Properties and action pattern of the recombinant mannuronan C-5-epimerase AlgE2

the mannuronan C-5-epimerase AlgE2 is one of a family of Ca2+-dependent epi merases secreted by Azotobacter vinelandii. These enzymes catalyze the conv ersion of beta-D-mannuronic acid residues (M) to alpha-L-guluronic acid res idues (G) in alginate. AlgE2 has been produced by fermentation with a recom binant strain of Escherichia coli, isolated and partially purified. Epimeri zation with AlgE2 increased the content of G-residues in different alginate s from starting values of 0-45% up to approximately 70%. The new G-residues were mainly present in short blocks. Although G-residues may be introduced next to pre-existing G-residues, AlgE2 was not able to epimerize strictly alternating MG-structures. The epimerization with AlgE2 was greatly affecte d by the concentration of Ca2+. The type of alginate used as substrate affe cted the reaction rate and the reaction pattern especially at low Ca2+ conc entration. AlgE2 appears to act by a preferred attack mechanism where the e nzyme associates with different sequences in the alginate depending on the concentration of Ca2+. During epimerization, AlgE2 occasionally causes clea vage of the alginate chain. The observed frequency corresponds to 1-3 break s per 1,000 M-units epimerized. (C) 1999 Elsevier Science Inc. All rights r eserved.