Cloning, Isolation, and Properties of a New Recombinant Tannase from the Aspergillus niger Fungus

A new recombinant Aspergillus niger tannase (tannin acyl hydrolase) produced by the Penicillium verruculosum fungus has been studied. A strain with a high level of extracellular tannase (TAN2) secretion (80% of the total extracellular protein) was obtained by cloning the tan2 gene (PDB Acc. no: MT828303) into the recipient strain. The tannase enzyme preparation degraded tannins in black tea extracts. TAN2 was isolated in homogeneous form using chromatographic methods; the enzyme had a high activity with respect to gallotannin (53 U/mg) and less activity against propyl gallate (4.7 U/mg). Homogeneous TAN2 showed temperature and pH optima of 45 degrees C and 3.5, respectively. At a temperature of 50 degrees C, TAN2 retained above 80% of its activity for 3 h; at 60 degrees C about 75% of its activity for 90 min was preserved; at 70 degrees C, the enzyme was completely inactivated within 10 min. Tannase was characterized by a high tolerance to NaC1, the activity with respect to gallotannin exceeded 50% of the initial value in solutions with a salt concentration of up to 5 M. The tannase activity was stimulated by Ca2+, mg(2+), zn(2+), mn(2+), Cu2+, Cd2+, a and Pb2+ by 3-64%, and inhibited by 4-65% in the presence of Co2+, Fe3+ and Fe2+ ions.