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 overall objective of this study is to evaluate the morphological [scanning electron microscopy (SEM)], physicochemical [differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), chemical composition analysis, Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR)], and biochemical properties of Candida rugosa lipase (CRL) immobilized on a natural biopolymer poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) in aqueous solution. CRL was immobilized by physical adsorption with efficiency of 30%. Compared with free CRL enzyme, there were slight changes in immobilized CRL activity as a function of temperature (from 37°C to 45°C), but a similar optimal pH value of 7.0. Inactivation rate constants for immobilized CRL enzyme were 0.009 and 0.334 h⁻¹, and half-lives were 77 and 2 h at 40°C and 60°C, respectively. Kinetic parameters obtained for immobilized CRL include the Michaelis-Menten constant of K(m) = 213.18 mM and maximum reaction velocity of V(max) = 318.62 U/g. The operational stability of immobilized CRL was tested repeatedly, and after 12 cycles of reuse, the enzyme retained 50% activity. Based on our results, we propose that PHBV-immobilized CRL could serve as a promising biocatalyst in several industrial applications.

Biocatalysis has significant advantages over chemocatalysis in the context of sustainability since environmentally compatible catalysts (enzymes) and mild reaction conditions are used. However, enzymes are labile macromolecules, and strategies such as the addition of cosolvents or additives or their immobilization in solid supports are the target of intensive investigation to improve the catalytic performance and recyclability potential. In this work, we investigated the use of phosphonium-based ionic liquids (ILs) on the activity of immobilized Burkholderia cepacia lipase (BCL) by two approaches: (i) use of ILs to prepare silica used as support and (ii) use of ILs during the enzyme immobilization process. Several phosphonium-based ILs were investigated, allowing us to address the cation alkyl side chain and anion nature effects. The enzymatic performance depends on the IL employed to prepare silica, with a positive effect observed when employing ILs comprising cations with longer alkyl side chains and more hydrophobic anions. The best identified IL, namely, [P 666(14) ][NTf 2 ], allows for a relative activity of 209.8% and immobilization yield of 77.3% and is capable of being recycled eight times (keeping more than 50% of the enzyme initial activity). When ILs are added during the BCL immobilization process, similar negative and beneficial effects are observed. With IL [P 666(14) ][NTf 2 ], the immobilized biocatalyst has a relative activity of 322.7%, a total activity recovery yield of 91.1%, and can be recycled 17 times (down to 50% of the enzyme initial activity). Finally, both approaches were combined; i.e., IL [P 666(14) ][NTf 2 ] was used both in the material preparation and immobilization process of the enzyme. This strategy allows for an increase in the relative activity up to 231%, an immobilization yield of 98%, and an increase of 9% in the enzyme relative activity. Although BCL activity is not significantly enhanced by this strategy, the combined use of the IL in silica preparation and during the enzyme immobilization process increased the recyclability potential of the immobilized biocatalyst material, capable of being recycled 26 times, while keeping more than 50% of the enzyme initial activity (equivalent to a half-life of 13 h). The results obtained in this work open the path to the efficient use of ILs, and particularly of the less explored phosphonium-based ILs, in the biocatalysis field.

A new source of lipase from Bacillus sp. ITP-001 was immobilized by physical adsorption on the polymer poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) in aqueous solution. The support and immobilized lipase were characterised, compared to the lyophilised lipase, with regard to the specific surface area, adsorption-desorption isotherms, pore volume (V(p)) and size (dp) by nitrogen adsorption, differential scanning calorimetry, thermogravimetric analysis, chemical composition analysis, Fourier transform infrared spectroscopy and biochemical properties. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0, whereas the optimum pH for the free enzyme was at pH 7.0; the optimum temperature of activity was 80 and 37 °C for the free and immobilized enzyme, respectively. The inactivation rate constant for the immobilized enzyme at 37 °C was 0.0038 h⁻¹ and the half-life was 182.41 h. The kinetic parameters obtained for the immobilized enzyme gave a Michaelis-Menten constant (K(m)) of 49.10 mM and a maximum reaction velocity (V(max)) of 205.03 U/g. Furthermore, the reuse of the lipase immobilized by adsorption allowed us to observe that it could be reused for 10 successive cycles, duration of each cycle (1 h), maintaining 33 % of the initial activity.