We report the characterization of tannase-encoding gene, AotanB, from Aspergillus oryzae and its recombinant enzyme expressed in Pichia pastoris. The gene except for the signal sequence was cloned into a vector pPICZ alpha A and the recombinant protein was secreted into the medium as an active enzyme. Recombinant AoTanB highly expressed in the incubation at 18 degrees C compared to 30 degrees C. Purified recombinant protein exhibited smeared band with molecular mass of approximately 90-120 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The recombinant protein yielded molecular mass of 65 kDa after N-deglycosylation. Purified recombinant enzyme had a pH and a temperature optima of 6.0 and 30-35 degrees C, respectively, and was stable up to 40 degrees C. Recombinant AoTanB was able to release gallic acid from natural substrates, such as (-)-catechin gallate, (-)-epicatechin gallate, (-)-gallochatechin gallate, and (-)epigallocatechin gallate. The enzyme also hydrolyzed ethyl protocatechuate. Meanwhile, no activity was detected toward ethyl 4-hydroxybenzoate. The activity of recombinant AoTanB was lower toward natural substrates compared to that of AoTanA from A. oryzae. The lower catalytic efficiency (k(at)/K-m value) toward ethyl protocatechuate was due to a combination of increased K-m and considerably decreased k(cat). Kinetic analysis of the recombinant AoTanB showed that kat values toward natural substrates decreased compared to those of recombinant AoTanA. Therefore, recombinant AoTanB showed a decrease in catalytic efficiency (k(cat)/K-m value) compared to recombinant AoTanA was due to considerably lower kat value. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.