Claudia Ortiz

A Novel Heterofunctional Epoxy‐Amino Sepabeads for a New Enzyme Immobilization Protocol: Immobilization‐Stabilization of β‐Galactosidase from Aspergillus oryzae

The properties of a new and commercially available amino‐epoxy support (amino‐epoxy‐Sepabeads) have been compared to conventional epoxy supports to immobilize enzymes, using the β‐galactosidase from Aspergillus oryzae as a model enzyme. The new support has a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. This support has both a great anionic exchanger strength and a high density of epoxy groups. Epoxy supports require the physical adsorption of the proteins onto the support before the covalent binding of the enzyme to the epoxy groups. Using conventional supports the immobilization rate is slow, because the adsorption is of hydrophobic nature, and immobilization must be performed using high ionic strength (over 0.5 M sodium phosphate) and a support with a fairly hydrophobic nature. Using the new support, immobilization may be performed at moderately low ionic strength, it occurs very rapidly, and it is not necessary to use a hydrophobic support. Therefore, this support should be specially recommended for immobilization of enzymes that cannot be submitted to high ionic strength. Also, both supports may be expected to yield different orientations of the proteins on the support, and that may result in some advantages in specific cases. For example, the model enzyme became almost fully inactivated when using the conventional support, while it exhibited an almost intact activity after immobilization on the new support. Furthermore, enzyme stability was significantly improved by the immobilization on this support (by more than a 12‐fold factor), suggesting the promotion of some multipoint covalent attachment between the enzyme and the support (in fact the enzyme adsorbed on an equivalent cationic support without epoxy groups was even slightly less stable than the soluble enzyme).

Reversible Immobilization of Invertase on Sepabeads Coated with Polyethyleneimine: Optimization of the Biocatalyst's Stability

Invertase from S. cerevisiae has been immobilized by ionic adsorption on Sepabeads fully coated with PEI. The enzyme was strongly adsorbed on the support (no desorption of the invertase was found under conditions in which all of the enzyme was released from conventional anionic exchanger supports (e.g., DEAE‐agarose)). Nevertheless, the enzyme could still be desorbed after its inactivation, and new fresh enzyme could be adsorbed on the supports without detrimental effects on enzyme loading. This is a multimeric enzyme, its minimal oligomerization active state being the dimer, but under certain conditions of pH and concentration it may give larger multimers. Very interestingly, results suggested that the adsorption of the enzyme on this large and flexible polymeric bed was able to freeze some of the different oligomeric structures of the enzyme. Thus, we have found that the enzyme immobilized at certain pH values (pH 8.5) and high enzyme concentration, in which the main enzyme structure is the tetramer, was more stable than immobilized preparations produced in conditions under which oligomerization was not favorable (dimers at low enzyme concentration) or it was too high (e.g., hexamers‐octamers at low pH value). The optimal enzyme preparation remained fully active after a 15‐day incubation at 50 °C and pH 4.5 (conditions of standard industrial use) and presented an optimal temperature approximately 5 °C higher than that of soluble enzyme.

Reversible and Strong Immobilization of Proteins by Ionic Exchange on Supports Coated with Sulfate-Dextran

New and strong ionic exchange resins have been prepared by the simple and rapid ionic adsorption of anionic polymers (sulfate‐dextran) on porous supports activated with the opposite ionic group (DEAE/MANAE). Ionic exchange properties of such composites were strongly dependent on the size of the ionic polymers as well as on the conditions of the ionic coating of the solids with the ionic polymers (optimal conditions were 400 mg of sulfate‐dextran 5000 kDa per gram of support). Around 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans could be adsorbed on these porous composites even at pH 7. This interaction was stronger than that using conventional carboxymethyl cellulose (CMC) and even others such as supports coated with aspartic‐dextran polymer. By means of the sequential use of the new supports and supports coated with polyethyleneimine (PEI), all proteins from crude extracts could be immobilized. In fact, a large percentage (over 50%) could be immobilized on both supports. Finally, some industrially relevant enzymes (β‐galactosidases from Aspergillus oryzae, Kluyveromyces lactis, and Thermus sp. strain T2, lipases from Candida antarctica A and B, Candida rugosa, Rhizomucor miehei, and Rhyzopus oryzae and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recoveries and immobilization rates. After enzyme inactivation, the protein could be fully desorbed from the support, and then the support could be reused for several cycles. Moreover, in some instances the enzyme stability was significantly improved, mainly in the presence of organic solvents, perhaps as a consequence of the highly hydrophilic microenvironment of the support.

Reversible immobilization of glucoamylase by ionic adsorption on sepabeads coated with polyethyleneimine.

