Immobilization of Eversa ® Transform 2.0 lipase on polymeric and biopolymeric matrices for packed-bed operation

Abstract

Immobilized lipases are key biocatalysts for advancing industrial biocatalysis. When integrated into packed-bed reactors (PBR), they combine low shear stress with catalyst retention and repeated reuse cycles under continuous operation. However, PBR performance is often constrained by mass-transfer phenomena and by how immobilization, support properties, and operating conditions interact along the bed. Building on a critical review of recent advances in PBR employing immobilized lipases (2019–2024), with emphasis on operational limitations and transport effects, this thesis develops a process-oriented basis for designing packed-bed biocatalysts containing the Eversa ® Transform 2.0 lipase (ETL) through (i) systematic characterization of ETL in its soluble form, (ii) development of a marine-derived biopolymeric support for ETL immobilization, and (iii) in situ immobilization under flow within the packed bed. The commercial ETL preparation exhibited a total protein concentration of 20.36 ± 1.09 mg mL -1 and an essentially single band by electrophoresis. Kinetic screening with p- nitrophenyl butyrate (pNPB) identified 50 mM as the best substrate concentration evaluated (103.36 ± 1.27 U) and 50 °C as the best reaction temperature evaluated,

increasing activity by 85.62% relative to 25 °C (216.71 ± 7.87 U). Thermal stability between 60 and 80 °C was described using the Sadana–Henley model (1987), revealing exceptional resistance at 60 °C (no measurable half-life over 288 h) and accelerated deactivation at higher temperatures; nonlinear Arrhenius analysis yielded a low apparent deactivation energy (5.05 kJ mol -1 ). Circular dichroism measurements supported preservation of α-helical structure across the pH range and the absence of a cooperative denaturation transition up to 95 °C, consistent with deactivation governed by aggregation and local rearrangements. The isoelectric point was pH 4.56, and DLS at pH 7.0 indicated a tendency toward aggregate formation. Next, iota-carrageenan (i-CA) was extracted from Solieria filiformis and optimized using a face-centered experimental design, achieving a maximum yield of 33.31% (100 °C, 4 h). The extracted polysaccharide was incorporated into a chitosan/i-CA blend (1:3, w/w) chemically crosslinked with glutaraldehyde (0.61%, v/v) and evaluated as an immobilization support (1 mg protein g -1 support, 1 h, pH 7.0). The support retained 37.3 ± 2.7% of the offered enzyme and exhibited 35.1 ± 0.9 U g -1 , corresponding to 106.9 ± 10.4% of the ETL activity at an equivalent soluble-enzyme concentration. Thus, enzymatic activity was preserved. Subsequently, ETL was immobilized in situ by hydrophobic adsorption onto Amberlite ™ XAD 1180 in a packed-bed reactor. Breakthrough-curve analysis (C/C 0 vs. bed volumes) and derived characteristic metrics demonstrated the influence of external and internal mass-transfer effects on front dynamics and effective loading. Increasing the feed concentration intensified adsorption but produced non-monotonic loading at a fixed immobilization time (maximum q = 23.95 mg g resin -1 at C 0 = 1.0 mg mL -1 ; q = 18.92 mg g resin -1 at C 0 = 2.0 mg mL -1 ), indicating accessibility limitations at high C 0 . Expressed activity reached a maximum at Q = 0.75 mL min -1 (A bed = 198.50 U g -1 ; A eff = 66.27 U mg -1 ). The immobilized bed was reused (stable hydrolysis over eight cycles) and maintained conversion in esterification after reuse. Solvent compatibility was supported by unchanged FTIR spectra and preservation of overall textural properties (S BET = 616-640 m 2 g -1 ). Overall, the results establish an integrated framework linking enzyme properties, support synthesis, and immobilization processes for the operation of ETL biocatalysts in packed-bed reactors, explicitly accounting for transport constraints and operational stability.