Purification,Crystallization and Characterization of a Nitrile Hydratase from <i>Rhodococcus ruber</i> CGMCC3090 Strain

doi:10.13523/j.cb.20171006

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China Biotechnology ›› 2017, Vol. 37 ›› Issue (10) : 42-52. DOI: 10.13523/j.cb.20171006

Purification,Crystallization and Characterization of a Nitrile Hydratase from Rhodococcus ruber CGMCC3090 Strain

WANG Shi-wei¹, WANG Min², WANG Qing-hui¹

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Abstract

Rhodococcus ruber CGMCC3090 was separated from soil polluted by nitriles at Tianjin suburb.The strain can transform nitriles into correspondent amides with broad substrate spectra and higher catalytic activity, stronger substrate tolerance, good regioselectivity especially to dinitrile substrates (e.g. adiponitrile).The new type of NHase was purified to homogenety after sonication, ammonium sulfate fractionation, hydrophobic, ion exchange and gel-filtration column chromatography. About 17.14-fold purification was achieved with 26.2% yield. The holoenzyme purified (Mr 105kDa;α₂β₂) consists of α subunit (Mr 24.3 kDa) and β subunit (Mr 28 kDa) respectively. The optimal temperature and pH was 30℃ and pH 7.5 respectively.The enzyme purified was inhibited or promoted by different metals in various degrees. The Km and Vmax values of purified NHase respectively were 178.8 mM and 209.1 μmole/min/mg protein using 3-cyanopyridine as substrate.The nitrile hydratase crystallization conditions were screened and optimized by using 6 crystallization reagents screened from 194 reagents from 4 set Kit(Hampton Research).A single crystal from the NHase was obtained at the optimum conditions(15 mg protein/ml,16℃,pH 7.5,30 d)by using 112-34# reagent (0.05mol/L cadmium sulfate hydrate,0.1mol/L HEPS and 1.0mol/L sodium acetate trihydrate). the Nitrile hydratase single crystal had a 3.7Å resolution by X ray diffraction analysis.It paved the way for in-depth study of the structure and function of the nitrile hydratase from Rhodococcus ruber CGMCC3090 strain.

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Rhodococcus ruber / Nitrile hydratase / Purification / Characterization / Crystallization

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WANG Shi-wei, WANG Min, WANG Qing-hui. Purification,Crystallization and Characterization of a Nitrile Hydratase from Rhodococcus ruber CGMCC3090 Strain[J]. China Biotechnology, 2017, 37(10): 42-52 https://doi.org/10.13523/j.cb.20171006

