Daniela Monti

Characterization of an industrial biocatalyst: Immobilized glutaryl‐7‐ACA acylase

A batch of the immobilized industrial biocatalyst glutaryl-7-ACA acylase (GA), one of the two enzymes involved in the biotransformation of cephalosporin C (CefC) into 7-aminocephalosporanic acid (7-ACA), was characterized. K(m) value for glutaryl-7-ACA was 5 mM. Enzyme activity was found to be optimal at pH between 7 and 9.5 and to increase with temperature and in buffered solutions. To avoid product degradation, optimal reaction conditions were obtained working at 25 degrees C using a 50-mM phosphate buffer, pH 8.0. Immobilized GA showed good stability at pH value below 9 and at temperature up to 30 degrees C. The inactivation of immobilized GA in the presence of different amounts of H(2)O(2), a side product that might be present in the plant-scale industrial solutions of glutaryl-7-ACA, was also investigated, but the deactivation rates were negligible at H(2)O(2) concentration that might be reached under operative conditions. Finally, biocatalyst performance in the complete two-step enzymatic conversion process from CefC to 7-ACA was determined on a laboratory scale. Following the complete conversion of a 75 mM solution of CefC into glutaryl-7-ACA catalyzed by an immobilized D-amino acid oxidase (DAAO), immobilized GA was used for the transformation of this intermediate into the final product 7-ACA. This reaction was repeated for 42 cycles. An estimation of the residual activity of the biocatalyst showed that 50% inactivation of immobilized GA was reached after approximately 300 cycles, corresponding to an enzyme consumption of 0.4 kU per kg of isolated 7-ACA.

Cascade Coupling of Ene Reductases with Alcohol Dehydrogenases: Enantioselective Reduction of Prochiral Unsaturated Aldehydes

The baker’s yeast (BY)‐mediated reduction of a set of α‐substituted derivatives of cinnamaldehyde to give the corresponding saturated alcohols was investigated. Our study demonstrates that the ene reductases of BY (OYE 2 or OYE 3) preferentially reduce the CC double bond of stereoisomers with the phenyl group trans to the carbonyl group. Moreover, the alcohol dehydrogenases (ADHs) chemoselectively reduce (oxidise) the carbonyl group (hydroxyl group) of saturated aldehydes (alcohols) if the side chain does not contain a heteroatom such as oxygen or sulphur and is longer than an ethyl group. Using isolated OYEs instead of BY, the saturated aldehydes partially lost their optical purity during the biotransformation, even with the in situ substrate feeding product removal (SFPR) technology. This limitation can be overcome by the combination of an isolated ene reductase (OYE 2 or OYE 3) together with an ADH (horse liver alcohol dehydrogenase) in a cascade system which allowed both yields and enantioselectivities to improve.

Enzymatic Kinetic Resolution of Silybin Diastereoisomers

In nature, the flavonolignan silybin (1) occurs as a mixture of two diastereomers, silybin A and silybin B, which in a number of biological assays exhibit different activities. A library of hydrolases (lipases, esterases, and proteases) was tested for separating the silybin A and B diastereomers by selective transesterification or by stereoselective alcoholysis of 23-O-acetylsilybin (2). Novozym 435 proved to be the most suitable enzyme for the preparative production of both optically pure silybins A and B by enzymatic discrimination. Gram amounts of the optically pure substances can be produced within one week, and the new method is robust and readily scalable to tens of grams.

Synthesis of Robalzotan, Ebalzotan, and Rotigotine Precursors via the Stereoselective Multienzymatic Cascade Reduction of α,β-Unsaturated Aldehydes

A stereoselective synthesis of bicyclic primary or secondary amines, based on tetralin or chroman structural moieties, is reported. These amines are precursors of important active pharmaceutical ingredients such as rotigotine (Neupro), robalzotan, and ebalzotan. The key step is based on a multienzymatic reduction of an α,β-unsaturated aldehyde or ketone to give the saturated primary or secondary alcohol, in a high yield and with a high ee. The catalytic system consists of the combination of an ene-reductase (ER; i.e., OYE2 or OYE3 belonging to the Old Yellow Enzyme family) with an alcohol dehydrogenase (ADH), applying the in situ substrate feeding product removal technology. By this system the formation of the allylic alcohol side product and the racemization of the chirally unstable α-substituted aldehyde intermediate are minimized. The primary alcohols were elaborated via a Curtius rearrangement. The combination of OYE2 with a Prelog or an anti-Prelog ADH allowed the preparation of the secondary alcohols with ee > 99% and de > 87%. The absolute configuration of the primary amines was unambiguously assigned by comparison with authentic samples. The stereochemistry of secondary alcohols was assigned by X-ray crystal structure and NMR analysis of Mosher esters.

Old Yellow Enzyme-mediated reduction of β-cyano-α,β-unsaturated esters for the synthesis of chiral building blocks: stereochemical analysis of the reaction

Bakers yeast and Old Yellow Enzyme-mediated reduction of the carbon–carbon double bonds of β-cyano-α,β-unsaturated esters was investigated, in order to broaden the applicability of this kind of reaction in the field of preparative organic chemistry. The synthetic significance of the enantioselective reduction of these difunctionalised substrates was shown by considering the conversion of saturated chiral cyanoesters into γ2-amino acid derivatives for foldamer chemistry applications. The stereochemical outcome of the biotransformations was carefully analysed by means of deuterium labeling experiments. The results of this analysis were employed to rationalise the effects of substrate-control on the stereoselectivity of a certain class of ene-reductase-mediated reduction reactions. A simple model was developed to describe the structural prerequisites for the optimal arrangement of the substrates within the binding site of OYE1-3 enzymes.

