Abstract
The nitrile hydratase (NHase, EC 4.2.1.84) genes (α and β subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (α subunit, Mr 23 kDa) and 218 amino acids (β subunit, Mr 24 kDa) and the NHase activator of 413 amino acids (Mr 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity was observed for aromatic compounds only with E values ranging 5–17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30°C and showed the highest stability at 4°C in thermal inactivation studies between 4°C and 50°C.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Banerjee A, Sharma R, Banerjee UC (2002) The nitrile-degrading enzymes: current status and future prospects. Appl Microbiol Biotechnol 60:33–44
Bornscheuer UT, Kazlauskas RJ (2006) Hydrolases in organic synthesis. Wiley, Weinheim
Chen CS, Fujimoto Y, Girdaukas G, Sih CJ (1982) Quantitative analyses of biochemical kinetic resolutions of enantiomers. J Am Chem Soc 104:7294–7299
Gilligan T, Yamada H, Nagasawa T (1993) Production of S-(+)-2-phenylpropionic acid from (R, S)-2-phenylpropionitrile by the combination of nitrile hydratase and stereoselective amidase in Rhodococcus equi TG328. Appl Microbiol Biotechnol 39:720–725
Gish W, States DJ (1993) Identification of protein coding regions by database similarity search. Nat Genet 3:266–272
Kobayashi M, Nagasawa T, Yamada H (1992) Enzymatic synthesis of acrylamide—a success story not yet over. Trends Biotechnol 10:402–408
Nagasawa T, Nanba H, Ryuno K, Takeuchi K, Yamada H (1987) Nitrile hydratase of Pseudomonas chlororaphis B23—purification and characterization. Eur J Biochem 162:691–698
Nojiri M, Yohda M, Odaka M, Matsushita Y, Tsujimura M, Yoshida T, Dohmae N, Takio K, Endo I (1999) Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine. J Biochem 125:696–704
Ochman H, Ajioka JW, Garza D, Hartl DL (1990) Inverse polymerase chain reaction. Biotechnology (N Y) 8:759–760
Okamoto S, Eltis LD (2007) Purification and characterization of a novel nitrile hydratase from Rhodococcus sp. RHA1. Mol Microbiol 65:828–838
Pitcher DG, Saunders NA, Owen RJ (1989) Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8:151–156
Prepachalova I, Martinkova L, Stolz A, Ovesna M, Bezouska K, Kopecky J, Kren V (2001) Purification and characterization of the enantioselective nitrile hydratase from Rhodococcus equi A4. Appl Microbiol Biotechnol 55:150–156
Shaw NM, Robins KT, Kiener A (2003) Lonza: 20 years of biotransformations. Adv Synth Catal 345:425–435
Song LY, Wang MX, Yang X, Qua A (2007) Purification and characterization of the enantioselective nitrile hydratase from Rhodococcus sp. AJ270. Biotechnol J 2:717–724
Song LY, Yuan HJ, Coffey L, Doran J, Wang MX, Qian SJ, O'Reilly C (2008) Efficient expression in E. coli of an enantioselective nitrile hydratase from Rhodococcus erythropolis. Biotechnol Lett 30:755–762
Stevens JM, Saroja NR, Jaouen M, Belghazi M, Schmitter JM, Mansuy D, Artaud I, Sari MA (2003) Chaperone-assisted expression, purification, and characterization of recombinant nitrile hydratase NI1 from Comamonas testosteroni. Protein Expr Purif 29:70–76
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Wu S, Fallon RD, Payne MS (1997) Over-production of stereoselective nitrile hydratase from Pseudomonas putida 5B in Escherichia coli: activity requires a novel downstream protein. Appl Microbiol Biotechnol 48:704–708
Yamada H, Shimizu S, Kobayashi M (2001) Hydratases involved in nitrile conversion: screening, characterization and application. Chem Rec 1:152–161
Author information
Authors and Affiliations
Corresponding author
Additional information
Kamila Rzeznicka and Sebastian Schätzle contributed equally to this work.
Rights and permissions
About this article
Cite this article
Rzeznicka, K., Schätzle, S., Böttcher, D. et al. Cloning and functional expression of a nitrile hydratase (NHase) from Rhodococcus equi TG328-2 in Escherichia coli, its purification and biochemical characterisation. Appl Microbiol Biotechnol 85, 1417–1425 (2010). https://doi.org/10.1007/s00253-009-2153-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-009-2153-y