RESUMEN
L-amino acid oxidase (LAAO) has important biological roles in many organisms, thus attracting great attention from researchers to establish its detection methods. In this study, a new quantitative in-gel determination of LAAO activity based on ferric-xylenol orange (Fe(III)XO) formation was established. This method showed that due to the conversion of Fe(II) to Fe(III) by H2O2 and subsequent formation of Fe(III)XO complex halo in agar medium, the logarithm of H2O2 concentration from 5 to 160 µM was linearly correlated to the diameter of purplish red Fe(III)XO halo. By extracting the LAAO-generated H2O2 concentration, the LAAO activity can be quantitatively determined. This Fe(III)XO agar assay is highly sensitive to detect H2O2 down to micromolar range. More importantly, it is easy to handle, cheap, reproducible, convenient and accurate. Coupled with SDS-PAGE, it can directly be used to determine the number and approximate molecular weight of LAAO in one assay. All these features make this in-gel Fe(III)XO assay useful and convenient as a general procedure for following enzyme purification, assaying fractions from a column, or observing changes in activity resulting from enzyme modifications, hence endowing this method with broad applications.
Asunto(s)
Pruebas de Enzimas/métodos , Compuestos Férricos/metabolismo , L-Aminoácido Oxidasa/metabolismo , Fenoles/metabolismo , Pseudoalteromonas/enzimología , Sulfóxidos/metabolismo , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Estándares de Referencia , Soluciones , Especificidad por SustratoRESUMEN
L-amino acid oxidase (LAAO) is attracting increasing attention due to its important functions. Diverse detection methods with their own properties have been developed for characterization of LAAO. In the present study, a simple, rapid, sensitive, cost-effective and reproducible method for quantitative in-gel determination of LAAO activity based on the visualization of Prussian blue-forming reaction is described. Coupled with SDS-PAGE, this Prussian blue agar assay can be directly used to determine the numbers and approximate molecular weights of LAAO in one step, allowing straightforward application for purification and sequence identification of LAAO from diverse samples.
Asunto(s)
Técnicas de Química Analítica/métodos , Peróxido de Hidrógeno/aislamiento & purificación , L-Aminoácido Oxidasa/metabolismo , Electroforesis en Gel de Poliacrilamida , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Oxidación-Reducción , Reacción del Azul Prusia , Especificidad por SustratoRESUMEN
Four new aspartic protease genes pepAa, pepAb, pepAc and pepAd from Aspergillus niger were identified using a comparative genomic approach. All four gene products have highly conserved attributes that are characteristic of aspartic proteases; however, each one has novel sequence features. The PEPAa protease appears to represent an ortholog of a pepsin-type aspartic protease previously identified from Talaromyces emersonii and Scleotinia sclerotiorum. The PEPAb protease appears to be an ortholog of an aspartic protease previously identified from BcAP1 of Botryotinia fuckeliana. The PEPAc protease also appears to be an ortholog of BcAP5 from B. fuckeliana. These four genes appear to be conserved in many species of filamentous fungi, all except PEPAb contain a predicted signal peptide. Transcriptome analysis revealed that transcripts of the pepAa gene of Aspergillus nidulans were significantly up-regulated due to recombinant chymosin secretion, suggesting that silencing these genes may lead to improved yields of secreted proteins. To establish the effects of reduced protease activity on the stabilities of secreted proteins, three of the four genes were individually disrupted by double crossover, although we were unable to disrupt the pepAc gene. The secretion level of heterologous laccase in the pepAa, pepAb and pepAd disruption mutants were increased by about 21%, 42% and 30%, respectively. And their total glucogenic enzymes secretion were also increased by about 18.7%, 37.0% and 5.20%, respectively.