Abstract: In this work, we investigated the effect of the Kex2 cleavage sites (K315-R316) on the enzymatic properties of tannase (AfTanA) from Aspergillus fumigates. We constructed a single amino acid mutant (AfTanR316A) and a double amino acid deletion mutant (AfTanΔKR). The two mutants were significantly enhanced for?resistance?to Kex2 proteases. Compared with the wild-type tannase (AfTanA), the mutants exhibited lower catalytic temperature and stronger stability. The optimum reaction temperatures of the mutants and the wild-type tannase were 20 and 40 ℃, respectively. After being treated for 1 h at pH 12.0, AfTanΔKR, AfTanR316A and AfTanA remained 85%, 60% and 0% of their original activity, respectively. After being treated for 1 h at 50 ℃, AfTanΔKR, AfTanR316A and AfTanA remained 40%, 21% and 2% of their original activity, respectively. The improved stability against pH and temperature were beneficial to the application of tannase at low temperatures and its production and storage. The Km and Vmax values of AfTanR316A were determined as 1.149 mmol/L and 10.427 mmol/(L·min). The Km and Vmax values of AfTanΔKR were determined as 1.46 mmol/L and 35.84 mmol/(L·min), respectively. In addition, compared with natural persimmon juice, juices treated separately with AfTanA and the mutants showed a 50% increase in the total phenolic concentration.?In?conclusion, the Kex2 protease cleavage sites (K315-R316) of AfTanA have a significant effect on the stability and catalytic efficiency, and AfTanΔKR could potentially be used in the food industry in the future.

Key words: tannase; site-directed mutation; Kex2 protease; stability; food enzymes