Micro-scale procedure for enzyme immobilization screening and operational stability assays
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A simple and inexpensive methodology, based on the use of micro-centrifuge filter tubes, is proposed for establishing the best enzyme immobilization conditions.
The immobilized biocatalyst is located inside the filter holder during the whole protocol, thus facilitating the incubations, filtrations and washings. This procedure minimizes the amount of enzyme and solid carrier needed, and allows exploring different immobilization parameters (pH, buffer concentration, enzyme/carrier ratio, incubation time, etc.) in a fast manner. The handling of immobilized enzymes using micro-centrifuge filter tubes can also be applied to assess the apparent activity of the biocatalysts, as well as their reuse in successive batch reaction cycles. The usefulness of the proposed methodology is shown by the determination of the optimum pH for the immobilization of an inulinase (Fructozyme L) on two anion-exchange polymethacrylate resins (Sepabeads EC-EA and Sepabeads EC-HA).
The micro-scale procedure described here will help to overcome the lack of guidelines that usually govern the selection of an immobilization method, thus favouring the development of stable and robust immobilized enzymes that can withstand harsh operating conditions in industry.
KeywordsBiotransformations Enzyme immobilization Enzyme carriers Immobilized biocatalysts Inulinase Operational stability
This work was supported by a Grant from the Spanish Ministry of Economy and Competitiveness (BIO2013-48779-C4-1-R). We thank the support of COST-Action CM1303 on Systems Biocatalysts. P. S-M. thanks the Spanish Ministry of Education for FPU Grant.
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