Characterization of the neoagarotetra-ase and neoagarobiase of Cytophaga flevensis.
The degradation of neoagarotetraose and neoagarobiose by Cytophaga flevensis was investigated. The organism possesses an enzyme that hydrolyzes the tetramer by cleavage of its central beta-galactosidic linkage. The product of this reaction, neoagarobiose, is further hydrolyzed enzymatically to D-galactose and 3,6-anhydro-L-galactose. Both enzyme activities were localized in the cytoplasm. Attempts were made to partially purify the respective enzymes and although a 30-40 fold-purification was achieved, the final preparation contained both neoagarotetra-ase and neoagarobiase activities. Evidence was obtained that these activities were due to different enzymes. Neoagarotetra-ase is highly specific for oligosaccharides containing neoagarobiose units; the rate of hydrolysis is greatest with neoagarotetraose. It cannot hydrolyze pyruvated neoagarotetraose. Optimal conditions for its activity were pH 7.0 and 25 C. Neoagarobiase hydrolyzes only neoagarobiose and neoagarobiitol and optimal conditions for activity were pH 6.75 and 25 C. Both enzymes were inhibited by Ag+, Hg2+ and Zn2+ ions and by p-CMB, which indicates that thiol groups are present in their active centres. Both enzymes were induced by neoagaro-oligosaccharides and melibiose and were repressed when glucose was added to the medium. Neoagarobiase was also induced by D-galacturonic acid. In continuous culture, the rate of enzyme production was maximal at a dilution rate of 0.1 h-1.
Characterization of the neoagarotetra-ase and neoagarobiase of Cytophaga flevensis. The degradation of neoagarotetraose and neoagarobiose by Cytophaga flevensis was investigated. The organism possesses an enzyme that hydrolyzes the tetramer by cleavage of its central beta-galactosidic linkage. The product of this reaction, neoagarobiose, is further hydrolyzed enzymatically to D-galactose and 3,6-anhydro-L-galactose. Both enzyme activities were localized in the cytoplasm. Attempts were made to partially purify the respective enzymes and although a 30-40 fold-purification was achieved, the final preparation contained both neoagarotetra-ase and neoagarobiase activities. Evidence was obtained that these activities were due to different enzymes. Neoagarotetra-ase is highly specific for oligosaccharides containing neoagarobiose units; the rate of hydrolysis is greatest with neoagarotetraose. It cannot hydrolyze pyruvated neoagarotetraose. Optimal conditions for its activity were pH 7.0 and 25 C. Neoagarobiase hydrolyzes only neoagarobiose and neoagarobiitol and optimal conditions for activity were pH 6.75 and 25 C. Both enzymes were inhibited by Ag+, Hg2+ and Zn2+ ions and by p-CMB, which indicates that thiol groups are present in their active centres. Both enzymes were induced by neoagaro-oligosaccharides and melibiose and were repressed when glucose was added to the medium. Neoagarobiase was also induced by D-galacturonic acid. In continuous culture, the rate of enzyme production was maximal at a dilution rate of 0.1 h-1.