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Purification and characterization of a membrane-bound linoleic acid isomerase from Clostridium sporogenes

Paper ID Volume ID Publish Year Pages File Format Full-Text
18046 42710 2007 9 PDF Available
Title
Purification and characterization of a membrane-bound linoleic acid isomerase from Clostridium sporogenes
Abstract

Clostridium sporogenes ATCC 25762 converted linoleic acid (c9,c12, 18:2) to c9,t11 conjugated linoleic acid (CLA, 18:2). The linoleic acid isomerase was membrane-associated and was very unstable, especially after being solubilized by detergents. Isomerase extraction, solubilization and stability were significantly improved by optimizing buffer composition and pH, and by minimizing detergent and protein precipitation. The isomerase was purified by DEAE, chromatofocusing and size exclusion column chromatography, achieving an overall purification of 364-fold and a specific activity of 400 nmol min−1 mg−1 protein. The purified enzyme was a single polypeptide band on native PAGE with an estimated molecular weight of about 190 kDa and a single band of around 45 kDa on SDS-PAGE, suggesting the enzyme is a homotetramer. The optimum pH for isomerase activity was about 7.5. No external cofactors or energy sources were required for catalysis. The isomerase had a definite bias toward substrates containing cis double bonds at the c9 and c12 positions of C18 polyunsaturated fatty acids. A free carboxyl group is absolutely necessary for isomerization. The Km for linoleic acid was 12 μM. The enzyme was subjected to substrate inhibition at linoleic concentrations above 20 μM. Oleic acid, palmitoleic acid and some linoleic acid derivatives also inhibited the isomerase.

Keywords
Biotransformation; Chromatography; CLA; Conjugated linoleic acid; Linoleic acid isomerase; Membrane protein purification
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Purification and characterization of a membrane-bound linoleic acid isomerase from Clostridium sporogenes
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 40, Issue 4, 5 March 2007, Pages 831–839
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us