Covalent Immobilisation of Different Enzymes on Hydroxyapatite, an Alternative Green Support

The implementation of biocatalysis in industrial processes has been hampered by poor long-term stability and difficult recyclability of enzymes. A viable solution to solve these problems is the immobilisation of the biocatalyst on insoluble supports, thus allowing also its implementation in combination with continuous flow technologies for process intensification. The push for a more sustainable Chemistry has fostered the research of greener alternatives compared to petro-based support usually employed in enzyme immobilisation that are also quite expensive. 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. As it can be sourced from waste, it fulfils also the circular economy principles. To test the possibility of using HAP for covalent immobilisation, three model enzymes were chosen: a vanadium-dependent chloroperoxidase from Curvularia inaequalis (CiVCPO), a L-tyrosine decarboxylase from Lactobacillus brevis (LbTDC) and an R-selective transaminase from Thermomyces stellatus (TsRTA). The immobilisation of these enzymes on other commercial supports has been reported in the context of producing valuable APIs or natural products, therefore the results on hydroxyapatite could be rigorously compared with literature data. The strategy tested consisted of a first derivatisation of HAP with a silanising agent ((3-aminopropyl)triethoxysilane, APTES) followed by the activation with glutaraldehyde and then the addition of the enzyme (Figure 1). Different experimental conditions and protein loading were tested to maximise the immobilisation yield and the recovered activity. Figure 1. Immobilisation strategy on HAP using glutaraldehyde All the enzymes tested were efficiently bound to the support, albeit with variable recovered activities depending both on the protein loading and the enzyme, with CiVCPO presenting the lowest values. The immobilised LbTDC and TsRTA were tested in multiple reaction cycles to assess their stability and reusability with promising results. Overall, it was confirmed the feasibility of hydroxyapatite as a support for covalent immobilisation of enzymes, expanding the scope of biocatalysts present in the literature.