Network


Latest external collaboration on country level. Dive into details by clicking on the dots.

Hotspot


Dive into the research topics where Giacomo Carrea is active.

Publication


Featured researches published by Giacomo Carrea.


Tetrahedron-asymmetry | 1992

Chloroperoxidase and hydrogen peroxide: An efficient system for enzymatic enantioselective sulfoxidations.

Stefano Colonna; Nicoletta Gaggero; Luigi Casella; Giacomo Carrea; Piero Pasta

Abstract High enantioselectivities were obtained in chloroperoxidase catalyzed oxidation of organic sulfides ( 99% ee in the case of methyl 2-pyridyl sulfide) with H 2 O 2 in aqueous buffer solution, pH 5, at 25°C. The kinetic parameters in the oxidation of a series of sulfides both with H 2 O 2 and tert -butyl hydroperoxide were determined and the data are consistent with enzymatic oxidation involving presumably a ternary complex, in all cases the reaction afforded the (R) sulfoxide as predominant or exclusive enantiomer.


Biochemistry | 2008

Kinetic mechanism of phenylacetone monooxygenase from Thermobifida fusca

Daniel E. Torres Pazmiño; Bert-Jan Baas; Dick B. Janssen; Marco W. Fraaije; Gonzalo de Gonzalo; Gianluca Ottolina; Giacomo Carrea

Phenylacetone monooxygenase (PAMO) from Thermobifida fusca is a FAD-containing Baeyer-Villiger monooxygenase (BVMO). To elucidate the mechanism of conversion of phenylacetone by PAMO, we have performed a detailed steady-state and pre-steady-state kinetic analysis. In the catalytic cycle ( k cat = 3.1 s (-1)), rapid binding of NADPH ( K d = 0.7 microM) is followed by a transfer of the 4( R)-hydride from NADPH to the FAD cofactor ( k red = 12 s (-1)). The reduced PAMO is rapidly oxygenated by molecular oxygen ( k ox = 870 mM (-1) s (-1)), yielding a C4a-peroxyflavin. The peroxyflavin enzyme intermediate reacts with phenylacetone to form benzylacetate ( k 1 = 73 s (-1)). This latter kinetic event leads to an enzyme intermediate which we could not unequivocally assign and may represent a Criegee intermediate or a C4a-hydroxyflavin form. The relatively slow decay (4.1 s (-1)) of this intermediate yields fully reoxidized PAMO and limits the turnover rate. NADP (+) release is relatively fast and represents the final step of the catalytic cycle. This study shows that kinetic behavior of PAMO is significantly different when compared with that of sequence-related monooxygenases, e.g., cyclohexanone monooxygenase and liver microsomal flavin-containing monooxygenase. Inspection of the crystal structure of PAMO has revealed that residue R337, which is conserved in other BVMOs, is positioned close to the flavin cofactor. The analyzed R337A and R337K mutant enzymes were still able to form and stabilize the C4a-peroxyflavin intermediate. The mutants were unable to convert either phenylacetone or benzyl methyl sulfide. This demonstrates that R337 is crucially involved in assisting PAMO-mediated Baeyer-Villiger and sulfoxidation reactions.


Biotechnology Letters | 1992

Effect of the enzyme form on the activity, stability and enantioselectivity of lipoprotein lipase in toluene

Gianluca Ottolina; Giacomo Carrea; Sergio Riva; L. Sartore; Francesco M. Veronese

SummaryThe properties of various forms of lipoprotein lipase (powder, adsorbed onto Celite, covalently linked to PEG, with additives) in toluene were investigated. The form of the enzyme dramatically influenced the activity and stability of the enzyme with the highest activity obtained with PEG-lipase and the highest stability with Celite-immobilized lipase. By contrast, the enantioselectivity was only marginally affected.


Biotechnology Letters | 1989

Subtilisin-catalyzed transesterification in supercritical carbon dioxide

Piero Pasta; G Mazzola; Giacomo Carrea; Sergio Riva

SummarySubtilisin Carlsberg was found to catalyze transesterification between N-acetyl-L-phenylalanine chloroethyl ester and ethanol in supercritical carbon dioxide. The effects of different temperatures and carbon dioxide/ethanol ratios on the reaction rate were investigated. A comparative study showed that enzymatic transesterification is faster in supercritical carbon dioxide than in anhydrous organic solvents.


Biotechnology and Bioengineering | 2000

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

Daniela Monti; Giacomo Carrea; Sergio Riva; Eva Baldaro; Giovanni Frare

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.


