Valerio Ferrario

Efficient immobilisation of industrial biocatalysts: criteria and constraints for the selection of organic polymeric carriers and immobilisation methods

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.

The Closure of the Cycle: Enzymatic Synthesis and Functionalization of Bio-Based Polyesters.

The polymer industry is under pressure to mitigate the environmental cost of petrol-based plastics. Biotechnologies contribute to the gradual replacement of petrol-based chemistry and the development of new renewable products, leading to the closure of carbon circle. An array of bio-based building blocks is already available on an industrial scale and is boosting the development of new generations of sustainable and functionally competitive polymers, such as polylactic acid (PLA). Biocatalysts add higher value to bio-based polymers by catalyzing not only their selective modification, but also their synthesis under mild and controlled conditions. The ultimate aim is the introduction of chemical functionalities on the surface of the polymer while retaining its bulk properties, thus enlarging the spectrum of advanced applications.

Endo- and exo-inulinases: enzyme-substrate interaction and rational immobilization.

Three‐dimensional models of exoinulinase from Bacillus stearothermophilus and endoinulinase from Aspergillus niger were built up by means of homology modeling. The crystal structure of exoinulinase from Aspergillus awamori was used as a template, which is the sole structure of inulinase resolved so far. Docking and molecular dynamics simulations were performed to investigate the differences between the two inulinases in terms of substrate selectivity. The analysis of the structural differences between the two inulinases provided the basis for the explanation of their different regio‐selectivity and for the understanding of enzyme‐substrate interactions. Surface analysis was performed to point out structural features that can affect the efficiency of enzymes also after immobilization. The computational analysis of the three‐dimensional models proved to be an effective tool for acquiring information and allowed to formulate an optimal immobilized biocatalyst even more active that the native one, thus enabling the full exploitation of the catalytic potential of these enzymes.

Towards feasible and scalable solvent-free enzymatic polycondensations: integrating robust biocatalysts with thin film reactions

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 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.

Enlarging the tools for efficient enzymatic polycondensation: structural and catalytic features of cutinase 1 from Thermobifida cellulosilytica

Cutinase 1 from Thermobifida cellulosilytica is reported for the first time as an efficient biocatalyst in polycondensation reactions. Under thin film conditions the covalently immobilized enzyme catalyzes the synthesis of oligoesters of dimetil adipate with different polyols leading to higher Mw (~1900) and Mn (~1000) if compared to lipase B from Candida antarctica or cutinase from Humicola insolens. Computational analysis discloses the structural features that make this enzyme readily accessible to substrates and optimally suited for covalent immobilization. As lipases and other cutinase enzymes, it presents hydrophobic superficial regions around the active site. However, molecular dynamics simulations indicate the absence of interfacial activation, similarly to what already documented for lipase B from Candida antarctica. Notably, cutinase from Humicola insolens displays a “breathing like” conformational movement, which modifies the accessibility of the active site. These observations stimulate wider experimental and bioinformatics studies aiming at a systematic comparison of functional differences between cutinases and lipases.

BioGPS descriptors for rational engineering of enzyme promiscuity and structure based bioinformatic analysis.

A new bioinformatic methodology was developed founded on the Unsupervised Pattern Cognition Analysis of GRID-based BioGPS descriptors (Global Positioning System in Biological Space). The procedure relies entirely on three-dimensional structure analysis of enzymes and does not stem from sequence or structure alignment. The BioGPS descriptors account for chemical, geometrical and physical-chemical features of enzymes and are able to describe comprehensively the active site of enzymes in terms of “pre-organized environment” able to stabilize the transition state of a given reaction. The efficiency of this new bioinformatic strategy was demonstrated by the consistent clustering of four different Ser hydrolases classes, which are characterized by the same active site organization but able to catalyze different reactions. The method was validated by considering, as a case study, the engineering of amidase activity into the scaffold of a lipase. The BioGPS tool predicted correctly the properties of lipase variants, as demonstrated by the projection of mutants inside the BioGPS “roadmap”.

Lipases Immobilization for Effective Synthesis of Biodiesel Starting from Coffee Waste Oils

Immobilized lipases were applied to the enzymatic conversion of oils from spent coffee ground into biodiesel. Two lipases were selected for the study because of their conformational behavior analysed by Molecular Dynamics (MD) simulations taking into account that immobilization conditions affect conformational behavior of the lipases and ultimately, their efficiency upon immobilization. The enzymatic synthesis of biodiesel was initially carried out on a model substrate (triolein) in order to select the most promising immobilized biocatalysts. The results indicate that oils can be converted quantitatively within hours. The role of the nature of the immobilization support emerged as a key factor affecting reaction rate, most probably because of partition and mass transfer barriers occurring with hydrophilic solid supports. Finally, oil from spent coffee ground was transformed into biodiesel with yields ranging from 55% to 72%. The synthesis is of particular interest in the perspective of developing sustainable processes for the production of bio-fuels from food wastes and renewable materials. The enzymatic synthesis of biodiesel is carried out under mild conditions, with stoichiometric amounts of substrates (oil and methanol) and the removal of free fatty acids is not required.

Enantioselective production of 3-hydroxy metabolites of tibolone by yeast reduction

The enantioselective reduction of tibolone into the corresponding 3alpha-hydroxy or 3beta-hydroxy metabolite can be controlled by choosing suited strains of yeasts and biotransformation conditions. A restricted screening performed among 52 yeasts showed that the 3alpha-epimer was preferentially obtained with high epimeric purity with various strains (i.e. with Kluyveromyces lactis CBS 2359), while only Saccharomyces cerevisiae CBS 3093 gave the 3beta-epimer as major product. The reduction of tibolone with K. lactis CBS 2359 and S. cerevisiae CBS 3093 was optimised. S. cerevisiae CBS 3093 furnished a 96:4 ratio of 3beta/3alpha with complete molar conversion within 72h when the initial concentration of substrate was below 2.5g/L. K. lactis CBS 2359 gave a 99:1 ratio of 3alpha/3beta with complete conversion in 64h.

Large scale applications of immobilized enzymes call for sustainable and inexpensive solutions: rice husks as renewable alternatives to fossil-based organic resins

Despite the extensive efforts of the scientific community towards the development of a vast variety of immobilization methods, there is a limited number of immobilized biocatalysts used at an industrial scale. Most often, cost issues prevent the transfer of methodologies to large scale but more recently sustainability criteria are also becoming increasingly relevant, so that petroleum based carriers for enzyme immobilization appear unsuitable for responding to the new challenges of green and renewable chemistry. Here we report, for the first time, a preliminary overview of the potential of rice husks as carriers 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 husks, along with their virtually unlimited availability worldwide, make this inexpensive natural matrix a promising candidate for replacing organic fossil-based carriers for enzyme immobilization.

Fully renewable polyesters via polycondensation catalyzed by Thermobifida cellulosilytica cutinase 1: an integrated approach

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.