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/mug protein with a 120000Da molecular mass. The enzyme activity was maximum at 35 degrees C in the presence of 250mM NaCl at pH 7.5. The enzyme activity was inhibited in the presence of Ba(2+), Hg(2+), Cd(2+), IAA and DEPC, and enhanced by the presence of Cu(2+), Fe(2+) 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 K(m) value of 0.026mM. 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.
Banga
Jaspreet
J
Fermentation Technology Division, Central Drug Research Institute, Post Box 173, Lucknow, U.P., 226001 India.
Tripathi
C K M
CK
D016954:Acinetobacter calcoaceticus / Q000201:enzymology*; D000596:Amino Acids / Q000494:pharmacology; D055162:Biocatalysis / Q000187:drug effects; D005591:Chemical Fractionation; D002845:Chromatography; D019763:Heparin Lyase / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D013058:Mass Spectrometry; D008670:Metals / Q000494:pharmacology; D010802:Phylogeny; D011108:Polymers / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
D000596:Amino Acids; D008670:Metals; D011108:Polymers; D019763:Heparin Lyase