A novel low molecular weight acido-thermophilic tannase from Enterobacter cloacae MTCC 9125

• Tannase from Enterobacter cloacae was purified and characterized.
• Temperature of 50 °C and pH 5.5 were found to be optimum.
• Km and Vmax of the enzyme were 0.00337 M and 3.401 U/ml, respectively.
• Methyl gallate and propyl gallate inhibited the enzyme strongly.

Thermophilic tannase from Enterobacter cloacae MTCC 9125 was purified using two-step purification strategy comprising of ultrafiltration and ion-exchange chromatography. A purification fold of 8.47 with 33.1% yield was obtained. The apparent molecular mass of tannase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 31±1 kDa. The temperature of 50 °C and pH 5.5 were found to be optimum for tannase activity. Tannase retained almost 50% activity after 2 and 4 h of incubation at its optimum pH (pH 5.5) and temperature (50 °C), respectively. Km of the enzyme for tannic acid was found to be 0.00337 M with Vmax 3.401 U/ml. Effects of several metal salts, chelators, surfactants, and typical enzyme inhibitors on tannase activity were evaluated. Inhibition by n-bromosuccinic acid and phenylmethylsulfonyl fluoride (PMSF) indicated that tryptophan and serine or cysteine residues play an important role in maintaining the active conformation of the enzyme. Among the divalent cations, Mg2+, Zn2+ and Mn2+ were found to be activators of the enzyme whereas Fe2+, Ba2+ and Cu2+ acted as inhibitors. Surfactant such as Tween 20, Tween 80, Triton X-100 and SDS resulted in considerable loss of enzyme activity.