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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

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Biosciences Doctoral Program, Universidad de La Sabana, km 7 Autopista Norte, Chía 250001, Colombia
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Agro-industrial Processes Research Group, Engineering Faculty, Universidad de La Sabana, km 7 Autopista Norte, Chía, Cundinamarca 250001, Colombia
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Science Faculty, Universidad Antonio Nariño, Calle 58 A # 37–94 Bogotá D.C.111511, Colombia
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Bioprospecting Research Group, Engineering Faculty, Universidad de La Sabana, km 7 Autopista Norte, Chía, Cundinamarca 250001, Colombia
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(11), 3925; https://doi.org/10.3390/ijms21113925
Received: 2 March 2020 / Revised: 23 May 2020 / Accepted: 26 May 2020 / Published: 30 May 2020
(This article belongs to the Section Biochemistry)
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. View Full-Text
Keywords: Pseudomonas aeruginosa lipase; lipase LipA; overexpression of E. coli SHuffle; lipase and foldase overexpression; expression of disulfide bond proteins Pseudomonas aeruginosa lipase; lipase LipA; overexpression of E. coli SHuffle; lipase and foldase overexpression; expression of disulfide bond proteins
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MDPI and ACS Style

Pulido, I.Y.; Prieto, E.; Pieffet, G.P.; Méndez, L.; Jiménez-Junca, C.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. Int. J. Mol. Sci. 2020, 21, 3925. https://doi.org/10.3390/ijms21113925

AMA Style

Pulido IY, Prieto E, Pieffet GP, Méndez L, Jiménez-Junca CA. 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. International Journal of Molecular Sciences. 2020; 21(11):3925. https://doi.org/10.3390/ijms21113925

Chicago/Turabian Style

Pulido, Ingrid Y.; Prieto, Erlide; Pieffet, Gilles P.; Méndez, Lina; Jiménez-Junca, Carlos A. 2020. "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" Int. J. Mol. Sci. 21, no. 11: 3925. https://doi.org/10.3390/ijms21113925

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