Purification and characterization of tannin acyl hydrolase from Aspergillus niger MTCC 2425.
The present investigation was carried out for increasing the yield of tannase of Aspergillus niger and the physico-chemical characterization of this enzyme. the extraction of enzyme protein. However, extraction of fungal pigments and proteins was observed to have high pH dependence, and maximum enzyme extraction was obtained at pH 5.5. The two-step purification protocol gave 51-fold purified enzyme with a yield of 20%. The total tannase activity was made up of nearly equal activity of esterase and depsidase. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of purified tannase protein indicated it to be made up of two polypeptides of molecular weight 102 and 83 kDa. Based on the Michaelis-Menten constant (Km) of tannase for three substrates tested, tannic acid was the best substrate with Km of 2.8 x 10(-4) M, followed by methyl gallate and propyl gallate. The inhibition was maximum for CaCl2 (58%) whereas EDTA had no modulatory effect on tannase activity. The inhibitor binding constant (KI) of CaCl2 was 5.9 x 10(-4) M Homogenization and detergent pretreatments did not have any remarkable effect on and the inhibition was of noncompetitive type.
Bhardwaj
Rita
R
Department of Chemistry & Biochemistry, College of Basic Sciences C. S. K., Himachal Pradesh Krishi Vishvavidyalaya, Palampur, H. P., India.
Singh
Birbal
B
Bhat
Tej K
TK
D001234:Aspergillus niger / Q000201:enzymology*; D002122:Calcium Chloride / Q000494:pharmacology; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D053630:Depsides; D003902:Detergents; D003956:Dialysis / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004952:Esters / Q000378:metabolism; D005612:Freeze Drying; D006863:Hydrogen-Ion Concentration; D062385:Hydroxybenzoates / Q000378:metabolism; D021122:Protein Subunits / Q000032:analysis; D013379:Substrate Specificity
D053630:Depsides; D003902:Detergents; D004791:Enzyme Inhibitors; D004952:Esters; D062385:Hydroxybenzoates; D021122:Protein Subunits; D002265:Carboxylic Ester Hydrolases; C022711:tannase; D002122:Calcium Chloride