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Membrane-bound Δ12 fatty acid desaturase (FAD12); From Brassica napus to E. coli expression system

Fatty acid desaturase catalyzes the desaturation reactions by insertion of double bonds into the fatty acyl chain, producing unsaturated fatty acids. Though soluble fatty acid desaturases have been studied widely in advanced organisms, there are very limited studies of membrane fatty acid desaturase...

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Main Authors: Abd Halim, Nur Farah Anis, Mohamad Ali, Mohd Shukuri, Leow, Adam Thean Chor, Raja Abd Rahman, Raja Noor Zaliha
Format: Article
Published: Elsevier 2021
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spelling oai:psasir.upm.edu.my:94146 http://psasir.upm.edu.my/id/eprint/94146/ Membrane-bound Δ12 fatty acid desaturase (FAD12); From Brassica napus to E. coli expression system Abd Halim, Nur Farah Anis Mohamad Ali, Mohd Shukuri Leow, Adam Thean Chor Raja Abd Rahman, Raja Noor Zaliha Fatty acid desaturase catalyzes the desaturation reactions by insertion of double bonds into the fatty acyl chain, producing unsaturated fatty acids. Though soluble fatty acid desaturases have been studied widely in advanced organisms, there are very limited studies of membrane fatty acid desaturases due to the difficulty of generating recombinant desaturase. Brassica napus is a rapeseed, which possesses a range of different membrane-bound desaturases capable of producing fatty acids including Δ3, Δ4, Δ8, Δ9, Δ12, and Δ15 fatty acids. The 1155 bp open reading frame of Δ12 fatty acid desaturase (FAD12) from Brassica napus codes for 383 amino acid residues with a molecular weight of 44 kDa. It was expressed in Escherichia coli at 37 °C in soluble and insoluble forms when induced with 0.5 mM IPTG. Soluble FAD12 has been purified using Ni2+-Sepharose affinity chromatography with a total protein yield of 0.728 mg/mL. Gas chromatography–mass spectrometry (GC–MS) analysis revealed that desaturase activity of FAD12 could produce linoleic acid from oleic acid at a retention time of 17.6 with a conversion rate of 47%. Characterization of purified FAD12 revealed the optimal temperature of FAD12 was 50 °C with 2 mM preferred substrate concentration of oleic acid. Analysis of circular dichroism (CD) showed FAD12 was made up of 47.3% and 0.9% of alpha-helix and β-sheet secondary structures. The predicted Tm value was 50.2 °C. Elsevier 2021-06-01 Article PeerReviewed Abd Halim, Nur Farah Anis and Mohamad Ali, Mohd Shukuri and Leow, Adam Thean Chor and Raja Abd Rahman, Raja Noor Zaliha (2021) Membrane-bound Δ12 fatty acid desaturase (FAD12); From Brassica napus to E. coli expression system. International Journal of Biological Macromolecules, 180. 242 - 251. ISSN 0141-8130 https://www.sciencedirect.com/science/article/pii/S0141813021005894?via%3Dihub 10.1016/j.ijbiomac.2021.03.072
institution UPM IR
collection UPM IR
description Fatty acid desaturase catalyzes the desaturation reactions by insertion of double bonds into the fatty acyl chain, producing unsaturated fatty acids. Though soluble fatty acid desaturases have been studied widely in advanced organisms, there are very limited studies of membrane fatty acid desaturases due to the difficulty of generating recombinant desaturase. Brassica napus is a rapeseed, which possesses a range of different membrane-bound desaturases capable of producing fatty acids including Δ3, Δ4, Δ8, Δ9, Δ12, and Δ15 fatty acids. The 1155 bp open reading frame of Δ12 fatty acid desaturase (FAD12) from Brassica napus codes for 383 amino acid residues with a molecular weight of 44 kDa. It was expressed in Escherichia coli at 37 °C in soluble and insoluble forms when induced with 0.5 mM IPTG. Soluble FAD12 has been purified using Ni2+-Sepharose affinity chromatography with a total protein yield of 0.728 mg/mL. Gas chromatography–mass spectrometry (GC–MS) analysis revealed that desaturase activity of FAD12 could produce linoleic acid from oleic acid at a retention time of 17.6 with a conversion rate of 47%. Characterization of purified FAD12 revealed the optimal temperature of FAD12 was 50 °C with 2 mM preferred substrate concentration of oleic acid. Analysis of circular dichroism (CD) showed FAD12 was made up of 47.3% and 0.9% of alpha-helix and β-sheet secondary structures. The predicted Tm value was 50.2 °C.
format Article
author Abd Halim, Nur Farah Anis
Mohamad Ali, Mohd Shukuri
Leow, Adam Thean Chor
Raja Abd Rahman, Raja Noor Zaliha
spellingShingle Abd Halim, Nur Farah Anis
Mohamad Ali, Mohd Shukuri
Leow, Adam Thean Chor
Raja Abd Rahman, Raja Noor Zaliha
Membrane-bound Δ12 fatty acid desaturase (FAD12); From Brassica napus to E. coli expression system
author_facet Abd Halim, Nur Farah Anis
Mohamad Ali, Mohd Shukuri
Leow, Adam Thean Chor
Raja Abd Rahman, Raja Noor Zaliha
author_sort Abd Halim, Nur Farah Anis
title Membrane-bound Δ12 fatty acid desaturase (FAD12); From Brassica napus to E. coli expression system
title_short Membrane-bound Δ12 fatty acid desaturase (FAD12); From Brassica napus to E. coli expression system
title_full Membrane-bound Δ12 fatty acid desaturase (FAD12); From Brassica napus to E. coli expression system
title_fullStr Membrane-bound Δ12 fatty acid desaturase (FAD12); From Brassica napus to E. coli expression system
title_full_unstemmed Membrane-bound Δ12 fatty acid desaturase (FAD12); From Brassica napus to E. coli expression system
title_sort membrane-bound δ12 fatty acid desaturase (fad12); from brassica napus to e. coli expression system
publisher Elsevier
publishDate 2021
_version_ 1819300701020356608
score 13.4562235

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