Reversible immobilization of rhamnulose-1-phosphate aldolase for biocatalysis: Enzyme loading optimization and aldol addition kinetic modeling

Reusability of the usually expensive immobilization supports by means of reversible immobilization techniques helps reduce the overall cost of the biocatalytic process. One of these techniques, the reversible binding of the histidine residues of proteins with the metal chelated in affinity supports, has been extensively exploited in purification of His-tagged proteins in one-step. The use of these supports for immobilization of His-tagged recombinant enzymes allows performing simultaneously the enzyme purification and immobilization, thus further reducing the number of process steps, enzyme losses and costs. In this work, His-tagged rhamnulose-1-phosphate aldolase (RhuA) was immobilized onto iminodiacetic acid-functionalized agarose charged with cobalt (Co-IDA) for the catalysis of aldol addition reactions of industrial interest. Enzyme loading was optimized to maximize the immobilized activity avoiding diffusion limitations for both the RhuA natural reaction and the synthetic aldol addition between dihydroxyacetone phosphate (DHAP) and (S)-Cbz-alaninal (10 AU/mL of support and 25 AU/mL of support, respectively). RhuA:Co-IDA biocatalysts with high retained activity (92%) and enhanced stability (37 days of half life time) were obtained. Kinetic modeling of the synthetic aldol addition between DHAP and (S)-Cbz-alaninal and the unwanted secondary reaction catalyzed by soluble RhuA was carried out. RhuA:Co-IDA catalysis of the same reactions was found to follow the same kinetic model.

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• A metal-chelate affinity cobalt support is used for enzyme immobilization.
• Immobilized rhamnulose-1-P aldolase exhibits high retained activity and stability.
• Optimal enzyme loading was determined for the natural reaction and a synthetic one.
• Kinetic modeling of the synthetic aldol addition and secondary reaction was performed.
• The model was validated for both soluble and immobilized RhuA catalysis.