Lipase and ω-Transaminase : Biocatalytic Investigations
In a lipase investigation, Candida antarctica lipase B (CALB) are explored for enzyme catalytic promiscuity. Enzyme catalytic promiscuity is shown by enzymes catalyzing alternative catalytic transformations proceeding via different transition state structures than normal. CALB normally performs hydr...
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ftkthstockholm:oai:DiVA.org:kth-13279 2023-05-15T14:01:36+02:00 Lipase and ω-Transaminase : Biocatalytic Investigations Svedendahl, Maria 2010 application/pdf http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-13279 eng eng KTH, Biokemi Stockholm : KTH Trita-BIO-Report, 1654-2312 2010:10 orcid:0000-0003-2371-8755 http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-13279 urn:isbn:978-91-7415-648-5 info:eu-repo/semantics/openAccess Candida antarctica lipase B enzyme catalysis enzyme catalytic promiscuity molecular modeling ω-transaminase Biochemistry and Molecular Biology Biokemi och molekylärbiologi Doctoral thesis, comprehensive summary info:eu-repo/semantics/doctoralThesis text 2010 ftkthstockholm 2022-08-11T12:33:15Z In a lipase investigation, Candida antarctica lipase B (CALB) are explored for enzyme catalytic promiscuity. Enzyme catalytic promiscuity is shown by enzymes catalyzing alternative catalytic transformations proceeding via different transition state structures than normal. CALB normally performs hydrolysis reactions by activating and coordinating carboxylic acid/ester substrates in an oxyanion hole prior to nucleophilic attack from an active-site serine resulting in acyl enzyme formation. The idea of utilizing the carbonyl activation oxyanion hole in the active-site of CALB to catalyze promiscuous reactions arose by combining catalytic and structural knowledge about the enzyme with chemical imagination. We choose to explore conjugate addition and direct epoxidation activities in CALB by combining molecular modeling and kinetic experiments. By quantum-chemical calculations, the investigated promiscuous reactions were shown to proceed via ordered reaction mechanisms that differ from the native ping pong bi bi reaction mechanism. The investigated promiscuous activities were shown to take place in the enzyme active-site by various kinetic experiments, but despite this, no enantioselectivity was displayed. The reason for this is unknown, but can be a result of a too voluminous active-site or the lack of covalent coordination of the substrates during enzyme-catalysis (Paper I-IV). Combining enzyme structural knowledge with chemical imagination may provide numerous novel enzyme activities to be discovered. In an ω-transaminase investigation, two (S)-selective ω-transaminases from Arthrobacter citreus (Ac-ωTA) and Chromobacterium violaceum (Cv-ωTA) are explored aiming to improve their catalytic properties. Structural knowledge of these enzymes was provided by homology modeling. A homology structure of Ac-ωTA was successfully applied for rational design resulting in enzyme variants with improved enantioselectivity. Additionally, a single-point mutation reversed the enantiopreference of the enzyme from (S) to (R), which ... Doctoral or Postdoctoral Thesis Antarc* Antarctica Royal Institute of Technology, Stockholm: KTHs Publication Database DiVA |
institution |
Open Polar |
collection |
Royal Institute of Technology, Stockholm: KTHs Publication Database DiVA |
op_collection_id |
ftkthstockholm |
language |
English |
topic |
Candida antarctica lipase B enzyme catalysis enzyme catalytic promiscuity molecular modeling ω-transaminase Biochemistry and Molecular Biology Biokemi och molekylärbiologi |
spellingShingle |
Candida antarctica lipase B enzyme catalysis enzyme catalytic promiscuity molecular modeling ω-transaminase Biochemistry and Molecular Biology Biokemi och molekylärbiologi Svedendahl, Maria Lipase and ω-Transaminase : Biocatalytic Investigations |
topic_facet |
Candida antarctica lipase B enzyme catalysis enzyme catalytic promiscuity molecular modeling ω-transaminase Biochemistry and Molecular Biology Biokemi och molekylärbiologi |
description |
In a lipase investigation, Candida antarctica lipase B (CALB) are explored for enzyme catalytic promiscuity. Enzyme catalytic promiscuity is shown by enzymes catalyzing alternative catalytic transformations proceeding via different transition state structures than normal. CALB normally performs hydrolysis reactions by activating and coordinating carboxylic acid/ester substrates in an oxyanion hole prior to nucleophilic attack from an active-site serine resulting in acyl enzyme formation. The idea of utilizing the carbonyl activation oxyanion hole in the active-site of CALB to catalyze promiscuous reactions arose by combining catalytic and structural knowledge about the enzyme with chemical imagination. We choose to explore conjugate addition and direct epoxidation activities in CALB by combining molecular modeling and kinetic experiments. By quantum-chemical calculations, the investigated promiscuous reactions were shown to proceed via ordered reaction mechanisms that differ from the native ping pong bi bi reaction mechanism. The investigated promiscuous activities were shown to take place in the enzyme active-site by various kinetic experiments, but despite this, no enantioselectivity was displayed. The reason for this is unknown, but can be a result of a too voluminous active-site or the lack of covalent coordination of the substrates during enzyme-catalysis (Paper I-IV). Combining enzyme structural knowledge with chemical imagination may provide numerous novel enzyme activities to be discovered. In an ω-transaminase investigation, two (S)-selective ω-transaminases from Arthrobacter citreus (Ac-ωTA) and Chromobacterium violaceum (Cv-ωTA) are explored aiming to improve their catalytic properties. Structural knowledge of these enzymes was provided by homology modeling. A homology structure of Ac-ωTA was successfully applied for rational design resulting in enzyme variants with improved enantioselectivity. Additionally, a single-point mutation reversed the enantiopreference of the enzyme from (S) to (R), which ... |
format |
Doctoral or Postdoctoral Thesis |
author |
Svedendahl, Maria |
author_facet |
Svedendahl, Maria |
author_sort |
Svedendahl, Maria |
title |
Lipase and ω-Transaminase : Biocatalytic Investigations |
title_short |
Lipase and ω-Transaminase : Biocatalytic Investigations |
title_full |
Lipase and ω-Transaminase : Biocatalytic Investigations |
title_fullStr |
Lipase and ω-Transaminase : Biocatalytic Investigations |
title_full_unstemmed |
Lipase and ω-Transaminase : Biocatalytic Investigations |
title_sort |
lipase and ω-transaminase : biocatalytic investigations |
publisher |
KTH, Biokemi |
publishDate |
2010 |
url |
http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-13279 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_relation |
Trita-BIO-Report, 1654-2312 2010:10 orcid:0000-0003-2371-8755 http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-13279 urn:isbn:978-91-7415-648-5 |
op_rights |
info:eu-repo/semantics/openAccess |
_version_ |
1766271524192387072 |