Bioconversion of indole-3-acetonitrile by the N2-fixing bacterium Ensifer meliloti CGMCC 7333 and its Escherichia coli-expressed nitrile hydratase


An N2-fixing bacterium, Ensifer meliloti CGMCC 7333, has been reported to degrade the cyano-containing neonicotinoid insecticides acetamiprid and thiacloprid using a nitrile hydratase (NHase). Here, the bioconversion of indole-3-acetonitrile (IAN) by E. meliloti, Escherichia coli overexpressing the NHase, and purified recombinant NHase was studied. E. meliloti converted IAN to the product indole-3-acetamide (IAM), and no nitrilase or amidase activities, or indole-3-acetic acid formation, were detected. Whole cells of E. meliloti converted IAN from the initial content of 6.41 to 0.06 mmol/L in 48 h. Meanwhile, forming 5.99 mmol/L IAM, the molar conversion of 94.4%. E. coli Rosetta overexpressing the NHase from E. meliloti produced 4.46 mmol/L IAM in 5 min, with a conversion rate of 91.1%. The purified NHase had a Vmax for IAN conversion of 294.28 U/mg. Adding 2% and 10% (v/v) dichloromethane to 50 mmol/L sodium phosphate buffer containing 200 mg/L IAN increased the NHase activity by 26.8% and 11.5% respectively, while the addition of 20% hexane had no inhibitory effect on IAN bioconversion. E. meliloti shows high NHase activity without forming a byproduct carboxylic acid, and its tolerance of dichloromethane and hexane increases its potential for application in the green biosynthesis of high-value amide compounds.

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  1. Asano Y, Fujishiro K, Tani Y, Yamada H (1982) Aliphatic nitrile hydratase from Arthrobacter sp. J-1 purification and characterization. Agric Biol Chem 46:1165–1174

    CAS  Google Scholar 

  2. Banerjee A, Sharma R, Banerjee UC (2002) The nitrile-degrading enzymes: current status and future prospects. Appl Microbiol Biotechnol 60:33–44.

    CAS  Article  PubMed  Google Scholar 

  3. Bauer R, Knackmuss H-J, Stolz A (1998) Enantioselective hydration of 2-arylpropionitriles by a nitrile hydratase from Agrobacterium tumefaciens strain d3. Appl Microbiol Biotechnol 49:89–95

    CAS  Article  Google Scholar 

  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    CAS  Article  Google Scholar 

  5. Cramp RA, Cowan DA (1999) Molecular characterisation of a novel thermophilic nitrile hydratase. Biochim Biophys Acta Protein Struct Mol Enzymol 1431:249–260

    CAS  Article  Google Scholar 

  6. de Bont JA (1998) Solvent-tolerant bacteria in biocatalysis. Trends Biotechnol 16:493–499

    Article  Google Scholar 

  7. Duca D, Lorv J, Patten CL, Rose D, Glick BR (2014) Indole-3-acetic acid in plant-microbe interactions. Antonie Van Leeuwenhoek 106:85–125.

    CAS  Article  PubMed  Google Scholar 

  8. Ge F, Zhou LY, Wang Y, Ma Y, Zhai S, Liu ZH, Dai YJ, Yuan S (2014) Hydrolysis of the neonicotinoid insecticide thiacloprid by the N2-fixing bacterium Ensifer meliloti CGMCC 7333. Int Biodeterior Biodegradation 93:10–17.

    CAS  Article  Google Scholar 

  9. Holtze MS, Sorensen SR, Sorensen J, Aamand J (2008) Microbial degradation of the benzonitrile herbicides dichlobenil, bromoxynil and ioxynil in soil and subsurface environments--insights into degradation pathways, persistent metabolites and involved degrader organisms. Environ Pollut 154:155–168.

