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Synthesis of enantio-pure amides by reversal of the Geobacillus pallidus RAPc8 amidase hydrolysis reaction in non-aqueous media
Amidases are hydrolytic enzymes that catalyze the hydrolysis of amides to their corresponding carboxylic acids and ammonia. Amidases are ubiquitous in nature, and they have been isolated from a wide range of microorganisms, the most common source being bacteria. Amidases are recognized as potential...
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| Format: | Thesis |
| Language: | English |
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Centre for Bioprocess Engineering Research
2016
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| _version_ | 1867613167640641536 |
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| access_status_str | Open Access |
| author | Gabathuse, Anne Onalethata |
| author2 | Burton, Stephanie |
| author_browse | Burton, Stephanie Gabathuse, Anne Onalethata |
| author_facet | Burton, Stephanie Gabathuse, Anne Onalethata |
| author_sort | Gabathuse, Anne Onalethata |
| collection | Thesis |
| description | Amidases are hydrolytic enzymes that catalyze the hydrolysis of amides to their corresponding carboxylic acids and ammonia. Amidases are ubiquitous in nature, and they have been isolated from a wide range of microorganisms, the most common source being bacteria. Amidases are recognized as potential industrial biocatalysts in processes that involve the synthesis of chiral compounds, mostly used in the pharmaceutical, agrochemical and food industries. The discovery of amidases from extremophiles has increased the potential for application of these enzymes for the development of new processes. In nonaqueous media, amidases have the ability to synthesize enantiopure amides due to the shift in thermodynamic equilibrium towards synthesis. For synthesis to occur, an acyl donor and an acyl acceptor are required, in which the acyl acceptor acts as a nucleophile. The applicability of amidases in non-aqueous media opens new possibilities for processes in which the enzyme can be used for the industrial synthesis of commercially relevant new products. A novel amidase was previously isOlated from a thermophilic Geobacillus species, and the amidase was cloned and expressed in an Escherichia coli BL21 strain. Also in previous studies, it was shown that the enzyme exhibits both amide hydrolysis and acyl transfer activities. The highest activity of the G. pallidus RAPc8 amidase was observed at 50°C in the presence of acetamide and substrate preference was towards aliphatic, short chain amides. Furthermore, the enzyme displayed enantioselectivity towards lactamide, which is a chiral compound. The amidase compound showed selectivity towards the D-isomer of lactamide and no detectable activity on the L-isomer. This study presents the investigation and development of a novel biocatalytic process that involves the synthesis of enantiopure amides in non-aqueous media, using the G. pal/idus RAPc8 amidase. The amidase was produced and expressed in E. coli BL21. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/16916 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:31:50.330Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2016 |
| publishDateRange | 2016 |
| publishDateSort | 2016 |
| publisher | Centre for Bioprocess Engineering Research |
| publisherStr | Centre for Bioprocess Engineering Research |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/16916 Synthesis of enantio-pure amides by reversal of the Geobacillus pallidus RAPc8 amidase hydrolysis reaction in non-aqueous media Gabathuse, Anne Onalethata Burton, Stephanie Bioprocess Engineering Amidases are hydrolytic enzymes that catalyze the hydrolysis of amides to their corresponding carboxylic acids and ammonia. Amidases are ubiquitous in nature, and they have been isolated from a wide range of microorganisms, the most common source being bacteria. Amidases are recognized as potential industrial biocatalysts in processes that involve the synthesis of chiral compounds, mostly used in the pharmaceutical, agrochemical and food industries. The discovery of amidases from extremophiles has increased the potential for application of these enzymes for the development of new processes. In nonaqueous media, amidases have the ability to synthesize enantiopure amides due to the shift in thermodynamic equilibrium towards synthesis. For synthesis to occur, an acyl donor and an acyl acceptor are required, in which the acyl acceptor acts as a nucleophile. The applicability of amidases in non-aqueous media opens new possibilities for processes in which the enzyme can be used for the industrial synthesis of commercially relevant new products. A novel amidase was previously isOlated from a thermophilic Geobacillus species, and the amidase was cloned and expressed in an Escherichia coli BL21 strain. Also in previous studies, it was shown that the enzyme exhibits both amide hydrolysis and acyl transfer activities. The highest activity of the G. pallidus RAPc8 amidase was observed at 50°C in the presence of acetamide and substrate preference was towards aliphatic, short chain amides. Furthermore, the enzyme displayed enantioselectivity towards lactamide, which is a chiral compound. The amidase compound showed selectivity towards the D-isomer of lactamide and no detectable activity on the L-isomer. This study presents the investigation and development of a novel biocatalytic process that involves the synthesis of enantiopure amides in non-aqueous media, using the G. pal/idus RAPc8 amidase. The amidase was produced and expressed in E. coli BL21. 2016-02-08T14:22:07Z 2016-02-08T14:22:07Z 2012 Master Thesis Masters MSc (Eng) http://hdl.handle.net/11427/16916 eng application/pdf Centre for Bioprocess Engineering Research Faculty of Engineering and the Built Environment University of Cape Town |
| spellingShingle | Bioprocess Engineering Gabathuse, Anne Onalethata Synthesis of enantio-pure amides by reversal of the Geobacillus pallidus RAPc8 amidase hydrolysis reaction in non-aqueous media |
| thesis_degree_str | Master's |
| title | Synthesis of enantio-pure amides by reversal of the Geobacillus pallidus RAPc8 amidase hydrolysis reaction in non-aqueous media |
| title_full | Synthesis of enantio-pure amides by reversal of the Geobacillus pallidus RAPc8 amidase hydrolysis reaction in non-aqueous media |
| title_fullStr | Synthesis of enantio-pure amides by reversal of the Geobacillus pallidus RAPc8 amidase hydrolysis reaction in non-aqueous media |
| title_full_unstemmed | Synthesis of enantio-pure amides by reversal of the Geobacillus pallidus RAPc8 amidase hydrolysis reaction in non-aqueous media |
| title_short | Synthesis of enantio-pure amides by reversal of the Geobacillus pallidus RAPc8 amidase hydrolysis reaction in non-aqueous media |
| title_sort | synthesis of enantio pure amides by reversal of the geobacillus pallidus rapc8 amidase hydrolysis reaction in non aqueous media |
| topic | Bioprocess Engineering |
| url | http://hdl.handle.net/11427/16916 |
| work_keys_str_mv | AT gabathuseanneonalethata synthesisofenantiopureamidesbyreversalofthegeobacilluspallidusrapc8amidasehydrolysisreactioninnonaqueousmedia |