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In vitro and in vivo examination of phosphate incorporation by Escherichia coli phosphorylase enzymes
Thesis (MScAgric)--Stellenbosch University, 2019.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2017
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| _version_ | 1867614029758857216 |
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| access_status_str | Open Access |
| author | Prince, Erica |
| author2 | Lloyd, James Richard |
| author_browse | Lloyd, James Richard Prince, Erica |
| author_facet | Lloyd, James Richard Prince, Erica |
| author_sort | Prince, Erica |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MScAgric)--Stellenbosch University, 2019. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/102589 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:45:32.686Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2017 |
| publishDateRange | 2017 |
| publishDateSort | 2017 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/102589 In vitro and in vivo examination of phosphate incorporation by Escherichia coli phosphorylase enzymes Prince, Erica Lloyd, James Richard Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics. Escherichia coli Glycogen phosphorylase Phosphorylase Starch -- Metabolism Phosphate Enzymes -- Analysis UCTD Thesis (MScAgric)--Stellenbosch University, 2019. ENGLISH ABSTRACT: Starch and glycogen are two storage polymers that are similar in structure, function and metabolism. Alterations in starch structure, including its phosphorylation, can alter its properties in a way that make it useful for industry. In this study the role of two Escherichia coli phosphorylase enzymes, glycogen phosphorylase (GlgP) and maltodextrin phosphorylase (MalP), in the incorporation of phosphate into glycogen is examined both in vitro and in vivo. In vitro analysis revealed that when MalP and GlgP were incubated with a primer and glucose-1,6-bisphosphate, glucose-6-phosphate was incorporated into glycogen. The Km’s of GlgP and MalP for glucose-1,6-bisphosphate were found respectively to be ~1.3 mM and ~0.7 mM when glycogen was used as a priming polymer, with a Vmax of ~0.08 μmol/μg/min and ~0.05 μmol/μg/min respectively. To examine if this in vitro activity is relevant biologically, their in vivo role was explored using knockout mutants. Glycogen extracted from wild type E. coli and mutants lacking either MalP, GlgP or both, contained similar amounts of covalently bound phosphate indicating that it is incorporated by another mechanism. AFRIKAANSE OPSOMMING: Stysel en glikogeen is twee stoorpolimere met gelyksoorte struktuur, funksie en metabolisme. Veranderinge in die struktuur van stylsel, insluitend deur middel van fosforilering, kan sy eienskappe sodoende verander wat dit meer toepaslik maak vir industriële gebruike. In hierdie studie word die rol van twee Escherichia coli fosforileerings-ensieme, glikogeenfosforilase (GlgP) en maltodekstrienfosforilase (MalP), in die inkorporering van fosfaat in glikogeen in vitro en in vivo ondersoek. In vitro analise het getoon dat beide MalP en GlgP, in die teenwoordigheid van 'n inleier en glukose 1,6-bisfosfaat, die inkorporering van glukose 6-fosfaat in glikogeen kon uitvoer. Die Km's van MalP en GlgP vir glukose 1,6-bisfosfaat is onderskeidelik gevind as ~0,7 mM en ~1,3 mM wanneer glikogeen as 'n inleier gebruik was, en die Vmax gevind as ~0.08 μmol/μg/min en ~0.05 μmol/μg/min. Om te ondersoek of hierdie in vitro aktiwiteite biologies van toepassing is, was hul rolle in vivo ondersoek met behulp van mutante wat die betrokke gene ontbreek. Glikogeen geïsoleerdvanuit wild tipe E. coli en mutante ontbreek inMalP, GlgP of beide die gene, het soortgelyke hoeveelhede kovalent gebonde fosfaat bevat, wat dus daarop dui dat dit deur 'n ander meganisme geïnkorporeer word. Masters 2017-11-21T13:11:34Z 2017-12-11T10:29:00Z 2020-07-21T03:00:13Z 2017-12 Thesis http://hdl.handle.net/10019.1/102589 en_ZA Stellenbosch University xiii, 51 pages : illustrations (some color) xiii, 51 pages : illustrations (some color) application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Escherichia coli Glycogen phosphorylase Phosphorylase Starch -- Metabolism Phosphate Enzymes -- Analysis UCTD Prince, Erica In vitro and in vivo examination of phosphate incorporation by Escherichia coli phosphorylase enzymes |
| title | In vitro and in vivo examination of phosphate incorporation by Escherichia coli phosphorylase enzymes |
| title_full | In vitro and in vivo examination of phosphate incorporation by Escherichia coli phosphorylase enzymes |
| title_fullStr | In vitro and in vivo examination of phosphate incorporation by Escherichia coli phosphorylase enzymes |
| title_full_unstemmed | In vitro and in vivo examination of phosphate incorporation by Escherichia coli phosphorylase enzymes |
| title_short | In vitro and in vivo examination of phosphate incorporation by Escherichia coli phosphorylase enzymes |
| title_sort | in vitro and in vivo examination of phosphate incorporation by escherichia coli phosphorylase enzymes |
| topic | Escherichia coli Glycogen phosphorylase Phosphorylase Starch -- Metabolism Phosphate Enzymes -- Analysis UCTD |
| url | http://hdl.handle.net/10019.1/102589 |
| work_keys_str_mv | AT princeerica invitroandinvivoexaminationofphosphateincorporationbyescherichiacoliphosphorylaseenzymes |