High-Throughput Screening of T7 Promoter Mutants for Soluble Expression of Cephalosporin C Acylase in E. coli.
Appl Biochem Biotechnol
; 190(1): 293-304, 2020 Jan.
Article
en En
| MEDLINE
| ID: mdl-31346919
ABSTRACT
Cephalosporin C acylase (CCA) is the key enzyme in the production of 7-aminocephalosporanic acid (7-ACA) via a one-step enzymatic process. To improve the soluble expression level of CCA in recombinant Escherichia coli at elevated temperatures, a library of T7 promoter mutants was created by site-saturation mutagenesis, and a series of mutated promoters were subsequently screened. Green fluorescent protein (GFP) was fused to the C-terminus of CCA to facilitate library screening, and the expression of the CCA and GFP fusion proteins was investigated under the control of the T7 promoter. Twenty-four mutants were selected by detecting the fluorescence intensity of colonies on agar plates to form a library with different expression levels. The enzyme activities of the mutants were positively correlated with their fluorescence intensities. The highest enzyme activity among these mutant promoters was 1.3-fold higher than the enzyme activity resulting from the wild-type promoter when the cells were cultured at 32 °C for 16 h. In addition, the transcription and expression levels of several typical promoters were discussed, and the effects of GFP fusion on the enzyme activity of CCA were investigated.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Contexto en salud:
3_ND
Problema de salud:
3_neglected_diseases
/
3_zoonosis
Asunto principal:
Cefalosporinas
/
Regiones Promotoras Genéticas
/
Bacteriófago T7
/
Escherichia coli
/
Ensayos Analíticos de Alto Rendimiento
/
Amidohidrolasas
/
Mutación
Tipo de estudio:
Diagnostic_studies
/
Screening_studies
Idioma:
En
Revista:
Appl Biochem Biotechnol
Año:
2020
Tipo del documento:
Article
País de afiliación:
China