Continuous flow synthesis of gamma-glutamyl peptides using GGT adsorbed on hydroxyapatite

Despite the many advantages presented by enzyme-catalysed reactions, their implementation at industrial level is hampered due to protein’s poor long-term stability under harsh conditions and difficult separation from the reaction mixture and consequently reuse. To address these problems, the immobilisation of the enzyme on an insoluble support is a possible solution. This strategy, in combination with continuous flow technologies, can lead to process intensification. In the pursuit of a more sustainable Chemistry, the research of the last years has been devoted to find greener alternatives compared to petro-based support usually employed in enzyme immobilisation. Among these, hydroxyapatite (HAP), the mineral component of mammalian bones, represents a suitable candidate thanks to its structural stability, non-toxicity, large surface area and ease of surface modification. Moreover, it can also be derived 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 immobilisation process on HAP. Enzyme immobilisation 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-ups 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-L-glutamyl-S-allyl-L-cysteine, a natural compound with flavour-enhancer 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 (Figure 1). Figure 1. Continuous flow synthesis of gamma-L-glutamyl-S-allyl-L-cysteine