Cargando…
Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference
[Image: see text] The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against...
Autores principales: | , , , , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American
Chemical Society
2021
|
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8461973/ https://www.ncbi.nlm.nih.gov/pubmed/34589311 http://dx.doi.org/10.1021/acssuschemeng.1c00699 |
_version_ | 1784572101390237696 |
---|---|
author | Bosch, Sandra Sanchez-Freire, Esther del Pozo, María Luisa C̆esnik, Morana Quesada, Jaime Mate, Diana M. Hernández, Karel Qi, Yuyin Clapés, Pere Vasić-Rački, Đurđa Findrik Blažević, Zvjezdana Berenguer, José Hidalgo, Aurelio |
author_facet | Bosch, Sandra Sanchez-Freire, Esther del Pozo, María Luisa C̆esnik, Morana Quesada, Jaime Mate, Diana M. Hernández, Karel Qi, Yuyin Clapés, Pere Vasić-Rački, Đurđa Findrik Blažević, Zvjezdana Berenguer, José Hidalgo, Aurelio |
author_sort | Bosch, Sandra |
collection | PubMed |
description | [Image: see text] The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better “evolvability” of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 °C higher melting temperature (T(m)) and 3-fold longer half-life at 65 °C compared to parental Hph5, with no changes in the steady-state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-keto-3-deoxy-l-rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 °C in T(m) compared to the wild-type enzyme but severely lower retro-aldol activities (150- and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a T(m) 8.9 °C higher, a 16-fold improvement in half-life at 60 °C and higher operational stability than the wild-type, without substantial modification of the kinetic parameters. |
format | Online Article Text |
id | pubmed-8461973 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American
Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-84619732021-09-27 Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference Bosch, Sandra Sanchez-Freire, Esther del Pozo, María Luisa C̆esnik, Morana Quesada, Jaime Mate, Diana M. Hernández, Karel Qi, Yuyin Clapés, Pere Vasić-Rački, Đurđa Findrik Blažević, Zvjezdana Berenguer, José Hidalgo, Aurelio ACS Sustain Chem Eng [Image: see text] The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better “evolvability” of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 °C higher melting temperature (T(m)) and 3-fold longer half-life at 65 °C compared to parental Hph5, with no changes in the steady-state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-keto-3-deoxy-l-rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 °C in T(m) compared to the wild-type enzyme but severely lower retro-aldol activities (150- and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a T(m) 8.9 °C higher, a 16-fold improvement in half-life at 60 °C and higher operational stability than the wild-type, without substantial modification of the kinetic parameters. American Chemical Society 2021-04-07 2021-04-19 /pmc/articles/PMC8461973/ /pubmed/34589311 http://dx.doi.org/10.1021/acssuschemeng.1c00699 Text en © 2021 American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Bosch, Sandra Sanchez-Freire, Esther del Pozo, María Luisa C̆esnik, Morana Quesada, Jaime Mate, Diana M. Hernández, Karel Qi, Yuyin Clapés, Pere Vasić-Rački, Đurđa Findrik Blažević, Zvjezdana Berenguer, José Hidalgo, Aurelio Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference |
title | Thermostability Engineering of a Class II Pyruvate
Aldolase from Escherichia coli by in Vivo Folding Interference |
title_full | Thermostability Engineering of a Class II Pyruvate
Aldolase from Escherichia coli by in Vivo Folding Interference |
title_fullStr | Thermostability Engineering of a Class II Pyruvate
Aldolase from Escherichia coli by in Vivo Folding Interference |
title_full_unstemmed | Thermostability Engineering of a Class II Pyruvate
Aldolase from Escherichia coli by in Vivo Folding Interference |
title_short | Thermostability Engineering of a Class II Pyruvate
Aldolase from Escherichia coli by in Vivo Folding Interference |
title_sort | thermostability engineering of a class ii pyruvate
aldolase from escherichia coli by in vivo folding interference |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8461973/ https://www.ncbi.nlm.nih.gov/pubmed/34589311 http://dx.doi.org/10.1021/acssuschemeng.1c00699 |
work_keys_str_mv | AT boschsandra thermostabilityengineeringofaclassiipyruvatealdolasefromescherichiacolibyinvivofoldinginterference AT sanchezfreireesther thermostabilityengineeringofaclassiipyruvatealdolasefromescherichiacolibyinvivofoldinginterference AT delpozomarialuisa thermostabilityengineeringofaclassiipyruvatealdolasefromescherichiacolibyinvivofoldinginterference AT cesnikmorana thermostabilityengineeringofaclassiipyruvatealdolasefromescherichiacolibyinvivofoldinginterference AT quesadajaime thermostabilityengineeringofaclassiipyruvatealdolasefromescherichiacolibyinvivofoldinginterference AT matedianam thermostabilityengineeringofaclassiipyruvatealdolasefromescherichiacolibyinvivofoldinginterference AT hernandezkarel thermostabilityengineeringofaclassiipyruvatealdolasefromescherichiacolibyinvivofoldinginterference AT qiyuyin thermostabilityengineeringofaclassiipyruvatealdolasefromescherichiacolibyinvivofoldinginterference AT clapespere thermostabilityengineeringofaclassiipyruvatealdolasefromescherichiacolibyinvivofoldinginterference AT vasicrackiđurđa thermostabilityengineeringofaclassiipyruvatealdolasefromescherichiacolibyinvivofoldinginterference AT findrikblazeviczvjezdana thermostabilityengineeringofaclassiipyruvatealdolasefromescherichiacolibyinvivofoldinginterference AT berenguerjose thermostabilityengineeringofaclassiipyruvatealdolasefromescherichiacolibyinvivofoldinginterference AT hidalgoaurelio thermostabilityengineeringofaclassiipyruvatealdolasefromescherichiacolibyinvivofoldinginterference |