Research on the NAMase of Ensifer meliloti 1021 and Regulation Mechanism of 3-Cyanopyridine

doi:10.13560/j.cnki.biotech.bull.1985.2019-0027

Abstract

Abstract: This work aims to clone and express the NAMase of Ensifer meliloti 1021,and to investigate its enzymatic properties and explore the enzymatic mechanisms regulated by 3-CP. PCR was used to amplify the gene nam of E. meliloti 1021 and the constructed recombinant nam-containing plasmid was imported to Escherichia coli Rosetta（DE3）cells for heterologous expression. Ni-NTA affinity chromatography was employed to purify the protein. The effects of temperature,pH,organic solvents and metal ions on NAMase activity were explored,and the immobilization of NAMase was further investigated. The results showed that the full-length of nam was 636 bp,encoded a 22.6 kD protein with PI of 5.5. The optimal pH for NAMase was 7,it retained over 97.1% of its initial activity after incubation at 30-50℃ for 2 h,and the optimal temperature ranged in 30-70℃. Ag²⁺and isoamyl alcohol demonstrated the highest inhibitory effect on NAMase activity. 3-CP inhibited nicotinic acid production during the transformation of nicotinamide by E. meliloti 1021 and over-expressed NAMase in E. coli Rosetta（DE3）,and this was not a substrate inhibition.

Key words: Ensifer meliloti 1021, resting conversion, NAMase, enzymatic properties

GUO Jing-jing, GUO Lei-lei, ZHAO Yun-xiu, DAI Yi-jun. Research on the NAMase of Ensifer meliloti 1021 and Regulation Mechanism of 3-Cyanopyridine[J]. Biotechnology Bulletin, 2019, 35(8): 51-58.

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