Chemo-enzymatic epoxidation: Activity and stability of Candida antarctica lipase B

Growing concerns about climate change and depleting fossil feedstocks are driving a shift within the chemical industry from energy-intensive processes to more environmentally sound and sustainable processing using renewable feedstocks. One such process is the utilization of lipases for the chemo-enzymatic epoxidation of vegetable oils and fatty acids. The lipase mediated route for production of fatty epoxides is a two-step process in which the enzyme catalyzes the first step; the formation of a peracid from a carboxylic acid and hydrogen peroxide. This thesis is based on research with the objective to develop a cost-effective, environmentally friendly enzyme-based industrial process for the production of fatty epoxides. A solvent-free process was first established using immobilized Candida antarctica lipase B (Novozym 435). Both rapeseed methyl ester and tall oil derivatives gave biodegradable epoxide products with high degree of epoxidation, but being a by-product of the pulp and paper industry, tall oil scored much better as a raw material for chemical production from an environmental point of view. The enzyme was prone to deactivation under the given conditions and hydrogen peroxide was found to cause by far the most severe deactivation of the enzyme. In order to prolong the lifetime of the enzyme, the hydrogen peroxide was added to the process continuously rather than all at once. It was found that maintaining the peroxide concentration at a low level in the reactor was an even better alternative for extending the enzyme lifetime. A thorough study of the enzyme was also performed, to shed light on the mechanism behind the deactivation. Since an industrial process would involve the immobilized enzyme, a method was developed to evaluate the changes occurring in Novozym 435 at the amino acid level upon treatment with hydrogen peroxide without detaching the enzyme from the carrier. All four methionines and two tryptophans present in the enzyme were oxidized. Moreover, cystine was oxidized to cysteic acid which implied the disruption of all three disulfide bridges in CalB. Circular dichroism and dynamic light scattering investigations verified that profound changes in the secondary structure occurred upon exposure to hydrogen peroxide. These results are laying the basis for mutagenesis experiments, recently initiated, with the aim of developing an improved variant of this enzyme