Sergio Águila

Production and Purification of Statins from Pleurotus ostreatus (Basidiomycetes) Strains

Pleurotus ostreatus strains were cultured in liquid medium and on wheat straw. The yields of lovastatin were compared

Stereoselective oxidation of R-(+)-limonene by chloroperoxidase from Caldariomyces fumago

The oxidation of R-(+)-limonene by chloroperoxidase (CPO) from Caldariomyces fumago is reported. The reaction was performed in 60 mM phosphate buffer at pH 3.0 and 6.0, and in the absence and in the presence of chloride ions. In the absence of chloride ions, at both pH values, the reaction was regio and stereoselective with a diasteromeric excess (de) >99% of (1S,2S)-4R-limonene-1,2-diol. On the other hand, when the reaction was carried out in the presence of chloride ions an enhancement in the reaction rate was observed, maintaining the regioselectivity, but not the stereoselectivity (de <5.4). The reaction products under these conditions were identified as (1S,2S)-4R-limonene-1,2-diol and (1R,2R)-4R-limonene-1,2-diol. It seems that in the absence of chloride ions the stereoselectivity is determined by stereospecific interaction of limonene with CPO active site, as supported by docking analysis, while in the presence of potassium chloride the limonene oxidation also occurs by the produced hypochlorite without stereoselectivity.

Peroxidase activity stabilization of cytochrome P450BM3 by rational analysis of intramolecular electron transfer

Combined quantum mechanical and molecular mechanical (QM/MM) calculations were used to explore the electron pathway involved in the suicide inactivation of cytochrome P450BM3 from Bacillus megaterium. The suicide inactivation is a common phenomenon observed for heme peroxidases, in which the enzyme is inactivated as a result of self-oxidation mediated by highly oxidizing enzyme intermediates formed during the catalytic cycle. The selected model was a mutant comprising only the heme domain (CYPBM3 21B3) that had been previously evolved to efficiently catalyze hydroxylation reactions with hydrogen peroxide (H2O2) as electron acceptor. An extensive mapping of residues involved in electron transfer routes was obtained from density functional calculations on activated heme (i.e. Compound I) and selected amino acid residues. Identification of oxidizable residues (electron donors) was performed by selectively activating/deactivating different quantum regions. This method allowed a rational identification of key oxidizable targets in order to replace them for less oxidizable residues by site-directed mutagenesis. The residues W96 and F405 were consistently predicted by the QM/MM electron pathway to hold high spin density; single and double mutants of P450BM3 on these positions (W96A, F405L, W96A/F405L) resulted in a more stable variants in the presence of hydrogen peroxide, displaying a similar reaction rate than P450BM3 21B3. Furthermore, mass spectrometry confirmed these oxidation sites and corroborated the possible routes described by QM/MM electron transfer (ET) pathways.

Prediction model based on decision tree analysis for laccase mediators

A Structure Activity Relationship (SAR) study for laccase mediator systems was performed in order to correctly classify different natural phenolic mediators. Decision tree (DT) classification models with a set of five quantum-chemical calculated molecular descriptors were used. These descriptors included redox potential (ɛ°), ionization energy (E(i)), pK(a), enthalpy of formation of radical (Δ(f)H), and OH bond dissociation energy (D(O-H)). The rationale for selecting these descriptors is derived from the laccase-mediator mechanism. To validate the DT predictions, the kinetic constants of different compounds as laccase substrates, their ability for pesticide transformation as laccase-mediators, and radical stability were experimentally determined using Coriolopsis gallica laccase and the pesticide dichlorophen. The prediction capability of the DT model based on three proposed descriptors showed a complete agreement with the obtained experimental results.

Enzyme orientation for direct electron transfer in an enzymatic fuel cell with alcohol oxidase and laccase electrodes.

