Biochemical characterisation of aconitase from Corynebacterium glutamicum

Journal of Biotechnology
Meike Baumgart, Michael Bott

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 Umg(-1)) and the lowest one with the coupled assay and citrate as substrate (0.134 ± 0.026 Umg(-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 K(m) values of 480 ± 200 μM for citrate, 552 ± 302 μM for isocitrate and 18.5 ± 3.4 μM for cis-aconitate. The highest V(max) was observed for the hydration of cis-aconitate with 40.6 Umg(-1). Aconitase was active over a wide pH and temperature range with maximal activity...Continue Reading

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Citations

Apr 15, 2016·Proceedings of the National Academy of Sciences of the United States of America·Andreas KüberlMichael Bott
Jun 24, 2015·Metabolic Engineering·Andreas OttenMichael Bott
Jul 3, 2013·Journal of Proteomics·Natalia Comba GonzálezDolly Montoya
Oct 4, 2014·Applied Microbiology and Biotechnology·Kiira S VuoristoRuud A Weusthuis
Jun 13, 2020·Biotechnology and Bioengineering·Xiulai ChenLiming Liu
Jun 6, 2020·Frontiers in Plant Science·Jayamini JayawardhaneAbir U Igamberdiev
Oct 28, 2021·ACS Synthetic Biology·Jing ZhangTao Chen

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