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ARTICLE This journal isAqueous two-phase systems (ATPS) are efficient, environmentally friendly, and "biocompatible" separation processes, which allow the recovery of enzymes. The most common systems are based on polymers and salts, and recently, to overcome the low polarity difference between the phases of the polymeric systems, ATPS based on ionic liquids (ILs) were proposed and have been successfully applied on the field. This work discusses the use of imidazolium-based ILs not as phase forming compounds but as adjuvants (5 wt%) in ATPS of polyethylene glycol systems (1500, 4000, 6000 and 8000 g.mol -1 ) with potassium phosphate buffer at pH 7, in the extraction and purification of a lipase produced by submerged fermentation by Bacillus sp. ITP-001. An initial optimization study was carried with the commercial lipase B from Candida antarctica (CaLB) allowing to further purify the commercial CaLB (Purification Factor = 5.2). Using the optimized conditions, a Purification Factor of 245 for the lipase from Bacillus sp. ITP-001 was achieved with 1-hexyl-3-methyl imidazolium chloride. The high purification factor is a consequence of the favorable interactions between the IL and the contaminant proteins that migrate for the PEG-rich phase, where the IL also concentrates preferentially, while the enzyme remains in the saltrich phase.

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In this work, the effect of several phosphonium‐based ionic liquids (ILs) on the activity of lipase from Burkholderia cepacia (BCL) was evaluated by experimental assays and molecular docking. ILs comprising different cations ([P4444]+, [P444(14)]+, [P666(14)]+) and anions (Cl−, Br−, [Deca]−, [Phosp]−, [NTf2]−) were investigated to appraise the individual roles of IL ions on the BCL activity. From the activity assays, it was found that an increase in the cation alkyl chain length leads to a decrease on the BCL enzymatic activity. ILs with the anions [Phosp]− and [NTf2]− increase the BCL activity, while the remaining [P666(14)]‐based ILs with the Cl−, Br−, and [Deca]− anions display a negative effect on the BCL activity. The highest activity of BCL was identified with the IL [P666(14)][NTf2] (increase in the enzymatic activity of BCL by 61% at 0.055 mol·L−1). According to the interactions determined by molecular docking, IL cations preferentially interact with the Leu17 residue (amino acid present in the BCL oxyanion hole). The anion [Deca]− has a higher binding affinity compared to Cl− and Br−, and mainly interacts by hydrogen‐bonding with Ser87, an amino acid residue which constitutes the catalytic triad of BCL. The anions [Phosp]− and [NTf2]− have high binding energies (−6.2 and −5.6 kcal·mol−1, respectively) with BCL, and preferentially interact with the side chain amino acids of the enzyme and not with residues of the active site. Furthermore, FTIR analysis of the protein secondary structure show that ILs that lead to a decrease on the α‐helix content result in a higher BCL activity, which may be derived from an easier access of the substrate to the BCL active site.

The comparative evaluation of distinct types of ionic liquid-based aqueous biphasic systems (IL-ABS) and more conventional polymer/salt-based ABS to the extraction of two antioxidants, eugenol and propyl gallate, is focused. In a first approach, IL-ABS composed of ILs and potassium citrate (C6H5K3O7/C6H8O7) buffer at pH 7 were applied to the extraction of two antioxidants, enabling the assessment of the impact of IL cation core on the extraction. The second approach uses ABS composed of polyethylene glycol (PEG) and potassium phosphate (K2HPO4/KH2PO4) buffer at pH 7 with imidazolium-based ILs as adjuvants. Their application to the extraction of the compounds allowed the investigation of the impact of the presence/absence of IL, the PEG molecular weight, and the alkyl side chain length of the imidazolium cation on the partition. It is possible to maximize the extractive performance of both antioxidants up to 100% using both types of IL-ABS. The IL enhances the performance of ABS technology. The data puts in evidence the pivotal role of the appropriate selection of the ABS components and design to develop a successful extractive process, from both environmental and performance points of view.

Alternative strategies are required to develop the optimized production of fatty acids using biocatalysis; molecular docking and response surface methodology are efficient tools to achieve this goal. In the present study, we demonstrate a novel and robust methodology for the sustainable production of fatty acids from Moringa oleifera Lam oil using lipase‐catalyzed hydrolysis (without the presence of emulsifiers or buffer solutions). Seven commercial lipases from Candida rugosa (CRL), Burkholderia cepacia (BCL), Thermomyces lanuginosus (TLL), Rhizopus niveus (RNL), Pseudomonas fluorescens (PFL), Mucor javanicus (MJL), and porcine pancreas (PPL) were used as biocatalysts. Initial screening showed that CRL had the highest hydrolytic activity (hydrolysis degree of 81%). Molecular docking analysis contributed to the experimental results, showing that CRL displays more stable binding free energy with oleic acid (C18:1), which is the fatty acid of highest concentration in Moringa oleifera Lam oil. To evaluate and optimize the hydrolysis process, response surface methodology (RSM) was used. The effect of temperature, mass ratio oil:water, and hydrolytic activity on enzymatic hydrolysis was evaluated by central composite design using RSM. Under the optimized conditions (temperature of 37 °C, mass ratio oil:water of 25%, and hydrolytic activity of 550 U goil−1), the maximum hydrolysis degree (100%) was achieved. The present study provides a robust method for the enzymatic hydrolysis of different oils for efficient and sustainable fatty acid production.

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