Cloning, expression and characterization of N-acetylornithine aminotransferase from Corynebacterium crenatum and its effects on L-arginine fermentation.
- Author:
Meijuan XU
1
;
Xian ZHANG
;
Zhiming RAO
;
Juan YANG
;
Wenfang DOU
;
Jian JIN
;
Zhenghong XU
Author Information
1. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
- Publication Type:Journal Article
- MeSH:
Arginine;
biosynthesis;
Cloning, Molecular;
Corynebacterium;
enzymology;
genetics;
Escherichia coli;
enzymology;
genetics;
Fermentation;
Industrial Microbiology;
methods;
Metabolic Engineering;
Transaminases;
biosynthesis;
genetics;
Transformation, Bacterial
- From:
Chinese Journal of Biotechnology
2011;27(7):1013-1023
- CountryChina
- Language:Chinese
-
Abstract:
N-Acetylornithine aminotransferase (EC 2.6.1.11, ACOAT) catalyzes the conversion of N-acetylglutamic semialdehyde to N-acetylornithine, the forth step involved in the L-arginine biosynthetic pathways. We studied the enzyme properties to set up reliable theoretical basis for the arginine fermentation optimization. ACOAT encoding gene argD was cloned from an industrial L-arginine producer Corynebacterium crenatum SYPA 5-5. Analysis of argD sequences revealed that only one ORF existed, which coded a peptide of 390 amino acids with a calculated molecular weight of 41.0 kDa. The argD gene from C. crenatum SYPA 5-5 was expressed both in Escherichia coli BL21 and C. crenatum SYPA. Then ACOAT was purified by Ni-NTA affinity chromatography and its specific enzyme activity was 108.2 U/g. Subsequently, the expression plasmid pJCtac-CcargD was transformed into C. crenatum SYPA and the specific activity of ACOAT was improved evidently in the recombinant C. crenatum CCD. Further fermentative character of CCD1 was also analyzed. The results showed that the L-arginine producing ability of the recombinant strain was 39.7 g/L improved by 14.7%.
