Lauzacco, Italy
Lauzacco, Italy

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Calleri E.,University of Pavia | Cattaneo G.,University of Pavia | Rabuffetti M.,University of Milan | Serra I.,University of Pavia | And 8 more authors.
Advanced Synthesis and Catalysis | Year: 2015

A purine nucleoside phosphorylase from Aeromonas hydrophyla (AhPNP) was covalently immobilized in a pre-packed stainless steel column containing aminopropylsilica particles via Schiff base chemistry upon glutaraldehyde activation. The resulting AhPNP-IMER (Immobilized Enzyme Reactor, immobilization yield ≈50%) was coupled on-line through a 6-way switching valve to an HPLC apparatus containing an analytical or a semi-preparative chromatographic column. The synthesis of five 6-modified purine ribonucleosides was carried out by continuously pumping the reaction mixture through the AhPNP-IMER until the highest conversion was reached, and then directing the reaction mixture to chromatographic separation. The conditions of the AhPNP-catalyzed transglycosylations (2:1 ratio sugar donor:base acceptor; 10 mM phosphate buffer; pH 7.5; temperature 37 °C, flow rate 0.5 mL min-1) were optimized by a fractional factorial experimental design. Coupling the bioconversion step with the product purification in such an integrated platform resulted in a fast and efficient synthetic process (yield=52-89%; <10 mg) where sample handling was minimized. To date, AhPNP-IMER has retained completely its activity upon 50 reactions in 10 months. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Ubiali D.,University of Pavia | Ubiali D.,Italian Biocatalysis Center | Serra C.D.,University of Milan | Serra I.,University of Pavia | And 9 more authors.
Advanced Synthesis and Catalysis | Year: 2012

Purine nucleoside phosphorylase (PNP) from Aeromonas hydrophila encoded by the deoD gene has been over-expressed in Escherichia coli, purified, characterized about its substrate specificity and used for the preparative synthesis of some 6-substituted purine-9-ribosides. Substrate specificity towards natural nucleosides showed that this PNP catalyzes the phosphorolysis of both 6-oxo- and 6-aminopurine (deoxy)ribonucleosides. A library of nucleoside analogues was synthesized and then submitted to enzymatic phosphorolysis as well. This assay revealed that 1-, 2-, 6- and 7-modified nucleosides are accepted as substrates, whereas 8-substituted nucleosides are not. A few transglycosylation reactions were carried out using 7-methylguanosine iodide (4) as a d-ribose donor and 6-substituted purines as acceptor. In particular, following this approach, 2- amino-6-chloropurine-9-riboside (2c), 6-methoxypurine- 9-riboside (2d) and 2-amino-6-(methylthio)purine- 9-riboside (2g) were synthesized in very high yield and purity. © 2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim.


Speranza G.,University of Milan | Speranza G.,Italian Biocatalysis Center | Speranza G.,CNR Institute of Molecular Science and Technologies | Morelli C.F.,University of Milan | Morelli C.F.,Italian Biocatalysis Center
Journal of Molecular Catalysis B: Enzymatic | Year: 2012

γ-Glutamyl derivatives of S-substituted cysteines are naturally occurring flavor enhancers found in garlic and in other plants of the genus Allium. In this paper we describe a straightforward enzymatic synthesis of γ-glutamyl-S-allylcysteine, γ-glutamyl-S-methylcysteine and γ-glutamylmethionine based on a transpeptidation reaction involving glutathione as the γ-glutamyl donor and catalyzed by a commercially available γ-glutamyl transpeptidase (EC 2.3.2.2). Reaction conditions have been optimized with respect to pH, temperature, reactants molar ratio and enzyme concentration using a method based on TLC technique and computer-assisted image analysis for the quantitative evaluation of the reaction mixtures composition. Preparative enzymatic syntheses of γ-glutamyl-S- allylcysteine, γ-glutamyl-S-methylcysteine and γ-glutamylmethionine were carried out at 1 mmol scale. The method is suited for the laboratory scale synthesis of reference compounds for quality control assay, in that it avoids laborious and low-yielding isolation procedures from natural sources or complicated protection and deprotection steps required for chemical synthesis. © 2012 Elsevier B.V. All rights reserved.


