Simultaneous Production and Sustainable Eutectic Mixture Based Purification of Narringinase With Bacillus Amyloliquefaciens by Valorization of Tofu Wastewater

Abstract

Sustainable one-pot production and purification of naringinase using natural deep eutectic solvent based extractive fermentation is being executed in the current investigation. Tofu wastewater was used as alternative low-cost media for sustainable production of naringinase. Five natural deep eutectic solvents were prepared and their physicochemical properties were determined as a function of temperature. Optimization of essential variables in extractive fermentation for determining effective concentration of NADES at (74.5% w/w), Na2SO4 (15% w/v) and tofu wastewater (1.5% w/w) to achieve optimal naringinase yield of (249.6 U/ml). Scale up of naringinase production with a 3 L custom made desktop bioreactor was accomplished and effective regeneration of NADES was established. Quantification of the resulting active enzyme was done by size exclusion chromatography (736.85 U/mg). Ultrapure enzyme fraction was obtained with anion exchange chromatography yielding maximum purity of (63.2) and specific enzyme activity of (3516 U/mg). The in-vitro debittering activity of resulting ultrapure naringinase was determined with grape juice.