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Improved biocatalysts based on Candida antarctica lipase B immobilization

(2009) Miletic, Nemanja

This thesis deals with an immobilization of the most widely used lipase, Candida antarctica lipase B (Cal-B), on different types of carriers. To this end, as starting carrier, a highly porous epoxy-containing copolymer was synthesized: poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [poly(GMA-co-EGDMA)]. The properties of immobilized enzymes are highly influenced by the copolymer properties, in particular, porosity characteristics.

The use of poly(GMA-co-EGDMA) was motivated by the presence of epoxy rings in its structure that makes this copolymer highly reactive. Hence, various modifications of epoxy-containing copolymer were carried out. The influence of the type of immobilization on enzyme loading and enzyme activity is assessed.

After modifying the starting epoxy-containing copolymer, we turned our attention to Cal-B itself and tried to develop carrierless immobilization methods. Since, it was proved that Cal-B may successfully react with epoxy groups from the poly(GMA-co-EGDMA), modification and cross-linking with epoxy-containing compounds were chosen. With these treatments, in most of cases, activity and thermal stability of the Cal-B derivatives were significantly improved.

The standard procedure to evaluate the surface topography of immobilized enzyme and to characterize the architecture down to the molecular level is enzyme immobilization on flat surfaces. In order to immobilize Cal-B on silicon surfaces, pre-treatments with aminopropyltriethoxysilane and subsequently with glutaraldehyde were performed. Final enzyme properties are strictly determined by the structure and architecture of the aminopropyltriethoxysilane film, and all subsequent steps cannot significantly change the immobilization protocol. We have found the reaction conditions that favour creation of the perfect fully-covered aminopropyltriethoxysilane monolayer.

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