Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution
To expand the functionality of lipase B from Candida antarctica (CALB) we have used directed evolution to create CALB mutants with improved resistance towards irreversible thermal inactivation. Two mutants, 23G5 and 195F1, were generated with over a 20‐fold increase in half‐life at 70°C compared wit...
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fthighwire:oai:open-archive.highwire.org:proeng:16/8/599 2023-05-15T13:46:15+02:00 Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution Zhang, Ningyan Suen, Wen-Chen Windsor, William Xiao, Li Madison, Vincent Zaks, Aleksey 2003-08-01 00:00:00.0 text/html http://peds.oxfordjournals.org/cgi/content/short/16/8/599 https://doi.org/10.1093/protein/gzg074 en eng Oxford University Press http://peds.oxfordjournals.org/cgi/content/short/16/8/599 http://dx.doi.org/10.1093/protein/gzg074 Copyright (C) 2003, Oxford University Press ORIGINAL ARTICLES TEXT 2003 fthighwire https://doi.org/10.1093/protein/gzg074 2007-06-25T01:41:24Z To expand the functionality of lipase B from Candida antarctica (CALB) we have used directed evolution to create CALB mutants with improved resistance towards irreversible thermal inactivation. Two mutants, 23G5 and 195F1, were generated with over a 20‐fold increase in half‐life at 70°C compared with the wild‐type CALB (WT‐CALB). The increase in half‐life was attributed to a lower propensity of the mutants to aggregate in the unfolded state and to an improved refolding. The first generation mutant, 23G5, obtained by error‐prone PCR, had two amino acid mutations, V210I and A281E. The second generation mutant, 195F1, derived from 23G5 by error‐prone PCR, had one additional mutation, V221D. Amino acid substitutions at positions 221 and 281 were determined to be critical for lipase stability, while the residue at position 210 had only a marginal effect. The catalytic efficiency of the mutants with p ‐nitrophenyl butyrate and 6,8‐difluoro‐4‐methylumbelliferyl octanoate was also found to be superior to that of WT‐CALB. Text Antarc* Antarctica HighWire Press (Stanford University) Protein Engineering Design and Selection 16 8 599 605 |
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HighWire Press (Stanford University) |
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English |
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ORIGINAL ARTICLES |
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ORIGINAL ARTICLES Zhang, Ningyan Suen, Wen-Chen Windsor, William Xiao, Li Madison, Vincent Zaks, Aleksey Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution |
topic_facet |
ORIGINAL ARTICLES |
description |
To expand the functionality of lipase B from Candida antarctica (CALB) we have used directed evolution to create CALB mutants with improved resistance towards irreversible thermal inactivation. Two mutants, 23G5 and 195F1, were generated with over a 20‐fold increase in half‐life at 70°C compared with the wild‐type CALB (WT‐CALB). The increase in half‐life was attributed to a lower propensity of the mutants to aggregate in the unfolded state and to an improved refolding. The first generation mutant, 23G5, obtained by error‐prone PCR, had two amino acid mutations, V210I and A281E. The second generation mutant, 195F1, derived from 23G5 by error‐prone PCR, had one additional mutation, V221D. Amino acid substitutions at positions 221 and 281 were determined to be critical for lipase stability, while the residue at position 210 had only a marginal effect. The catalytic efficiency of the mutants with p ‐nitrophenyl butyrate and 6,8‐difluoro‐4‐methylumbelliferyl octanoate was also found to be superior to that of WT‐CALB. |
format |
Text |
author |
Zhang, Ningyan Suen, Wen-Chen Windsor, William Xiao, Li Madison, Vincent Zaks, Aleksey |
author_facet |
Zhang, Ningyan Suen, Wen-Chen Windsor, William Xiao, Li Madison, Vincent Zaks, Aleksey |
author_sort |
Zhang, Ningyan |
title |
Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution |
title_short |
Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution |
title_full |
Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution |
title_fullStr |
Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution |
title_full_unstemmed |
Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution |
title_sort |
improving tolerance of candida antarctica lipase b towards irreversible thermal inactivation through directed evolution |
publisher |
Oxford University Press |
publishDate |
2003 |
url |
http://peds.oxfordjournals.org/cgi/content/short/16/8/599 https://doi.org/10.1093/protein/gzg074 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_relation |
http://peds.oxfordjournals.org/cgi/content/short/16/8/599 http://dx.doi.org/10.1093/protein/gzg074 |
op_rights |
Copyright (C) 2003, Oxford University Press |
op_doi |
https://doi.org/10.1093/protein/gzg074 |
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Protein Engineering Design and Selection |
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16 |
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8 |
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599 |
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605 |
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1766238922370711552 |