Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (5): 98-107.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1149

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Biochemical Characterization and Structural Analysis of N-acetylornithine Transaminase from Synechocystis sp. PCC6803

BAI Fu-mei1(), LI Zhi-min2, WANG Xiao-qin1, HU Zi-wei1, BAO Ling-ling1, LI Zhi-min1,3()   

  1. 1. College of Bioscience and Bioengineering,Jiangxi Agricultural University,Nanchang 330045
    2. College of Science,Jiangxi Agricultural University,Nanchang 330045
    3. Jiangxi Engineering Laboratory for the Development and Utilization of Agricultural Microbial Resources,Jiangxi Agricultural University,Nanchang 330045
  • Received:2020-09-09 Online:2021-05-26 Published:2021-06-11
  • Contact: LI Zhi-min E-mail:bfm18270860135@163.com;zmlizm@126.com

Abstract:

The aim of this study is to characterize the biochemical and structural properties of N-acetylornithine transaminase encoded by slr1022 gene from Synechocystis sp. PCC6803,for laying theoretical foundation in further study on the catalytic function and mechanism of the enzyme. The slr1022 gene was obtained through PCR amplification using the genomic DNA of Synechocystis sp. PCC6803 as template,and then was linked to the expression vector pET-28a. The constructed plasmid was transformed into Escherichia coli strain BL21(DE3). The recombinant Slr1022 protein was purified by Ni-NTA affinity chromatography after the expressing strain was induced by IPTG. The catalytic function of the Slr1022 protein was characterized preliminarily through UV spectrophotometric methods,and the structure of the Slr1022 protein was analyzed by bioinformatics software. The recombinant expressing plasmid of the pET28a-slr1022 was constructed and the Slr1022 protein was expressed successfully. The molecular weight of the protein by SDS-PAGE was about 50 kD,which was consistent with the theoretical size. The binding constant Km and maximum reaction velocity Vmax of the Slr1022 protein with substrate N-acetylornithine were 0.12 mmol/L and 0.60 μmol/(L·s),respectively,and the Km and Vmax of Slr1022 protein with α-ketoglutarate were 0.039 mmol/L and 0.65 μmol/(L·s),respectively. Slr1022 protein had the highest catalytic activity at pH 8.5. The amino acid sequence of Slr1022 protein and other N-acetylornithine transaminase from different sources shared certain homology,and the amino acid residues of the active site were highly conserved. In conclusion,the slr1022 gene is successfully cloned and the Slr1022 protein is expressed and purified,enzymatic properties and bioinformatics analysis demonstrates that Slr1022 protein is N-acetylornithine transaminase.

Key words: N-acetylornithine transaminase, prokaryotic expression, biochemical characterization, structural analysis, Synechocystis sp. PCC6803