Victoria Gascón

Designing Functionalized Mesoporous Materials for Enzyme Immobilization: Locating Enzymes by Using Advanced TEM Techniques

One of the widely accepted uses of ordered mesoporous materials is as supports of enzymes for biotechnological applications. Enzymes have been trapped, anchored, or encapsulated in organized porous networks of the mesoporous range (2–50 nm). The reactivity of the surface of mesoporous materials has enabled the synthesis of various supports by using different forces for the immobilization process. To design catalysts for specific applications, we have developed functionalized mesoporous materials with tunable hydrophobicity for the immobilization of lipase. More recently, we moved to the immobilization of laccase with amino‐functionalized ordered mesoporous materials. In this case, it is required to use pore expanders along with optimized functionalization techniques. Advanced TEM techniques have been applied to locate not only the functional groups but also the macromolecules inside the silica matrix.

Mesoporous Silicas with Tunable Morphology for the Immobilization of Laccase

Siliceous ordered mesoporous materials (OMM) are gaining interest as supports for enzyme immobilization due to their uniform pore size, large surface area, tunable pore network and the introduction of organic components to mesoporous structure. We used SBA-15 type silica materials, which exhibit a regular 2D hexagonal packing of cylindrical mesopores of uniform size, for non-covalent immobilization of laccase. Synthesis conditions were adjusted in order to obtain supports with different particle shape, where those with shorter channels had higher loading capacity. Despite the similar isoelectric points of silica and laccase and the close match between the size of laccase and the pore dimensions of these SBA-15 materials, immobilization was achieved with very low leaching. Surface modification of macro-/mesoporous amorphous silica by grafting of amine moieties was proved to significantly increase the isoelectric point of this support and improve the immobilization yield.

Hybrid periodic mesoporous organosilica designed to improve the properties of immobilized enzymes

Authors thank the Spanish MINECO for the nancial support through the project MAT 2012-31127. We thank D. Ramiro Martinez (Novozymes, Spain) for his kind help with the supply of enzymes. V. G. acknowledges Ministerio de Educacion, Cultura y Deporte for a FPU PhD fellowship (AP2010-2145).Two types of highly ordered periodic mesoporous organosilicas have been synthesized as tailor-made supports to immobilize two different enzymes, lipase and laccase. These materials provide an environment where abundant organic groups in close vicinity to the enzyme surface generate a high chemical affinity, which results in high values of enzyme loading, catalytic activity and stabilization. A hydrophobic periodic mesoporous organosilica support (PMO) with a highly ordered hexagonal arrangement of parallel pore channels with a diameter around 7 nm, containing framework hydrocarbon groups (ethylene), was used for lipase immobilization. A novel periodic mesoporous aminosilica (PMA), containing secondary amine groups in its framework and having expanded pores was synthesized and studied for immobilization of a larger enzyme, namely laccase. The synthesis conditions were adjusted, using bis[(3-trimethoxysilyl)propyl]amine and 1,2-bis(trimethoxysilyl)ethane as framework co-precursors and 1,3,5-triisopropylbenzene as micelle expander for producing large pores (>10 nm). The properties of this multifunctional PMA having hydrophilic amino groups and hydrophobic ethylene/propylene groups within the framework were studied. This work compares the confinement of lipase and laccase enzymes in the pores of these hybrid organosilica materials and its effect on immobilization and stabilization parameters. Laccase immobilized on PMA and lipase immobilized on PMO exhibited higher stability in solvents (ethanol and methanol, respectively) compared to enzymes supported on functionalized silica materials with pending organic groups on the surface. High retention of enzymes inside the pores of these materials has been achieved and leaching has been fully prevented. These results can be attributed to the different interactions (hydrophobic, electrostatic and hydrogen bonding) established between the surfaces of the enzyme and the PMO/PMA support, which are enhanced by an optimum pore size adjusted to the enzyme dimensions.

Oriented Coimmobilization of Oxidase and Catalase on Tailor-Made Ordered Mesoporous Silica

Mesoporous silica materials are promising carriers for enzyme immobilization in heterogeneous biocatalysis applications. By tailoring their pore structural framework, these materials are designable for appropriate enzyme binding capacity and internal diffusivity. To supply O2 efficiently to solid-supported immobilized enzymes represents a core problem of heterogeneously catalyzed oxidative biotransformations. In this study, therefore, we synthesized and compared three internally well-ordered and two amorphous silica materials as enzyme carriers, each of those with pore sizes of ≥10 nm, to enable the coimmobilization of d-amino-acid oxidase (79 kDa) and catalase (217 kDa). Both enzymes were fused to the silica-binding module Zbasic2 to facilitate their selective and oriented immobilization directly from crude protein mixtures on native silica materials. Analyzing the effects of varied pore architecture and internal surface area on the performance of the immobilized bienzymatic system, we showed that a uniform pore structural framework was beneficial for enzyme loading (≥70 mg protein/g carrier), immobilization yield (≥90%), surface and pore volume filling without hindered adsorption, and catalytic effectiveness (≥60%) of the coimmobilizate. Using the best carrier LP-SBA-15, we obtained a solid oxidase-catalase preparation with an activity of 2000 μmol/(min g_material) that was recyclable and stable during oxidation of d-methionine. These results affirm a strategy of optimizing immobilized O2-dependent enzymes via tunable internal structuring of the silica material used as carrier. They thus make a significant advance toward the molecular design of heterogeneous oxidation biocatalysts on mesoporous silica supports.

Location of laccase in ordered mesoporous materials

The functionalization with amine groups was developed on the SBA-15, and its effect in the laccase immobilization was compared with that of a Periodic Mesoporous Aminosilica. A method to encapsulate the laccase in situ has now been developed. In this work, spherical aberration (Cs) corrected scanning transmission electron microscopy combined with high angle annular dark field detector and electron energy loss spectroscopy were applied to identify the exact location of the enzyme in the matrix formed by the ordered mesoporous solids.