Impact of Metal Ions on Catalytic Kinetics, Stability, and Reactivation of Purified Tannase from Aspergillus niger

This study explores the purification and impact of metal ions on Aspergillus niger tannase, a metalloenzyme. Gel permeation chromatography achieved a 22.03-fold purification, yielding a specific activity of 106 U/mg. The enzyme, confirmed as a homodimer (100 kDa native, 50 kDa SDS PAGE), exhibited enhanced activity in the presence of calcium ions, reaching 163.46 U/mg, a 1.42-fold increase. UV absorption with 6 µM Ca ions indicated structural stability. Kinetic studies revealed a sigmoidal Michaelis–Menten graph with calcium ions, suggesting allosteric effects. The Lineweaver–Burk plot showed increased Km (14.04 to 36.39 mM) with mercuric ions, indicating competitive inhibition, while Vmax remained stable. Reactivation studies with DTT and different metal ions showed maximum reactivation with Hg (88.8%) and Ag (81.4%), implicating binding with cysteine residue in inactivation. EDTA-induced reactivation displayed maximum reactivation with Cu ions (67.6%) and minimum with Hg (22.5%), indicating Cu ions form complexes with amino acid groups. In contrast, Hg forms a coordination sphere with the thiol group of a cysteine residue. This comprehensive examination provides insights for optimizing tannase-based processes in various industries and expanding its biotechnological applications.