Kaewmanee T.,University of Genoa | Bagnasco L.,University of Genoa | Bagnasco L.,University of Milan | Benjakul S.,Prince of Songkla University | And 6 more authors.
Food Chemistry | Year: 2014

The chemical composition, physicochemical, functional and sensory properties of mucilages, extracted from seven Italian flax cultivars, were evaluated. All samples were composed of neutral and acidic sugars, with a low protein content. From the NMR data, a rhamnogalacturonan backbone could be inferred as a common structural feature for all the mucilages, with some variations depending on the cultivar. All the suspensions showed a poor stability, which was consistent with a low zeta potential absolute value. The viscosity seemed to be positively correlated with the neutral sugars and negatively with the amount of proteins. Functional properties were dependent on the cultivar. The sensory analysis showed that most mucilages are tasteless. All these outcomes could support the use of flaxseed mucilages for industrial applications. In particular, Solal and Festival cultivars could be useful as thickeners, due to their high viscosity, while Natural, Valoal and Kaolin as emulsifiers for their good surface-active properties. © 2013 Elsevier Ltd. All rights reserved.


Bagnasco L.,University of Genoa | Pappalardo V.M.,University of Milan | Meregaglia A.,University of Milan | Kaewmanee T.,University of Genoa | And 7 more authors.
Food Research International | Year: 2013

The effects of enzymatic hydrolysis induced by food-grade exo- plus endo-proteases (i.e. Umamizyme and Flavourzyme) on the sensory characteristics and functional properties of rice middlings were investigated. Enzymatic hydrolysis was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), by monitoring the rate of hydrolysis with the Coefficient of Protein Degradation (CPD). The presence of medium-low size polypeptides at the end point of 24. h was detected, resulting from 24.84 ± 1.28 % and 67.04 ± 0.55 % hydrolysis for Flavourzyme and Umamyzyme, respectively. Sensory and functional properties, including emulsifying and foaming properties, were evaluated on hydrolysates obtained after 24. h incubation. The sensory analysis described the umami taste as the most intense attribute for all the products and good emulsifying and foaming properties were observed, mainly in Umamizyme hydrolysates. Then the hydrolysis procedure here described could be convenient to provide ingredients characterized by a good taste profile and by functional properties useful for industrial food processing, storage and consumption. © 2012 Elsevier Ltd.


Bagnasco L.,University of Genoa | Cosulich M.E.,University of Genoa | Cosulich M.E.,University of Pavia | Speranza G.,University of Milan | And 4 more authors.
Food Chemistry | Year: 2014

The relationships between sensory attribute and analytical measurements, performed by electronic tongue (ET) and near-infrared spectroscopy (NIRS), were investigated in order to develop a rapid method for the assessment of umami taste. Commercially available umami products and some aminoacids were submitted to sensory analysis. Results were analysed in comparison with the outcomes of analytical measurements. Multivariate exploratory analysis was performed by principal component analysis (PCA). Calibration models for prediction of the umami taste on the basis of ET and NIR signals were obtained using partial least squares (PLS) regression. Different approaches for merging data from the two different analytical instruments were considered. Both of the techniques demonstrated to provide information related with umami taste. In particular, ET signals showed the higher correlation with umami attribute. Data fusion was found to be slightly beneficial - not so significantly as to justify the coupled use of the two analytical techniques. © 2014 Elsevier Ltd. All rights reserved.


