Enzymes are not only essential for the workings of every cells in our bodies. They are also becoming more important for our daily life as they play an increasing role in production of and as part of everyday items. Thus, increasing the efficiently of these enzymes can result in better and possible cheaper products. One aspect that can have a large impact on the efficiency of an enzyme is its stability. By increasing the enzyme stability production cost and time can be reduced, and consumers will have a better product with longer activity.
In the past it was only possible to increasing enzymes stability by randomly generate mutants and lengthy screening processes to identify the best new mutants. However, with the increase in available genomic sequences of thermophilic or hyperthermophilic organisms a world of enzymes with intrinsic high stability are now available. As these organisms are adapted to life at high temperatures so are their enzymes, as a result the high stability is accompanied by low activity at moderate temperatures. Thus, much effort had been put into decoding the mechanisms behind the high stability of the thermophilic enzymes. The hope is to enable scientist to design enzymes with high stability and activity at a target temperature. This thesis presents an investigation of an hyperthermophilic esterase, and how post-translational modifications (PTM) and other factors affect the stability of this enzyme. As part on the ongoing effort to understanding the mechanisms involved in the high stability of hyperthermophilic enzymes.
The thesis starts with an introduction to the field of protein and enzyme stability with special focus on the thermophilic and hyperthermophilic enzymes and proteins. After the introduction three original research manuscripts present the experimental data related to this study. In the first manuscript, the effect of lysine methylation on enzyme stability is investigated. This study makes use of two different methods to acquire enzyme without the native lysine methylation. The effect of the methylation is subsequently evaluated by testing the stability of the different versions of the esterase. Here we show that methylation plays a minimal, yet significant, role in stabilising the enzyme. We also show that if the esterase is produced from a mesophilic host, an organism that lives at moderate temperatures, in this case E. coli, that additional factors that serves to stabilise the enzyme is either missing or that factors from E. coli destabilises the enzyme. We were however unable to identify these factors. The second manuscript present an investigation of the importance of dimer formation for the stability of the esterase investigated in manuscript one. By the introduction of a novel cross dimer disulphide bond we demonstrated a stabilysing effect of dimer formation by increasing the half-life of the esterase by 1.62 fold at a temperature of 90°C. However, efforts to disrupt the native dimer formation by targeting the core elements of the dimer interface failed. The mutants were however compromised in catalytic stability at a temperature of 90°C. The third manuscript details problem area and makes suggestions for better design of esterase activity assay for use at temperature up to 90°C.