats et des réactifs. Cette diffusion a étémise à profit pour réaliser des réactions enzymatiques en cascade. Ces matériaux se positionnentcomme des biocatalyseurs très prometteurs pour de nombreuses applications<br>Although porous materials are numerous in nature, the laboratory synthesis of materials withmulti-scale or hierarchical porosity is always difficult. By using soft matter (concentrated emulsions,self-assemblies, lyotropic mesophases, etc.) and the sol-gel process, it is possible to obtaina wide variety of monolithic materials with hierarchical porosity composed of a silicic skeleton.The porosity of these materials can be optimized by playing with the nature of the emulsion,the surfactant used, or with the addition of external agents such as salt. By combining these methods,materials with hexagonal mesoporosity have been obtained. Thanks to their silanol-richsurface, these porous materials have been functionalized by post-synthesis grafting of organicmolecules. Therefore, the immobilization of biological entities such as enzymes within the porousstructure has made it possible to use these materials for hydrolysis, synthesis or discolorationreactions in aqueous media in a "green chemistry" approach. Finally, microorganisms were trappedin these porous materials which were covered with a silica shell. Microorganisms can growthere without restriction and their growth is very different from that observed in conventionalcultures. The silica shell formed on the surface is therefore impermeable to the passage of bacteria(micrometric size) but permeable to diffusion of substrates and reagents. This diffusion wasused to carry out cascade enzymatic reactions. These materials are positioned as very promisingbiocatalysts for many applications
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SILVA, Barbara Dumas Santos. "Ação antimicrobiana de enzimas hidrolíticas produzidas por Trichoderma asperellum e imobilizadas em blendas de polímeros biodegradáveis." Universidade Federal de Goiás, 2011. http://repositorio.bc.ufg.br/tede/handle/tde/1254.
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Previous issue date: 2011-01-28<br>The hydrolytic activity of enzymes produced by Trichoderma asperellum, immobilized biodegradable films, as growth inhibitor of microorganisms was tested. The inhibitory activity was demonstrated on Aspergillus niger, Penicillium sp. and Sclerotinia sclerotiorum, microorganisms usually related to the attack and/or food contamination at the field or packaged. We used two polymer blends with different compositions, cassava starch and poly-butylene adipate-co-terephthalate (Ecoflex®, BASF Chemical Company) and other composed for polyvinyl alcohol (PVA) and polysaccharide cashew gum (PEJU). T. asperellum was induced to produce enzymes involved in the attack mycoparasite (N-acetylglucosaminidases, β-1,3-glucanases, chitinases and proteases) by the addition of crude chitin in the growth medium. The enzymes produced in major quantity were N-acetylglucosaminidase and chitinase. The pool of enzymes produced in the experiments was then used for immobilization tests. The immobilization process was performed in films by two methods: covalent and ionic bonding. In both methods, the presence of immobilized hydrolytic enzymes resulted in reduced growth of microorganisms, but the covalent immobilization of the results were more expressive. S.sclerotiorum was the microorganism most sensitive, followed by A. niger and Penicillium sp. To confirm the action of hydrolytic enzymes produced by T. asperellum and evaluate the effects they produce cell wall of microorganisms and other structures, the films with enzyme immobilized by covalent bonding were subjected to scanning electron microscopy. The structures most affected were hyphae and spores. Overall, the synergistic action of all enzymes produced by T. asperellum, reduced the growth of microorganisms when immobilized on the surface of the films Starch-Ecoflex® and PVA-PEJU. Moreover, the polymer blends tested exhibited desirable characteristics for future use in food packaging and most importantly, also provide efficient systems for the immobilization of enzymes.<br>O trabalho foi conduzido com o objetivo de testar a ação de enzimas hidrolíticas produzidas por Trichoderma asperellum, imobilizadas em filmes biodegradáveis, como agente inibidor do crescimento de microrganismos. A atividade inibitória foi testada sobre Aspergillus niger, Penicillium sp. e Sclerotinia sclerotiorum, microrganismos geralmente relacionados com o ataque e, ou, contaminação de alimentos, seja no campo ou embalados. Foram utilizadas duas blendas poliméricas de diferentes composições, uma a base de amido de mandioca e poli-butileno-adipato-co-tereftalato (Ecoflex®; BASF Chemical Company) e outra a base de álcool polivinílico (PVA) e polissacarídeo de caju (PEJU). T. asperellum foi induzido a produzir as enzimas envolvidas no processo de ataque micoparasita (N-acetilglicosaminidases, β-1,3-glicanases, quitinases e proteases) pela adição de quitina bruta no meio de crescimento. As enzimas produzidas em maior quantidade foram N-acetilglicosaminidases e quitinases. O pool de enzimas produzidas foi então utilizado nos experimentos de imobilização. O processo de imobilização foi realizado nos filmes por dois métodos: ligação covalente ou ligação iônica. Em ambos os métodos, a presença de enzimas hidrolíticas imobilizadas resultou em redução do crescimento dos microorganismos, porém na imobilização por ligação covalente os resultados foram mais expressivos. S.sclerotiorum foi o microrganismo mais sensível, seguido por A. niger e Penicillium sp. Para confirmar a ação hidrolítica das enzimas produzidas por T. asperellum e avaliar os efeitos por elas produzidos na parede celular e demais estruturas dos microrganismos, os filmes com enzima imobilizada por ligação covalente foram submetidos à microscopia eletrônica de varredura. As estruturas mais afetadas foram hifas e esporos. De forma geral, a ação sinérgica de todas as enzimas produzidas por T. asperellum reduziu visivelmente o crescimento dos microrganismos quando imobilizadas na superfície dos filmes Amido-Ecoflex® e PVA-PEJU. Além disso, as blendas poliméricas testadas apresentaram características desejáveis para futuro uso em embalagens de alimentos e principalmente, também constituem sistemas eficientes para imobilização de enzimas.
