Purification and characterization of tannase from Paecilomyces variotii: hydrolysis of tannic acid using immobilized tannase.
An extracellular tannase (tannin acyl hydrolase) was isolated from Paecilomyces variotii and purified from cell-free culture filtrate using ammonium sulfate precipitation followed by ion exchange and gel filtration chromatography. Fractional precipitation of the culture filtrate with ammonium sulfate yielded 78.7% with 13.6-folds purification, and diethylaminoethyl-cellulose column chromatography and gel filtration showed 19.4-folds and 30.5-folds purifications, respectively. Molecular mass of tannase was found 149.8 kDa through native polyacrylamide gel electrophoresis (PAGE) analysis. Sodium dodecyl sulphate-PAGE revealed that the purified tannase was a monomeric enzyme with a molecular mass of 45 kDa. Temperature of 30 to 50 degrees C and pH of 5.0 to 7.0 were optimum for tannase activity and stability. Tannase immobilized on alginate beads could hydrolyze tannic acid even after extensive reuse and retained about 85% of the initial activity. Thin layer chromatography, high performance liquid chromatography, and (1)H-nuclear magnetic resonance spectral analysis confirmed that gallic acid was formed as a byproduct during hydrolysis of tannic acid.
Mahendran
B
B
Department of Earth and Environmental Sciences, Korea University, Seoul, Republic of Korea. bmahesh16@yahoo.com
Raman
N
N
Kim
D-J
DJ
D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004800:Enzymes, Immobilized / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008970:Molecular Weight; D010143:Paecilomyces / Q000201:enzymology*; D013634:Tannins / Q000737:chemistry*
D004800:Enzymes, Immobilized; D013634:Tannins; D002265:Carboxylic Ester Hydrolases; C022711:tannase