Alessandra Basso

Biocatalysis in non-conventional media—ionic liquids, supercritical fluids and the gas phase

During the last decades much attention has been paid, by both academia and industry, to the development of new solvents. The introduction of ILs, sc-fluids and solid–gas interphase reactions has definitely opened new perspectives. ILs have been introduced into organic chemistry to meet the increasing demand for clean technologies in industrial processes. However, the toxicology of ILs remains unclear and further studies are necessary to assess their sustainability. Use of supercritical fluids is already a mature technique and their power lies less in their number but more in the variety of characteristics that one sc-fluid can have depending on the pressure. Enzyme-catalysed reactions at the solid–gas interphase have undergone a rapid development, and this will definitely find a place in the production of high value added compounds, such as fragrances.

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.

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.

Synthesis of octyl glucopyranoside by almond β-glucosidase adsorbed onto Celite R-640®

Abstract The synthesis of octyl glucoside from p -nitrophenyl glucopyranoside ( p -NPG) and 1-octanol was carried out with almond β-glucosidase adsorbed onto Celite R-640®. The influence of the amount of water added to the system as well as the addition of co-solvents have been studied. The presence of small percentages of DMF has a strong denaturing effect on the enzyme adsorbed onto Celite R-640®. Denaturation is less pronounced with other co-solvents such as acetonitrile, 2-methyl-2-butanol or ethyl acetate.

Fructose Production by Inulinase Covalently Immobilized on Sepabeads in Batch and Fluidized Bed Bioreactor

The present work is an experimental study of the performance of a recently designed immobilized enzyme: inulinase from Aspergillus sp. covalently immobilized on Sepabeads. The aim of the work is to test the new biocatalyst in conditions of industrial interest and to assess the feasibility of the process in a fluidized bed bioreactor (FBBR). The catalyst was first tested in a batch reactor at standard conditions and in various sets of conditions of interest for the process. Once the response of the catalyst to different operating conditions was tested and the operational stability assessed, one of the sets of conditions tested in batch was chosen for tests in FBBR. Prior to reaction tests, preliminary fluidization tests were realized in order to define an operating range of admissible flow rates. As a result, the FBR was run at different feed flow rates in a closed cycle configuration and its performance was compared to that of the batch system. The FBBR proved to be performing and suitable for scale up to large fructose production.

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.

Controlling the hydration of covalently immobilised penicillin G amidase in low-water medium: properties and use of Celite R-640

Abstract The study describes how Celite R-640 adsorbs liquid water in toluene and vapour water from a gas phase. In toluene, Celite R-640 is able to maintain the water activity ( a w ) constant within broad ranges of water concentrations. The a w values are strongly related to the original hydration of the Celite batches, but prolonged drying confers comparable and reproducible properties to the different batches. The use of Celite R-640 in controlling the hydration and activity of covalently immobilised PGA in toluene is reported.