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Enantiopure epichlorohydrin (ECH) is an important building block for the production of chiral pharmaceuticals and agrochemicals such as L- carnitine, β-blockers and pheromones. ECH is also used as an intermediate in the preparation of paper chemicals, synthetic rubbers, epoxy resins, as a starting material in the production of adhesives, surfactants, insecticides, coatings, plasticizers and solvents. Conventional ways of producing ECH have disadvantages such as relatively low reaction yields (around 50 %), high energy consumption and use of a metal catalyst (cobalt-salen complexes) that lead to environmental pollution. Because all mentioned, biocatalytic methods for the preparation of chiral ECH are getting much attention. The advantages of using biocatalytic methods are high enantioselectivity, low production cost, extensive enzyme sources and environmentally friendly conditions. Halohydrin dehalogenases (HHDHs) can be used as biocatalysts for the ring-closure of a haloalcohol to an epoxide (Figure 1). HHDHs belong to the superfamily of the short-chain dehydrogenase/reductase proteins and structurally they are homotetramers. HHDHs are divided into subtypes from letter A to G. These enzymes are easily obtained from different bacterial hosts and can be expressed in recombinant form in Escherichia coli. In this study, enzymes from Corynebacterium sp. N- 1074 (HheB) and modified HHDH from Mycobacterium sp. GP1 (HheB2-T120A), belonging to group B, were utilised for (R)-ECH synthesis (Figure 1). The kinetic characterisation was done with both enzymes and kinetic models for enzymatic synthesis of (R)-ECH from 1, 3-dichloro- 2-propanol (DCP) were developed. The kinetic characterisation is a crucial step in the development of a mathematical model as it offers a better understanding of reaction which leads to the optimization of the reaction conditions. Except kinetic characterization, the stability of enzymes during incubation with different concentrations of (R)- ECH and DCP was assessed. The experiments were done by incubating the enzymes with a different substrate (DCP) or product ((R)-ECH) concentration over 24 hours period. Enzyme activity assay was performed spectrophotometrically through absorption differences between para-nitro-2-bromo- 1- phenylethanol and para-nitrostyrene oxide. It was found that (R)-ECH has no considerable effect on enzyme stability, while DCP decreased activity of both enzymes in concentration-dependent manner. Subsequently, validation of models was performed in batch reactor experiments. As implied by monitoring enzyme stability during incubation, it was confirmed within validation experiments that substrate deactivates the enzyme. The deactivation rate constants were obtained through batch reactor experiments and it was concluded that the deactivation rate is dependent upon the concentration of the substrate. Mathematical modelling is an essential step in the optimization of a process. The model can be used to predict reaction profiles in various reactor types and under different conditions. That information can be used for selection of the appropriate reactor type and process conditions. With this approach, mathematical modelling can also be used for the scale-up of the process. Acknowledgements: Work was supported by CAT PHARMA (KK.01.1.1.04.0013), a project co-financed by the Croatian Government and the European Union through the European Regional Development Fund - the Competitiveness and Cohesion Operational Programme. |
