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Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay
Published on: May 1, 2019
Purification and characterization of a novel heparin degrading enzyme from Aspergillus flavus (MTCC-8654)
Jaspreet Banga1, C K M Tripathi
1Fermentation Technology Division, Central Drug Research Institute, P.O. Box 173, Lucknow U. P., India 226001.
Abstract:
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.
Insights
Researchers isolated Aspergillus flavus, a fungus producing intracellular heparinase. This purified heparinase enzyme exhibits specific activity and optimal function at 30°C and pH 7.0, with enhanced activity in the presence of certain ions and reducing agents.
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- Heparinase enzymes are crucial for depolymerizing heparin, a vital anticoagulant.
- Characterizing novel heparinase sources like fungi can lead to new biotechnological applications.
Purpose of the Study:
- To isolate and taxonomically characterize a heparinase-producing fungus.
- To purify and biochemically analyze the intracellular heparinase enzyme.
- To investigate the enzyme's substrate specificity and kinetic properties.
Main Methods:
- Fungal isolation and identification using morphophysiological and 26S rRNA gene sequencing.
- Multi-step column chromatography for enzyme purification (DEAE-Sephadex A-50, CM-Sephadex C-50, Sephadex G-100).
- Enzyme activity assays, molecular weight determination (SDS-PAGE), peptide mass spectrometry, and kinetic analysis (Km, Vmax).
Main Results:
- Aspergillus flavus was identified as the heparinase-producing fungus.
- Purified heparinase (40.5-fold) showed specific activity of 44.6 IU/µg protein and a molecular weight of 24 kDa.
- Optimal activity at 30°C, pH 7.0, with 300 mM NaCl; activity enhanced by Co²⁺, Mn²⁺, and reducing agents; inhibited by iodoacetic acid and DEPC, suggesting cysteine and histidine involvement.
- High affinity for heparin (Km = 2.2 x 10⁻⁵ M) and specific substrate preference for heparin and related polysaccharides.
- Depolymerization yielded heparin disaccharides.
Conclusions:
- A novel intracellular heparinase from Aspergillus flavus has been purified and characterized.
- The enzyme's properties suggest the necessity of cysteine and potentially histidine residues at the active site.
- The enzyme demonstrates high specificity for heparin, producing heparin disaccharides, indicating potential therapeutic and research applications.
