Sustainable production and preparative purification of thermostable alkaline α-amylase by Bacillus simplex (ON754233) employing natural deep eutectic solvent-based extractive fermentation.

Using PEG-based deep eutectic solvents (PDES), the current study proposes extractive fermentation as a sustainable process integration for the production and purification of α-amylase from Bacillus simplex (ON754233). Glucose: PEG 400 outperformed five PDES in terms of tie lie length (58) and slope value (1.23) against sodium sulphatt. Apple cider pomace was used as a low-cost, sustainable carbon source to produce-amylase, with a maximum enzyme production of 2200.13 U/mL. PDES concentration (20% w/v), salt (12.75 w/v), and apple waste (2.75 g/mL) were all optimized using response surface methodology. When scaled upto 3 L benchtop bioreactor, extractive fermentation was proved to be better technology with maximum recovery of 92.4% with highest partition coefficient (3.59). The partially purified enzyme was further purified using a Sephadex G 100 followed by DEAE-Sephadex anion exchange chromatography with a purity fold of 33. The enzyme was found to be thermostable at the temperature (60 °C), remains alkaline (pH 8), and the activity was stimulated in the presence of Mg2+ ions. With SDS PAGE electrophoresis, the molecular weight was found to be around 140 kDa. Finally, the enzyme kinetics parameters were evaluated with observed Km (0.00396 mM) and Vmax (37.87 U/mL). Thus scaling up extractive fermentation entails increasing production capacity with improved extraction efficiency using green solvents.