Composite particles

As it was shown, either inorganic or organic supports possess advantages and disadvantages. The appropriate immobilization method and support should be selected to maximize the advantages and minimize the disadvantages. It looks obvious that a combination of both support types could merge the advantages and lead to increased catalytic efficiency of the immobilized enzyme. Manufacture of such hybrid inorganic/organic supports is usually realized by coating inorganic particles with organic layers containing reactive functional groups or groups suitable to be activated for attachment of the enzyme. There are two composite particle types used for preparation of hybrid supports: magnetic particles and silica particles.

Polymer-coated magnetic beads

Magnetic microbeads can be produced in different ways, but the usual methodology involves the coating of magnetically susceptible particles with synthetic polymers having convenient sites for attachment of enzymes. Alternatively, conventional supports can be post-magnetized by treatment with magnetic ferro fluids, without loss of subsequent binding activity (Demirel et al., 2004).

To increase the loading amount of enzymes on magnetic particles and improve the stability of immobilized enzyme, various polymers have been grafted to functionalize the surface of magnetic particles. The obtained carriers contain active groups like epoxy, amino, etc., which could efficiently bind the enzyme, without or with a cross-linking agent.

Sureshkumar and Lee used chitin as a protective and dispersive matrix for the preparation of magnetic granules in the co-precipitation process. The magnetic-chitin particles with average size about 1.5 µm were modified with dopamine, followed by self-polymerization of dopamine and simultaneous coating onto the magnetic-chitin particles, offering adherent surface for enzymes. α-Amylase was immobilized on the polydopamine coated magnetic-chitin particles. Probably, self-oxidation of dopamine generated quinone groups in polydopamine layer, which initiated nucleophilic addition reaction with free –SH and – NH2 groups of the enzyme, leading to immobilization. Pretreatment of the polydopamine surface with glutaraldehyde enhanced the enzyme immobilization efficiency (Sureshkumar and Lee, 2011).

Magnetic hydrogel microspheres were synthesized by copolymerization of N-isopropylacrylamide, methacrylic acid and N,N’-methylen-bis-acrylamide in presence of magnetite particles (10-20 nm diameter). Trypsin was covalently immobilized on this carrier in high yield, using carbodiimide as activator (Kondo and Fukuda, 1997).

Bayramoğlu et al. obtained magnetic poly(glycidylmethacrylate-methylmethacrylate-ethyleneglycol dimethacrylate), p(GMA-MMA-EGDMA) carrier for immobilization of chloroperoxidase (Fig. 1.8). The epoxy groups of the ferric beads resulted in the first step were converted into amino groups during the next step by ammonolysis. The resulted aminated magnetic beads were used for the covalent immobilization of the enzyme via glutaraldehyde coupling. The maximum amount of immobilized chloroperoxidase on the magnetic beads was not high, 2.94 mg/g support (Bayramoğlu et al., 2008).

H2

Fig. 1.8. Reaction scheme of chloroperoxidase immobilization onto magnetic poly(glycidylmethacrylate-methylmethacrylate-ethyleneglycol dimethacrylate beads

(Bayramoğlu et al., 2008)

Another carrier with epoxy groups, based on a magnetic nanoparticles grafted copolymer, was obtained from 2-hydroxyethyl methacrylate and dimethyl diallyl ammonium chloride, by surface-initiated radical polymerization on Fe3O4 nanoparticles, previously derivatized with vinyl-triethoxysilane. The diameter of the resulted magnetic polymer particles was 80-100 nm. The amino groups of lipase reacted with the reactive epoxy groups of this composite support in mild conditions. In the best conditions, the loading amount of

Candida rugosa lipase was 68 mg/g support, and the activity retention about 60%, compared to the free enzyme (Liu et al. 2011).

Tanyolac and Özdural prepared uniform and spherical magnetic particles in the diameter range of 125-250 µm, from nitrocellulose and magnetite through a modified solvent evaporation technique. Hydroxyl groups on the particle surface were activated with glutaraldehyde for covalent immobilization of glucoamylase, obtaining very high recovery (94.7%) of the enzymatic activity (Tanyolac and Özdural, 2000). The same enzyme, glucoamylase, was immobilized by Bahar and Celebi on a different polymer-coated magnetic support, magnetic polystyrene, using the same solvent evaporation technique.