Serra I.,University of Pavia | Conti S.,University of Pavia | Conti S.,Gedeon Richter Italia srl | Piskur J.,Lund University | And 6 more authors.
Advanced Synthesis and Catalysis | Year: 2014

Fruit fly (Drosophila melanogaster) deoxyribonucleoside kinase (DmdNK; EC: 2.7.1.145) was characterized for its substrate specificity towards natural and non-natural nucleosides, confirming its potential in the enzymatic synthesis of modified nucleotides. DmdNK was adsorbed on a solid ion exchange support (bearing primary amino groups) achieving an expressed activity >98%. Upon cross-linking with aldehyde dextran, expressed activity was 30-40%. Both biocatalysts (adsorbed or cross-linked) were stable at pH 10 and room temperature for 24 h (about 70% of retained activity). The cross-linked DmdNK preparation was used for the preparative synthesis of arabinosyladenine monophosphate (araA-MP) and fludarabine monophosphate (FaraAMP). Upon optimization of the reaction conditions (50 mM ammonium acetate, substrate/ATP ratio= 1:1.25, 2 mM MgCl2, 378C, pH 8) immobilized DmdNK afforded the title nucleotides with high conversion (>90%), whereas with the soluble enzyme lower conversions were achieved (78-87%). Arabinosyladenine monophosphate was isolated in 95% yield and high purity (96.5%). © 2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim.


Bavaro T.,University of Pavia | Bavaro T.,Italian Biocatalysis Center | Ubiali D.,University of Pavia | Ubiali D.,Italian Biocatalysis Center | And 12 more authors.
Biocatalysis and Biotransformation | Year: 2010

Commercial lipases from the yeast Candida rugosa have been compared with two recombinant C. rugosa lipases, rCRL1 and rCRL1lid3, with respect to their immobilization and exploitation in biotransformations aimed at the synthesis of pyrimidine nucleosides. Immobilization on octyl-agarose and decaoctyl-Sepabeads but not on Eupergit® C gave comparable results to commercial lipases for rCRL1, while only a low percentage (12%) of rCRL1lid3 was efficiently immobilized. When immobilized on decaoctyl-Sepabeads, rCRL1 showed a markedly higher stability to chemical inactivation, since it could maintain 100% activity after 180 h incubation in 30% (v/v) acetonitrile. Hydrolysis of peracylated uridine and cytidine and their fluorinated counterparts proceeded with high regioselectivity and good yield, and even improved when rCRL1 was immobilized on decaoctyl-Sepabeads. © 2010 Informa UK Ltd.


Temporini C.,University of Pavia | Temporini C.,Italian Biocatalysis Center | Bonomi P.,University of Pavia | Bonomi P.,Italian Biocatalysis Center | And 12 more authors.
Biomacromolecules | Year: 2010

An innovative approach to determine the orientation of penicillin G acylase (PGA) from Escherichia coli covalently immobilized onto solid supports has been developed. This method is based on tryptic digestion of immobilized PGA followed by HPLC-MS analysis of the released peptides which are supposed to be only those exposed toward the reaction medium and not directly bound to the solid support. To this purpose, PGA was immobilized on Eupergit C (acrylic hydrophobic resin) and glyoxyl-agarose (hydrophilic resin) functionalized with epoxy and aldehyde groups, respectively, both involving the Lys residues of the protein. The peptide maps obtained were analyzed to derive the orientation of immobilized PGA, as the position of the detected Lys gave indication concerning the accessibility of the different areas of the protein. The results indicate that PGA immobilization on both supports involves mainly Lys located near the binding pocket (70%). Some differences in the enzyme orientation on the two supports can be deduced by the presence of different unbound Lys residues in the released peptides, specific to each support (Lys 117R for PGA-Eupergit C; Lys 163R and Lys 165R for PGA-glyoxylagarose). These results have been correlated with the data obtained in the kinetically controlled synthesis and indicate that the orientation of PGA on both supports is partially unfavorable, driving the active site near the support surface. This type of orientation of the enzyme enhances the effect of the nature of the support and of the binding chemistry on the catalytic properties. The information obtained indicated the most suitable support and activation strategy to design an immobilized acylase with good synthetic properties for preparative processes. The glyoxyl-Eupergit C support with enhanced porosity synergically combines the mechanical stability and synthetic performances of immobilized PGA and was successfully used in the synthesis of several cephalosporins. © 2010 American Chemical Society.

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