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.

There is an enormous potential for synthesizing novel bio-based functionalized polyesters under environmentally benign conditions by exploiting the catalytic efficiency and selectivity of enzymes. Despite the wide number of studies addressing in vitro enzymatic polycondensation, insufficient progress has been documented in the last two decades towards the preparative and industrial application of this methodology. The present study analyses bottlenecks hampering the practical applicability of enzymatic polycondensation that have been most often neglected in the past, with a specific focus on solvent-free processes. Data here presented elucidate how classical approaches for enzyme immobilization combined\ud with batch reactor configuration translate into insufficient mass transfer as well as limited recyclability of the biocatalyst. In order to overcome such bottlenecks, the present study proposes thin-film processes employing robust covalently immobilized lipases. The strategy was validated experimentally by carrying out the solvent-free polycondensation of esters of adipic and itaconic acids. The results open new perspectives for enlarging the applicability of biocatalysts in other viscous and solvent-free syntheses

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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Despite the extensive efforts of scientific community towards the development of a vast variety of immobilization methods, there is a limited number of immobilized biocatalysts used at industrial scale. Most often, cost issues prevent the transfer of methodologies to large scale but more recently also sustainability criteria are becoming increasingly relevant, so that petroleum based carriers for enzyme immobilization appear unsuitable for responding to new challenges of green and renewable chemistry. Here we report, for the first time, a preliminary overview of the potential of rice husk as carrier to be employed for both physical and covalent immobilization of enzymes. The data indicate that the chemical versatility of this lignocellulosic biomass, containing also silica, opens wide scenarios for optimizing different immobilization procedures requiring minimal pre-treatments and applicable to various enzymes and process conditions. The mechanical and chemical robustness of rice husk, along with its virtual unlimited availability worldwide, make this inexpensive natural matrix a promising candidate for replacing organic fossil-based carriers for enzyme immobilization.Computational construction of 3D models and surface analysis. Protein structures were visualized and recorded using the PyMOL software. The 3D-structures used for the hydrophobicity comparisons were retrieved from the PDB with the codes 1TCA for CaLB, 5DPF for Thermolysin and 4EQV for Invertase

The present study addresses comprehensively the problem of producing polyesters through sustainable processes while using fully renewable raw materials and biocatalysts. Polycondensation of bio-based dimethyl adipate with different diols was catalyzed by cutinase 1 from Thermobifida cellulosilytica (Thc_cut1) under solvent free and thin-film conditions. The biocatalyst was immobilized efficiently on a fully renewable cheap carrier based on milled rice husk. A multivariate factorial design demonstrated that Thc_cut1 is less sensitive to the presence of water in the system and it works efficiently under milder conditions (50 °C; 535 mbar) when compared to lipase B from Candida antarctica (CaLB), thus enabling energy savings. Experimental and computational investigations of cutinase 1 from Thermobifida cellulosilytica (Thc_cut1) disclosed structural and functional features that make this serine-hydrolase efficient in polycondensation reactions. Bioinformatic analysis performed with the BioGPS tool pointed out functional similarities with CaLB and provided guidelines for future engineering studies aiming, for instance, at introducing different promiscuous activities in the Thc_cut1 scaffold. The results set robust premises for a full exploitation of enzymes in environmentally and economically sustainable enzymatic polycondensation reactions

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