Comparative oil extraction from mutt (Myliobatis goodei) liver by enzymatic hydrolysis: free versus immobilized biocatalyst.

BACKGROUND: The development and fine-tuning of biotechnological processes for fish oil extraction constitute a very important focus to contribute to the development of a food industry based on fish consumption. This work lies in a comparative analysis of the oil extraction yield of Myliobatis goodei livers using free and immobilized enzymes. RESULTS: An immobilized biocatalyst was designed from the cell-free extract of a Bacillus sp. Mcn4. A complete factorial design was used to study the components of the bacterial culture medium and select the condition with the highest titers of extracellular enzymatic activities. Wheat bran had a significant effect on the culture medium composition for enzymatic production. The immobilized biocatalyst was designed by covalent binding of the proteins present in the cocktail retaining a percentage of different types of enzymatic activities (Mult.Enz@MgFe2 O4 ). Among the biocatalyst used, Alcalase® 2.4 L and Purazyme® AS 60 L (free commercial proteases) showed extraction yields of 87.39% and 84.25%, respectively, while Mult.Enz@MgFe2 O4 achieved a better one of 89.97%. The oils obtained did not show significant differences in their physical-chemical properties while regarding the fatty acid content, the oil extracted with Purazyme® AS 60 L showed a comparatively lower proportion of polyunsaturated fatty acids. CONCLUSIONS: Our results suggest that the use of by-products of M. goodei is a valid alternative and encourages the use of immobilized multienzyme biocatalysts for the treatment of complex substrates in the fishing industry. © 2023 Society of Chemical Industry.

Interfacial Hyperactivation of Candida rugosa Lipase onto Ca2Fe2O5 Nanoparticles: pH and Ionic Strength Fine-Tuning to Modulate Protein-Support Interactions.

The current study provides a comprehensive look of the adsorption process of Candida rugosa lipase (CRL) on Ca2Fe2O5 iron oxide nanoparticles (NPs). Protein-support interactions were identified across a broad range of pH and ionic strengths (mM) through a response surface methodology, surface charge determination, and spectroscopic and in silico analyses. The maximum quantity of immobilized protein was achieved at an ionic strength of 50 mM and pH 4. However, this condition did not allow for the greatest hydrolytic activity to be obtained. Indeed, it was recorded at acidic pH, but at 150 mM, where evaluation of the recovered activity revealed hyperactivation of the enzyme. These findings were supported by adsorption isotherms performed under different conditions. Based on zeta potential measurements, electrostatic interactions contributed differently to protein-support binding under the conditions tested, showing a strong correlation with experimentally determined immobilization parameters. Raman spectra revealed an increase in hydrophobicity around tryptophan residues, whereas the enzyme immobilization significantly reduced the phenylalanine signal in CRL. This suggests that this residue was involved in the interaction with Ca2Fe2O2 and molecular docking analysis confirmed these findings. Fluorescence spectroscopy showed distinct behaviors in the CRL emission patterns with the addition of Ca2Fe2O5 at pH 4 and 7. The calculated thermodynamic parameters indicated that the contact would be mediated by hydrophobic interactions at both pHs, as well as by ionic ones at pH 4. In this approach, this work adds to our understanding of the design of biocatalysts immobilized in iron oxide NPs.

Tuning surface interactions on MgFe2O4 nanoparticles to induce interfacial hyperactivation in Candida rugosa lipase immobilization.

Lipase adsorption on solid supports can be mediated by a precise balance of electrostatic and hydrophobic interactions. A suitable fine-tuning could allow the immobilized enzyme to display high catalytic activity. The objective of this work was to investigate how pH and ionic strength fluctuations affected protein-support interactions during immobilization via physical adsorption of a Candida rugosa lipase (CRL) on MgFe2O5. The highest amount of immobilized protein (IP) was measured at pH 4, and an ionic strength of 90 mM. However, these immobilization conditions did not register the highest hydrolytic activity (HA) in the biocatalyst (CRLa@MgFe2O4), finding the best values also at acidic pH but with a slight shift towards higher values of ionic strength around 110 mM. These findings were confirmed when the adsorption isotherms were examined under different immobilization conditions so that the maximum measurements of IP did not coincide with that of HA. Furthermore, when the recovered activity was examined, a strong interfacial hyperactivation of the lipase was detected towards acidic pH and highly charged surrounding environments. Spectroscopic studies, as well as in silico molecular docking analyses, revealed a considerable involvement of surface hydrophobic protein-carrier interactions, with aromatic aminoacids, especially phenylalanine residues, playing an important role. In light of these findings, this study significantly contributes to the body of knowledge and a better understanding of the factors that influence the lipase immobilization process on magnetic inorganic oxide nanoparticle surfaces.