Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme
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© Springer-Verlag 1996
O R I G I N A L PA P E R
C. G. van Ginkel · G. B. Rikken · A. G. M. Kroon · S. W. M. Kengen
Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme
Received: 9 April 1996 / Accepted: 12 August 1996
Abstract A novel enzyme that catalyzes the disproportionation of chlorite into chloride and oxygen was purified from a gram-negative bacterium, strain GR-1 to homogeneity. A four-step purification procedure comprising QSepharose, hydroxyapatite, and phenyl-Superose chromatography and ultrafiltration resulted in a 13.7-fold purified enzyme with a final specific activity of 2.0 mmol min–1 (mg protein)–1. The dismutase obeyed MichaelisMenten kinetics. The Vmax and Km calculated for chlorite were 2,200 U (mg protein)–1 and 170 µM, respectively. Dismutase activity was inhibited by hydroxylamine, cyanide, and azide, but not by 3-amino-1,2,4-triazole. Chlorite dismutase had a molecular mass of 140 kDa and consisted of four 32-kDa subunits. The enzyme was red-colored and had a Soret peak at 392 nm. Per subunit, it contained 0.9 molecule of protoheme IX and 0.7 molecule of iron. Chlorite dismutase displayed maxima for activity at pH 6.0 and 30° C. Key words Chlorite · Oxygen · Chloride · Chlorite dismutase · Homotetramer · Heme iron · Disproportionation
Introduction Chloro-oxo acids, i.e., perchlorate, chlorate, chlorite, and hypochlorite, are produced for a variety of purposes. Perchlorate serves as an oxidant in the aerospace and armament industries. Chlorate is used as a herbicide, and
C. G. van Ginkel (Y) · G. B. Rikken · A. G. M. Kroon Akzo Nobel Central Research, Analytical and Environmental Chemistry Department, Velperweg 76, 6800 SB Arnhem, The Netherlands Tel. +31-26-366-2634; Fax +31-26-366-2528 e-mail: [email protected] S. W. M. Kengen Wageningen Agricultural University, Department of Microbiology, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands
hypochlorite is widely employed as a disinfectant and as an antifouling agent in cooling-water systems. Chlorite and hypochlorite are bleaching agents used in the textile, pulp and paper industries. A result of the wide variety of applications is that chloro-oxo acids are present in water as contaminants. During the last few years, efforts have, therefore, been made to remove chloro-oxo acids from waste water and drinking water by either physicochemical or biotechnological methods (Griese et al. 1991; Malmqvist et al. 1991; Ataway and Smith 1993; Gonce and Voudrias 1994). Interest in biotechnological water treatment has resulted in the isolation of facultatively anaerobic microorganisms that can utilize perchlorate and chlorate as sole electron acceptor (Korenkov et al. 1976; Stepanyuk et al. 1992; Malmqvist et al. 1994). Among the few (per)chlorate-reducing organisms available in pure culture, only strain GR-1 has been studied in detail (Rikken et al. 1996). This organism, originally isolated from activated sludge used to treat domestic waste water, can grow on many fatty acids and dicarb
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