Production, Purification and Characterization of Extracellular Tannase from a Newly Isolated Yeast, Geotrichum cucujoidarum

Abstract

With an aim to isolate a tannase positive organism, the microbial mat growing on the stored areca extract leachate surface was screened. Once the tannase positive organism was isolated, it was identified by ITS/18S rRNA gene sequencing. Further, the enzyme was purified and examined for its biochemical properties. A potent extracellular tannase-producing yeast was isolated and was identified as Geotrichum cucujoidarum. After the shake flask studies, the enzyme activity of 4.42 U/ml and specific activity of 29.86 U/mg were achieved in a medium with tannic acid as an inducer. Later, ethanol (70%) precipitation followed by purification through FPLC using SEC 650 column resulted in 166.37 U/mg specific activity and a recovery of 50.54%. The purified enzyme was a monomer with a molecular weight of 63 kDa. The optimum pH and the temperature of the enzyme were found to be 5.0 and 30°C, respectively. The Michaelis-Menten constant (Km) was found to be 2.9 mM, and the turn over number (kcat) and catalytic efficiency (kcat/km) of the purified tannase were 102 S-1 and 35.17 mM-1S-1 respectively. Temperature and pH stability profiles of the enzyme, influence of various metal ions, chelators and surfactants on enzyme activity and kinetic constants of enzyme shows that the tannase produced from Geotrichum cucujoidarum is unique and is a potential candidate for further studies.