PURIFICATION OF THE COENZYME B12-CONTAINING 2-METHYLENEGLUTARATE MUTASE FROM CLOSTRIDIUM-BARKERI BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Two methods are described by which the enzymes 2-methyleneglutarate mutase and 3-methylitaconate DELTA-isomerase from Closiridium barkeri have been separated by high-performance liquid chromatography on a much larger scale than reported previously. First, the mutase eluted before the DELTA-isomerase after incubation with the mild detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulphonate (CHAPS) followed by high-performance anion-exchange chromatography on Mono Q in the presence of the same detergent. Second, an even better separation, although with a lower yield of mutase, was obtained by hydrophobic interaction chromatography on phenyl-Sepharose HiLoad, whereby the enzymes were eluted in the reverse order. Final high-performance anion-exchange chromatography of the latter preparation on Mono Q at pH 8 gave highly purified 2-methyleneglutarate mutase (> 95% purity) which had a pink-orange colour (lambda(max) 280, 375, 470 and 532 nm). The enzyme was active in the absence of coenzyme B-12 (adenosylcobalamin) and contained 2.1 mol of this coenzyme per homotetramer (molecular mass, m = 300 kilodalton).