Oxidation of galactomannan by laccase plus TEMPO yields an elastic gel

Chemical modifications of galactomannans are applied to improve and/or modify their solubility, rheological and functional properties, but have limited specificity and are often difficult to control. Enzymatic reactions, catalyzed under mild process conditions, such as depolymerization, debranching and oxidation, represent a viable and eco-friendly alternative. In this study, we describe oxidation of guar galactomannan primary hydroxyl groups by a fungal laccase using the stable radical TEMPO as mediator. Four fungal laccases were investigated from: Trametes versicolor, Myceliophthora thermophila, Thielavia arenaria, Cerrena unicolor. The laccase from T. versicolor was found to efficiently oxidize TEMPO and to be free of mannanase side activity. Oxidation of galactomannan with this enzyme plus TEMPO brought about a ten-fold increase in viscosity of a guar galactomannan solution and altered its rheological profile, by converting a viscous polysaccharide solution into an elastic gel. This structural modification is presumably due to formation of inter-chain hemiacetalic bonds between newly generated carbonyl groups and free OH groups, yielding a cross-linked gel. These findings could be of practical importance, considering that polysaccharides with high viscosity, gelling and elastic properties can find interesting and novel applications as thickeners, viscosifiers and emulsion stabilizers in several industrial applications such as: personal care, oil operations, paper coating, paints, construction and mining.

Discovery and characterization of thermophilic limonene-1,2-epoxide hydrolases from hot spring metagenomic libraries.

The epoxide hydrolases (EHs) represent an attractive option for the synthesis of chiral epoxides and 1,2‐diols which are valuable building blocks for the synthesis of several pharmaceutical compounds. A metagenomic approach has been used to identify two new members of the atypical EH limonene‐1,2‐epoxide hydrolase (LEH) family of enzymes. These two LEHs (Tomsk‐LEH and CH55‐LEH) show EH activities towards different epoxide substrates, differing in most cases from those previously identified for Rhodococcus erythropolis (Re‐LEH) in terms of stereoselectivity. Tomsk‐LEH and CH55‐LEH, both from thermophilic sources, have higher optimal temperatures and apparent melting temperatures than Re‐LEH. The new LEH enzymes have been crystallized and their structures solved to high resolution in the native form and in complex with the inhibitor valpromide for Tomsk‐LEH and poly(ethylene glycol) for CH55‐LEH. The structural analysis has provided insights into the LEH mechanism, substrate specificity and stereoselectivity of these new LEH enzymes, which has been supported by mutagenesis studies.

New insights on the features of the vinyl phenol reductase from the wine-spoilage yeast Dekkera/Brettanomyces bruxellensis

Vinyl phenol reductase activity was assayed in extracts from 19 strains of Dekkera bruxellensis isolated from wine. In all strains, vinyl phenol reductase activity was insensitive to the presence/absence of 4-vinyl guaiacol, confirming that expression is not related to the presence of the substrate. D. bruxellensis CBS 4481 showed the highest vinyl phenol reductase activity toward 4-vinyl guaiacol. Vinyl phenol reductase from D. bruxellensis CBS 4481 was purified to mass spectrometric homogeneity, and sequenced by trypsinolysis and mass spectrometry. The sequence of the purified protein showed convincing homology with a Cu/Zn superoxide dismutase in the D. bruxellensis AWRI 1499 genome, and indeed it was found to possess both vinyl phenol reductase and superoxide dismutase activities. A bioinformatics analysis of the sequence of vinyl phenol reductase/superoxide dismutase from D. bruxellensis CBS 4481 reveals the presence in this protein of cofactor-binding structural features, that are absent in sequences of superoxide dismutases from related microorganisms, that do not display vinyl phenol reductase activity.

Cascade Coupling of Ene‐Reductases and ω‐Transaminases for the Stereoselective Synthesis of Diastereomerically Enriched Amines

One‐pot sequential and cascade processes performed by employing ene‐reductases (ERs) together with ω‐transaminases (ω‐TAs) for the obtainment of diastereomerically enriched (R)‐ and (S)‐amine derivatives containing an additional stereocenter were investigated. By using either α‐ or β‐substituted unsaturated ketones as substrates and by coupling purified ERs belonging to the Old Yellow Enzyme (OYE) family with a panel of commercially available ω‐TAs, the desired products were obtained in up to >99 % conversion and >99 % de. The sequential reactions were performed in a one‐pot fashion with no need to adapt the reaction conditions to the reductive amination step or to purify the reaction intermediate. Moreover, high chemoselectivity of the tested ω‐TAs for the saturated ketones was shown in the cascade reactions.

The Quest for New Mild and Selective Modifications of Natural Structures: Laccase‐Catalysed Oxidation of Ergot Alkaloids Leads to Unexpected Stereoselective C‐4 Hydroxylation

Laccase-catalysed oxidation of ergot alkaloids in the absence of chemical mediators allowed the unexpected isolation of the mono-hydroxylated derivatives of compounds 2-7. Structure determination by NMR techniques clearly indicated that hydroxylation took place at the C-4 benzylic position. Quite notably, the proposed protocol allowed, for the first time, functionalisation at the C-4 position of the ergoline skeleton. Depending on the absence or on the presence of a C-10 α-methoxy substituent, hydroxylation was either stereoselective (furnishing C-4α OH derivatives) or gave rise to a C-4α/C-4β OH mixture in a 2:1 ratio, respectively.