Cynthia Ebert

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.

Stability and activity of immobilised penicillin G amidase in ionic liquids at controlled aw

Despite the great interest in ionic liquids as novel solvents for biocatalysis, there is still no clear idea of their influence on the stability and the activity of enzymes. Here we analysed the activity and stability of PGA in six different ionic liquids, having different cations ([bmim] and [omim]) and anions (CH3OSO3−, PF6− and BF4−). To be active in ionic liquids, PGA-450 requires an acceptable hydration (aw > 0.60), as in organic solvents. PGA is highly stable in [bmim][PF6] and [bmim][BF4], and catalytic activity, assayed by studying the synthesis of the amide of L-phenylglycine methyl ester with methyl phenylacetate, in these ILS is comparable to that obtained in toluene.

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.

d-Phenylglycine and d-4-hydroxyphenylglycine methyl esters via penicillin G acylase catalysed resolution in organic solvents

Abstract Penicillin G acylase in organic solvents catalyses specifically the acylation of the l -enantiomers of methyl esters of phenylglycine and 4-hydroxyphenylglycine. Hydrolytic reactions are prevented by controlling the water activity of the system and no excess of acylating agent is required. The process leads to the facile isolation of the enantiomerically pure d -enantiomer, which is of practical use for the preparation of β-lactam antibiotics. Electrospray mass spectroscopy has been applied to the study of the enantioselectivity of the enzyme.

CONTROL OF ENZYME HYDRATION IN PENICILLIN AMIDASE CATALYSED SYNTHESIS OF AMIDE BOND

Penicillin amidase catalyses the synthesis of amide bond in very high yield (>98%), using equimolar concentrations of the amine and the phenylacetic components. In situ hydrated phosphates were employed for controlling the water activity in a benzene/water system (97:3 v/v), where the water is taken up by the salt with formation of the hydrated species.

Penicillin G amidase in low-water media: immobilisation and control of water activity by means of celite rods

Abstract Penicillin G amidase (PGA) adsorbed on Celite rods (Celite R-640 from Fluka) catalyses, in toluene, the synthesis of amide bonds with yields >98% using equimolar concentrations of reactants. The method allows the easy recovery of the product and the recycling of the catalyst. Experimental data have pointed out that Celite rods adsorb water in a unusual but useful way, maintaining the water activity of the reaction system constant within defined ranges of water concentrations. Adsorption isotherms of Celite rods are reported and further applications of the method are proposed.

Organically modified xerogels as novel tailor-made supports for covalent immobilisation of enzymes (penicillin G acylase)

Abstract A novel application of organically modified silicates for covalent immobilisation of penicillin G acylase is reported. The immobilisation is efficient and the enzymatic preparation shows high specific activity and thermal stability. The technique opens new perspectives for the preparation of innovative tailor-made supports matching specific requirements of enzymatic processes.

A novel support for enzyme adsorption: properties and applications of aerogels in low water media

Abstract Aerogels, because of their porosity, have a great ability to adsorb water, and this characteristic was exploited to extend the applicability of aerogels for the adsorption and entrapment of hydrolases to be used in organic media. The applicability of aerogels as solid supports for immobilisation of the enzymes PGA, thermolysin and α-chymotrypsin was assayed. By controlling the distribution of water between the catalyst, the support and the reaction medium, aerogel preserves the catalytic activity of the enzymes and prevents hydrolytic reactions.

Activity of covalently immobilised PGA in water miscible solvents at controlled aw

Covalently immobilised penicillin G acylase is active in both apolar and water miscible solvents. Hydrated Celite R-640 added to water miscible solvents prevents the medium from stripping the water off the enzyme. The porous siliceous matrix has been used also for the dehydration and storage of the enzyme in apolar media.