RESUMEN
Tyrosinases catalyze oxidation of phenols with a formation of biphenols, quinones, and highly polymerized melanins. Tyrosinases have prospects for industrial use to remove phenols, also in biosensors, in bioorganic synthesis, and for a production of biocompatible adhesives (medical glues). Despite growing fields of potential applications, a selection of commercially available tyrosinases are currently limited to a single enzyme which is isolated from fruiting bodies of mushrooms. This article describes a preparation of recombinant tyrosinase from a bacterium Verrucomicrobium spinosum using a heterologous expression in Escherichia coli. Recombinant V. spinosum tyrosinase has high specific activity (13,200 U/mg). A resistance of the enzyme was investigated to chemical agents used to denature proteins and keep poorly solvable proteins in a solution. The enzyme preserves activity in the presence of urea and retains at least a fraction of its enzymatic activity at concentrations of urea up to 4.5 M. An addition of sodium lauroyl sarcosinate to 1 or 2% activates the tyrosinase. Novel means of quantitatively expressing tyrosinase activity is described in this article. The method uses a set of parameters obtained from non-linear estimation of the progress curves and is suitable for enzymatic reactions which do not comply with Michaelis-Menten kinetics. Tyrosinase may be used to introduce into proteins a post-translational modification which is a conversion of tyrosine residues (Tyr) into residues of 3,4-dioxyphenylalanine (DOPA). The presence of DOPA provides the polypeptides with a capability of strong molecular adhesion. Co-expression of tyrosinase and a recombinant protein mimicking marine mussel-encoded adhesive proteins resulted in obtaining of the protein in which at least a part of Tyr residues had been converted to DOPA. The DOPA-containing protein had high adhesion strength (2.5 MPa).
Asunto(s)
Monofenol Monooxigenasa/metabolismo , Proteínas/metabolismo , Verrucomicrobia/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , Dihidroxifenilalanina/química , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/genética , Desnaturalización Proteica , Procesamiento Proteico-Postraduccional , Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sarcosina/análogos & derivados , Sarcosina/química , Urea/químicaRESUMEN
A rapid method for detection of the surface lipopolysaccharide antigen and the cells of the causative agent of bovine brucellosis was developed. The method represents a sandwich format immunochromatographic assay. The contact between the sample and the test strip with immobilized immunoreagents initiates the fluid movement along the membrane components of the test strip, immunochemical reactions, and the formation of colored bands. The novel method requires 10 minutes to determine the lipopolysaccharide antigen of the cell wall of the brucellosis causative agent at concentrations down to 10 ng/mL and the Brucella abortus cells at concentrations down to 10(6) cells/mL (5 x 10(4) cells in the sample). The specificity of the immunodetection was confirmed. The designed test system can be used for the rapid field diagnosis of brucellosis in cattle.