Enzyme adsorption on hydroxyapatite to enable continuous flow biocatalysis

Biocatalysis has emerged in the last time as an interesting tool for organic synthesis: in fact, the use of enzymes provides high efficiency and selectivity, mild operational conditions, and lower hazards thanks to their non- toxicity. However, the high cost of these catalysts often hampers their application at an industrial level. To tackle this issue, their recoverability and reusability must be granted, and this can be achieved through enzyme immobilization on a solid support. Compared to free enzymes in solution, the immobilized ones are more robust, possess higher operational and storage stability as well as higher tolerance to organic solvents, and can be integrated in continuous flow reactors to achieve process intensification. Among the possible carriers for enzyme immobilization, hydroxyapatite (HAP), the inorganic component of bones, is emerging for its many favourable features, such as non-toxicity, high structural stability, large surface area, and ease in surface modification. Moreover, it can be obtained from waste such as ashes from waste-to-energy plants, the fish supply chain, the avian supply chain, etc., in agreement with circular economy principles. Gamma-Glutamyl transferase from Escherichia coli (EcGGT) was chosen as the model enzyme to study the immobilization process on HAP. Enzyme immobilization was carried out by adsorption, simply by mixing an enzyme solution and a hydroxyapatite suspension under controlled conditions (pH, temperature). Different particle sizes and experimental set-up were investigated and, after assessing that the enzyme did not desorb under reaction conditions, the supported GGT was tested as biocatalysts in the enzymatic synthesis of gamma- glutamyl-S-allyl-L-cysteine, a natural compound with flavor-enhancing properties. The enzyme reusability was tested and its storage stability was verified. After assessing the applicability of the HAP-adsorbed GGT in batch conditions, the same enzyme was adsorbed in continuous flow conditions in a packed-bed reactor made of HAP. Different residence times were studied and the obtained reactor was used in the same model reaction (Scheme 1). Scheme 1 In flow gamma-glutamylation of S-allyl-L-cysteine