Hanna Gustafsson

Enzymes Immobilized in Mesoporous Silica: a Physical-Chemical Perspective

Mesoporous materials as support for immobilized enzymes have been explored extensively during the last two decades, primarily not only for biocatalysis applications, but also for biosensing, biofuels and enzyme-controlled drug delivery. The activity of the immobilized enzymes inside the pores is often different compared to that of the free enzymes, and an important challenge is to understand how the immobilization affects the enzymes in order to design immobilization conditions that lead to optimal enzyme activity. This review summarizes methods that can be used to understand how material properties can be linked to changes in enzyme activity. Real-time monitoring of the immobilization process and techniques that demonstrate that the enzymes are located inside the pores is discussed by contrasting them to the common practice of indirectly measuring the depletion of the protein concentration or enzyme activity in the surrounding bulk phase. We propose that pore filling (pore volume fraction occupied by proteins) is the best standard for comparing the amount of immobilized enzymes at the molecular level, and present equations to calculate pore filling from the more commonly reported immobilized mass. Methods to detect changes in enzyme structure upon immobilization and to study the microenvironment inside the pores are discussed in detail. Combining the knowledge generated from these methodologies should aid in rationally designing biocatalyst based on enzymes immobilized in mesoporous materials.

Immobilization of lipase from Mucor miehei and Rhizopus oryzae into mesoporous silica—The effect of varied particle size and morphology

Immobilization of enzymes usually improves the recyclability and stability and can sometimes also improve the activity compared to enzymes free in solution. Mesoporous silica is a widely studied material as host for immobilized enzymes because of its large internal surface area and tunable pores. It has previously been shown that the pore size is critical both for the loading capacity and for the enzymatic activity; however, less focus has been given to the influence of the particle size. In this work the effect of particle size and particle morphology on the immobilization of lipase from Mucor miehei and Rhizopus oryzae have been investigated. Three kinds of mesoporous silica, all with 9 nm pores but with varying particle size (1000 nm, 300 nm and 40 nm) have been synthesized and were used as host for the lipases. The two lipases, which have the same molecular size but widely different isoelectric points, were immobilized into the silica particles at varied pH values within the interval 5-8. The 300 nm particles were proven to be the most suitable carrier with respect to specific activity for both enzymes. The lipase from M. miehei was more than four times as active when immobilized at pH 8 compared to free in solution whereas the difference was less pronounced for the R. oryzae lipase.

A comparison of lipase and trypsin encapsulated in mesoporous materials with varying pore sizes and pH conditions

Immobilized enzymes have an advantage over enzymes free in solution in that they are easily recovered after completed reaction. In addition, immobilization often gives enhanced stability. Entrapment of an enzyme in the pores of a mesoporous material is an attractive procedure since the enzyme is immobilized without any covalent bonding to a support which may be detrimental to the catalytic performance. The objective of this work is to compare the encapsulation and catalytic performance of lipase from Mucor miehei and trypsin from bovine pancreas, two hydrolases with rather dissimilar properties and structures. We also demonstrate the importance of the pore dimensions and the pH for proper function of the encapsulated enzyme. Mesoporous silica particles (SBA-15) with three different pore sizes (50 Å, 60 Å and 89 Å) were synthesized and hexagonal structures with narrow pore size distributions were confirmed with TEM, SAXS and N(2)-adsorption. Lipase and trypsin were encapsulated separately in the silica particles and the results indicate distinct differences between the two enzymes, both in loading capacity and catalytic activity. For trypsin the encapsulation rate and the loading capacity were large with a maximum reached at pH 7.6. The largest product yield was obtained with the particles with 60 Å pores, however, the yield was significantly lower than with free trypsin. For lipase optimal encapsulation rate and loading capacity were reached with the particles with 89 Å pores at pH 6.0 but were low compared to trypsin. However, the catalytic activity of the encapsulated lipase was more than twice as large as for free lipase, which can be explained by an interfacial activation of lipase at the silica surface.

