Purification and characterization of tannase from Paecilomyces variotii: hydrolysis of tannic acid using immobilized tannase |
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Authors: | B Mahendran N Raman D-J Kim |
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Institution: | (1) Department of Earth and Environmental Sciences, Korea University, Seoul, 136-701, Republic of Korea;(2) Centre for Advanced studies in Botany, University of Madras, Guindy Campus, Chennai, 600 025, India |
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Abstract: | 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°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 1H-nuclear magnetic resonance spectral analysis confirmed that gallic acid was formed as a byproduct during hydrolysis of tannic
acid. |
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