A Modular Toolkit to Enable Intein-mediated Enzyme Fusions
| dc.contributor.author | Storm, Linus | |
| dc.contributor.department | Chalmers tekniska högskola / Institutionen för life sciences | sv |
| dc.contributor.department | Chalmers University of Technology / Department of Life Sciences | en |
| dc.contributor.examiner | Larsbrink, Johan | |
| dc.contributor.supervisor | Widén, Tove | |
| dc.date.accessioned | 2024-05-14T14:14:39Z | |
| dc.date.available | 2024-05-14T14:14:39Z | |
| dc.date.issued | 2024 | |
| dc.date.submitted | ||
| dc.description.abstract | In second generation biorefineries, lignocellulosic biomass is fermented by microbes to higher-value products. Hydrolysis of the substrate is necessary to break it into fragments that the microbes can utilize. Enzymatic hydrolysis is often preferred, but a bottleneck is the high costs of enzyme cocktails since lignocellulosic polymers require several types of enzymes to be degraded. Fusion enzymes have been produced to increase conversation rates, but the methods for creating them are either complex or limited. This project aimed to circumvent some of these limitations by creating a modular toolkit to produce any fusion enzymes, where the fusion is split intein-mediated in vitro. For evaluation of the toolkit, an α-xylosidase (BoGH3B) and a β-glucosidase (BoGH31A) involved in xyloglucan (XyG) degradation, were chosen. The toolkit was developed to completion and consists of eight types of parts, excluding the protein of interest. Three assemblies were confirmed by sequencing, proving that the toolkit works. Aside from the toolkit-mediated assemblies, the enzymes on their own were expressed and tested in different combinations for XyG degradation. This was done to compare with the split intein-fused enzymes. However, when it came to expressing the protein assemblies the yield was low, and no fusion could be observed. Thus, the possibly synergistic effect of fusing BoGH3B and BoGH31A could not be investigated. A lot of research is still needed to determine the usefulness of split inteins for creating enzyme fusions. For future work, some experiments need redoing, and new experiments with different linkers and configurations of the assemblies could be performed. | |
| dc.identifier.coursecode | BBTX60 | |
| dc.identifier.uri | http://hdl.handle.net/20.500.12380/307686 | |
| dc.language.iso | eng | |
| dc.setspec.uppsok | LifeEarthScience | |
| dc.subject | Modular cloning | |
| dc.subject | DNA assembly | |
| dc.subject | inteins | |
| dc.subject | split inteins | |
| dc.subject | fusion enzymes | |
| dc.subject | xyloglucan | |
| dc.title | A Modular Toolkit to Enable Intein-mediated Enzyme Fusions | |
| dc.type.degree | Examensarbete för masterexamen | sv |
| dc.type.degree | Master's Thesis | en |
| dc.type.uppsok | H | |
| local.programme | Biotechnology (MPBIO), MSc |