CONICET | Buscador de Institutos y Recursos Humanos

In the last few years, synthesis of carrier-free immobilized biocatalysts by cross-linking of enzyme aggregates hasappeared as a promising technique. Cross-linked enzyme aggregates (CLEAs) present several interesting advantagesover carrier-bound immobilized enzymes, such as highly concentrated enzymatic activity, high stability of theproduced superstructure, important production costs savings by the absence of a support, and the fact that noprevious purification of the enzyme is needed. However, the published literature evidences that a) much specificnon-systematic exploratory work is being done and, b) recovered activity calculations in CLEAs still need to beoptimized. In this context, this contribution presents results of an optimized procedure for the calculation of theactivity retained by CLEAs, based on the comparison of their specific activity relative to their free enzymecounterparts. The protocol implies determination of precipitable protein content in commercial enzymepreparations through precipitation with ammonium sulphate and a protein co-feeder. The identification of linearranges of activity versus concentration/amount of protein in the test reaction is also required for proper specificactivity determinations. By use of mass balances that involve the protein initially added to the synthesis medium,and the protein remaining in the supernatant and washing solutions (these last derived from activitymeasurements), the precipitable protein present in CLEAs is obtained, and their specific activity can be calculated.In the current contribution the described protocol was applied to CLEAs of Thermomyces lanuginosa lipase, whichshowed a recovered specific activity of 11.1% relative to native lipase. The approach described is simple and caneasily be extended to other CLEAs and also to carrier-bound immobilized enzymes for accurate determination oftheir retained activity.

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