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Enhanced Soluble Expression of Linoleic Acid Isomerase by Coordinated Regulation of Promoter and Fusion Tag in Escherichia coli

PAI is a linoleic acid isomerase from Propionibacterium acnes and is the key enzyme in the synthesis of trans10, cis12-conjugated linoleic acid. However, the majority of the expressed PAI in Escherichia coli occurs in its nonfunctional form in inclusion bodies, limiting the biosynthesis of conjugate...

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Autores principales: Zhang, Baixi, Zhu, Tong, Huang, Xintian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9141242/
https://www.ncbi.nlm.nih.gov/pubmed/35627089
http://dx.doi.org/10.3390/foods11101515
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author Zhang, Baixi
Zhu, Tong
Huang, Xintian
author_facet Zhang, Baixi
Zhu, Tong
Huang, Xintian
author_sort Zhang, Baixi
collection PubMed
description PAI is a linoleic acid isomerase from Propionibacterium acnes and is the key enzyme in the synthesis of trans10, cis12-conjugated linoleic acid. However, the majority of the expressed PAI in Escherichia coli occurs in its nonfunctional form in inclusion bodies, limiting the biosynthesis of conjugated linoleic acid. In an attempt to improve the solubility of recombinant PAI in Escherichia coli, three promoters representing different transcriptional strengths (T7, CspA, and Trc), paired with three fusion tags, (His6, MBP, and Fh8), respectively, were investigated in this study. Among the nine recombinant strains, Escherichia coli BL21 (DE3) (pET24a-Mpai), containing the T7 promoter and MBP fusion tag, led to a considerable increase in PAI solubility to 86.2%. MBP-PAI was purified 41-fold using affinity column chromatography. The optimum catalytical conditions of MBP-PAI were 37 °C and pH 7.5 with the addition of 1 mmol/L Tween-20. Most of the tested metal ions inhibited MBP-PAI activity. The apparent kinetic parameters (Km and Vmax) were measured with linoleic acid concentrations ranging from 71 μM to 1428 μM. The substrate linoleic acid did not exert any inhibitory effect on MBP-PAI. The Km of MBP-PAI was 253.9 μmol/L, and the Vmax was 2253 nmol/min/mg. This study provided a new method for improving the solubility of the recombinant linoleic acid isomerase in Escherichia coli.
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spelling pubmed-91412422022-05-28 Enhanced Soluble Expression of Linoleic Acid Isomerase by Coordinated Regulation of Promoter and Fusion Tag in Escherichia coli Zhang, Baixi Zhu, Tong Huang, Xintian Foods Article PAI is a linoleic acid isomerase from Propionibacterium acnes and is the key enzyme in the synthesis of trans10, cis12-conjugated linoleic acid. However, the majority of the expressed PAI in Escherichia coli occurs in its nonfunctional form in inclusion bodies, limiting the biosynthesis of conjugated linoleic acid. In an attempt to improve the solubility of recombinant PAI in Escherichia coli, three promoters representing different transcriptional strengths (T7, CspA, and Trc), paired with three fusion tags, (His6, MBP, and Fh8), respectively, were investigated in this study. Among the nine recombinant strains, Escherichia coli BL21 (DE3) (pET24a-Mpai), containing the T7 promoter and MBP fusion tag, led to a considerable increase in PAI solubility to 86.2%. MBP-PAI was purified 41-fold using affinity column chromatography. The optimum catalytical conditions of MBP-PAI were 37 °C and pH 7.5 with the addition of 1 mmol/L Tween-20. Most of the tested metal ions inhibited MBP-PAI activity. The apparent kinetic parameters (Km and Vmax) were measured with linoleic acid concentrations ranging from 71 μM to 1428 μM. The substrate linoleic acid did not exert any inhibitory effect on MBP-PAI. The Km of MBP-PAI was 253.9 μmol/L, and the Vmax was 2253 nmol/min/mg. This study provided a new method for improving the solubility of the recombinant linoleic acid isomerase in Escherichia coli. MDPI 2022-05-23 /pmc/articles/PMC9141242/ /pubmed/35627089 http://dx.doi.org/10.3390/foods11101515 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/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 (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Zhang, Baixi
Zhu, Tong
Huang, Xintian
Enhanced Soluble Expression of Linoleic Acid Isomerase by Coordinated Regulation of Promoter and Fusion Tag in Escherichia coli
title Enhanced Soluble Expression of Linoleic Acid Isomerase by Coordinated Regulation of Promoter and Fusion Tag in Escherichia coli
title_full Enhanced Soluble Expression of Linoleic Acid Isomerase by Coordinated Regulation of Promoter and Fusion Tag in Escherichia coli
title_fullStr Enhanced Soluble Expression of Linoleic Acid Isomerase by Coordinated Regulation of Promoter and Fusion Tag in Escherichia coli
title_full_unstemmed Enhanced Soluble Expression of Linoleic Acid Isomerase by Coordinated Regulation of Promoter and Fusion Tag in Escherichia coli
title_short Enhanced Soluble Expression of Linoleic Acid Isomerase by Coordinated Regulation of Promoter and Fusion Tag in Escherichia coli
title_sort enhanced soluble expression of linoleic acid isomerase by coordinated regulation of promoter and fusion tag in escherichia coli
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9141242/
https://www.ncbi.nlm.nih.gov/pubmed/35627089
http://dx.doi.org/10.3390/foods11101515
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