Enhancing hydrolase activity and selectivity by medium, substrate, and protein engineering
Researchers use enzymes for enantio- and regioselective reactions because of their high selectivity and activity toward natural substrates. However, researchers sometimes need to modify the reaction system or the enzyme itself to get reliable selectivity and activity when they deal with unnatural su...
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ftcanadathes:oai:collectionscanada.gc.ca:QMM.83088 2023-05-15T14:02:04+02:00 Enhancing hydrolase activity and selectivity by medium, substrate, and protein engineering Park, Seongsoon Kazlauskas, Romas J. (advisor) Doctor of Philosophy (Department of Chemistry.) 2003 application/pdf http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=83088 en eng McGill University alephsysno: 001985058 proquestno: AAINQ88548 Theses scanned by UMI/ProQuest. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=83088 All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. Chemistry Biochemistry Organic Electronic Thesis or Dissertation 2003 ftcanadathes 2014-02-16T00:53:03Z Researchers use enzymes for enantio- and regioselective reactions because of their high selectivity and activity toward natural substrates. However, researchers sometimes need to modify the reaction system or the enzyme itself to get reliable selectivity and activity when they deal with unnatural substrates. To obtain researcher's need, one can change the solvent, modify the substrates, or alter the enzyme itself. These processes are called medium, substrate, and protein engineering, respectively. This thesis deals with hydrolases, which are classified by EC 3. We applied the proper approach to improve their activity and selectivity depending on the reactions. For the first approach, highly polar ionic liquids were applied to lipase-catalyzed acylation. Ionic liquids worked reliably in enantio- and regioselective lipase-catalyzed reactions. In particular, ionic liquids dissolved polar substrates such as glucose and L-ascorbic acid, thereby facilitating their acylations. In the second approach to improving enantioselectivity of CAL-B (Candida antarctica lipase B) in beta-lactam ring opening reactions, we changed the nucleophile from water to a range of alcohols. Longer, secondary alcohols increased the reaction rate as well as the enantioselectivity. Molecular modeling revealed that the high enantioselectivity of CAL-B and the critical role of alcohols. For the last approach, structure-guided random mutagenesis was applied to increase the enantioselectivity of PFE ( Pseudomonas fluorescens esterase) toward MBMP (methyl 3-bromo-2-methylpropionate). The homology model was used to select amino acid residues for mutagenesis near the stereocenter of the docked tetrahedral intermediate of the substrate. Randomization of these residues yielded a Val122Ser mutant with E increased to 61 (from 12 of wild type enzyme), as well as a Val122Met mutant to 36. Thesis Antarc* Antarctica Theses Canada/Thèses Canada (Library and Archives Canada) |
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Theses Canada/Thèses Canada (Library and Archives Canada) |
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English |
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Chemistry Biochemistry Organic |
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Chemistry Biochemistry Organic Park, Seongsoon Enhancing hydrolase activity and selectivity by medium, substrate, and protein engineering |
topic_facet |
Chemistry Biochemistry Organic |
description |
Researchers use enzymes for enantio- and regioselective reactions because of their high selectivity and activity toward natural substrates. However, researchers sometimes need to modify the reaction system or the enzyme itself to get reliable selectivity and activity when they deal with unnatural substrates. To obtain researcher's need, one can change the solvent, modify the substrates, or alter the enzyme itself. These processes are called medium, substrate, and protein engineering, respectively. This thesis deals with hydrolases, which are classified by EC 3. We applied the proper approach to improve their activity and selectivity depending on the reactions. For the first approach, highly polar ionic liquids were applied to lipase-catalyzed acylation. Ionic liquids worked reliably in enantio- and regioselective lipase-catalyzed reactions. In particular, ionic liquids dissolved polar substrates such as glucose and L-ascorbic acid, thereby facilitating their acylations. In the second approach to improving enantioselectivity of CAL-B (Candida antarctica lipase B) in beta-lactam ring opening reactions, we changed the nucleophile from water to a range of alcohols. Longer, secondary alcohols increased the reaction rate as well as the enantioselectivity. Molecular modeling revealed that the high enantioselectivity of CAL-B and the critical role of alcohols. For the last approach, structure-guided random mutagenesis was applied to increase the enantioselectivity of PFE ( Pseudomonas fluorescens esterase) toward MBMP (methyl 3-bromo-2-methylpropionate). The homology model was used to select amino acid residues for mutagenesis near the stereocenter of the docked tetrahedral intermediate of the substrate. Randomization of these residues yielded a Val122Ser mutant with E increased to 61 (from 12 of wild type enzyme), as well as a Val122Met mutant to 36. |
author2 |
Kazlauskas, Romas J. (advisor) |
format |
Thesis |
author |
Park, Seongsoon |
author_facet |
Park, Seongsoon |
author_sort |
Park, Seongsoon |
title |
Enhancing hydrolase activity and selectivity by medium, substrate, and protein engineering |
title_short |
Enhancing hydrolase activity and selectivity by medium, substrate, and protein engineering |
title_full |
Enhancing hydrolase activity and selectivity by medium, substrate, and protein engineering |
title_fullStr |
Enhancing hydrolase activity and selectivity by medium, substrate, and protein engineering |
title_full_unstemmed |
Enhancing hydrolase activity and selectivity by medium, substrate, and protein engineering |
title_sort |
enhancing hydrolase activity and selectivity by medium, substrate, and protein engineering |
publisher |
McGill University |
publishDate |
2003 |
url |
http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=83088 |
op_coverage |
Doctor of Philosophy (Department of Chemistry.) |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_relation |
alephsysno: 001985058 proquestno: AAINQ88548 Theses scanned by UMI/ProQuest. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=83088 |
op_rights |
All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
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