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Functional Heterologous Expression of Mature Lipase LipA from Pseudomonas aeruginosa PSA01 in Escherichia coli SHuffle and BL21 (DE3): Effect of the Expression Host on Thermal Stability and Solvent Tolerance of the Enzyme Produced

This study aimed to express heterologously the lipase LipA from Pseudomonas aeruginosa PSA01 obtained from palm fruit residues. In previous approaches, LipA was expressed in Escherichia coli fused with its signal peptide and without its disulfide bond, displaying low activity. We cloned the mature L...

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Autores principales: Pulido, Ingrid Yamile, Prieto, Erlide, Pieffet, Gilles Paul, Méndez, Lina, Jiménez-Junca, Carlos A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312249/
https://www.ncbi.nlm.nih.gov/pubmed/32486240
http://dx.doi.org/10.3390/ijms21113925
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author Pulido, Ingrid Yamile
Prieto, Erlide
Pieffet, Gilles Paul
Méndez, Lina
Jiménez-Junca, Carlos A.
author_facet Pulido, Ingrid Yamile
Prieto, Erlide
Pieffet, Gilles Paul
Méndez, Lina
Jiménez-Junca, Carlos A.
author_sort Pulido, Ingrid Yamile
collection PubMed
description This study aimed to express heterologously the lipase LipA from Pseudomonas aeruginosa PSA01 obtained from palm fruit residues. In previous approaches, LipA was expressed in Escherichia coli fused with its signal peptide and without its disulfide bond, displaying low activity. We cloned the mature LipA with its truncated chaperone Lif in a dual plasmid and overexpressed the enzyme in two E. coli strains: the traditional BL21 (DE3) and the SHuffle(®) strain, engineered to produce stable cytoplasmic disulfide bonds. We evaluated the effect of the disulfide bond on LipA stability using molecular dynamics. We expressed LipA successfully under isopropyl β-d-1-thio-galactopyranoside (IPTG) and slow autoinducing conditions. The SHuffle LipA showed higher residual activity at 45 °C and a greater hyperactivation after incubation with ethanol than the enzyme produced by E. coli BL21 (DE3). Conversely, the latter was slightly more stable in methanol 50% and 60% (t½: 49.5 min and 9 min) than the SHuffle LipA (t½: 31.5 min and 7.4 min). The molecular dynamics simulations showed that removing the disulfide bond caused some regions of LipA to become less flexible and some others to become more flexible, significantly affecting the closing lid and partially exposing the active site at all times.
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spelling pubmed-73122492020-06-26 Functional Heterologous Expression of Mature Lipase LipA from Pseudomonas aeruginosa PSA01 in Escherichia coli SHuffle and BL21 (DE3): Effect of the Expression Host on Thermal Stability and Solvent Tolerance of the Enzyme Produced Pulido, Ingrid Yamile Prieto, Erlide Pieffet, Gilles Paul Méndez, Lina Jiménez-Junca, Carlos A. Int J Mol Sci Article This study aimed to express heterologously the lipase LipA from Pseudomonas aeruginosa PSA01 obtained from palm fruit residues. In previous approaches, LipA was expressed in Escherichia coli fused with its signal peptide and without its disulfide bond, displaying low activity. We cloned the mature LipA with its truncated chaperone Lif in a dual plasmid and overexpressed the enzyme in two E. coli strains: the traditional BL21 (DE3) and the SHuffle(®) strain, engineered to produce stable cytoplasmic disulfide bonds. We evaluated the effect of the disulfide bond on LipA stability using molecular dynamics. We expressed LipA successfully under isopropyl β-d-1-thio-galactopyranoside (IPTG) and slow autoinducing conditions. The SHuffle LipA showed higher residual activity at 45 °C and a greater hyperactivation after incubation with ethanol than the enzyme produced by E. coli BL21 (DE3). Conversely, the latter was slightly more stable in methanol 50% and 60% (t½: 49.5 min and 9 min) than the SHuffle LipA (t½: 31.5 min and 7.4 min). The molecular dynamics simulations showed that removing the disulfide bond caused some regions of LipA to become less flexible and some others to become more flexible, significantly affecting the closing lid and partially exposing the active site at all times. MDPI 2020-05-30 /pmc/articles/PMC7312249/ /pubmed/32486240 http://dx.doi.org/10.3390/ijms21113925 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Pulido, Ingrid Yamile
Prieto, Erlide
Pieffet, Gilles Paul
Méndez, Lina
Jiménez-Junca, Carlos A.
Functional Heterologous Expression of Mature Lipase LipA from Pseudomonas aeruginosa PSA01 in Escherichia coli SHuffle and BL21 (DE3): Effect of the Expression Host on Thermal Stability and Solvent Tolerance of the Enzyme Produced
title Functional Heterologous Expression of Mature Lipase LipA from Pseudomonas aeruginosa PSA01 in Escherichia coli SHuffle and BL21 (DE3): Effect of the Expression Host on Thermal Stability and Solvent Tolerance of the Enzyme Produced
title_full Functional Heterologous Expression of Mature Lipase LipA from Pseudomonas aeruginosa PSA01 in Escherichia coli SHuffle and BL21 (DE3): Effect of the Expression Host on Thermal Stability and Solvent Tolerance of the Enzyme Produced
title_fullStr Functional Heterologous Expression of Mature Lipase LipA from Pseudomonas aeruginosa PSA01 in Escherichia coli SHuffle and BL21 (DE3): Effect of the Expression Host on Thermal Stability and Solvent Tolerance of the Enzyme Produced
title_full_unstemmed Functional Heterologous Expression of Mature Lipase LipA from Pseudomonas aeruginosa PSA01 in Escherichia coli SHuffle and BL21 (DE3): Effect of the Expression Host on Thermal Stability and Solvent Tolerance of the Enzyme Produced
title_short Functional Heterologous Expression of Mature Lipase LipA from Pseudomonas aeruginosa PSA01 in Escherichia coli SHuffle and BL21 (DE3): Effect of the Expression Host on Thermal Stability and Solvent Tolerance of the Enzyme Produced
title_sort functional heterologous expression of mature lipase lipa from pseudomonas aeruginosa psa01 in escherichia coli shuffle and bl21 (de3): effect of the expression host on thermal stability and solvent tolerance of the enzyme produced
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312249/
https://www.ncbi.nlm.nih.gov/pubmed/32486240
http://dx.doi.org/10.3390/ijms21113925
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