The stabilisation of epoxide hydrolase activity

Abstract

Biocatalysis and enzyme technology represent significant research topics of contemporary biotechnology. The immobilisation of these catalysts on or in static supports serves the purpose of transforming the catalyst into a particle that can be handled through effortless mechanical operations, while the entrapment within a membrane or capsule leads to the restraint of the enzyme to a distinct space. This confinement leads to a catalyst with a superior stability, and cell durability under reaction conditions. Epoxide hydrolase is a widely available co-factor independent enzyme, which is known to have remarkable chemio-, regio- and stereoselectivity for a wide range of substrates. Recently it was found that certain yeasts, including Rhodosporidium toruloides, contain this enzyme and are able to enantioselectively catalyse certain hydrolysis reactions. The objective of this project was four-sided: a) to immobilise Rhodospridium toruloides in an optimised immobilisation matrix (calcium alginate beads), for the kinetic resolution of 1.2- epoxyoctane in order to obtain an optically pure epoxide and its corresponding vicinal diol, b) to determine the effect of immobilisation on activity as well as stability of the enzyme and gain better understanding of the parameters that influence enzyme activity in a support, c) to determine the effect of formulation parameters on some of the bead characteristics and, d) to gain some insight in the distribution of epoxide and diol in the water and bead phases and the formulation parameters that have an effect thereon. Rhodospridium toruloides was immobilised in calcium alginate beads consisting of different combinations of alginate and CaCl2 concentrations. Best results were obtained with a combination of 0,5 % (m/v) alginate and 0,2 M CaC12. The immobilised cells exhibited lower initial activity. but more than 40 times the residual activity of that of the free cells after a 12-hour storage period. Both the immobilised and free cells exhibited an increase in reaction rate (V) with an increase in substrate concentration. An increase in the alginate concentration lead to the formation of smaller beads, but a decrease in enzume activity, while an increase in the CaCl2 solution concentration had no effect on bead diameter or enzyme activity. Epoxide diffused preferentially into the beads (± 96 %), and the diol into the water phase, which leads to the natural separation of the epoxide and the diol. The CaCl2 concentration affected epoxide diffusion with no effect on diol diffusion, which opens up the possibility to regulate the diffusion of epoxide into the beads. Although only a very small fraction of the epoxide inside the beads could be extracted, the alginate proved to be chirally selective for the (R)-epoxide, improving the reaction efficiency by increasing the % ee, of the epoxide extracted from the beads between 26 % and 43 %. The possibility to develop a system where the product is formed, purified and concentrated in a one-step reaction by extracting the product from the bead phase was clearly demonstrated.