Improving enzyme properties through directed evolution

Abstract

Enzymes are specific and economical biocatalysts and as such are highly desirable synthetic tools. While there are many examples of successful applications of enzymes into industrial or commercial processes, the vast potential of enzyme technology is yet to be reached. Protein engineering, specifically directed evolution, is a powerful tool for generating enzymes tailored for specific industrial tasks. The ease of this process dictates the availability of enzymes and their diversity as biocatalysts. This thesis is concerned with the use of directed evolution to enhance the physical and catalytic properties of dienelactone hydrolase, a small monomeric alpha/beta hydrolase fold enzyme. Solubility is an important property dictating the suitability of enzymes as industrial biocatalysts in terms of both cost and functional utility. This thesis describes the adaption and use of the dihydrofolate reductase fusion reporter system to select for more soluble dienelactone hydrolase variants. The selection system was modified to incorporate a pre-culturing period, then used to identify mutations in solvent accessible locations that appeared to offer increased expression and solubility. While the original system was capable of selecting for improvements to the solubility of inherently insoluble proteins, the modified system can be used to select for further improvements. This work provides a solid foundation for the continued use of the modified dihydrofolate reductase fusion reporter system. This thesis also discusses linked directed evolution experiments that were designed to rapidly alter and enhance the substrate specificity of dienelactone hydrolase in favour of non-physiological p-nitrophenyl ester substrates. The best variants possessed in excess of 2000-fold improvements in kcat/Km compared to the native enzyme. Active site mutations were able to accumulate rapidly, despite most being detrimental to the overall stability of the enzyme, due to constant monitoring and maintenance of enzyme stability. The roles of six of the seven active site mutations were elucidated with the use of substrate docking and structural analysis. Additional work focused on the surface mutations that seemed to provide compensatory stabilising effects. These mutations were made individually and in combination to determine the roles they served in the evolution process. Two of the three mutations were shown to be thermally and chemically stabilising while the third was destabilising individually but exhibited epistasis in combination with the former mutations. This work is of interest due to the evolutionary strategy but more importantly for the insight into the evolution and relationship between sequence, structure and function of alpha/beta hydrolase fold enzymes. Work investigating and improving the stability of dienelactone hydrolase in the presence of organic co-solvents is also discussed. Eight rounds of directed evolution yielded a variant with 7 surface mutations that provided increased thermal and chemical stability. These experimental results corroborated with computational predictions, which provide promise for an alternative method to rapidly engineer chemical stability. Additional work analysing the effects of crystal packing on protein structure is also discussed. It was found that the crystalline environment caused small changes to side chain orientation, mostly at crystallographic interfaces and flexible surface loops, with only minor changes to the protein backbone.