Partial purification and characterization of a thermostable mushroom tannase induced during solid state fermentation of Toxicodendron vernicifluum stem bark by Fomitella fraxinea

Abstract

A thermostable extracellular tannase induced during solid state fermentation of the stem bark of Toxicodendron vernicifluum (Stokes) F.A. Barkley (Anacardiaceae) with Fomitella fraxinea was homogeneously isolated through ammonium sulfate precipitation and purified by DEAE-cellulose, and Sephadex G‒100 gel chromatography. It was then characterized for optimal reaction temperature and pH, thermal and pH stability, effects of metal ions as well as its hydrolysis patterns using 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG) as a substrate. The enzyme was purified 103.9-fold with 2.92% recovery and a single protein band corresponding to it was observed at 44.9 kDa during SDS-PAGE analysis. Optimal temperature and pH were found to be 50–70 °C and 5.5, respectively. This enzyme was substantially stable at temperatures below 70 °C and pH values between 5.0 and 7.0. Its activity was inhibited by Fe2+ ion (66%) and Cu2+ (39%) at a concentration of 5 mM. It also showed a different hydrolysis pattern from that of commercial Aspergillus oryzae tannase when PGG was used as a substrate. The tannase purified from the fermented plant material in this study unusually produced oligomeric galloylglucoses such as digalloylglucose (DGG), trigalloylglucose (TGG) and tetragalloylglucose (TeGG) from PGG while the Asp. oryzae tannase produced gallic acid and methyl gallate as final products. Therefore, this enzyme can be more preferably applied for production of bioactive oligomeric galloylglucoses such as DGG, TGG and TeGG from PGG or tannic acid.