References

[1] Prasad S, Misra A, Jangir V P, et al. A propionitrile-induced nitrilase of Rhodococcus sp. NDB 1165 and its application in nicotinic acid synthesis. World Journal of Microbiology and Biotechnology,2007, 23(3):345-353.
[2] Cantarella L, Gallifuoco A, Malandra A, et al. High-yield continuous production of nicotinic acid via nitrile hydratase-amidase cascade reactions using cascade CSMRs. Enzyme and Microbial Technology, 2011,48(4-5):345-350.
[3] Pratush A, Seth A, Bhalla T C.Cloning, sequencing, and expression of nitrile hydratase gene of mutant 4D strain of Rhodococcus rhodochrous PA 34 in E. coli. Applied Biochemstry Biotechnology, 2012,168(3):465-486.
[4] Prasad S, Bhalla T. Nitrile hydratases (NHases):At the interface of academia and industry. Biotechnology Advances, 2010, 28(6):725-741.
[5] Sharma M, Sharma N N, Bhalla TC. Amidases:versatile enzymes in nature. Rev Environ Sci Biotechnol, 2009, 8(4):343-366.
[6] Ma Y, Yu H, Pan W, et al. Identification of nitrile hydratase-producing Rhodococcus ruber TH and characterization of an amiE-negative mutant. Bioresource Technology,2010,101(1):285-291.
[7] Jun Y, Wang Z Q, Liu R C, et al. Screening, cultivation, and biocatalytic performance of Rhodococcus boritolerans FW815 with strong 2,2-dimethylcyclopropanecarbonitrile hydratase activity.Journal of Industrial Microbiology & Biotechnology,2012, 39(3):409-417.
[8] Zhang J L, Wang M, Sun H, et al. Isolation and characterization of Rhodococcus ruber CGMCC3090 that hydrolyzes aliphatic, aromatic and heterocyclic nitriles. African Journal of Biotechnology, 2009, 8(20):5467-5486.
[9] Zhong L P, Zhang J L, Song Y, et al. Study on screening, identification and culture conditions of a strain with nitrile hydratase. Journal of Tianjin University of Science and Technology, 2008, 23(4):35-39.
[10] Zhang J L, Wang M, Zhong L P, et al.Identification and study on enzymic activity of Rhodococcus sp.TCCC 28001 with nitrile hydratase activity. Journal of Shandong Agricultural University (Natural Science), 2010, 41(1):11-16.
[11] Song Y, Zhong L P, Zhang J L, et al. Effect of metal ions on Rhrodococcus sp. nitrile hydratase activity. Journal of Tianjin University of Science and Technology, 2009, 24(1):11-14,19.
[12] Wang S W, Wang M, Wang Q H. Breeding of NHase hyper-producing Rhodococcus ruber strain LUV30-06 and verification of mutants by RAPD. American Journal of Molecular Biology, 2013, 3(2):108-114.
[13] Wang S W, Wang Q H, Zhai Y, et al. Screening of NHase high-producing Rhodococcus ruber strain TQD-58 by both parents inactivated protoplast fusion. 2012 International Symposium on Information Technology in 279 Medicine and Education (ITME2012), Hokkaido, Japan.2012, 737-740.
[14] Zhang J L, Wang M, Zhong L P, et al. Identification and study on enzymic activity of Rhodcoccus sp.TCCC28001 with nitrile hydratase activity. Journal of Shangdong Agricultural University (Natural science), 2010,41(1):11-16.
[15] Shen Y B, Wang M, Li X D, et al.Highly efficient synthesis of 5-cyanovaleramide by Rhodococcus ruber CGMCC3090 resting cells.Journal of Chemical Technology and Biotechnology, 2012, 87(10):1396-1400.
[16] Zhai Y, Shen Y B, Tang R, et al.Biotransformation of cinnamonitrile into cinnamamide with Rhodococcus ruber CGMCC3090.Journal of Tianjin University of Science and Technology, 2014, 29(4):11-15.
[17] Wang S W, Dai Y J, Wang J X, et al.Molecular insights into substrate specificity of Rhodococcus ruber CGMCC3090 by gene cloning and homology modeling.Enzyme and Microbial Technology, 2013, 52(2):111-117.
[18] Wang S W, Wang M. Research process on microbial diversity of producing-nitrile hydratase and study on nitrile hydratase. China Biotechnology, 2011, 31(9);117-123.
[19] Davis B, DISC J.Electrophoresis-Ⅱ. method and application to human serum proteins.Annals of the New York Academy of Sciences, 1964, 121(2):404-427.
[20] Laemmli U K.Cleavage of structural protein during the assembly of the head of bacteriophage T4.Nature, 1970, 227:680-685.
[21] Okamoto S, Eltis L D.Purification and characterization of a novel nitrile hydratase from Rhodococcus sp. RHA1.Molecular Microbiology, 2007, 65(3):828-838.
[22] Prasad S, Raj J, Bhalla T C.Purification of a hyperactive nitrile hydratase from resting cells of Rhodococcus rhodochrous PA-34.Indian J Microbiol, 2009, 49(3):237-242.
[23] Wang S W, Wang Q H, Wang W Y, et al. Research process on protein purification of nitrile hydratase. Journal of Science of Teachers College and University, 2013, 33(1):50-53.
[24] Kamila Rzeznicka, Sebastian Schätzle, Dominique Böttcher, et al.Cloning and functional expression of a nitrile hydratase(NHase)from Rhodococcus equi TG328-2 in Escherichia coli, its purification and biochemical characterization.Appl Microbiol Biotechno, 2010, 85(5):1417-1425.
[25] Cramp R A, Cowan D A. Molecular characterization of a novel thermophilic nitrile hydratase.Biochem Biophys Acta,1999, 1431(1):249-260.
[26] Li Q J,Liu Z Q,Zhang Y G,et al.Identification and characterization of Serratia marcescens ZJB-09104, a nitrile-converting bacterium.World Journal of Microbiology and Biotechnology,2010, 26, (5):817-823.
[27] Okamoto S, Eltis L D. Purification and characterization of a novel nitrile hydratase from Rhodococcus sp. RHA 1. Mol Microbiol, 2007, 65(3):828-838.
[28] Wieser M, Takeuchi K, Wada Y, et al.Low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1:purification, substrate specificity and comparison with the analogous high-molecular-mass enzyme.FEMS Microbiology Letters, 1998, 169(1):17-22.
[29] Mauger J, Nagasawa T, Yamada H. Synthesis of various aromatic amide derivatives using nitrile hydratase of Rhodococcus rhodochrous J1.Tetrahedron, 1989, 45(5):1347-1354.
[30] Mauger J, Nagasawa T, Yamada H. Nitrile hydratase-catalyzed production of isonicotinamide, picolinamide and pyrazineamide from 4-cyanopyridine, 2-cyanopyridine and cyanopyrazine in Rhodococcus rhodochrous J1.Journal of Biotechnology,1988, 8(1):87-95.