RESUMO
As a result of the very attractive pleiotropic properties of the heme-enzymes, three P450 cytochrome isoforms (P4501A2, P4502B4, P450SCC) have been utilized to identify a general optimal procedure to biodevice assembly for sensing a wide range of organic substances. The Langmuir-Blodgett films appears to yield the best stable working conditions as shown by UV-vis spectrophotometry, nanogravimetry, circular dichroism, and electrochemical characterization, to identify the ordered nanostructures of P450 cytochromes optimal for clozapine, styrene, and cholesterol sensing. Only in the presence of low purity grade protein, as in the case of P4501A2, a gel-matrix was needed to warrant the optimal clozapine sensing. By the combination of proper immobilization, transducer and nanostructured mutants of high-grade stable and selective P450-based sensors appear capable to detect the interaction with a wide range of organic substrates such as fatty acids, drugs, and toxic compounds.
Assuntos
Técnicas Biossensoriais/métodos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Colesterol/análise , Clozapina/análise , Sistema Enzimático do Citocromo P-450/genética , Enzimas Imobilizadas/genética , Microscopia Eletrônica de Varredura , Estrutura Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise EspectralRESUMO
Molecular modeling and protein engineering were synergically employed to improve the fabrication of cytochrome P450scc mutant nanostructures for biodevice assembly. The optimization of protein three-dimensional structure by molecular modeling was performed using two models: in vacuum and simulating the presence of a polar solvent. Calculations were performed on a model to predict a P450scc mutant which could improve the process of molecules' immobilization onto solid supports. Engineerized cytochrome P450scc thin films were prepared and characterized by various biophysical techniques such as pi-A isotherms, surface potential measurements, Brewster angle microscopy, UV-vis spectroscopy, circular dichroism, nanogravimetry, and electrochemical analysis. This paper takes into consideration biomolecules modified by protein engineering that represent a new and powerful approach for obtaining synthetic simpler artificial structures with new or improved properties (i.e., specificity, stability, sensitivity, etc.) useful for biosensors development.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/química , Modelos Moleculares , Nanotecnologia/métodos , Engenharia de Proteínas/métodos , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Substituição de Aminoácidos , Clonagem Molecular , Simulação por Computador , Sistema Enzimático do Citocromo P-450/genética , Eletroquímica/métodos , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Melhoramento Genético/métodos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Relação Estrutura-Atividade , TemperaturaRESUMO
Cytochrome P450sccK201E, mutated form of cytochrome P450scc native recombinant (P450sccNR), was employed to study the enzyme-substrate interaction. The detection of the cholesterol was performed by electrochemical method using cyclic voltammetry (CV) and chronoamperometry measurements. The biochemical analysis was realized to observe the electrochemical responses of the engineerized enzyme to three different forms of cholesterol: free, low-density lipoprotein (LDL) and high-density lipoproteins (HDL). Compared to cytochrome P450sccNR, the cytochrome P450sccK201E displays a different behavior in the interaction with the substrate detection. The results show that the engineerized enzyme can be utilized for the cholesterol detection in biosensor field.