[Expression and characterization of NADPH-cytochrome P450 reductase from Trametes versicolor in Escherichia coli].
Sheng Wu Gong Cheng Xue Bao
; 34(7): 1156-1168, 2018 Jul 25.
Article
em Zh
| MEDLINE
| ID: mdl-30058314
ABSTRACT
Trametes versicolor has strong ability to degrade environmental organic pollutants. NADPH-cytochrome P450 reductase (CPR) of T. versicolor transfers electron to cytochrome P450s (CYPs) and participates in the degradation process of organic pollutants. Sequence analysis showed that the genome of T. versicolor contains 1 potential CPR and multiple potential CYP sequences. To further study the molecular mechanism for the involvement of T. versicolor CPR in the cellular degradation of organic pollutants, a CPR gene from T. versicolor was cloned and heterologously expressed in Escherichia coli. Subsequently, the main properties of the recombinant enzyme were investigated. A truncated CPR protein lacking the predicted membrane anchor region (residues 1-24), named CPRΔ24, was overexpressed as a soluble form in E. coli. The recombinant CPRΔ24 protein showed a molecular weight consistent with the theoretical value of 78 kDa. Recombinant CPRΔ24 was purified using a Ni²âº-chelating column followed by size exclusion chromatography. The specific activity of the purified CPRΔ24 was 5.82 U/mg. The CPRΔ24 enzyme displayed the maximum activity at 35 â and pH 8.0. It has different degrees of tolerance against several types of metal ions and organic solvents. The apparent Km and kcat values of recombinant CPRΔ24 for NADPH were 19.7 µmol/L and 3.31/s, respectively, and those for the substrate cytochrome c were 25.9 µmol/L and 10.2/s, respectively, under conditions of 35 â and pH 8.0. The above research provides the basis for exploring the functional mechanism of T. versicolor CPR in the degradation pathway of environmental organic pollutants.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
NADPH-Ferri-Hemoproteína Redutase
/
Trametes
Idioma:
Zh
Revista:
Sheng Wu Gong Cheng Xue Bao
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
China