Purification and characterization of a novel heparin degrading enzyme from Aspergillus flavus (MTCC-8654).
A heparinase-producing fungus was isolated, and the strain was taxonomically characterized as Aspergillus flavus by morphophysiological and 26S rRNA gene homology studies. The culture produced intracellular heparinase enzyme, which was purified 40.5-fold by DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-100 column chromatography. Specific activity of the purified enzyme was found to be 44.6 IU/microg protein and the molecular weight of native as well as reduced heparinase was 24 kDa, showing a monomeric unit structure. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. The enzyme activity was maximum at 30 degrees C in the presence of 300 mM NaCl at pH 7.0. In the presence of Co2+, Mn2+ ions, and reducing agents (beta-mercaptoethanol, dithiothreitol), enzyme activity was enhanced and inhibited by iodoacetic acid. These observations suggested that free sulfohydryl groups of cysteine residues were necessary for catalytic activity of the enzyme. The enzyme was also inhibited by histidine modifier, DEPC, which suggests that along with cysteine, histidine may be present at its active site. The enzyme showed a high affinity for heparin as a substrate with K (m) and V (max) as 2.2 x 10(-5 )M and 30.8 mM min(-1), respectively. The affinity of the enzyme for different glycosaminoglycans studied varied, with high substrate specificity toward heparin and heparin-derived polysaccharides. Depolymerization of heparin and fractionation of the oligosaccharides yielded heparin disaccharides as main product.
Banga
Jaspreet
J
Fermentation Technology Division, Central Drug Research Institute, P.O. Box 173, Lucknow U. P., India 226001.
Tripathi
C K M
CK
D000818:Animals; D001231:Aspergillus flavus / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000378:metabolism; D019763:Heparin Lyase / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D013696:Temperature
D002413:Cations, Divalent; D006493:Heparin; D019763:Heparin Lyase