Immobilization of Eversa Lipase on Octyl Agarose Beads and Preliminary Characterization of Stability and Activity Features

Abstract

Eversa is an enzyme recently launched by Novozymes to be used in a free form as biocatalyst in biodiesel production. This paper shows for first time the immobilization of Eversa (a commercial lipase) on octyl and aminated agarose beads and the comparison of the enzyme properties to those of the most used lipase, the isoform B from Candida antarctica (CALB) immobilized on octyl agarose beads. Immobilization on octyl and aminated supports of Eversa has not had a significant effect on enzyme activity versus p-nitrophenyl butyrate (pNPB) under standard conditions (pH 7), but immobilization on octyl agarose beads greatly enhanced the stability of the enzyme under all studied conditions, much more than immobilization on aminated support. Octyl-Eversa was much more stable than octyl-CALB at pH 9, but it was less stable at pH 5. In the presence of 90% acetonitrile or dioxane, octyl-Eversa maintained the activity (even increased the activity) after 45 days of incubation in a similar way to octyl-CALB, but in 90% of methanol, results are much worse, and octyl-CALB became much more stable than Eversa. Coating with PEI has not a clear effect on octyl-Eversa stability, although it affected enzyme specificity and activity response to the changes in the pH. Eversa immobilized octyl supports was more active than CALB versus triacetin or pNPB, but much less active versus methyl mandelate esters. On the other hand, Eversa specificity and response to changes in the medium were greatly modulated by the immobilization protocol or by the coating of the immobilized enzyme with PEI. Thus, Eversa may be a promising biocatalyst for many processes different to the biodiesel production and its properties may be greatly improved following a suitable immobilization protocol, and in some cases is more stable and active than CALB.

Immobilization of lipases on hydrophobic supports: immobilization mechanism, advantages, problems, and solutions

“…However, it seems that the stabilized open form of the lipase exhibited after lipase immobilization on hydrophoboic supports causes this enzyme to be specially sensitive to the negative effects of phosphate anions and to the positive effect of some cations (Ca 2+ and Mg 2+ ) on enzyme stability (Arana-Peña et al, 2018;Cipolatti et al, 2018;Fernandez-Lopez et al, 2015, 2016Zaak et al, 2017b).…”

“…This causes that in most instances, multipoint immobilized lipase biocatalysts are more stable in aqueous/organic solvents than the just interfacially activated enzyme on hydrophobic supports (dos Santos et al, 2015e, 2015d). Even ionically exchanged lipases may become more stable than the interfacially activated enzymes in these systems (Arana-Peña et al, 2018). It should be remarked that this problem is mainly related to systems consisting of aqueous medium containing high concentrations of organic cosolvent miscible with water,…”

Effects of Enzyme Loading and Immobilization Conditions on the Catalytic Features of Lipase From Pseudomonas fluorescens Immobilized on Octyl-Agarose Beads

“…The use of high ionic strength should have the contrary effect, favoring the closed form of the lipase but making the adsorption stronger. Ca 2+ has been also used, as it can stabilize some lipases immobilized on these supports (Fernandez-Lopez et al, 2015Arana-Peña et al, 2018;Cipolatti et al, 2018).…”

“…On the other hand, phosphate anions have been described to have a very significant destabilizing effect on lipases immobilized on these supports (Zaak et al, 2017a;Arana-Peña et al, 2018), including PFL (Rios et al, 2019c). Comparing two enzyme loads, it may be possible to determine if the enzyme-enzyme interactions can alter the effect of the immobilization conditions on the biocatalysts performance.…”

Recent Advances on Biocatalysis and Metabolic Engineering for Biomanufacturing

“…An optimized procedure of fluorescein diacetate hydrolysis for quantifying total enzymatic activity in the whole biofilm on the carrier without disturbing immobilization was reported by Dzionek et al, which can serve as a promising method to evaluate the physiological state of immobilized bacterial cells [8]. Additionally, Arana-Peña et al reported the immobilization of Eversa lipase on octyl and aminated agarose beads for the first time, which greatly enhanced the stability of the enzyme [9]. The immobilized enzymes prepared by the cross-linked enzyme aggregates (CLEA) have become more attractive due to their simple preparation and high catalytic efficiency.…”