Academic literature on the topic 'Immobilization (Enzymes)'

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.

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.

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.

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.

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.

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.

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.