ENHANCEMENT OF LIPASE EVERSA® TRANSFORM 2.0 IMMOBILIZATION USING THE PROTIC IONIC LIQUID [MEA][AC] IN ALGINATE MATRIX

ENHANCEMENT OF LIPASE EVERSA® TRANSFORM 2.0 IMMOBILIZATION USING THE PROTIC IONIC LIQUID [MEA][AC] IN ALGINATE MATRIX

^1,2MARASCA,N.; ²CARNEIRO,R.L. ; ²GUILHERME,E.P.X.; ^1,2FORTE,M.B.S.

¹UNICAMP, Interinstitucional Graduate Program in Bioenergy (USP/UNICAMP/UNESP) – São Paulo, [email protected]; [email protected]

² UNICAMP, Bioprocess and Metabolic Engineering Laboratory (LEMeB) – São Paulo

The incorporation of the protic ionic liquid monoethanolamide acetate ([Mea][Ac]) into alginate beads was investigated as a strategy to enhance the immobilization of Eversa® Transform 2.0 lipase and its catalytic performance, aiming at more robust and sustainable industrial applications. The objective was to assess how [Mea][Ac] affects immobilization yield and recovered activity of the immobilization process, mechanical strength of the biocatalyst, and conformational state of the enzyme.

Beads were produced with 2% (w/w) alginate, 10% (w/w) lipase, and 0.5% (w/w) [Mea][Ac], dripped by a peristaltic pump into CaCl₂, freeze-dried for 48 h, and stored in a desiccator; control samples without ionic liquid were prepared in parallel. Protein content was quantified by the Bradford assay, and enzymatic activity was determined via p-nitrophenyl palmitate hydrolysis. Immobilization yield and recovered activity were calculated from mass balances and activity measurements. Mechanical resistance of wet beads was measured on a TA.XT plus texture analyzer. Thermogravimetric analysis (TGA) was performed on a Shimadzu TGA-50 (N₂ 50 mL·min⁻¹, 10 °C·min⁻¹, 27–800 °C). Steady-state fluorescence spectroscopy (FS) was conducted on an ISS K2 (excitation 280 nm; emission 300–400 nm).

Adding [Mea][Ac] increased enzymatic activity by 36% and immobilization yield by 15.6%, indicating a more favorable microenvironment for entrapment and catalysis. Mechanical strength rose from 5.98 to 8.18 N, supporting improved structural integrity under operation. Although recovered activity remained <30%, the ionic-liquid beads outperformed controls, suggesting superior preservation of catalytic function. TGA showed ~86% total mass loss for [Mea][Ac] beads versus ~82% for controls (≈4% additional), consistent with ionic-liquid incorporation into the matrix. FS indicated an intensity gain upon immobilization, but the addition of [Mea][Ac] favored lid opening and raised microenvironmental polarity around Tryptophane residues. The resulting PIL–water milieu produced quenching (lower intensity), in agreement with prior studies showing that intrinsically more polar media dampen lipase fluorescence.

Overall, incorporating [Mea][Ac] into alginate encapsulation simultaneously improved catalytic performance and mechanical robustness, advancing the practical viability of immobilized biocatalysts under near-industrial conditions. Future work should standardize long-term metrics (reactivation/reuse cycles), explore continuous operation, and optimize ionic-liquid loading to maximize recovered activity without compromising structural stability.