Engineering industrial enzymes to function at extreme conditions

Lignocellulosic biomass is the most abundantly available raw material in the world. Converting all molecular components of lignocellulose to valuable products has gained significant interest. These conversion processes often use enzymes. Xylanases are essential enzymes in the bioconversion of hemicellulose and they are successfully utilized in various industrial processes, especially in pulp bleaching and food industry. This thesis focused on determination of the alteration of enzymatic properties in engineered GH11 xylanases to generate understanding of structure-function relationships at extreme conditions. First, the extremely thermostable D. thermophilum GH11 xylanase (DtXYNB) was further stabilized to improve its function at ~100oC by introducing an N-terminal disulphide bridge. Then, the study of xylanase kinetic parameters indicated that substrate binding starts to weaken before the enzyme loses its catalytic ability at high temperatures. Concurrently, analysis of the end-products of xylan hydrolysis showed how the ability of enzyme to bind shorter substrates decreased. In this aspect, increasing the enzyme thermostability increases its ability to bind the substrate at 50-100oC due to the enzyme maintaining its folded structure. The active site mutations of T. flexuosa XYN11A shifted the pH optimum to be more acidic, but the tightly packed GH11 active site may cause steric and other problems for the mutations. However, extreme conditions may limit the utilization of GH11 xylanases in industrial applications. In this study, the performance of engineered GH11 xylanases at extreme conditions was elucidated. High pressure is one of the extreme conditions inactivating enzymes. Over a limited range, it may also increase enzyme stability. With a series of thermostability mutants of T. reesei xylanase (TrXYNII) it was found that pressure stability and thermostability largely correlate. The stabilizing TrXYNII mutations increase the stability against heat inactivation under high pressure. Thermal inactivation was found to dominate in pressure inactivation. Nevertheless, the extremophilic DtXYNB was extremely stable at 80oC at 500 MPa. This study provides evidence that even the most thermophilic enzymes may still be improved for industrial applications. However, in applications using ionic liquids, it is essential to note that polar ionic liquids used to dissolve lignocellulose may destabilize the enzymes. The DtXYNB is almost fully inactivated in the presence of 25% of ionic liquid ([EMIM][OAc]). The interaction of enzyme active site with the substrate is affected by [EMIM][OAc]. This study indicated that high thermostability is not the only factor necessary when using this enzyme in applications with polar ionic liquids. Another essential factor is the inhibition of enzyme activity caused by ionic liquids. The present study showed that one key feature in hydrolyzing biomass is the ability of the enzyme, in the conditions applied, to minimize the effect of inhibiting molecules and conditions on the catalytic reaction.