Glucoamylase (GA) from Aspergillus niger was immobilized via ionic adsorption onto DEAE‐agarose, Q1A‐Sepabeads, and Sepabeads EC‐EP3 supports coated with polyethyleneimine (PEI). After optimization of the immobilization conditions (pH, polymer size), it was observed that the adsorption strength was much higher in PEI‐Sepabeads than in Q1A‐Sepabeads or DEAE‐supports, requiring very high ionic strength to remove glucoamylase from the PEI‐supports (e.g., 1 M NaCl at pH 5.5). Thermal stability and optimal temperature was marginally improved by this immobilization. Recovered activity depended on the substrate used, maltose or starch, except when very low loading was used. The optimization of the loading allowed the preparation of derivatives with 750 IU/g in the hydrolysis of starch, preserving a high percentage of immobilized activity (around 50%).

Very Strong But Reversible Immobilization of Enzymes on Supports Coated With Ionic Polymers

In this chapter, the properties of tailor-made anionic exchanger resins based on films of large polyethylenimine polymers (e.g., molecular weight 25,000) as supports for strong but reversible immobilization of proteins are shown. The polymer is completely coated, via covalent immobilization, the surface of different porous supports. Proteins can interact with this polymeric bed, involving a large percentage of the protein surface in the adsorption. Different enzymes have been very strongly adsorbed on these supports, retaining enzyme activities. On the other hand, adsorption is very strong and the derivatives may be used under a wide range of pH and ionic strengths. These supports may be useful even to stabilize multimeric enzymes, by involving several enzyme subunits in the immobilization.

Síntesis de nanopartículas de ácido poli-láctico cargadas con antibióticos y su actividad antibacteriana contra Escherichia coli O157:H7 y Staphylococcus aureus resistente a meticilina

Introduccion. Las nanoparticulas polimericas constituyen una herramienta nanotecnologica que podria ayudar a combatir los microorganismos patogenos que han desarrollado resistencia a los antibioticos convencionales. Objetivo. Sintetizar nanoparticulas de acido polilactico cargadas con ofloxacina y vancomicina, y determinar su actividad antibacteriana frente a Escherichia coli O157:H7 y Staphylococcus aureus resistente a la meticilina (SARM). Materiales y metodos. Las nanoparticulas de acido polilactico cargadas con ofloxacina y vancomicina se sintetizaron utilizando el metodo de emulsion y evaporacion de solvente. Se caracterizaron mediante dispersion de luz en modo dinamico, electroforesis Doppler con laser y microscopia electronica de barrido (S-TEM). Se evaluo la actividad antibacteriana in vitro de las nanoparticulas de acido polilactico con ofloxacina contra E. coli O157:H7 y nanoparticulas de acido polilactico con vancomicina contra SARM, mediante el metodo de microdilucion en caldo. Resultados. Se obtuvieron nanoparticulas polimericas con tamanos inferiores a 379 nm y carga superficial positiva de hasta 21 mV. Las nanoparticulas cargadas con ofloxacina presentaron una concentracion inhibitoria minima (CIM50) de 0,001 μg/ml frente a E. coli O157:H7, valor 40 veces menor que la concentracion de antibiotico libre necesaria para lograr el mismo efecto (CIM50=0,04 μg/ml). Para SARM, las nanoparticulas mejoraron la potencia farmacologica in vitro de la vancomicina al exhibir una MIC50 de 0,005 μg/ml, comparada con la de 0,5 μg/ml del antibiotico libre. Conclusiones. Se mejoro el efecto antibacteriano de la ofloxacina y la vancomicina incorporadas en la matriz polimerica de acido polilactico. Las nanoparticulas polimericas constituirian una alternativa para el control de cepas bacterianas de interes en salud publica.INTRODUCTIONnPolymeric nanoparticles are promising nanotechnology tools to fight pathogenic bacteria resistant to conventional antibiotics.nnnOBJECTIVEnTo synthesize polylactic acid nanoparticles loaded with ofloxacin and vancomycin, and to determine their antibacterial activity against Escherichia coli O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA).nnnMATERIALS AND METHODSnWe synthesized ofloxacin or vancomycin loaded polylactic acid nanoparticles by the emulsification-solvent evaporation method, and characterized them by dynamic light scattering, laser Doppler electrophoresis and scanning electron microscopy. We evaluated in vitro antibacterial activity of ofloxacin- and vancomycin-loaded polylactic acid nanoparticles against E. coli O157:H7 and MRSA using the broth microdilution method.nnnRESULTSnOfloxacin- and vancomycin-loaded polylactic acid nanoparticles registered a positive surface charge density of 21 mV and an average size lower than 379 nm. In vitro minimum inhibitory concentration (MIC50) of ofloxacin-polylactic acid nanoparticles was 0,001 μg/ml against E. coli O157:H7, i.e., 40 times lower than the free ofloxacin (MIC50: 0.04 μg/ml), indicating enhanced antibacterial activity while the in vitro MIC50 of vancomycin-polylactic acid nanoparticles was 0,005 μg/ml against MRSA, i.e., 100 times lower than that of free vancomycin (MIC50: 0.5 μg/ml).nnnCONCLUSIONnPolylactic acid nanoparticles loaded with ofloxacin and vancomycin showed a higher antibacterial activity. Polymeric nanoparticles are a possible alternative for drug design against pathogenic bacterial strains of public health interest.