Abstract
Materials and procedures are described for the overproduction and purification of acetate kinase from Methanosarcina thermophila. Methods are detailed for large-scale preparation of the unaltered enzyme and for overproduction and one-step purification of the histidine-tagged recombinant acetate kinase. Also included is a method for routine assay of the enzyme, and enzymelinked assay procedures for obtaining kinetic constants in the forward and reverse reaction directions. Finally, a method is included for the overproduction and purification of a variant of the acetate kinase with an altered Km for acetate. A method is presented utilizing the variant for determination of acetate levels in biological samples.
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© 2005 Humana Press Inc.
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Iyer, P., Ferry, J.G. (2005). Acetate Kinase From Methanosarcina thermophila, a Key Enzyme for Methanogenesis. In: Barredo, J.L. (eds) Microbial Enzymes and Biotransformations. Methods in Biotechnology, vol 17. Humana Press. https://doi.org/10.1385/1-59259-846-3:239
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DOI: https://doi.org/10.1385/1-59259-846-3:239
Publisher Name: Humana Press
Print ISBN: 978-1-58829-253-7
Online ISBN: 978-1-59259-846-5
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