Simultaneous production and sustainable eutectic mixture based purification of narringinase with Bacillus amyloliquefaciens by valorization of tofu wastewater

Abstract

The current investigation is being executed for sustainable one-pot production and purification of naringinase using natural deep eutectic solvent-based extractive fermentation. Five natural deep eutectic solvents were prepared and their physicochemical properties were determined as a function of temperature. Tofu wastewater was used as a low-cost substrate for naringinase production and simultaneous in-situ purification of the enzyme was accomplished by employing NADES. Optimal conditions of influential factors like concentrations of NADES (74.5% w/w), Na2SO4 (15% w/v) and tofu wastewater (1.5% w/w) resulted in an effective yield of naringinase (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. NADES exhibits selectivity during extraction even after the fifth cycle proving it to be tailor-made. The resulting active enzyme was quantified by size exclusion chromatography (736.85 U/mg). Ultrapure enzyme fraction was obtained with anion exchange chromatography yielding maximum purity of (63.2 U/ml) and specific naringinase activity of (3516 U/mg). The in-vitro debittering activity of the resulting ultrapure enzyme fraction was determined with grape juice resulting in naringin and limonin removal of [23.4% (w/w)] and [64.3% (w/w)] respectively.