    CAS  Article  PubMed  Google Scholar 

  10. Kamble AL, Banoth L, Meena VS, Singh A, Chisti Y, Banerjee UC (2012) Nitrile hydratase of Rhodococcus erythropolis: characterization of the enzyme and the use of whole cells for biotransformation of nitriles. 3 Biotech 3:319–330.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Kaminskaia NV, Ullmann GM, Fulton DB, Kostic NM (2000) Spectroscopic, kinetic, and mechanistic study of a new mode of coordination of indole derivatives to platinum (II) and palladium (II) ions in complexes. Inorg Chem 39:5004–5013

    CAS  Article  Google Scholar 

  12. Kobayashi M, Suzuki T, Fujita T, Masuda M, Shimizu S (1995) Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium. Proc Natl Acad Sci 92:714–718

    CAS  Article  Google Scholar 

  13. Kobayashi M, Fujita T, Shimizu S (1996) Hyperinduction of nitrile hydratase acting on indole-3-acetonitrile in Agrobacterium tumefaciens. Appl Microbiol Biotechnol 45:176–181

    CAS  Article  Google Scholar 

  14. Laane C, Boeren S, Vos K, Veeger C (1987) Rules for optimization of biocatalysis in organic solvents. Biotechnol Bioeng 30:81–87

    CAS  Article  Google Scholar 

  15. Mathew CD, Nagasawa T, Kobayashi M, Yamada H (1988) Nitrilase-catalyzed production of nicotinic acid from 3-cyanopyridine in Rhodococcus rhodochrous J1. Appl Environ Microbiol 54:1030–1032

    CAS  Article  Google Scholar 

  16. Nagasawa T, Kobayashi M, Yamada H (1988) Optimum culture conditions for the production of benzonitrilase by Rhodococcus rhodochrous J1. Arch Microbiol 150:89–94

    CAS  Article  Google Scholar 

  17. Nagasawa T, Nakamura T, Yamada H (1990) ε-Caprolactam, a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase. Arch Microbiol 155:13–17

    CAS  Article  Google Scholar 

  18. Oelgen S, Coban T (2002) Synthesis and antioxidant properties of novel N-substituted indole-2-carboxamide and indole-3-acetamide derivatives. Arch Pharm 335:331–338

    Article  Google Scholar 

  19. Okamoto S, Eltis LD (2007) Purification and characterization of a novel nitrile hydratase from Rhodococcus sp. RHA1. Mol Microbiol 65:828–838

    CAS  Article  Google Scholar 

  20. Vega-Hernández MC, León-Barrios M, Pérez-Galdona R (2002) Indole-3-acetic acid production from indole-3-acetonitrile in Bradyrhizobium. Soil Biol Biochem 34:665–668

    Article  Google Scholar 

  21. Wang Y-J, Zheng Y-G, Xue J-P, Shen Y-C (2006) Microbial transformation of indole-3-acetonitrile to indole-3-acetamide by Nocardia sp. 108. Process Biochem 41:1746–1750.

    CAS  Article  Google Scholar 

  22. Zhou LY, Zhang LJ, Sun SL, Ge F, Mao SY, Ma Y, Liu ZH, Dai YJ, Yuan S (2014) Degradation of the neonicotinoid insecticide acetamiprid via the N-carbamoylimine derivate (IM-1-2) mediated by the nitrile hydratase of the nitrogen-fixing bacterium Ensifer meliloti CGMCC 7333. J Agric Food Chem 62:9957–9964.

    CAS  Article  PubMed  Google Scholar 

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The authors would like to thank James Allen, DPhil, from Liwen Bianji, Edanz Group China (, for editing the English text of a draft of this manuscript.


This research was financed by the National Science Foundation of China (grant number 31570104), the Program for Jiangsu Excellent Scientific and Technological Innovation Team (17CXTD00014), and the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.

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Correspondence to Yi-Jun Dai.

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Zhao, YX., Guo, LL., Sun, SL. et al. Bioconversion of indole-3-acetonitrile by the N2-fixing bacterium Ensifer meliloti CGMCC 7333 and its Escherichia coli-expressed nitrile hydratase. Int Microbiol 23, 225–232 (2020).

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  • Dichloromethane
  • Ensifer meliloti
  • Nitrile hydratase
  • Indole-3-acetonitrile
  • Indole-3-acetamide