Construction and characterization of Escherichia coli D-3-phosphoglycerate dehydrogenase mutants with feedback-inhibition relief / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 468-477, 2016.
Artigo
em Chinês
| WPRIM
| ID: wpr-337450
ABSTRACT
3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) is the key enzyme in L-serine biosynthesis and its coding gene is serA. PGDH is feedback inhibited by L-serine. In order to relieve the feedback-inhibition of PGDH by L-serine, H344 or D346 or D364 were chosen for site directed mutagenesis. The mutants were generated by the standard QuikChange mutagenesis, further subcloned into expression vector pT7-7 and transformed into Escherichia coli BL21 (DE3) cells. The recombinant cells were collected after cultured in LB media post induced by isopropyl beta-Dthiogalactopyranoside. The enzymes were purified by anion exchange chromatography, and SDS-PAGE showed that the purified enzymes were homogenous. Enzyme characterization indicated that the mutant enzyme showed similar activity, optimal temperature, and optimal pH as that of the wild-type enzyme. Moreover, feedback inhibition study showed that the activity of the double mutant (N346A/H344A) could remain 96% in the presence of serine up to 160 mmol/L, whereas the activity of the wild-type enzyme remains only 50% in the presents of serine of 7 μmol/L, thus successfully relieving the feedback inhibition of PGDH with its activity remained.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Serina
/
Microbiologia Industrial
/
Mutagênese Sítio-Dirigida
/
Proteínas de Escherichia coli
/
Eletroforese em Gel de Poliacrilamida
/
Escherichia coli
/
Fosfoglicerato Desidrogenase
/
Genética
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2016
Tipo de documento:
Artigo
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