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

Abstract

The mannuronan C-5-epimerase AlgE2 is one of a family of Ca 2+-dependent epimerases secreted by Azotobacter vinelandii. These enzymes catalyze the conversion of β- d-mannuronic acid residues (M) to α- l-guluronic acid residues (G) in alginate. AlgE2 has been produced by fermentation with a recombinant strain of Escherichia coli, isolated and partially purified. Epimerization with AlgE2 increased the content of G-residues in different alginates 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 affected by the concentration of Ca 2+. The type of alginate used as substrate affected the reaction rate and the reaction pattern especially at low Ca 2+ concentration. AlgE2 appears to act by a preferred attack mechanism where the enzyme associates with different sequences in the alginate depending on the concentration of Ca 2+. During epimerization, AlgE2 occasionally causes cleavage of the alginate chain. The observed frequency corresponds to 1–3 breaks per 1,000 M-units epimerized .