A new full enzymatic fuel cell was built and characterized. Both enzymatic electrodes were molecularly oriented to enhance the direct electron transfer between the enzyme active site and the electrode surface. The anode consisted in immobilized alcohol oxidase on functionalized carbon nanotubes with 4-azidoaniline, which acts as active-site ligand to orientate the enzyme molecule. The cathode consisted of immobilized laccase on functionalized graphite electrode with 4-(2-aminoethyl) benzoic acid. The enzymatic fuel cell reaches 0.5 V at open circuit voltage with both, ethanol and methanol, while in short circuit the highest current intensity of 250 μA cm(-2) was obtained with methanol. Concerning the power density, the methanol was the best substrate reaching 60 μW cm(-2), while with ethanol 40 μW cm(-2) was obtained.

Substitution of the Catalytic Metal and Protein PEGylation Enhances Activity and Stability of Bacterial Phosphotriesterase

Phosphotriesterase, a pesticide-degrading enzyme, from Flavobacterium sp. was cloned and expressed in Escherichia coli. The catalytic zinc ions were replaced by cobalt atoms increasing the catalytic activity of phosphotriesterase on different pesticides. This metal substitution increased the catalytic activity from 1.4 times to 4 times according to the pesticide. In order to explain this catalytic increase, QM/MM calculations were performed. Accordingly, the HOMO energy of the substrate is closer to the LUMO energy of the cobalt-substituted enzyme. The chemical modification of the enzyme surface with poly(ethylene glycol) increased the thermostability and stability against metal chelating agents of both metal phosphotriesterase preparations.

Lovastatin Production by Pleurotus ostreatus: Effects of the C:N Ratio

Abstract The types of carbon source and nitrogen source used as well as the C:N ratio in the medium influenced lovastatin production by Pleurotus ostreatus. The maximum value of the lovastatin yield was obtained in a medium that contained organic nitrogen

REGIO AND STEREOSELECTIVE HYDROXYLATION OF A-AGAROFURAN BY BIOTRANSFORMATION OF RHIZOPUS NIGRICANS

A new synthesis of 9 alpha-hydroxy-alpha-agarofuran (6 alpha) is described, using a microbiological hydroxylation alpha-agarofuran (5) as the key reaction. The stereochemistry of the biohydroxylation was determined on the basis of a NOESY-experiment and GIAO calculations at the B3LYP/cc-pVDZ level. A strong gamma-effect was observed at C15 of the agarofuran ring which was correctly predicted by the GIAO-B3LYP calculations

Production of Exopolysaccharides by a Submerged Culture of an Entomopathogenic Fungus, Paecilomyces sp.

Exopolysaccharide basic was obtained from a submerged culture of a native Paecilomyces sp. strain isolated from Chilean soil

Selective and colorimetric detection of Ba2+ ions in aqueous solutions using 11-mercaptoundecylphosphonic acid functionalized gold nanoparticles

In this paper we describe a simple colorimetric method for the sensitive and selective detection of Ba2+ ions, using 11-mercaptoundecylphosphonic acid functionalized gold nanoparticles (AuNPs–MPA) in aqueous solution. With increasing concentration, Ba2+ ions induced aggregation of AuNPs–MPA, causing a change in color of the colloidal solution from pink to blue. The detection result was monitored using the naked eye and UV-Vis spectroscopy. The maximum absorption was located at 625 nm. The stability of AuNPs–MPA and detection of Ba2+ by AuNPs–MPA was investigated at different pH values (pH = 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Results indicated that AuNPs–MPA at pH 6 were the optimal choice for detecting Ba2+ ions. The detection limit for Ba2+ was found to be 43.27 μmol L−1 (R2 = 0.9901) within a range of 20–120 μmol L−1. In addition, the selectivity of this method was investigated using other metal ions. The AuNPs–MPA was selective for Ba2+ compared with other metal ions (Li+, Na+, K+, Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Sb3+, Bi3+) leading to a remarkable color change. Moreover, the developed cost-effective probe has been successfully applied to real drinking and tap water samples which demonstrated the potential for field applications.