Abstract
Cross-linked enzyme aggregates (CLEA®) technology is a well-established method in the current literature for the low-cost and effective immobilization of several enzymes. The main advantage of this particular method is the simplicity of the process, since it consists of only two steps. However, CLEA immobilization must be carefully designed for each desired enzyme, since the optimum conditions for enzymes can vary significantly, according to their physicochemical properties. In the present study, an investigation of the optimum CLEA immobilization conditions was carried out for eight feruloyl esterase preparations. Feruloyl esterases are a very important enzyme group in the valorization of lignocellulosic biomass, since they act in a synergistic way with other enzymes for the breakdown of plant biomass. Specifically, we investigated the type and concentration of precipitant and the crosslinker concentration, for retaining optimal activity. FAE68 was found to be the most promising enzyme for CLEA immobilization, since in this case, the maximum retained activity, over 98%, was observed. Subsequently, we examined the operational stability and the stability in organic solvents for the obtained CLEA preparations, as well as their structure. Overall, our results support that the maximum activity retaining and the stability properties of the final CLEAs can vary greatly in different FAE preparations. Nevertheless, some of the examined FAEs show a significant potential for further applications in harsh industrial conditions.
Highlights
The immobilization of enzymes using cross-linked enzyme aggregates (CLEA) technology has gained significant attention in the last 20 years, with a wide variety of enzymes and enzyme mixtures being immobilized with standard or modified CLEA methodologies
The selection of the precipitating agent is of crucial importance for CLEA preparations, because it should not have a detrimental effect on the enzyme activity
It is obvious that the precipitating agent is of crucial importance for the residual activity recovered after CLEA formation
Summary
The immobilization of enzymes using cross-linked enzyme aggregates (CLEA) technology has gained significant attention in the last 20 years, with a wide variety of enzymes and enzyme mixtures being immobilized with standard or modified CLEA methodologies. The popularity of CLEA immobilization is mainly due to the simplicity and the low cost of the process. CLEA immobilization consists of only two steps: the precipitation and the cross-linking step [1]. The overall process does not require any solid support, minimizing the cost of the procedure and making it competitive with other immobilization methods, where a solid matrix is required, which often has to be tailored to the individual nature of the target enzyme (such as size, hydrophilicity, etc.) [2]. Due to the very low specificity of the process, the optimal conditions
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