RESUMO
Candida rugosa lipase crude preparations (CRL) catalyse the regioselective acylation of methyl 6-O-trytil beta-d-glucopyranoside in organic solvents, using vinyl acetate as acyl donor. The ratio of the two products formed, namely methyl 2-O acetyl 6-O-trytil beta-d-glucopyranoside and methyl 3-O acetyl 6-O-trytil beta-d-glucopyranoside was found to be markedly affected by the nature of the reaction medium. In hydrophobic solvents values up to 80% of the monoacetylated product in position C-3 were obtained compared to less than 30% in solvents with low hydrophobicity. Computational studies were carried out to simulate the interactions between methyl 6-O-trytil beta-d-glucopyranoside and both CRL and the solvents, in order to rationalize the experimental results.
Assuntos
Candida/enzimologia , Glucosídeos/metabolismo , Lipase/metabolismo , Modelos Moleculares , Acilação , Interações Hidrofóbicas e Hidrofílicas , Solventes/química , Compostos de Vinila/metabolismoRESUMO
(+/-)-2-(6-Methoxy-2-naphthyl)propionic acid methyl ester (methyl ester of Naproxen), the precursor of therapeutically important nonsteroidal anti-inflammatory drugs (NSAIDs) was enantioselectively hydrolysed using as biocatalyst Candida rugosa lipase. In research aimed at studying the structure-activity relationship (SAR), NMR spectroscopy methods were employed to identify which Naproxen molecular moiety was essential to the substrate-enzyme interaction. The experimental results, in agreement with previous computer modelling studies and reported kinetic data, gave new information on the enzyme-substrate complex formation in solution.
Assuntos
Ésteres/metabolismo , Lipase/metabolismo , Naproxeno/metabolismo , Propionatos/metabolismo , Candida/enzimologia , Ésteres/química , Hidrólise , Cinética , Naproxeno/química , Ressonância Magnética Nuclear Biomolecular/métodos , Propionatos/química , Prótons , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Within a research project aimed at probing the substrate specificity and the enantioselectivity of Candida rugosa lipase (CRL), computer modeling studies of the interactions between CRL and methyl (+/-)-2-(3-benzoylphenyl)propionate (Ketoprofen methyl ester) have been carried out in order to identify which amino acids are essential to the enzyme/substrate interaction. Different binding models of the substrate enantiomers to the active site of CRL were investigated by applying a computational protocol based on molecular docking, conformational analysis, and energy minimization procedures. The structural models of the computer generated complexes between CRL and the substrates enabled us to propose that Phe344 and Phe345, in addition to the residues constituting the catalytic triad and the oxyanion hole, are the amino acids mainly involved in the enzyme-ligand interactions. To test the importance of these residues for the enzymatic activity, site-directed mutagenesis of the selected amino acids has been performed, and the mutated enzymes have been evaluated for their conversion and selectivity capabilities toward different substrates. The experimental results obtained in these biotransformation reactions indicate that Phe344 and especially Phe345 influence CRL activity, supporting the findings of our theoretical simulations.
Assuntos
Candida/genética , Lipase/genética , Propionatos/metabolismo , Sequência de Bases , Sítios de Ligação , Candida/enzimologia , Ésteres/metabolismo , Cetoprofeno/química , Lipase/metabolismo , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Naproxeno/química , Proteínas Recombinantes/genética , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Racemic arylpropionic esters 1-3, precursors of therapeutically important non-steroidal antiinflammatory drugs, were subjected to hydrolyses in the presence of either Candida rugosa or Rhizomucor miehei crude lipases. The hydrolyses of 1 and 2 proved to be highly enantioselective, whereas 3 was not transformed at all. Both the substrate specificity and the enantioselectivity of these lipases were explained through a molecular modeling study involving docking experiments between 1-3 and the amino acids forming the enzymes active-sites, whose three dimensional structures were obtained from X-ray crystallographic data, followed by extensive conformational analysis on their computer-generated complexes. The results of this study also account for the high enantioselective and good yielding hydrolysis of 3 (as the corresponding 2-chloroethyl ester) catalyzed by CRL pretreated with 2-propanol, recently reported in the literature, and lead to admit that such a treatment may operate very deep conformational changes on the amino acids of the enzyme active-site.