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Dominick, Wendy D. "Enzyme immobilization of poly(methyl methacrylate) (PMMA) surfaces." Cincinnati, Ohio : University of Cincinnati, 2006. http://rave.ohiolink.edu/etdc/view.cgi?acc_num=ucin1148253649.
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Thesis (Ph. D.)--University of Cincinnati, 2006.<br>Advisor: Patrick Alan Limbach. Title from electronic thesis title page (viewed Apr. 3, 2009). Keywords: enzyme immobilization; proteins; mass spectrometry; microchip. Includes abstract. Includes bibliographical references.
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Dominick, Wendy D. "Enzyme Immobilization on Poly(methyl methacrylate) (PMMA) Surfaces." University of Cincinnati / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1148253649.
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Dewa, Andrew Steven. "A silicon-based enzyme biosensor utilizing Langmuir-Blodgett film immobilization." Case Western Reserve University School of Graduate Studies / OhioLINK, 1993. http://rave.ohiolink.edu/etdc/view?acc_num=case1057002686.
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Gairola, Priyanka. "Association of Metal-Organic Framework and Transaminase for chemoenzymatic production of amines." Electronic Thesis or Diss., Sorbonne université, 2019. http://www.theses.fr/2019SORUS107.
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Du fait de la raréfaction des ressources fossiles, l’industrie chimique doit aujourd’hui évoluer pour se tourner vers de nouvelles sources de matière première. A cela s’ajoutent les pressions environnementales toujours croissantes qui imposent une réduction de l’impact écologique et énergétique des procédés. Répondre à ces enjeux technologiques majeurs implique la conception de nouveaux procédés chimiques rendant notamment possible la transformation massive des ressources chimiques d’origine naturelle (cellulose, lignine, algues...etc) en produits chimiques à haute valeur ajoutée et répondant aux critères dits de « chimie verte ». , Dans ce contexte, la catalyse hétérogène est un outil incontournable puisqu’elle permet d’accélérer les réactions chimiques, dans des conditions durables, en rendant possible le recyclage des phases actives. Le développement de procédés toujours plus adaptés aux enjeux industriels et environnementaux actuels nécessite cependant l’élaboration de nouveaux catalyseurs, notamment capables de réduire toujours davantage la consommation d’énergie, de faire des économie d’atomes et de réduire autant que possible les quantités de réactifs et de solvants utilisés ainsi que les déchets produits. Pour ce faire, les catalyseurs hétérogènes capables de catalyser plusieurs réactions chimiques en une seule étape « en cascade » sont particulièrement prometteurs. [...] L’objectif global de [ma] thèse était de fabriquer un système chimioenzymatique capable de réaliser une cascade de deux réactions permettant la transformation d’alcools en amines. Pour cela il était proposé d’immobiliser sur un matériau hybride organique-inorganique poreux cristallin appelé MOF (Metal-Organic Framework), un catalyseur chimique, responsable d’une première étape d'oxydation d'alcool en composé carbonylé, et une enzyme transaminase catalysant l’étape ultérieure de transfert d'amine. La mise en œuvre d'un tel système sophistiqué était un réel défi, notamment parce qu’il s’agissait de trouver des conditions de réaction (solvant, température, pH, et choix des réactifs chimiques) qui soient compatibles avec les conditions de travail des transaminases (températures de réaction douces ≤ 60 ° C, solvants au moins partiellement aqueux). Ceci était un pré-requis nécessaire à la réalisation des synthèses "one-pot", où les deux réactions visées devaient être catalysées consécutivement par le catalyseur chimique et l’enzyme dans le même milieu réactionnel sans isolement du carbonylé intermédiaire. Il fallait également s’assurer de la stabilité du MOF dans le milieu réactionnel, et notamment de l’intégrité de sa structure dans des solvants contenant les solutions tampons aqueuses nécessaires à la stabilité des enzymes. [...]