Co-immobilization of enzymes with the help of a dendronized polymer and mesoporous silica nanoparticles

The two enzymes Aspergillus sp. glucose oxidase (GOD) and horseradish peroxidase (HRP) were co-immobilized on solid silica supports in a spatially controlled way by using mesoporous silica nanoparticles (Hiroshima Mesoporous Materials, HMM) and a polycationic dendronized polymer (denpol). The silica support was first coated with the denpol, followed by the deposition of the mesoporous silica nanoparticles into which - in a next step - GOD was adsorbed. Finally, the GOD-loaded silica nanoparticles were coated with a denpol-HRP conjugate constituting of several HRP molecules which were covalently bound to the denpol via bis-aryl hydrazone (BAH) bonds. The entire immobilization process was followed in real time with quartz crystal microbalance with dissipation monitoring (QCM-D). The activities and storage stabilities of the co-immobilized enzymes were determined by analyzing a two-step cascade reaction involving the two immobilized enzymes GOD and HRP. d-glucose and o-phenylenediamine (OPD) were used as substrates for GOD and HRP, respectively. The cascade reaction - in which intermediate hydrogen peroxide was formed from d-glucose and dissolved O2 with GOD - was shown to take place. The immobilized enzymes remained fairly stable for at least 2 weeks if stored in contact with an aqueous solution of pH = 7 at 4 °C. If, however, denpol-BAH-GOD coated HRP-loaded mesoporous silica nanoparticles were used (the reversed situation), the cascade reaction was not effective. This was probably due to slow diffusion of hydrogen peroxide from the surface-exposed GOD to the particle-trapped HRP, and/or due to an inefficient loading of active HRP inside the particles. Overall, the combination of two enzyme immobilization methodologies - enzymes adsorbed within mesoporous silica nanoparticles and enzymes adsorbed as denpol-BAH-enzyme conjugates - allows the spatially controlled localization of different types of enzymes in a simple way. Possible applications of the concept are in the field of bioelectrode fabrication.

Mesoporous silica nanoparticles with controllable morphology prepared from oil-in-water emulsions

Mesoporous silica nanoparticles are an important class of materials with a wide range of applications. This paper presents a simple protocol for synthesis of particles as small as 40nm and with a pore size that can be as large as 9nm. Reaction conditions including type of surfactant, type of catalyst and presence of organic polymer were investigated in order to optimize the synthesis. An important aim of the work was to understand the mechanism behind the formation of these unusual structures and an explanation based on silica condensation in the small aqueous microemulsion droplets that are present inside the drops of an oil-in-water emulsion is put forward.

Emulsion-based synthesis of porous silica

We review the use of various types of emulsions as media for synthesis of porous silica particles. The use of high internal phase emulsions, i.e. emulsions with a high ratio of dispersed to continuous phase, is an approach that has attracted considerable attention. Polymerization of the continuous phase followed by removal of the dispersed phase leads to a material with an even distribution of pores if the emulsion droplets are uniform in size. Another route is to use particle stabilized emulsions as template. This will lead to either hydrophilic or hydrophobic porous silica particles depending on whether the templating emulsion is oil-in-water or water-in-oil, respectively. Use of double emulsions as templates is a way to obtain porous particles with hierarchical porosity, usually both macropores and mesopores. Templating amphiphiles, which are often polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymers, are needed in order to obtain materials with highly ordered pore structure. Non-ordered mesoporous silica with small particle size and relatively large pores can be obtained by emulsifying a silica precursor together with an inert solvent in water and allowing the silica to gel within the drops. Hollow silica spheres, i.e. spherical particles with a void in the middle, can be prepared by using emulsion drops as templates around which silica polymerizes. The particles have nanometer-sized pores penetrating the shell.

Fighting Panic with Haptics

This paper presents some of the findings from the master’s thesis project Fighting Panic with Haptics! (Gustafsson and Yxhage 2016) which aimed to answer the question of if and how one could make a product to help people suffering from panic attacks and panic disorder. Through the use of design and human factors tools such as personas, user journeys and user testing we came to the conclusion that it is possible to create a physical product that can help against panic attacks. Furthermore, our findings suggest that such a product should be designed to help the persons suffering from panic attacks break their internal focus by including the world around them since internal focus on thoughts and bodily reactions was found to be one of the core triggers of the attacks. Moreover, we could conclude that design theories and methodologies are effective tools also when trying to solve complex problems outside of the conventional product design sphere.