Assuntos
Candida/enzimologia , Lipase/metabolismo , Mucorales/enzimologia , Propionatos/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Cinética , Lipase/química , Modelos Moleculares , Estrutura Molecular , Propionatos/química , Conformação Proteica , Especificidade por SubstratoRESUMO
The preparation of new stationary phases of cross-linked polyvinyl alcohol esterified with various linear fatty acids is described. The physico-chemical properties of these polymers are reported, including electron microscopy and swelling measurements. Batch adsorption experiments were performed in order to characterize the basic separative properties of these phases. Cross-linked polyvinyl alcohol esterified with dodecanoic acid was used for hydrophobic interaction chromatography of a commercial crude preparation of Candida rugosa lipase. Characterization of the purified fractions was carried out via native electrophoresis and sodium dodecyl sulfate-polyacrylamide electrophoresis.
Assuntos
Candida/enzimologia , Lipase/isolamento & purificação , Álcool de Polivinil/química , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Microscopia Eletrônica de Varredura , PolímerosRESUMO
The enzyme catalyzed hydrolysis of esters 1-3, precursors of therapeutically important non-steroidal antiinflammatory drugs, in the presence of the lipase from Candida rugosa was studied and the relative rates of the enzymatic hydrolysis were determined. With the exception of 3, which was not transformed under the reaction conditions, all transformations proved to be highly enantiospecific. Usually the mechanism of enantiorecognition is probed by substrate mapping. Although more theoretical approaches are existing, these all require knowledge of the three-dimensional structure of the enzyme. A model capable of correlating the extent of substrate hydrolysis as well as the initial reaction rates with their stereoelectronic properties has been developed by a Comparative Molecular Field Analysis (CoMFA) approach. This model does not require detailed knowledge of the three-dimensional structure of the enzyme and proved to be highly predictive. It is possible that this kind of approach holds promise for future work on enzyme-substrate interactions.
Assuntos
Candida/enzimologia , Lipase/metabolismo , Modelos Moleculares , Fenilpropionatos/metabolismo , Hidrólise , Modelos Químicos , Especificidade por SubstratoRESUMO
A simple HPLC assay for serum guanase based on the direct determination of enzymatically formed xanthine was applied to normal and pathological sera. The procedure is sensitive, precise (CV below 5%) and suitable for routine purposes, and the method requires only 50 microL of sample. Using this method the reference range as determined from the sera of 40 healthy adult controls is 0-1.1 U/L. In patients with various liver diseases serum guanase activities were found to be increased 5- to 50-fold compared with the normal mean value.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Guanina Desaminase/sangue , Hepatopatias/enzimologia , Adulto , Feminino , Humanos , Masculino , Reprodutibilidade dos Testes , Espectrofotometria UltravioletaRESUMO
Lipolytic enzymes represent an important class of biocatalysts and are widely used in the resolution of racemic mixtures. The activity of lipase from Candida cylindracea at different temperatures has been studied by NMR determination of the enantiomeric excess in the enantioselective hydrolysis of 2-arylpropionic acid esters of pharmacological interest. At a purpose, a system based on Europium (III) chiral shift reagent has been settled and utilized.
Assuntos
Candida/enzimologia , Lipase/metabolismo , Catálise , Lipase/química , Espectroscopia de Ressonância Magnética/métodos , Estereoisomerismo , TemperaturaRESUMO
Thermodynamic characteristics pertinent to the formation equilibria of two ternary systems: 1) Copper(II), 4,9-diazadodecane-1,12-diamine (spermine, Spe), and adenosine 5'-triphosphate (ATP) and 2) Copper(II), Spe, and tripolyphosphate (TPP) have been determined by means of potentiometric and calorimetric techniques, together with the parent binary complex characteristics. Ternary complexes involving ATP can give information useful in the interpretation of bioenergetic reactions and of biological interactions between nucleic acids and polyamines. As a model system, the TPP-containing ternary complexes have been studied, together with the parent binary complexes. The thermodynamic study of these systems is very important because it can give information about the structural environment of the complexes; moreover, it can help in outlining different noncovalent interactions such as coulombic forces and hydrogen bonds.
Assuntos
Trifosfato de Adenosina/metabolismo , Cobre/metabolismo , Polifosfatos/metabolismo , Espermina/metabolismo , Calorimetria , Ligantes , Potenciometria , TermodinâmicaRESUMO
Thermodynamic parameters pertinent to the equilibria of ternary system copper(II), adenosine 5'-monophosphate (AMP), and 4,9-diazadodecane-1,12-diamine (Spermine, Spe) were determined by means of potentiometric and calorimetric techniques, together with the corresponding parent binary complex parameters. This system could be considered as a model in the study of more complicated biological systems. In particular, it could give useful evidence in the investigation of the interaction between tetramine and DNA. The experimental results seem to suggest that the major contribution in DNA stabilization by means of tetramines is due to the electrostatic interactions occurring between the protonated nitrogen atoms of the amine molecules and the phosphate groups of nucleotides in the nucleic chains.