Optimization and partial purification of tannase from Bacillus licheniformis AS1 under submerged fermentation and its application

Tannase has vital importance in several industries. However, tannase production employing tannic acid as a substrate is costly. This study optimized medium components, concentration of medium components and physical parameters to yield maximum tannase production from Bacillus licheniformis AS1 in submerged fermentation utilizing low-cost agri-waste Citrus limetta (Mosambi) peels as a substrate. Tannase activity and stability parameters were also optimized. The produced crude tannase and partially purified tannase were used to reduce the bitterness (tannin content) from pomegranate juice and to remove the dye. B. licheniformis AS1 produced 0.361 U/mL under un-optimized conditions. During screening of medium components, Mosambi peels, yeast extract, potassium nitrate and sodium chloride was selected. The concentration of medium components (0.8% Mosambi peels, 0.2% yeast extract, 0.12% potassium nitrate, and 0.06% sodium chloride) was optimized using central composite design which yielded tannase up to 27.809 U/mL. Then, physical conditions were optimized (agitation, 100 µl inoculum size, 40 °C temperature, pH 3, and 72 h of incubation) and yielded tannase up to 43.83 ± 0.82 U/mL. The optimal conditions for tannase activity appeared at pH 8, at 40 °C, and 10 min incubation period with 0.3% substrate concentration. The tannase showed highest stability at 40 ºC and at pH 7. The maximum partial purified tannase activity was recorded at pH 8 and at 40 °C, while enzyme stability was at pH 7 and a temperature of 40 °C. The reduction in tannin content of pomegranate juice was noted after 2 h incubation at 37 ºC. This enzyme was also effective for the partial removal of crystal violet dye. The tannase produced in this study was cost-effective due to utilization of low cost agri-waste and showed potential in various industrial applications.