Continuous Flow Mode Biocatalytic Transamination Using Macrocellular Silica Monoliths: Optimizing Support Functionalisation and Enzyme Grafting

Abstract

<div>Transaminases are immobilized onto macrocellular silica monoliths and used for carrying a continuous flow mode transamination reaction. Monoliths were prepared via an emulsion-templated sol-gel method and functionalized by amino-moieties (APTES) in order to covalently immobilize the enzymes, using glutaraldehyde as a cross-linking agent. In order to obtain higher performance and improved reproducibility, we investigate the key parameters of APTES functionalization and of enzyme grafting. Four functionalization protocols were studied. It is shown that controlling the moisture levels in monolith and in the functionalisation solution led to a 3-fold increase in activity as compared to the previously reported data, and greatly improved the reproducibility. Additionally, we report a strong beneficial effect of running the enzyme immobilization at room temperature instead of 4°C, further enhancing the obtained activity. Finally, the popular method which consists in stabilizing the covalent attachment of the enzyme by reducing the imine bonds formed between the enzyme and the functionalized surface was investigated. We highlight a strong enzyme deactivation caused by cyanoborohydride, making this strategy irrelevant in this case. All in all, the improvements presented here for enzyme immobilization in macrocellular silica monoliths, lead to the preparation of more active materials for continuous flow mode biocatalysis.<br></div>

Experimental and modeling of tetracycline degradation in water in a flow-through enzymatic monolithic reactor

“…Similarly, Barrios-Estrada et al, (2018) found that only 33% of bisphenol-A was degraded in 24 h with an immobilized laccase on the same type of membranes. To overcome some of these disadvantages, meso-/macroporous monoliths have been recently applied for enzyme immobilization, the objective is to provide a big surface area to immobilize a large amount of enzymes, together with a macroporosity which allows low pressure drop while avoiding clogging (Ahmad et al, 2021;Biggelaar et al, 2019;Sebai et al, 2022). Moreover, as far as substrates are forced to flow through the monolith porosity the probability of contact with the biocatalyst is enhanced while allowing a precise control of the contact time (Sanchez Marcano, 2019).…”

“…This configuration is called Flow-Trough-Reactor (FTR). Silica monoliths prepared by emulsion templating and presenting a large distribution of macropores (50 nm to 6 μm) have been recently used as supports in biocatalysis (Biggelaar et al, 2017(Biggelaar et al, , 2019. Another type of silica monoliths have been prepared by spinodal decomposition.…”