Extractive Fermentation for Recovery of Bacteriocin-Like Inhibitory Substances Derived from Lactococcus lactis Gh1 Using PEG2000/Dextran T500 Aqueous Two-Phase System

This work aimed to optimize the parameters affecting partitioning of a bacteriocin-like inhibitory substances (BLIS) from Lactococcus lactis Gh1 in extractive fermentation using polyethylene glycol (PEG)/dextran aqueous two-phase system (ATPS). This system was developed for the simultaneous cell cultivation and downstream processing of BLIS. Results showed that the molecular weight of PEG, PEG concentration, and dextran T500 affect the partition coefficient (K), purification factor (PF), and yield of BLIS partitioning. ATPS composed of 10% (w/w) PEG2000 and 8% (w/w) dextran T500, provided the greatest conditions for the extractive BLIS production. The K (1.00 ± 0.16), PF (2.92 ± 0.37) and yield (77.24 ± 2.81%) were increased at selected orbital speed (200 rpm) and pH (pH 7). Sustainable growth of the cells in the bioreactor and repeated fermentation up to the eighth extractive batch were observed during the scale up process, ensuring a continuous production and purification of BLIS. Hence, the simplicity and effectiveness of ATPS in the purification of BLIS were proven in this study.

Switchable deep eutectic solvent driven micellar extractive fermentation of ultrapure fibrin digesting enzyme from Bacillus subtilis

Fibrinolytic protease (FLP) is a therapeutic enzyme used in the treatment of thrombolytic diseases. The present study proposed the concept of pH-driven swappable micellar two-phase extraction for the concurrent production and purification of FLP from Bacillus subtilis at cloud point extraction. Extractive fermentation was carried out with a pH swap mechanism, and FLP was extracted to the top phase by surfactant deep eutectic solvents (SADES). Shrimp waste was chosen as a sustainable low-cost substrate that yielded a maximum protease of 185 U/mg. Six SDESs were synthesized with nonionic surfactants as hydrogen bond donors and quaternary ammonium salts as hydrogen bond acceptors, and their association was confirmed by H1 NMR. Thermophysical investigation of the synthetic SADES was accomplished as a function of temperature. Response surface methodology for extractive fermentation was performed with the concentration of SADES (35% w/v), Na2SO4 (15% w/v) and pH (6.3) as variables and the enzyme activity (248 IU/mg) as a response. Furthermore, purification using gel filtration chromatography was used to quantify the amount of enzyme obtained in the extraction phase (849 IU/ml). After final purification with an anion exchange column, the maximum purity fold (22.32) with enzyme activity (1172 IU/ml) was achieved. The in vitro fibrinolytic activity was confirmed using a fibrin plate assay.