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1.3.2. Particulate forms of supports applied in biocatalysis

1.3.2.2. Microspheres and nanospheres

Polymeric microspheres are considered excellent supports for immobilization of enzymes, because they can be produced easily in a wide variety of compositions, and are suitable for functionalization by a variety of activation methods. Monodisperse micron-sized microspheres are characterized by large specific surface area, high absorption capacity, and excellent surface reactivity. Dispersion polymerization is the common method for preparing non-porous microspheres of narrow size distribution in a single step. Polymeric microspheres containing functional groups such as aldehydes, chloromethyls, oxiranes, hydroxyls, and thiols have been used for covalent binding, via various activation methods.

Moreover, it was an increasing interest to modify the surface of microspheres, without changing their bulk properties, since changing the surface composition and wetability properties could improve immobilization. Surface modification has been accomplished by different methods: high-energy radiation, ozone exposure, graft polymerization on core microspheres, etc. (Omer-Mizrahi and Margel, 2010).

Li et al. used poly(methyl methacrylate) microspheres prepared and functionalized with aldehyde groups for immobilization of pepsin. To obtain the functionalized microspheres, methyl methacrylate was polymerized at 70°C for 10 h, then 8% acrolein was added as functional monomer and the mixture was kept for another 10 h in the same conditions.

Aldehyde groups can react under mild conditions with primary amino groups of the enzyme, forming Schiff bases (Fig. 1.9). Modified microspheres were approximately mono-sized and about 7 µm in average diameter. The maximum enzyme loading on this carrier was 76.8 mg × g−1, higher than on other supports. The relative activity of immobilized enzyme, calculated as the ratio of Vmax kinetic constant values of immobilized and free enzyme, was 74% (Li et al., 2004).

Fig. 1.9. Covalent immobilization of enzymes on poly(methyl methacrylate) microspheres modified with aldehyde groups (Li et al., 2004)

In a second report of the same group, the particle size distribution of the functionalized poly(methyl methacrylate) microspheres, determined by Malvern size analyzer, showed a very narrow size distribution. The obtained uniformity coefficient of particles was 0.2003.

Uniformity is a measure calculated by the Malvern Mastersizer software (Malvern Instruments) and represents the measure of absolute deviations from the median.

Uniformity provides measure of spread of the distribution, or in this case, the degree of sorting. Samples with high uniformity values have a broad range of particle sizes while lower uniformity values mean smaller range of particle sizes (Alderliesten, 1991). The average diameter, determined by light scattering, was 6.1 µm, and specific surface area of particles was up to 0.51 m2/g (Wu et al., 2005).

Omer-Mizrahi and Margel obtained micrometer sized, uniform polyepoxide particles by redox graft polymerization of glycidyl methacrylate on oxidized polystyrene and polydivinylbenzene microspheres. Hydroperoxide conjugated polystyrene and poly(divinyl benzene) microspheres were produced by controlled ozonolysis of the appropriate microspheres, obtained by usual polymerization techniques. Polyepoxide conjugated microspheres were then formed by redox graft polymerization of glycidyl methacrylate on the hydro peroxide-conjugated microspheres. In this manner, microspheres with different properties, e.g., size, size distribution, shape, surface morphology, surface area, etc., have been prepared by changing the reaction parameters. SEM analysis demonstrated that the porous structure, shape, surface morphology, size (3.12 ± 0.08 µm), and size distribution of poly(divinylbenzene) microspheres (Fig. 1.10A) were not significantly influenced following the ozonolysis process (Fig. 1.10B). Similarly, the size of particles was preserved by the graft polymerization of lower amount of glycidyl methacrylate (3.13 ± 0.08 µm, Fig. 1.10C), and slightly increased by the graft polymerization of higher glycidyl

methacrylate amount (3.2 ± 0.09 µm, Fig. 1.10D). This small size change was explained by graft polymerization of glycidyl methacrylate only within the pores the oxidized poly(divinylbenzene) particles in the first case, and also on the surface of particles in the second. Increased roughness of the observed composite particles was also observed at increased glycidyl methacrylate amount (Omer-Mizrahi and Margel, 2010).

Fig. 1.10. SEM photomicrographs of the poly(divinylbenzene) microspheres (A), oxidized poly(divinylbenzene) microspheres (B) and grafted composite microspheres prepared by redox graft polymerization of lower (C) and higher (D) amount of glycidyl methacrylate on

the oxidized microspheres (Omer-Mizrahi and Margel, 2010)

Trypsin was covalently bound to polyepoxide conjugated microspheres by reaction of epoxide groups of the particles with primary amino groups of enzyme. Immobilization on polystyrene-polyglicidylmethacrylate composite microspheres resulted in up to 435 µg bound trypsin on 1 mg microspheres (87% efficiency), with 40% relative activity compared to the free enzyme (Omer-Mizrahi and Margel, 2010).

Several reports were issued on the topic of immobilization of enzymes on non-magnetic nanoparticles, as reviewed by Ansari and Husain (Ansari and Husain, 2012). Miletic et al.

have found that immobilization of lipase on polystyrene nanoparticles resulted in a remarkably increase of activity. Polystyrene nanoparticles were prepared by a nanoprecipitation process, and immobilization of Candida antarctica B lipase has been accomplished by adsorption. The shape of polystyrene nanoparticles was spherical (Fig.

1.11), and hydrophobic interactions were the driving forces of immobilization. The highest

enzyme loading, 248 µg/g support was observed at pH 6.8, while the activity of the immobilized enzyme was 1.8 fold higher, compared to the free enzyme (Miletic et al., 2010).

Fig. 1.11. Scanning electron micrograph images of polystyrene nanoparticles utilized for lipase immobilization (Miletic et al., 2010)

A quite unusual support, 5-8 nm sized thiolated gold nanoparticles, has been studied for immobilization of glucose oxidase by covalent binding via N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. Gold nanoparticles have been identified as a biocompatible material with interesting optical and electronic properties, providing a compatible environment for enzymes, as well. Glucose oxidase immobilized on thiolated gold nanoparticles could have biosensor applications (Pandey et al., 2007).