RESUMO
The aim of our work was to develop a method for the determination of six organophosphorous pesticides (Ops) (azynphos-ethyl (AZP), diazinon (DZN), chlorpyrifos (CLP), chlorfenvinfos (CLF), parathion-ethyl (PRT) and quinalphos (QLP)) in whole blood using microextraction by packed sorbent (MEPS) and analysis by gas chromatography-tandem mass spectrometry (GC-MS/MS). The optimization of the MEPS procedure was performed using a design of experiments (DOE) approach, assessing different factors that significantly affected the extraction efficiency. Ultimately, the number of sample strokes, wash volume, percentage of 2-propanol in the wash solvent and the number of elution strokes were successfully optimized using a response surface methodology (RSM). The developed and optimized method was fully validated according to international guidelines. Linearity was established from 2.5 to 50 µg/mL for AZP and from 0.5 to 50 µg/mL for the remaining compounds, with coefficients of determination (R2) higher than 0.99 in all cases. The lower limit of quantification were 2.5 µg/mL (AZP) and 0.5 µg/mL (remaining compounds). Recoveries ranged from 61% to 77%. Intra- and inter-day precision and accuracy were considered adequate according to the guidelines. This is the first method employing MEPS as a sample preparation procedure for the analysis of these OPs in whole blood.
Assuntos
Organofosfatos/sangue , Compostos Organotiofosforados/sangue , Praguicidas/sangue , 2-Propanol/química , Métodos Analíticos de Preparação de Amostras , Bancos de Sangue , Calibragem , Estabilidade de Medicamentos , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Análise dos Mínimos Quadrados , Limite de Detecção , Estrutura Molecular , Organofosfatos/química , Organofosfatos/isolamento & purificação , Compostos Organofosforados/sangue , Compostos Organofosforados/química , Compostos Organofosforados/isolamento & purificação , Compostos Organotiofosforados/química , Compostos Organotiofosforados/isolamento & purificação , Praguicidas/química , Praguicidas/isolamento & purificação , Reprodutibilidade dos Testes , Microextração em Fase Sólida , Solventes/química , Espectrometria de Massas em TandemRESUMO
Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is an enzyme that catalyzes the methylation of catechol substrates, and while structural and functional studies of its membrane-bound isoform (MBCOMT) are still hampered by low recombinant production, Pichia pastoris has been described as an attractive host for the production of correctly folded and inserted membrane proteins. Hence, in this work, MBCOMT biosynthesis was developed using P. pastoris X33 and KM71H cells in shake flasks containing a semidefined medium with different methanol concentrations. Moreover, after P. pastoris glass beads lysis, biologically and immunologically active hMBCOMT was found mainly in the solubilized membrane fraction whose kinetic parameters were identical to its correspondent native enzyme. In addition, mixed feeds of methanol and glycerol or sorbitol were also employed, and its levels quantified using liquid chromatography coupled to refractive index detection. Overall, for the first time, two P. pastoris strains with opposite phenotypes were applied for MBCOMT biosynthesis under the control of the strongly methanol-inducible alcohol oxidase (AOX) promoter. Moreover, this eukaryotic system seems to be a promising approach to deliver MBCOMT in high quantities from fermentor cultures with a lower cost-benefit due to the cheaper cultivation media coupled with the higher titers tipically achieved in biorreactors, when compared with previously reported mammallian cell cultures.
Assuntos
Catecol O-Metiltransferase/biossíntese , Proteínas de Membrana/biossíntese , Pichia/enzimologia , Proteínas Recombinantes/biossíntese , Oxirredutases do Álcool/metabolismo , Catecol O-Metiltransferase/genética , Técnicas de Cultura de Células , Fermentação , Glicerol/química , Proteínas de Membrana/metabolismo , Metanol/metabolismo , Fenótipo , Pichia/genética , Proteínas Recombinantes/genéticaRESUMO
Catechol-O-methyltransferase (COMT, EC 2.1.1.6) plays a crucial role in dopamine metabolism which has intimately linked this enzyme to some neurodegenerative diseases, such as Parkinson's disease. In recent years, in the attempt of developing new therapeutic strategies for Parkinson's disease, there has been a growing interest in the search for effective COMT inhibitors. In order to do so, large amounts of COMT in an active form are needed, and the best way to achieve this is by up-scaling its production through biotechnological processes. In this work, a fed-batch process for the biosynthesis of the soluble isoform of COMT in Escherichia coli is proposed. This final process was selected through the evaluation of the effect of different dissolved oxygen concentrations, carbon and nitrogen source concentrations and feeding profiles on enzymatic production and cell viability, while controlling various parameters (pH, temperature, starting time of the feeding and induction phases and carbon source concentration) during the process. After several batch and fed-batch experiments, a final specific COMT activity of 442.34 nmol/h/mg with approximately 80% of viable cells at the end of the fermentation were achieved. Overall, the results described herein provide a great improvement on hSCOMT production in recombinant bacteria and provide a new and viable option for the use of a fed-batch fermentation with a constant feeding profile to the large scale production of this enzyme.
