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Structural and functional characterization of a novel acetyl xylan esterase from a desert soil metagenome
Thesis (PhD)--University of Pretoria, 2017.
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| Format: | Thesis |
| Language: | English |
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University of Pretoria
2018
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| _version_ | 1867613453136429056 |
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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 | © 2018 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, 2017. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/65881 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:36:23.211Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2018 |
| publishDateRange | 2018 |
| publishDateSort | 2018 |
| 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:2263/65881 Structural and functional characterization of a novel acetyl xylan esterase from a desert soil metagenome Cowan, Don A u13337115@tuks.co.za Makhalanyane, Thulani Peter Schubert, Wolf-Dieter Adesioye, Fiyinfoluwa Adenike UCTD Thesis (PhD)--University of Pretoria, 2017. A Namib Desert soil hypolith metagenomic dataset was screened, in silico, for novel acetyl xylan esterase (AcXE) - encoding genes. AcXEs hydrolyse ester bonds to liberate acetic acid in acetylated polymeric xylan and xylooligosaccharides during bioconversion of lignocellulosic biomass for sustainable biofuel production. One of the identified genes (NaMet1) was synthesized, cloned and expressed to produce a ~36 kDa protein. This protein, NaM1, was confirmed to be functional and was purified and characterized. NaM1, a carbohydrate esterase (CE) 7 enzyme, was optimally active on para-nitrophenol acetate at pH 8.5 and 30 oC, and remained active in up to 5 M NaCl and 65% DMSO. The specific activity and catalytic efficiency were 488.9 Umg-1 and 3.26x106 M-1s-1, respectively. NaM1 deacetylated para-nitrophenol acetate and butyrate, 7-aminocephalosporanic acid and acetylated xylan. Most investigations of CE7 esterases have been carried out using structural information from thermostable members of this family and little is known about thermolabile members. A 2.03 Å crystal structure of native NaM1, the first CE structure of metagenomic origin to be submitted to the Protein data bank, was solved. The structure was compared with those of thermostable CE7 enzymes and used to study the thermal stability determinants of this enzyme family. This comparison showed strong structural conservation between both enzyme types and suggested that differences in several key residues, as well as, packing within the core, were responsible for thermal stability. Directed evolution (DE) of NaM1 yielded thermostable variants, including a variant with 10oC improved stability. Analyses of the kinetic and putative structural characteristics of selected variants in comparison with those of the wild-type provided insights to the role of residues influencing the thermal stability, substrate specificity and activity of NaM1. A single substitution was found to expand acyl moiety specificity and improve both thermal stability and activity of NaM1. Knowledge of key residues identified during NaM1 DE is useful for the future engineering of CE7 and ?/? hydrolase enzymes in order to improve catalytic turnover, substrate specificity and thermal stability. Genetics PhD Unrestricted 2018-07-25T09:00:45Z 2018-07-25T09:00:45Z 2018/04/26 2017 Thesis Adesioye, FA 2017, Structural and functional characterization of a novel acetyl xylan esterase from a desert soil metagenome, PhD Thesis, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/65881> A2018 http://hdl.handle.net/2263/65881 en © 2018 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 Structural and functional characterization of a novel acetyl xylan esterase from a desert soil metagenome |
| title | Structural and functional characterization of a novel acetyl xylan esterase from a desert soil metagenome |
| title_full | Structural and functional characterization of a novel acetyl xylan esterase from a desert soil metagenome |
| title_fullStr | Structural and functional characterization of a novel acetyl xylan esterase from a desert soil metagenome |
| title_full_unstemmed | Structural and functional characterization of a novel acetyl xylan esterase from a desert soil metagenome |
| title_short | Structural and functional characterization of a novel acetyl xylan esterase from a desert soil metagenome |
| title_sort | structural and functional characterization of a novel acetyl xylan esterase from a desert soil metagenome |
| topic | UCTD |
| url | http://hdl.handle.net/2263/65881 |