Production and investigation of highly thermophilic multi-domain carbohydrate-active enzymes
Doctoral thesis, 2021
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.
plant biomass degradation
Chalmers, Biology and Biological Engineering, Industrial Biotechnology
Investigation of a thermostable multi-domain xylanase-glucuronoyl esterase enzyme from Caldicellulosiruptor kristjanssonii incorporating multiple carbohydrate-binding modules
Biotechnology for Biofuels,; Vol. 13(2020)p. 1-13
Structural and Functional Analysis of a Multimodular Hyperthermostable Xylanase-Glucuronoyl Esterase from Caldicellulosiruptor kristjansonii
Biochemistry,; Vol. 60(2021)p. 2206-2220
Characterization of a novel multidomain CE15-GH8 enzyme encoded by a polysaccharide utilization locus in the human gut bacterium Bacteroides eggerthii
Scientific Reports,; Vol. 11(2021)
Creation of large multidomain proteins using split intein technology
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.
Development of thermostable enzyme cocktails
Swedish Energy Agency (Dnr 2016‑011207), 2018-01-01 -- 2019-12-31.
Formas (Dnr 2016-01065), 2017-05-01 -- 2020-12-31.
Biochemistry and Molecular Biology
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5036
Chalmers University of Technology
10-an, Forskarhus 1, Kemigården 4, Chalmers
Opponent: Prof. Maija Tenkanen, University of Helsinki, Finland