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| Purification,Crystallization and Characterization of a Nitrile Hydratase from Rhodococcus ruber CGMCC3090 Strain |
| WANG Shi-wei1, WANG Min2, WANG Qing-hui1 |
1. College of Life Science of Qiqihar University, Qiqihar 161006, China;
2. Key Laboratory of Fermentation Microbiology, Tianjin University of Science and Technology, Ministry of Education, Tianjin 300457, China |
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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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Received: 27 June 2017
Published: 25 October 2017
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