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- Kmezik, Cathleen, 1991, et al.
(author)
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Characterization of a novel multidomain CE15-GH8 enzyme encoded by a polysaccharide utilization locus in the human gut bacterium Bacteroides eggerthii
- 2021
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 11:1
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Journal article (peer-reviewed)abstract
- Bacteroidetes are efficient degraders of complex carbohydrates, much thanks to their use of polysaccharide utilization loci (PULs). An integral part of PULs are highly specialized carbohydrate-active enzymes, sometimes composed of multiple linked domains with discrete functions—multicatalytic enzymes. We present the biochemical characterization of a multicatalytic enzyme from a large PUL encoded by the gut bacterium Bacteroides eggerthii . The enzyme, Be CE15A-Rex8A, has a rare and novel architecture, with an N-terminal carbohydrate esterase family 15 (CE15) domain and a C-terminal glycoside hydrolase family 8 (GH8) domain. The CE15 domain was identified as a glucuronoyl esterase (GE), though with relatively poor activity on GE model substrates, attributed to key amino acid substitutions in the active site compared to previously studied GEs. The GH8 domain was shown to be a reducing-end xylose-releasing exo-oligoxylanase (Rex), based on having activity on xylooligosaccharides but not on longer xylan chains. The full-length Be CE15A-Rex8A enzyme and the Rex domain were capable of boosting the activity of a commercially available GH11 xylanase on corn cob biomass. Our research adds to the understanding of multicatalytic enzyme architectures and showcases the potential of discovering novel and atypical carbohydrate-active enzymes from mining PULs.
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| 2. |
- Krska, Daniel, 1989, et al.
(author)
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Investigation of a thermostable multi-domain xylanase-glucuronoyl esterase enzyme from Caldicellulosiruptor kristjanssonii incorporating multiple carbohydrate-binding modules
- 2020
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In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 13:1, s. 1-13
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Journal article (peer-reviewed)abstract
- Background Efficient degradation of lignocellulosic biomass has become a major bottleneck in industrial processes which attempt to use biomass as a carbon source for the production of biofuels and materials. To make the most effective use of the source material, both the hemicellulosic as well as cellulosic parts of the biomass should be targeted, and as such both hemicellulases and cellulases are important enzymes in biorefinery processes. Using thermostable versions of these enzymes can also prove beneficial in biomass degradation, as they can be expected to act faster than mesophilic enzymes and the process can also be improved by lower viscosities at higher temperatures, as well as prevent the introduction of microbial contamination. Results This study presents the investigation of the thermostable, dual-function xylanase-glucuronoyl esterase enzyme Ck Xyn10C-GE15A from the hyperthermophilic bacterium Caldicellulosiruptor kristjanssonii . Biochemical characterization of the enzyme was performed, including assays for establishing the melting points for the different protein domains, activity assays for the two catalytic domains, as well as binding assays for the multiple carbohydrate-binding domains present in Ck Xyn10C-GE15A. Although the enzyme domains are naturally linked together, when added separately to biomass, the expected boosting of the xylanase action was not seen. This lack of intramolecular synergy might suggest, together with previous data, that increased xylose release is not the main beneficial trait given by glucuronoyl esterases. Conclusions Due to its thermostability, Ck Xyn10C-GE15A is a promising candidate for industrial processes, with both catalytic domains exhibiting melting temperatures over 70 °C. Of particular interest is the glucuronoyl esterase domain, as it represents the first studied thermostable enzyme displaying this activity.
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| 3. |
- Krska, Daniel, 1989
(author)
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Production and investigation of highly thermophilic multi-domain carbohydrate-active enzymes
- 2021
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Doctoral thesis (other academic/artistic)abstract
- 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.
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| 4. |
- Krska, Daniel, 1989, et al.
(author)
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Structural and Functional Analysis of a Multimodular Hyperthermostable Xylanase-Glucuronoyl Esterase from Caldicellulosiruptor kristjansonii
- 2021
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In: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 60:27, s. 2206-2220
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Journal article (peer-reviewed)abstract
- The hyperthermophilic bacterium Caldicellulosiruptor kristjansonii encodes an unusual enzyme, CkXyn10C-GE15A, which incorporates two catalytic domains, a xylanase and a glucuronoyl esterase, and five carbohydrate-binding modules (CBMs) from families 9 and 22. The xylanase and glucuronoyl esterase catalytic domains were recently biochemically characterized, as was the ability of the individual CBMs to bind insoluble polysaccharides. Here, we further probed the abilities of the different CBMs from CkXyn10C-GE15A to bind to soluble poly- and oligosaccharides using affinity gel electrophoresis, isothermal titration calorimetry, and differential scanning fluorimetry. The results revealed additional binding properties of the proteins compared to the former studies on insoluble polysaccharides. Collectively, the results show that all five CBMs have their own distinct binding preferences and appear to complement each other and the catalytic domains in targeting complex cell wall polysaccharides. Additionally, through renewed efforts, we have achieved partial structural characterization of this complex multidomain protein. We have determined the structures of the third CBM9 domain (CBM9.3) and the glucuronoyl esterase (GE15A) by X-ray crystallography. CBM9.3 is the second CBM9 structure determined to date and was shown to bind oligosaccharide ligands at the same site but in a different binding mode compared to that of the previously determined CBM9 structure from Thermotoga maritima. GE15A represents a unique intermediate between reported fungal and bacterial glucuronoyl esterase structures as it lacks two inserted loop regions typical of bacterial enzymes and a third loop has an atypical structure. We also report small-angle X-ray scattering measurements of the N-terminal CBM22.1-CBM22.2-Xyn10C construct, indicating a compact arrangement at room temperature.
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