<br>Due to the scarcity of fossil resources, the chemical industry must today evolve to turn to new sources of raw material. Added to this are the ever-increasing environmental pressures that impose a reduction in the ecological and energy impact of the processes. Responding to these major technological challenges involves the design of new chemical processes making it possible, in particular, for the massive transformation of natural chemical resources (cellulose, lignin, algae, etc.) into high value-added chemicals that meet the so-called "Green chemistry" i, ii. In this context, heterogeneous catalysis is an essential tool since it makes it possible to accelerate the chemical reactions under sustainable conditions by making it possible to recycle the active phases ii. The development of processes that are increasingly adapted to today's industrial and environmental challenges, however, requires the development of new catalysts, in particular capable of reducing energy consumption even more, of saving atoms and of reducing as much as possible the quantities of reagents and solvents used as well as the waste produced. To do this, heterogeneous catalysts capable of catalyzing several chemical reactions in one step "in cascade" are particularly promising. [...] The overall goal of this thesis was to build a chemoenzymatic system capable of carrying out a cascade of two reactions allowing the transformation of alcohols into amines. For that it was proposed to immobilize on a crystalline organic-inorganic hybrid material called MOF (Metal-Organic Framework), a chemical catalyst, responsible for a first step of oxidation of alcohol to carbonyl compound, and a transaminase enzyme catalyzing the subsequent amine transfer step. The implementation of such a sophisticated system was a real challenge, especially because it was a question of finding reaction conditions (solvent, temperature, pH, and choice of chemical reagents) that are compatible with the working conditions of transaminases (mild reaction temperatures ≤ 60 ° C, at least partially aqueous solvents). This was a prerequisite for carrying out "one-pot" syntheses, where the two targeted reactions were to be catalyzed consecutively by the chemical catalyst and the enzyme in the same reaction medium without isolation of the intermediate carbonyl. It was also necessary to ensure the stability of the MOF in the reaction medium, and in particular the integrity of its structure in solvents containing the aqueous buffer solutions necessary for the stability of the enzymes. [...]
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Song, Yeqing. "Enzyme immobilization by adsorption of phenylbutyramidinated protein on porous polymer beads /." The Ohio State University, 1989. http://rave.ohiolink.edu/etdc/view?acc_num=osu148767263159891.
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Kimmins, Scott David. "Emulsion-templated porous polymers as support materials for covalent enzyme immobilization." Thesis, Durham University, 2011. http://etheses.dur.ac.uk/902/.
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It has been observed that poly(High Internal Phase Emulsion) (polyHIPE) materials can be used as a biocatalysts, via the covalent immobilization of Candida Antarctica Lipase B (CAL-B). Recently, it has been shown that polyHIPEs can be prepared with epoxy functionality, which show potential for the covalent immobilization of enzymes. The aims of our work were, firstly, to produce an open-void glycidyl methacrylate (GMA)-based polyHIPE material. Secondly, these materials were then to be developed for use within a continuous flow set-up. Thirdly, the post-polymerisation of these materials was to be investigated. Finally, these materials were to be used as a support for the covalent immobilization of enzymes. Highly porous, open-void GMA-based polyHIPE materials were accomplished via the photo- initiation, rather than thermal initiation of the continuous phase of the emulsion. The rapid cure of the emulsion effectively ‘locks’ the emulsion morphology, prior to emulsion destabilisation, that is more prominent in the slower thermally initiated HIPEs. Photopolymerised GMA-based polyHIPE materials were further developed for use within a continuous flow-set up. GMA-based polyHIPE materials were functionalized post-polymerisation with tris(2-aminoethyl)amine, morpholine and O,O’-bis(3-aminopropyl)polyethylene glycol. The functionalization of these GMA-based materials was observed via a number of analysis techniques, such as FT-IR spectroscopy, XPS spectroscopy, elemental analysis, Fmoc number determination, 1H HR-MAS NMR spectroscopy, and the covalent attachment of ninhydrin and FITC. Elemental analysis of the morpholine and tris(2-aminoethyl)amine polyHIPE showed that a near quantitative conversion, of 72 and 82 % respectively, was accomplished via the reaction being conducted at reflux for 24 hours. The enzymes, Lipase from Candida Antarctica and Proteinase K from Tritirachium album were immobilized either directly onto the polyHIPE material or via a hydrophilic spacer group, O,O’-bis(3-aminopropyl)polyethylene glycol. CAL was immobilized with a loading of between 5.4 and 7.5 wt. % per g of polyHIPE material.