Production and investigation of highly thermophilic multi-domain carbohydrate-active enzymes
Doktorsavhandling, 2021

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.

protein structure


carbohydrate esterase

multidomain enzymes

carbohydrate-active enzymes

thermostable enzymes

plant biomass degradation


10-an, Forskarhus 1, Kemigården 4, Chalmers
Opponent: Prof. Maija Tenkanen, University of Helsinki, Finland


Daniel Krska

Chalmers, Biologi och bioteknik, Industriell bioteknik

To avoid the worsening effects of climate change, it is essential to transition from fossil to renewable resources and more sustainable energy and material production. One way to approach this is to use tools from nature known as carbohydrate-active enzymes (CAZymes). Microorganisms growing on plant biomass use CAZymes to deconstruct biomass. Many different CAZymes exist, and have complementary roles, with different enzymes degrading different parts of plant biomass. CAZymes are also used for human needs, to deconstruct renewable plant biomass into base components, to be converted to plastics, fuels, and other products.

In some cases, different CAZymes are tethered together in “multicatalytic” enzymes that can perform different functions simultaneously. Such multicatalytic enzymes are often very efficient because of the complementary functions of each part. However, they have not been previously extensively studied.

In this work, multicatalytic enzymes from different organisms were investigated for their ability to degrade plant biomass and to withstand extreme temperatures. Surprisingly, these enzymes did not appear to behave synergistically under the studied conditions. This work also builds a foundation for constructing non-natural multicatalytic enzymes, which may prove more useful in industrial processes than natural versions.

Overall, this work expands our understanding of multicatalytic enzymes, and has implications for biomass degradation in industrial biorefineries.

Utveckling av värmetåliga enzymblandningar

Formas (Dnr 2016-01065), 2017-05-01 -- 2020-12-31.

Energimyndigheten (Dnr 2016‑011207), 2018-01-01 -- 2019-12-31.


Hållbar utveckling


Biokemi och molekylärbiologi



Grundläggande vetenskaper



Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5036



10-an, Forskarhus 1, Kemigården 4, Chalmers


Opponent: Prof. Maija Tenkanen, University of Helsinki, Finland

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