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Structure-function analysis of a novel multi-functional glycoside hydrolase
Thesis (PhD)--University of Pretoria, 2018.
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| Format: | Thesis |
| Language: | English |
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University of Pretoria
2020
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| _version_ | 1867613596636151808 |
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| access_status_str | Open Access |
| author2 | Cowan, Don A. |
| author_browse | Cowan, Don A. |
| author_facet | Cowan, Don A. |
| collection | Thesis |
| dc_rights_str_mv | © 2020 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Thesis (PhD)--University of Pretoria, 2018. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/77859 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:38:39.945Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/77859 Structure-function analysis of a novel multi-functional glycoside hydrolase Cowan, Don A. u14455642@tuks.co.za Schultheiss, W. Mosina, Leticia Leticia UCTD Thesis (PhD)--University of Pretoria, 2018. The present study presents the research findings of the first ever multi-modular Paenibacillus mucilaginosus glycoside hydrolase (PmGH). Furthermore, we report the successful crystallisation of a multi-modular GH. The GH is composed of two catalytic modules (GH5 and GH6) and two carbohydrate binding modules (both CBM3). Functional analysis demonstrated that the cellulase, mannanase and xylanase activities of PmGH (130 kDa) were attributed to the GH5 catalytic domain. The presence of the GH6 catalytic domain resulted in slightly increased cellulase activity in PmGH. Optimal PmGH activity and functional stability was highest at pH 4-6 and at 40-60°C The structural properties of PmGH that determine its robust nature were further investigated. Homology modelling of PmGH showed the GH5 and GH6 domains to be independent but provided no structural information for the CBMs and linker regions. However, successful homology modelling of the individual domains indicated that the combination of the modules makes PmGH structurally and functionally novel. Glycoside hydrolases occur as independent modules or as part of a multi-modular protein with other catalytic and/or non-catalytic modules. Multiple combinations of these modules can occur in nature resulting in novel proteins such as PmGH. In an attempt to determine the PmGH crystal structure, a range of crystallisation conditions were tested. After extensive screening and optimisation, multiple PmGH crystals were diffracted, using both local diffraction and Synchrotron radiation sources (ESRF, France). Overall ~90% of the PmGH protein crystals did not diffract and of the remaining ~10% yielded unsatisfactory data. Phasing by molecular replacement also yielded no structural solutions. Alternative phasing methods such as multi-wavelength anomalous dispersion were also unsuccessful due to the quality of the diffraction data collected. Given the severe lack of multi-modular GH crystal structures in protein structure databases, the present study highlights the major limitations in structural studies of these important enzymes. Genetics PhD Unrestricted 2020-12-29T11:50:57Z 2020-12-29T11:50:57Z 2020/04/30 2019 Thesis Mosina, LL 2019, Structure-function analysis of a novel multi-functional glycoside hydrolase, PhD Thesis, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/77859> A2020 http://hdl.handle.net/2263/77859 en © 2020 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf University of Pretoria |
| spellingShingle | UCTD Structure-function analysis of a novel multi-functional glycoside hydrolase |
| title | Structure-function analysis of a novel multi-functional glycoside hydrolase |
| title_full | Structure-function analysis of a novel multi-functional glycoside hydrolase |
| title_fullStr | Structure-function analysis of a novel multi-functional glycoside hydrolase |
| title_full_unstemmed | Structure-function analysis of a novel multi-functional glycoside hydrolase |
| title_short | Structure-function analysis of a novel multi-functional glycoside hydrolase |
| title_sort | structure function analysis of a novel multi functional glycoside hydrolase |
| topic | UCTD |
| url | http://hdl.handle.net/2263/77859 |