Rapid purification and characterization of a novel heparin degrading enzyme from Acinetobacter calcoaceticus

An intracellularly produced constitutive heparinase was isolated from the periplasmic space of Acinetobacter calcoaceticus by freeze fracturing and purified 51.2-fold by ion exchange and gel filtration chromatography. Specific activity of the purified enzyme was found to be 41 IU/μg protein with a 120 000 Da molecular mass. The enzyme activity was maximum at 35 °C in the presence of 250 mM NaCl at pH 7.5. The enzyme activity was inhibited in the presence of Ba2+, Hg2+, Cd2+, IAA and DEPC, and enhanced by the presence of Cu2+, Fe2+ ions and reducing agents. Inhibition of enzyme activity by iodoacetic acid and enhancement of enzyme activity in the presence of reducing agents indicated that free sulfohydryl groups of cysteine residues were necessary for catalytic activity of the enzyme. The affinity of the enzyme for different glycosaminoglycans studied varied and showed high affinity for heparin with a Km value of 0.026 mM. In situ gel digestion of the purified protein with trypsin did not show any homology with heparinase I. Depolymerization of heparin and fractionation of the oligosachharides yielded heparin disaccharides as main product. This suggests a catalytic similarity and structural dissimilarity of heparinase from Acinetobacter with heparinase I.