Evolving an Enzyme for Industrial Application

Abstract

Enzymes are very efficient biocatalysts. While there are many examples of applications of enzymes in commercial or industrial processes, the true potential of enzyme technology is yet to be reached. Protein engineering, particularly directed evolution, is a powerful tool for tailoring enzymes for specific tasks. This thesis is concerned with the use of directed evolution to enhance the physical and catalytic properties of a lipase, Lip 3 that was originally identified in Drosophila melanogaster. The gene for Lip 3, CG8823, had been synthesized with codons optimized for expression in E. coli. It expressed very well, but very little of the protein was soluble. Biochemically, Lip 3 was poorly characterized. However, there was good reason to believe that it, like most lipases, it possessed an alpha/beta hydrolase fold that formed the catalytic core onto which was grafted a cap domain responsible for binding substrates. Lip 3 was not an enzyme that was well suited for an industrial application. The idea was to see if I could bring about significant changes with directed evolution so that it could be considered a candidate for inclusion in a commercial product. That is one of the uses of directed evolution, to evolve enzymes for industrial purposes, rather than looking for naturally occurring enzymes that had suitable properties. There are very few naturally occurring enzymes that are stable in laundry powders or dishwashing detergents. They represent a harsh and challenging environment and their components, surface active agents and proteases as well as oxidizing agents are reagents that inactivated Lip 3 and most other enzymes. This thesis describes the process by which the catalytic properties of Lip 3 were enhanced as was the tolerance for surface active agent, proteases and oxidizing agents. The project was divided into three parts: 1) Evolution of stability, solubility and increased activity towards short chain esters; 2) Evolution of increased activity towards naturally occurring oils as well as tolerance to surface active agents and proteases and 3) Tolerance to hydrogen peroxide. At the end of each stage, variants were characterized with respect to physical and catalytic properties. The changes identified in selected variants were mapped onto a model of the Lip 3 structure to gain some insight into the relationship between sequence, structure and catalytic function.