Enzyme and Microbial Technology | 2003

Cloning and expression in Escherichia coli of the gene encoding Streptomyces PMF PLD, a phospholipase D with high transphosphatidylation activity

Carlo Zambonelli; Paola Morandi; Maria A. Vanoni; Gabriella Tedeschi; Stefano Servi; Bruno Curti; Giacomo Carrea; Rossana Di Lorenzo; Daniela Monti

Abstract Phospholipases D (PLDs) hydrolyze phospholipids to yield phosphatidic acid and the corresponding alcohol and catalyze a transesterification (transphosphatidylation) reaction when alcohol is present as a nucleophilic donor. Bacterial forms of PLDs have shown to be suitable as biocatalysts for the synthesis of phospholipid derivatives of industrial interest. Recently, PLD from Streptomyces PMF, an enzyme with a high transphosphatidylation activity, was purified and its crystallographic structure was solved at 1.4xa0A. A 315-bp fragment of the pld gene of Streptomyces PMF was amplified by PCR using chromosomal DNA as a template and a pair of heterologous primers based on Streptomyces antibioticus pld gene sequence. The complete pld gene was isolated by colony hybridization and sequenced. DNA sequence analysis revealed a significant similarity with known pld gene sequences and showed the presence of highly conserved sequence motifs, namely the HKD motifs, shared by other members of the PLD superfamily. In order to promote the secretion of the protein into the medium, the mature PLD gene was fused in a pET derivative with the PelB signal sequence. Expression was performed in Escherichia coli BL21(DE3)pLysE cells after induction with IPTG, yielding 0.5xa0mg PLD/l of culture medium after protein purification to homogeneity. An inactive PLD variant, generated by site-directed mutagenesis experiments, was produced in large amounts using the same expression system, thus confirming the correlation between the effect of toxicity of PLD activity in E. coli cells and the low levels of enzyme production. Western blot analyses were used to detect PLD production in the culture supernatant, the recombinant protein was purified to homogeneity and structural and functional analyses confirmed its identity with the Streptomyces PLD.


Tetrahedron-asymmetry | 1991

Chemoenzymatic synthesis of the enantiomers of iopanoic acid

Mario Colombo; M. De Amici; C. De Micheli; Davide Pitrè; Giacomo Carrea; Sergio Riva

The two enantiomers of Iopanoic acid 1 were prepared in enantiomeric excess higher than 90% by enzyme-catalyzed hydrolysis of precursors (±)-2a and (±)-3a, followed by standard chemical transformations. Among the tested enzymes, chymotrypsin and Lipase PS proved to be the most selective catalysts. The stereochemical outcome of the lipase-catalyzed hydrolyses of esters (±)-2a-d is strictly dependent upon both the size of the alkyl group attached to the chiral center and the substituent in the aromatic ring. The enantioselectivity of the reactions was evaluated by chiral HPLC and the configurations of the new products were assigned by chemical correlations.


Tetrahedron-asymmetry | 1998

Chemoenzymatic synthesis of the enantiomers of desoxymuscarine

Paola Conti; Clelia Dallanoce; Marco De Amici; Carlo De Micheli; Giacomo Carrea; Francesca Zambianchi

Abstract Two different chemoenzymatic approaches allowed the preparation of the enantiomers of desoxymuscarine 5 , a muscarinic receptor agonist. Transesterification of racemic 5-hexen-2-ol 7 with vinyl butyrate under the catalysis of Candida antarctica B lipase was the key step for the preparation of (−)- 5 (2 R ,5 R ). On the other hand, lipase PS-catalyzed hydrolysis of iodo butyrate (±)- 14 was utilized to obtain (+)- 5 (2 S ,5 S ). Both enantiomers were prepared with enantiomeric excesses higher than 98%.


Biotechnology Letters | 1996

Enantioselective oxidations catalyzed by diketocamphane monooxygenase from Pseudomonas putida with macromolecular NAD in a membrane reactor

Piero Pasta; Giacomo Carrea; Nicoletta Gaggero; Gideon Grogan; Andrew Willetts

SummarySeveral sulfides and bicyclo[3.2.0]hept-2-en-6-one were enantioselectively oxidized to the corresponding sulfoxides and oxa lactones by a crude preparation of the two diketocamphane monooxygenases from Pseudomonas putida. The reactions were carried out in a membrane reactor with the use of poly(ethylene glycol)-N6-(2-aminoethyl)-NAD and coenzyme regeneration by the formate/formate dehydrogenase system.


Biocatalysis and Biotransformation | 1999

Enzymatic Resolution Of 3-Butene-1, 2-Diol In Organic Solvents And Optimization Of Reaction Conditions

Francesco Secundo; M.L. Oppizzi; Giacomo Carrea; M. De Amici; Clelia Dallanoce

Lipases from different sources were tested in the kinetic resolution of 2-hydroxy-3-butenyl butanoate [(R, S)-2] carried out by transesterification of the secondary alcohol. The influence of organic solvent, acyl donor and temperature on the enantioselectivity and activity of lipases was also investigated. Our study showed that both R- (+)-2 and S-(-)-2 could be obtained in high enantiomeric purity (ee ≥ 99%) and satisfactory yield (29% and 27%, respectively). Among the enzymes tested, lipase from Candida antarctica B (CALB) showed the highest preference for the (R)-enantiomer (E=26 at -13°C), whereas lipase from Pseudomonas fluorescens (lipase AK) acylated the (S)-enantiomer preferentially (E= 18 at -9°C).

Collaboration


Dive into the Giacomo Carrea's collaboration.

Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Researchain Logo
Decentralizing Knowledge