Immobilization of Pseudomonas stutzeri lipase through Cross-linking Aggregates (CLEA) for reactions in Deep Eutectic Solvents.

The use of deep eutectic solvents (DES) with buffer as cosolvent (up to 10 % v/v) leads to low-viscous media in which lipases can perform synthetic reactions, instead of hydrolysis. This paper explores the immobilization of Pseudomonas stutzeri lipase (TL) in cross-linking aggregates (CLEA) to deliver robust derivatives that are active in media like choline chloride - glycerol DES with buffer as cosolvent. While the free TL enzyme was markedly inactive in these media, TL-CLEA derivatives perform esterifications and can be reused several times. Overall, results are consistent with previous experiments reported for other lipases in these DES-water media and confirm that CLEA immobilization turns out a very useful and straightforward alternative for generating active (bio)catalysts for DES-aqueous media systems. Immobilized systems open the possibility of performing continuous processes in low-viscous DES-buffer media.

Immobilized lipase-CLEA aggregates encapsulated in lentikats® as robust biocatalysts for continuous processes in deep eutectic solvents.

The immobilization of lipases in cross linked aggregates (CLEA) leads to a robust biocatalyst that remains very stable in low viscous non-conventional Deep Eutectic Solvents - Buffer mixtures. To reinforce that stability, and to facilitate the biocatalyst recovery, this paper explores the immobilization of Lipase-CLEA derivatives in Lentikats®. This double immobilization can be successfully used in esterifications in DES-buffer media, using substrates of unpaired solubilities (e.g. benzoic acid and glycerol), in batch and continuous processes, and reaching full conversion. Under these conditions, the derivatives display an improved stability (compared to the Lipase-CLEA derivatives) and enable the reuse of the reaction media in continuous devices for at least 6 cycles under non-optimized conditions, accumulating 10 g product L-1, enhancing the productivity, and opening exciting future options for sustainable chemistry.

Aroma Release in Wine Using Co-Immobilized Enzyme Aggregates.

Aroma is a remarkable factor of quality and consumer preference in wine, representing a distinctive feature of the product. Most aromatic compounds in varietals are in the form of glycosidic precursors, which are constituted by a volatile aglycone moiety linked to a glucose residue by an O-glycosidic bond; glucose is often linked to another sugar (arabinose, rhamnose or apiose). The use of soluble ß-glycosidases for aroma liberation implies the addition of a precipitating agent to remove it from the product and precludes its reuse after one batch. An attractive option from a technological perspective that will aid in removing such constraints is the use of immobilized glycosidases. Immobilization by aggregation and crosslinking is a simple strategy producing enzyme catalysts of very high specific activity, being an attractive option to conventional immobilization to solid inert supports. The purpose of this work was the evaluation of co-immobilized ß-glycosidases crosslinked aggregates produced from the commercial preparation AR2000, which contains the enzymes involved in the release of aromatic terpenes in Muscat wine (α-l-arabinofuranosidase and ß-d-glucopyranosidase). To do so, experiments were conducted with co-immobilized crosslinked enzyme aggregates (combi-CLEAs), and with the soluble enzymes, using an experiment without enzyme addition as control. Stability of the enzymes at the conditions of winemaking was assessed and the volatiles composition of wine was determined by SPE-GC-MS. Stability of enzymes in combi-CLEAs was much higher than in soluble form, 80% of the initial activity remaining after 60 days in contact with the wine; at the same conditions, the soluble enzymes had lost 80% of their initial activities after 20 days. Such higher stabilities will allow prolonged use of the enzyme catalyst reducing its impact in the cost of winemaking. Wine treated with combi-CLEAs was the one exhibiting the highest concentration of total terpenes (18% higher than the control) and the highest concentrations of linalool (20% higher), nerol (20% higher) and geraniol (100% higher), which are the most important terpenes in determining Muscat typicity. Co-immobilized enzymes were highly stable at winemaking conditions, so their reutilization is possible and technologically attractive by reducing the impact of enzyme cost on winemaking cost.