Engineering and application of a recombinant Aspergillus niger CECT 2907 tannase to enhance the antioxidant activity of black tea.

BACKGROUND

Tannases (tannin acyl hydrolases, EC 3.1.1.20) catalyze the hydrolysis of galloylated polyphenols, releasing gallic acid and other bioactive compounds with antioxidant properties. These enzymes have a wide range of applications in the food and beverage industry, particularly for improving tea quality. In this study, a tannase gene from Aspergillus niger CECT 2907 was cloned and heterologously expressed in Komagataella phaffii. Two recombinant variants were generated: a double-chain enzyme (AnTanDC) and a single-chain variant (AnTanSC) obtained by mutating two Kex2 protease cleavage sites. Both enzymes were biochemically characterized, and the potential of AnTanSC to enhance the antioxidant properties of black tea was evaluated.

RESULTS

Both recombinant tannases were efficiently secreted and produced in 5 L bioreactors, reaching activities of 4238 ± 16 U L-1 (AnTanDC) and 6505 ± 362 U L-1 (AnTanSC). AnTanDC was proteolytically processed into two subunits, whereas AnTanSC remained as a single-chain enzyme. Both variants showed similar pH and temperature activity profiles, with maximal activity around pH 6.0 and 40 °C. AnTanSC showed improved thermal stability compared with AnTanDC, although lower activity toward tannic acid was observed. Treatment of black tea infusions with AnTanSC increased soluble phenolic content 1.94-fold and enhanced antioxidant capacity 5.76-fold and 3.12-fold according to TEAC and FRAP assays, respectively.

CONCLUSION

The engineered single-chain tannase AnTanSC combined improved thermal stability with effective enhancement of the antioxidant properties of black tea, supporting its potential application as a biocatalyst for the processing of tea beverages and other polyphenol-rich foods. © 2026 Society of Chemical Industry.