Hafiz Shakir - Institute of Zoology, Faculty of Life Sciences, University of the Punjab, Lahore, Pakistan
Muhammad Khan - Institute of Zoology, Faculty of Life Sciences, University of the Punjab, Lahore, Pakistan
Muhammad Irfan - Department of Biotechnology, University of Sargodha, Sargodha, Pakistan
Shaukat Ali - Department of Zoology, Faculty of Sciences, Government College University, Lahore, Pakistan
Muhammad Yousaf - Institute of Zoology, Faculty of Life Sciences, University of the Punjab, Lahore, Pakistan
Iqra Javed - Institute of Zoology, Faculty of Life Sciences, University of the Punjab, Lahore, Pakistan
Javed Qazi - Institute of Zoology, Faculty of Life Sciences, University of the Punjab, Lahore, Pakistan
Syed Shahid Bukhari - Department of Zoology, Faculty of Sciences, Government College University, Lahore, Pakistan

Introduction: Tannase (tannin acyl hydrolase EC۳.۱.۱.۲۰) is an industrially important enzyme with extensive applications. The current study aimed to optimize the tannase production employing corn leaves as substrate and characterize tannase activity.Materials and Methods: Tannase producing bacterial strains were isolated from Catla catla fish gut. The highest enzyme-producing bacterial strain was identified as Bacillus subtilis using ۱۶S rDNA sequencing.Results: Fermentation parameters and additional medium components were optimized with the application of one variable at a time and enhanced tannase was obtained with ۵۰% substrate moisture, acetate buffer (pH ۴.۰) as enzyme extraction medium with ۲ ml volume, ۴۵ °C incubation temperature, pH ۵, ۲% inoculum size, ۲۴ h incubation time, ۱۵۰ rpm agitation, large-sized substrate particles (۴.۰ mm), enzyme extraction without centrifugation and medium components (MgSO۴, ۴% tannic acid and yeast extract). The central composite design was employed to optimize the concentrations of optimal medium components, which were found as ۴.۰% tannic acid, ۰.۵% MgSO۴ and ۱.۵% yeast extract for the highest tannase production (۲۱۱.۹۷±۰.۰۸ U/ml). Tannase characterization revealed the maximum tannase activity at pH ۸, ۳۰ °C (with ۳۰ min incubation period and ۰.۳۵% substrate concentration).Conclusions: The results of the present study revealed the potential of utilization of agricultural resources (corn leaves) as a low-cost substrate to reduce the production cost of tannase. 

Bacillus subtilis, Tannase, production, Central composite design, Characterization