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Biochemical characterisation of aconitase from Corynebacterium glutamicum

Paper ID Volume ID Publish Year Pages File Format Full-Text
23954 43484 2011 8 PDF Available
Title
Biochemical characterisation of aconitase from Corynebacterium glutamicum
Abstract

In this work, aconitase of the biotechnologically relevant microorganism Corynebacterium glutamicum was characterised. The specific activity of aconitase in extracts of glucose-grown cells was determined by four different assays. In three of them the formation or disappearance of cis-aconitate was measured, whereas in the fourth assay the aconitase reaction was coupled with isocitrate dehydrogenase. The highest activity was determined with cis-aconitate as substrate (0.433 ± 0.054 U mg−1) and the lowest one with the coupled assay and citrate as substrate (0.134 ± 0.026 U mg−1). Only the latter assay covers the complete aconitase reaction and thus gives the most relevant information on in vivo activity. For the determination of kinetic constants, aconitase was heterologously overproduced, purified, reactivated and biochemically characterised. Size exclusion chromatography indicated that the protein is monomeric. The enzyme showed Michaelis–Menten kinetics with Km values of 480 ± 200 μM for citrate, 552 ± 302 μM for isocitrate and 18.5 ± 3.4 μM for cis-aconitate. The highest Vmax was observed for the hydration of cis-aconitate with 40.6 U mg−1. Aconitase was active over a wide pH and temperature range with maximal activity between pH 7.5 and 7.75 and at about 50 °C.

Keywords
Aconitase; Citrate cycle; Corynebacterium glutamicum; Glyoxylate shunt; Methylcitrate cycle
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Biochemical characterisation of aconitase from Corynebacterium glutamicum
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 154, Issues 2–3, 10 July 2011, Pages 163–170
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