Production and investigation of highly thermophilic multi-domain carbohydrate-active enzymes

Sammanfattning: With the looming threat of climate change caused largely by an excess of carbon dioxide in the atmosphere, recent scientific efforts have focused on the substitution of fossil fuels and other polluting compounds with more environmentally conscious choices. To this end, the investigation of biomass as both a renewable source of energy and as a chemical basis to produce high-value products is being extensively investigated. Although plant biomass is complex, it is also an extremely rich carbon source, and microorganisms in a plethora of environments have evolved to exploit it. These microorganisms produce carbohydrate-active enzymes (CAZymes) to degrade the plant biomass into components that can be utilized for their growth. The deeper study of these enzymes, especially those containing multiple enzyme domains, can elucidate their mechanisms of action, and guide their exploitation for industrial purposes. This thesis consists of the characterization of two different multicatalytic CAZymes from different bacteria found in extremely different environments. The enzymes both contain CE15 (carbohydrate esterase family 15) domains, which have not previously been studied in a multicatalytic context. CkXyn10C-GE15A from the hyperthermophilic Caldicellulosiruptor kristjanssonii consists of a GH10 (glycoside hydrolase family 10) xylanase linked to a CE15 enzyme, and additionally contains two CBM22 (carbohydrate binding module family 22) and three CBM9 domains. A second enzyme, BeCE15A-Rex8A from the gut bacterium Bacteroides eggerthii, consisting of a GH8 xylan-targeting domain and a CE15 domain was also investigated. Although the catalytic domains in both enzymes were active, no synergy was seen between them, respectively. As these enzymes were difficult to produce recombinantly, a new technique using split intein-mediated fusions to produce multicatalytic enzymes was investigated, with results showing that the produced enzymes remain catalytically active after the fusion event. The work presented in this thesis contributes to the understanding of multidomain enzymes and the synergy (or lack thereof) of xylanases in combination with CE15 domains. It also provides structural insights into a number of highly thermophilic CAZyme domains, and has implications for industrial biorefinery applications.