Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 4 de 4
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Enzyme Microb Technol ; 170: 110287, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37487431

RESUMO

L-glutamate oxidase (LGOX, EC: 1.4.3.11) is an oxidoreductase that catalyzes L-glutamate deamination. LGOX from Streptomyces sp. X-119-6 is used widely for L-glutamate quantification in research and industrial applications. This enzyme encoded as a single precursor chain that undergoes post-translational cleavage to four fragments by an endogenous protease to become highly active. Efficient preparation of active LGOX by heterologous expression without proteolysis process should be indispensable for wide application of this enzyme. Thus, developing an LGOX that requires no protease treatment should expand the potential applications of recombinant LGOX. In this report, we succeeded in obtaining an active single-chain LGOX by connecting the four fragments of the mature form with insertion of flexible linkers. The most active single-chain mutant showed the similar activity to that of the mature form from Streptomyces sp. X-119-6. The structure of this mutant was determined at 2.9 Å resolution by X-ray crystallography. It was revealed that this single-stranded mutant had the similar conformation to that of mature form. This single-chain LGOX can be produced efficiently and should expand LGOX applications.

2.
Anal Biochem ; 587: 113447, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31562850

RESUMO

Glycine analysis is important in research fields such as physiology and healthcare because the concentration of glycine in human plasma has been reported to change with various disorders. Glycine oxidase from Bacillus subtilis (GlyOX) is useful for quantitative analysis of glycine. However, GlyOX is not sufficiently stable for use in physiology-based research or clinical settings. In this report, site-directed mutagenesis was used to engineer a GlyOX mutant suitable for glycine analysis. The GlyOX triple-mutant (T42 A/C245 S/L301V) retained most of its enzymatic activity during storage for over a year at 4 °C. A colorimetric enzyme analysis protocol was established using the GlyOX triple-mutant to determine glycine concentrations in human plasma. The analysis showed high accuracy (-5.4 to 3.5% relative errors when compared with the results from an amino acid analyzer, and 96.0-98.7% recoveries) and high precision (<4% between-run variation). Sample pretreatments of deproteinization and derivatization were not required. Therefore, this novel enzymatic analysis offers an effective and useful method for determining glycine concentrations in physiology related research and the healthcare field.


Assuntos
Aminoácido Oxirredutases/genética , Análise Química do Sangue , Colorimetria , Glicina/sangue , Aminoácido Oxirredutases/metabolismo , Engenharia Genética , Humanos , Mutação
3.
Anal Biochem ; 570: 13-20, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30586560

RESUMO

l-Histidine analysis is essential in physiological research and clinical applications because l-histidine concentrations in biofluids are associated with various diseases. However, an enzymatic method for l-histidine quantitation has not yet been established. Here, we describe a novel l-histidine quantitation assay using a combination of histidine decarboxylase (HDC) and histamine dehydrogenase (HDH) enzymes. Wild-type HDC is unstable and completely lost its activity within 50 days of storage at 4 °C in solution. We rationally designed a HDC C57S mutant with markedly improved stability (storage at 4 °C for over 200 days) without altering the enzyme's substrate specificity. Together with HDH, the HDC C57S mutant was applied to quantify l-histidine concentrations in human plasma. The assay showed high precision (<2.0% inter-assay variation) and high accuracy (<5.8% deviation from the results of LC/MS).


Assuntos
Histidina Descarboxilase/metabolismo , Histidina/sangue , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Cromatografia Líquida de Alta Pressão , Histidina/metabolismo , Histidina Descarboxilase/genética , Humanos , Cinética , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Photobacterium/enzimologia , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato
4.
J Biochem ; 164(5): 359-367, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30053101

RESUMO

l-Tryptophan oxidase, VioA from Chromobacterium violaceum, which has a high substrate specificity for tryptophan, is useful for quantitative assay of tryptophan. However, stability of wild type VioA is not enough for its application in clinical or industrial use. To improve the thermal stability of the enzyme, we developed a VioA (C395A) mutant, with higher stability than wild type VioA. The VioA (C395A) exhibited similar specificity and kinetic parameter for tryptophan to wild type. Conventionally, the quantity of tryptophan is determined by instrumental methods, such as high-performance liquid chromatography (HPLC) after pre-column-derivatization. Using the mutant enzyme, we succeeded in the tryptophan quantification in human plasma samples, to an accuracy of <2.9% when compared to the instrumental method, and to a precision of CV <3.2%. To analyse the improvement in storage stability and substrate specificity, we further determined the crystal structures of VioA (C395A) complexed with FAD, and with FAD and tryptophan at 1.8 Å resolution.


Assuntos
Engenharia de Proteínas , Temperatura , Triptofano Oxigenase/química , Triptofano Oxigenase/metabolismo , Cromatografia Líquida de Alta Pressão , Chromobacterium/enzimologia , Estabilidade Enzimática , Conformação Proteica , Triptofano Oxigenase/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...