Since polystyrene does not contain active groups for covalent binding of enzymes, it was mixed with an additive polymer containing reactive aldehyde groups (Bahar and Celebi, 1999). The activity of immobilized glucoamylase was 70% of the free enzyme, while the activity half life of the bound enzyme was 190 h, compared to 34 h for the free enzyme (Bahar and Celebi, 1998).

Arica et al. utilized as composite support magnetic poly(methylmethacrylate) microspheres, holding a 6-carbon spacer-arm (hexamethylene diamine) attached to the carbonyl groups of the magnetic polymer particles. Glucoamylase was covalently immobilized through this spacer-arm, using carbodiimide or cyanogen bromide as coupling agent. The activity yield of the immobilized glucoamylase was 57% for carbodiimide coupling and 73% for cyanogen bromide coupling (Arica et al., 2000).

Commercial paramagnetic, nonporous, polyacrolein beads (Magna-bind) preactivated with aldehyde groups for coupling, were also tested as supports for immobilization of glucose oxidase, urease, and α-amylase. Up to 15 µg active enzyme loading was achieved on 1 mg coated beads (Varlan et al., 1996).

The applications of magnetic support to enzyme immobilization are mainly based on the magnetic properties of the solid-phase that enables a rapid separation in a magnetic field.

Moreover, the magnetic supports can be easily stabilized in a fluidized bed reactor for continuous operation of enzyme by applying an external magnetic field and the use of magnetic supports can also reduce capital and operation costs (Xie et al., 2009). Utilization of coated magnetic carriers for enzyme immobilization is a promising method, but there are several technical problems that still restrict application of such a technology at industrial scale. The magnetic carriers prepared could have abnormal shape, wide size distribution and nonhomogeneous content of magnetic core. In many cases, impurities such

as emulsions are mixed in the shell during the preparation process. The reported results were obtained at laboratory scale, while large scale preparation of magnetic carriers and modification of their surface still need to be handled before industrial-scale implementation (Zhang et al., 2007).

Coated porous silica particles

Porous silica is one of the most widely used immobilization carriers, as it is commercially available in a wide variety of shapes and sizes with different porosities and contains superficial Si-OH groups that can be modified by organosilane-coupling agents or by coating with different polymers.

The methodology to obtain silica-gel particles coated with polymers was already available, as such composites, particularly polyamines, were intensively studied as stationary phases for chromatography (Gailliez-Degremont et al., 1997). The polymers employed as coating materials should have appropriate structure, chemical composition and suitable molecular weight, since all these parameters may influence the coating efficiency. Functional groups of polymers must interact strongly with the silica surface by formation of hydrogen bonds or van der Waals interactions. In optimal conditions, the obtained polymer-silica particles may combine the advantageous properties of both inorganic support (mechanical strength and high specific surface) and bound polymer (functionality and selectivity). Polymers utilized for surface modification of silica particles were polyethyleneimine (poly(4-vinylpyridine), copolymers of 1-vinyl-2-pyrrolidone and maleic anhydride, polyacrylonitrile, etc. Godjevargova et al., immobilized glucose oxidase on acrylonitrile copolymer/silica gel hybrid supports obtained from Silica gel 40 and Silica gel 100 coated with acrylonitrile copolymer (LukOil-Neftochim) functionalized with ethylenediamine.

The best covalently bound enzyme (using 0.5% glutaraldehyde as cross-linking agent) preserved 67% of the free enzyme activity, but was more stable compared to glucose oxidase immobilized on pure silica gel and the free enzyme (Godjevargova et al., 2005).

The same research group investigated several other polyacrylonitrile copolymers for coating the surface of silica gel: acrylonitrile-vinyl pyridine, acrylonitrile-vinylimidazole, N,N-dimethylaminoethylmethacrylate, acrylonitrileacrylic acid, acrylonitrile-2,6-dichlorphenilmaleimide, acrylonitrile-maleic anhydride, acrylonitrile-hydroxyethyl-methacrylate, and acrylonitrile-methylmethacrylate and Na-vinylsulfonate. Covalent

binding and ionic adsorption were employed as immobilization methods, depending on the polymer structure. The highest relative activity (97%) was obtained for glucose oxidase immobilized on silica gel coated with poly(acrylonitrile-co-vinylimidazole) by ionic adsorption (Godjevargova et al., 2006).