Characterization of a novel Aspergillus oryzae tannase expressed in Pichia pastoris

Abstract

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 pPICZαA and the recombinant protein was secreted into the medium as an active enzyme. Recombinant AoTanB highly expressed in the incubation at 18°C compared to 30°C. Purified recombinant protein exhibited smeared band with molecular mass of approximately 90-120kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The recombinant protein yielded molecular mass of 65kDa after N-deglycosylation. Purified recombinant enzyme had a pH and a temperature optima of 6.0 and 30-35°C, respectively, and was stable up to 40°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 (kcat/Km value) toward ethyl protocatechuate was due to a combination of increased Km and considerably decreased kcat. Kinetic analysis of the recombinant AoTanB showed that kcat values toward natural substrates decreased compared to those of recombinant AoTanA. Therefore, recombinant AoTanB showed a decrease in catalytic efficiency (kcat/Km value) compared to recombinant AoTanA was due to considerably lower kcat value.