Efficient Decolorization of Molasses Wastewater by Thermophilic Carboxyl Ester Hydrolase: A New Immobilization Method

Natural deep eutectic solvents (NADESs), as green solvents with design flexibility and economic viability, were explored as innovative enhancers for lipase TLL immobilization. An economically efficient immobilization strategy, the precipitation-covalent cross-linking method, was developed. Through systematic process optimization, NADESs incorporation elevated the enzyme activity recovery of NADES-IM-TLL to 63.53%, representing an 11.76% improvement over conventional IM-TLL (51.77%). After 12 h of continuous operation under simulated extreme industrial conditions, including high temperatures, extreme pH, organic solvents, and repeated agitation, the biocatalyst retained residual activity above 45%, significantly outperforming the control. In decolorizing three distinct molasses wastewater (MWW) sources, NADES-IM-TLL achieved an average initial efficiency of 84.89%, markedly surpassing IM-TLL (72.04%); even after 5 days of continuous operation, the relative decolorization efficiency remained at 68.57%. Compared with commercially available immobilized lipases, NADES-IM-TLL demonstrated superior decolorization performance and catalytic stability. This research thus establishes an efficient, stable, and economical protocol for immobilized enzyme preparation, providing theoretical foundations and technical support for the industrial application of lipase TLL.