RESUMO
A recombinant esterase from Lactobacillus plantarum was immobilized on hydrophobic support polypropylene Accurel MP1000 by adsorption. Adsorption efficiency was 83%, and the immobilized protein was 12.4 mg/g of support. Esterase activity was determined using p-nitrophenyl butyrate as substrate, and highest activities were observed at 50 °C for immobilized enzyme and 30 °C for free enzyme extract. Concerning thermal stability, after enzyme incubation at 80 °C for 30 min, immobilized and free enzyme retained 91% and 56% of initial activity, respectively. Immobilized enzyme presented lower V(max) and higher K(m) than free enzyme. Protein was not released from the support, and esterase activity increased after 3 cycles of reuse.
Assuntos
Butiratos/metabolismo , Enzimas Imobilizadas/metabolismo , Esterases/metabolismo , Proteínas Recombinantes/metabolismo , Adsorção , Enzimas Imobilizadas/química , Escherichia coli , Esterases/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Lactobacillus plantarum/enzimologia , Polipropilenos/química , Proteínas Recombinantes/genética , TemperaturaRESUMO
Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis. Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and the recombinant histidine-tagged lipases were purified by immobilized metal ion affinity chromatography. The enzyme activity was determined using p-nitrophenyl butyrate as substrate and secondary structure content was evaluated by circular dichroism. MUT-Lys and MUT-Asp presented significant increase of lipase activity (P < 0.05) in comparison to WT-Val, although highest activities for the three enzymes were observed at the same pH and temperature (pH 9.0 and 42 degrees C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80 degrees C all enzymes retained their initial activities.
Assuntos
Proteínas de Bactérias/química , Lipase/química , Lipase/genética , Proteínas Recombinantes/química , Staphylococcus/enzimologia , Substituição de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Estabilidade Enzimática , Escherichia coli , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Mutação , Mutação Puntual , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , TemperaturaRESUMO
The synthetic n-alkyl esters of gallic acid (GA), also known as gallates, especially propyl, octyl and dodecyl gallates, are widely employed as antioxidants by food and pharmaceutical industries. The inhibitory effects of GA and 15 gallates on Herpes Simplex Virus type 1 (HSV-1) and Human Immunodeficiency Virus (HIV-1) replication were investigated here. After a preliminary screening of these compounds, GA and pentyl gallate (PG) seemed to be the most active compounds against HSV-1 replication and their mode of action was characterized through a set of assays, which attempted to localize the step of the viral multiplication cycle where impairment occurred. The detected anti-HSV-1 activity was mediated by the inhibition of virus attachment to and penetration into cells, and by virucidal properties. Furthermore, an anti-HIV-1 activity was also found, to different degrees. In summary, our results suggest that both compounds could be regarded as promising candidates for the development of topical anti-HSV-1 agents, and further studies concerning the anti-HIV-1 activity of this group of molecules are merited.
Assuntos
Antivirais/farmacologia , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacologia , HIV-1/efeitos dos fármacos , Herpesvirus Humano 1/efeitos dos fármacos , Animais , Fármacos Anti-HIV/farmacologia , Bovinos , Chlorocebus aethiops , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
The synthetic n-alkyl esters of gallic acid (GA), also known as gallates, especially propyl, octyl and dodecyl gallates, are widely employed as antioxidants by food and pharmaceutical industries. The inhibitory effects of GA and 15 gallates on Herpes Simplex Virus type 1 (HSV-1) and Human Immunodeficiency Virus (HIV-1) replication were investigated here. After a preliminary screening of these compounds, GA and pentyl gallate (PG) seemed to be the most active compounds against HSV-1 replication and their mode of action was characterized through a set of assays, which attempted to localize the step of the viral multiplication cycle where impairment occurred. The detected anti-HSV-1 activity was mediated by the inhibition of virus attachment to and penetration into cells, and by virucidal properties. Furthermore, an anti-HIV-1 activity was also found, to different degrees. In summary, our results suggest that both compounds could be regarded as promising candidates for the development of topical anti-HSV-1 agents, and further studies concerning the anti-HIV-1 activity of this group of molecules are merited.