Fermentative glycolysis with purified Escherichia coli enzymes for in vitro ATP production and evaluating an engineered enzyme.
J Biotechnol
; 157(1): 113-23, 2012 Jan.
Article
em En
| MEDLINE
| ID: mdl-21963590
ABSTRACT
Each of the twelve enzymes for glycolytic fermentation, eleven from Escherichia coli and one from Saccharomyces cerevisiae, have been over-expressed in E. coli and purified with His-tags. Simple assays have been developed for each enzyme and they have been assembled for fermentation of glucose to ethanol. Phosphorus-31 NMR revealed that this in vitro reaction accumulates fructose 1,6-bisphosphate while recycling the cofactors NAD(+) and ATP. This reaction represents a defined ATP-regeneration system that can be tailored to suit in vitro biochemical reactions such as cell-free protein synthesis. The enzyme from S. cerevisiae, pyruvate decarboxylase 1 (Pdc1; EC 4.1.1.1), was identified as one of the major 'flux controlling' enzymes for the reaction and was replaced with an evolved version of Pdc1 that has over 20-fold greater activity under glycolysis reaction conditions. This substitution was only beneficial when the ratio of glycolytic enzymes was adjusted to suit greater Pdc1 activity.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes de Fusão
/
Trifosfato de Adenosina
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Proteínas de Escherichia coli
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Escherichia coli
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article