Efficient immobilisation protocols are the result of perfect matching of factors depending on the enzyme, the process and the support for immobilisation. Physical-chemical phenomena, such as partition, solvation and diffusion, strongly affect the efficiency of the biocatalyst in each specific reaction system. Therefore, tailored solutions must be developed for each specific process of interest. Indeed, direct investigation of what occurs at the molecular level in a reaction catalysed by an immobilised enzyme is a quite formidable task and observed differences in the performance of immobilised biocatalysts must be interpreted very carefully. In any study dealing with enzyme immobilisation the prerequisite is the rigorous planning and reporting of experiments, being aware of the complexity of these multi-phase systems.

Itaconic acid is ac hemically versatile unsaturated diacid that can be produced by fermentation and potentially it can replace petrol-based monomers such as maleic and fumarica cids in the production of curable polyesters or new biocompatible functionalized materials.U nfortunately,d ue to the presence of the unsaturated C=Cb ond, polycondensation of itaconica cid is hampered by cross reactivity and isomerization. Therefore,e nzymatic polycondensations wouldr espondt ot he need of mild and selective synthetic routes for the production of novelb io-based polymers.T he present work analyses the feasibility of enzymatic polycondensation of diethyli taconate and, for the first time,p rovidesc omprehensives olutions embracing botht he formulation of the biocata-lyst, the reaction conditions and the choice of the comonomers.C omputational docking was used to disclose the structural factorsresponsible for the low reactivity of dimethyl itaconate and to identify possible solutions.S urprisingly,e xperimentala nd computational analyses revealed that 1,4-butanediol is an unsuitable co-monomer for the polycondensation of dimethyli taconate whereast he cyclica nd rigid 1,4-cyclohexanedimethanol promotes the elongation of the oligomers.

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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.

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