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This tutorial review focuses on recent advances in technologies for enzyme immobilisation, enabling their cost-effective use in the bio-based economy and continuous processing in general.

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This study presents a computational analysis of the structures of lipase B from Candida antarctica (CalB) and two penicillin G acylases (PGAs), from eukaryotic and prokaryotic sources, respectively. Molecular simulations were used to point out the regions of the enzymes that are prone to interact with immobilisation supports. In order to evaluate the accessibility of the active site, the location of the amino acid residues involved in the formation of covalent bonds with the polymers was visualised. The mapping of the distribution of hydrophobic and hydrophilic regions on the enzyme surface provided a view of the areas of the protein that can establish either hydrophobic or hydrophilic interactions with the carriers. Experimental data obtained from the immobilisation of the enzymes on supports bearing different chemical functionalities suggest the involvement of the glycan moiety in enzyme-polymer interactions. In the case of PGA the glycan moiety can constitute an extra site for the covalent linkage of the enzyme on the polymer.

In solid-phase peptide synthesis, dichloromethane is the predominant solvent used to incorporate the first amino acid on a 2-chlorotrityl chloride resin (via nucleophilic substitution) and Wang resin (via activation with carbodiimide in the presence of 4-dimethylaminopyridine). However, legal authorities have restricted the use of this solvent, as it is considered hazardous and a potential occupational carcinogen. Therefore, there is a need for an alternative that is easy to handle and poses less risk for the environment and more importantly for human health. Herein, we describe 2-methyltetrahydrofuran as a greener alternative for the incorporation of the first protected amino acids on both 2-chlorotrityl chloride and Wang resins. The amounts of several amino acids loaded on 2-chlorotrityl chloride and Wang resins using dry dichloromethane or 2-methyltetrahydrofuran were comparable. In addition, the use of 2-methyltetrahydrofuran rendered acceptable racemization and dipeptide formation in the case of Wang resin. This solvent can also be used to replace diethyl ether and tert-butyl methyl ether in the peptide precipitation step performed after final global deprotection. On the basis of our findings, 2-methyltetrahydrofuran emerges as a good alternative to the hazardous solvents currently used for amino acid incorporation and peptide precipitation. Furthermore, taking into account that 2-methyltetrahydrofuran proved effective for the elongation of the peptide in a solid-phase mode, we propose 2-methyltetrahydrofuran as a universal solvent for all solid-phase peptide synthesis steps, namely, incorporation of the first amino acid, assembly of the peptide chain, and precipitation of the final peptide.