Sustainable liquid supports for laccase immobilization and reuse: Degradation of dyes in aqueous biphasic systems

Abstract

Novel liquid supports for enzyme immobilization and reuse based on aqueous biphasic systems (ABS) constituted by cholinium-based ionic liquids (ILs) and polymers for the degradation of dyes are here proposed. The biocatalytic reaction for dye decolorization using laccase occured in the biphasic medium, with the enzyme being "supported" in the IL-rich phase and the dye and degradation products being enriched in the polymer-rich phase. An initial screening of the laccase activity in aqueous solutions of ABS constituents, namely cholinium dihydrogen citrate ([Ch][DHC]), cholinium dihydrogen phosphate ([Ch][DHP]), cholinium acetate ([Ch][Acet]), polypropylene glycol 400 (PPG 400), polyethylene glycol 400 (PEG 400) and K 2 HPO 4 was carried out. Compared to the buffered control, a relative laccase activity of up to 170%, 257%, and 530% was observed with PEG 400, [Ch][DHP], and [Ch][DHC], respectively. These ABS constituents were then investigated for the in situ enzymatic biodegradation of the Remazol Brilliant Blue R (RBBR) dye. At the optimized conditions, the ABS constituted by PPG 400 at 46 wt% and [Ch][DHC]at 16 wt% leads to the complete degradation of the RBBR dye, further maintaining the enzyme activity. This ABS also allows an easy immobilization, recovery, and reuse of the biocatalyst for six consecutive reaction cycles, achieving a degradation yield of the dye of 96% in the last cycle. In summary, if properly designed, high enzymatic activities and reaction yields are obtained with ABS as liquid supports, while simultaneously overcoming the safety and environmental concerns of conventional organic solvents used in liquid-liquid heterogeneous reactions, thus representing more sustainable biocatalytic processes.

New liquid supports in the development of integrated platforms for the reuse of oxidative enzymes and polydopamine production

“…Enzyme reuse and substrate recovery need to be considered when developing a sustainable and economical process. The reuse of enzymes using liquid supports can provide a novel opportunity to surpass the major challenge of biocatalyst recovery and reuse while improving its stability, activity, biocompatibility and reaction rates ( Ferreira et al, 2021 ; Muñiz-Mouro et al, 2021 ; Baptista et al, 2022 ). To this end, ABS approach was chosen as excellent liquid supports to develop an integrated and sustainable platform for enzymatic catalysis.…”

“…Thus, it was firstly evaluated the EE PDA %, EE Laccase %, and EE Dopamine % in five different ABS composed of PPG 400 and [Ch][DHP], [Ch][DHC], [Ch][Acet], PEG 400 or K 2 HPO 4. The ternary mixture compositions of the ABS were selected according to the literature ( Ferreira et al, 2021 ) and correspond to a mixture point that leads to a biphasic system with an approximately equal ratio of both phases. Thus, it is possible to ensure enough mass in each phase for laccase reaction and reuse and product recovery.…”

“…For the study of recovery and reuse of laccase, four consecutive cycles of dopamine polymerization were performed using the optimised ABS composed of 33 wt% PPG 400 + 6.4 wt% K₂HPO₄ + 20 wt% dopamine solution (3.00 mg ml −1 ) + 40.6 wt% laccase solution (1.00 mg ml −1 ). After the first reaction cycle, the PDA i -enriched top phase was removed, and the same volume of a solution containing the respective top polymer-rich phase constituents (determined by the tie-line) ( Ferreira et al, 2021 ) was added to the remaining laccase-rich bottom phase. Dopamine solution was added to reach a final concentration of 0.60 mg ml −1 in the ABS and a new biocatalytic cycle started.…”

“…ABS consist of two immiscible aqueous-rich phases formed when water-soluble components are mixed above given concentrations ( Freire et al, 2012 ; Pereira et al, 2020 ). Recently, ABS systems have been investigated as reaction media and liquid supports for enzymes since they provide a suitable and friendly environment for the maintenance of the enzymatic activity and its reuse ( Ferreira et al, 2018 ; Capela et al, 2020 ; Ferreira et al, 2021 ; Muñiz-Mouro et al, 2021 ). Another advantage and application of this approach is the possibility of simultaneous extract and concentrate the reaction product by manipulating the volume of the ABS phases, using the tie-line of binodal curves ( Dinis et al, 2018 ), while maintaining the enzyme in the opposite phase.…”

“…In a recent study assessed by. Ferreira et al (2021) it was demonstrated the recovery and reuse of laccase in an integrated reaction-separation process by applying thermoreversible ABS. No losses in the enzymatic activity were observed for at least five consecutive cycles of enzymatic reaction with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS).…”