ISOLATION AND CHARACTERIZATION OF AN EXTRACELLULAR GLYCOSYLATED PROTEIN COMPLEX FROM CLOSTRIDIUM-THERMOSACCHAROLYTICUM WITH PECTIN METHYLESTERASE AND POLYGALACTURONATE HYDROLASE ACTIVITY

M VANRIJSSEL, GJ GERWIG, TA HANSEN

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    Abstract

    An extracellular protein complex was isolated from the supernatant of a pectin-limited continuous culture 0 Clostridium thermosaccharolyticum Haren. The complex possessed both pectin methylesterase (EC 3.1.1.11) and exo-poly-alpha-galacturonate hydrolase (EC 3.2.1.82) activity and produced digalacturonate from the nonreducing end of the pectin chain. The protein consisted of 230- and 25-kDa subunits. The large subunit contained 10% (wt/wt) sugars (N-acetylgalactosamine and galactose). Under physiological conditions both activities acted in a coordinated manner: the ratio between methanol and digalacturonate released during degradation was constant and equal to the degree of esterification of the pectin used. Prolonged incubation of the enzyme with pectin led to a nondialyzable fraction that was enriched in neutral sugars, such as arabinose, rhamnose, and galactose; the high rhamnose/galacturonic acid ratio was indicative of hairy region-like structures. The smallest substrate utilized by the hydrolase was a tetragalacturonate. V(max) with oligogalacturonates increased with increasing chain length. The K(m) and V(max) values for the polygalacturonate hydrolase with citrus pectate as a substrate were 0.8 g liter-1 and 180 mumol min-1 mg of protein-1, respectively. The K(m) and V(max) for the esterase with citrus pectin as a substrate were 1.2 g liter-1 and 440 mumol min-1 mg of protein-1, respectively. The temperature optima for the hydrolase and esterase were 70 and 60-degrees-C, respectively. Both enzyme activities were stable for more than I h at 70-degrees-C. The exo-polygalacturonate hydrolase of Clostridium thermosulfurogenes was partially purified while the methylesterase was also copurified.

    Original languageEnglish
    Pages (from-to)828-836
    Number of pages9
    JournalApplied and environmental microbiology
    Volume59
    Issue number3
    Publication statusPublished - Mar-1993

    Keywords

    • PURIFICATION
    • ENZYMES
    • THERMOSULFUROGENES
    • THERMOCELLUM
    • FERMENTATION
    • PHYSIOLOGY
    • BACTERIA
    • GELS

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