One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase
tEnzymes active at low temperature are of great interest for industrial bioprocesses due to their highefficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes istheir increased enzymatic activity at low temperature, however the low thermostability presente...
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ftunivchile:oai:repositorio.uchile.cl:2250/168933 2023-05-15T13:56:31+02:00 One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase Acevedo, Juan Pablo Reetz, Manfred T. Asenjo de Leuze, Juan Parra, Loreto P. 2017 application/pdf https://doi.org/10.1016/j.enzmictec.2017.02.005 https://repositorio.uchile.cl/handle/2250/168933 en eng Elsevier Enzyme and Microbial Technology 100 (2017) 60–70 18790909 01410229 doi:10.1016/j.enzmictec.2017.02.005 https://repositorio.uchile.cl/handle/2250/168933 Attribution-NonCommercial-NoDerivs 3.0 Chile http://creativecommons.org/licenses/by-nc-nd/3.0/cl/ CC-BY-NC-ND Enzyme and Microbial Technology Cold-active enzymes Directed evolution epPCR Thermostability Xylanase Artículo de revista 2017 ftunivchile https://doi.org/10.1016/j.enzmictec.2017.02.005 2023-01-22T01:03:09Z tEnzymes active at low temperature are of great interest for industrial bioprocesses due to their highefficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes istheir increased enzymatic activity at low temperature, however the low thermostability presented inthis type of enzymes is still a major drawback for their application in biocatalysis. Directed evolutionof cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill thestability and activity requirements for the industry. This paper describes the recombinant expression andcharacterization of a new and highly active cold-adapted xylanase from the GH-family 10 (Xyl-L), andthe use of a novel one step combined directed evolution technique that comprises saturation mutage-nesis and focused epPCR as a feasible semi-rational strategy to improve the thermostability. The Xyl-Lenzyme was cloned from a marine-Antarctic bacterium, Psychrobacter sp. strain 2–17, recombinantlyexpressed in E. coli strain BL21(DE3) and characterized enzymatically. Molecular dynamic simulationsusing a homology model of the catalytic domain of Xyl-L were performed to detect flexible regions andresidues, which are considered to be the possible structural elements that define the thermolability ofthis enzyme. Mutagenic libraries were designed in order to stabilize the protein introducing mutationsin some of the flexible regions and residues identified. Twelve positive mutant clones were found toimprove the T5015value of the enzyme, in some cases without affecting the activity at 25◦C. The bestmutant showed a 4.3◦C increase in its T5015. The efficiency of the directed evolution approach can alsobe expected to work in the protein engineering of stereoselectivity. Article in Journal/Newspaper Antarc* Antarctic Universidad de Chile: Repositorio académico Antarctic Enzyme and Microbial Technology 100 60 70 |
institution |
Open Polar |
collection |
Universidad de Chile: Repositorio académico |
op_collection_id |
ftunivchile |
language |
English |
topic |
Cold-active enzymes Directed evolution epPCR Thermostability Xylanase |
spellingShingle |
Cold-active enzymes Directed evolution epPCR Thermostability Xylanase Acevedo, Juan Pablo Reetz, Manfred T. Asenjo de Leuze, Juan Parra, Loreto P. One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase |
topic_facet |
Cold-active enzymes Directed evolution epPCR Thermostability Xylanase |
description |
tEnzymes active at low temperature are of great interest for industrial bioprocesses due to their highefficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes istheir increased enzymatic activity at low temperature, however the low thermostability presented inthis type of enzymes is still a major drawback for their application in biocatalysis. Directed evolutionof cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill thestability and activity requirements for the industry. This paper describes the recombinant expression andcharacterization of a new and highly active cold-adapted xylanase from the GH-family 10 (Xyl-L), andthe use of a novel one step combined directed evolution technique that comprises saturation mutage-nesis and focused epPCR as a feasible semi-rational strategy to improve the thermostability. The Xyl-Lenzyme was cloned from a marine-Antarctic bacterium, Psychrobacter sp. strain 2–17, recombinantlyexpressed in E. coli strain BL21(DE3) and characterized enzymatically. Molecular dynamic simulationsusing a homology model of the catalytic domain of Xyl-L were performed to detect flexible regions andresidues, which are considered to be the possible structural elements that define the thermolability ofthis enzyme. Mutagenic libraries were designed in order to stabilize the protein introducing mutationsin some of the flexible regions and residues identified. Twelve positive mutant clones were found toimprove the T5015value of the enzyme, in some cases without affecting the activity at 25◦C. The bestmutant showed a 4.3◦C increase in its T5015. The efficiency of the directed evolution approach can alsobe expected to work in the protein engineering of stereoselectivity. |
format |
Article in Journal/Newspaper |
author |
Acevedo, Juan Pablo Reetz, Manfred T. Asenjo de Leuze, Juan Parra, Loreto P. |
author_facet |
Acevedo, Juan Pablo Reetz, Manfred T. Asenjo de Leuze, Juan Parra, Loreto P. |
author_sort |
Acevedo, Juan Pablo |
title |
One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase |
title_short |
One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase |
title_full |
One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase |
title_fullStr |
One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase |
title_full_unstemmed |
One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase |
title_sort |
one-step combined focused eppcr and saturation mutagenesis for thermostability evolution of a new cold-active xylanase |
publisher |
Elsevier |
publishDate |
2017 |
url |
https://doi.org/10.1016/j.enzmictec.2017.02.005 https://repositorio.uchile.cl/handle/2250/168933 |
geographic |
Antarctic |
geographic_facet |
Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_source |
Enzyme and Microbial Technology |
op_relation |
Enzyme and Microbial Technology 100 (2017) 60–70 18790909 01410229 doi:10.1016/j.enzmictec.2017.02.005 https://repositorio.uchile.cl/handle/2250/168933 |
op_rights |
Attribution-NonCommercial-NoDerivs 3.0 Chile http://creativecommons.org/licenses/by-nc-nd/3.0/cl/ |
op_rightsnorm |
CC-BY-NC-ND |
op_doi |
https://doi.org/10.1016/j.enzmictec.2017.02.005 |
container_title |
Enzyme and Microbial Technology |
container_volume |
100 |
container_start_page |
60 |
op_container_end_page |
70 |
_version_ |
1766264020394835968 |